Wearable SERS (Surface Enhanced Raman Scattering) sensing / medicine carrying patch for diabetic foot ulcer wound surface

Abstract

The invention relates to a wearable SERS (Surface Enhanced Raman Scattering) sensing / drug-loading patch for diabetic foot ulcer wounds, which comprises a flexible transparent substrate, a sensing area and a drug-loading area are arranged on the surface of one side of the flexible transparent substrate, and an active oxygen response group of an active oxygen response ratio type SERS nanoprobe contained in the sensing area has a Raman characteristic peak of which the intensity is reduced along with the increase of the active oxygen concentration; the medicine carrying area comprises a coaxial electrostatic spinning film, a shell layer of the coaxial electrostatic spinning film is made of a near-infrared light response type photo-thermal melting material, and the coaxial electrostatic spinning film can absorb light energy under irradiation of near-infrared light and convert the light energy into heat energy, so that the shell layer is melted and broken; the core layer comprises a water-soluble biodegradable matrix and an active oxygen scavenger dispersed in the water-soluble biodegradable matrix. When it is detected that the active oxygen concentration of the diabetic foot ulcer wound surface exceeds a threshold value, near-infrared light irradiation is started to enable the shell layer of the coaxial electrostatic spinning membrane to be quantitatively fused, the needed release amount of the active oxygen scavenger is released, and therefore the active oxygen concentration is controlled within the range of 20-100 micromole / liter.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a wearable surface-enhanced Raman scattering (SERS) sensing / drug-loaded patch for diabetic foot ulcer wounds. Background Technology

[0002] Diabetic foot ulcer (DFU) is a serious complication of diabetes. Its healing is a dynamic and orderly physiological process, including four consecutive stages: hemostasis, inflammation, cell proliferation, and tissue remodeling. Reactive oxygen species (ROS) concentrations in the range of 20–100 μmol / L have a beneficial effect on promoting DFU wound healing. However, the excessive oxidative stress induced by the high-glucose microenvironment of DFU produces excessive ROS, causing impaired cell and cytokine function and leading to DFU stagnation in the inflammatory stage.

[0003] Traditional wound dressings primarily provide physical protection and basic care, such as antibacterial dressings containing silver ions, alginate dressings, or hydrogel dressings. Their main function is to absorb exudate, prevent infection, or maintain wound moisture.

[0004] Existing wound patches based on the principle of scavenging reactive oxygen species can temporarily relieve inflammatory responses caused by oxidative stress, but they are prone to excessively reducing reactive oxygen species, which is not conducive to the healing of diabetic foot ulcers in the long run. Summary of the Invention

[0005] To address the issue that wound patches based on the principle of scavenging reactive oxygen species (ROS) can easily over-reduce ROS, this invention provides a wearable SERS-sensing / drug-loaded patch for diabetic foot ulcers. This patch can adjust the release amount of ROS scavenger according to the concentration of ROS in the diabetic foot ulcer wound, thereby controlling ROS within a certain range and accelerating wound healing.

[0006] The technical solution provided by this invention is as follows:

[0007] In a first aspect, the present invention provides a wearable SERS sensing / drug-loaded patch for diabetic foot ulcer wounds, comprising a flexible transparent substrate, wherein a sensing region and a drug-loaded region are disposed on one side surface of the flexible transparent substrate; wherein:

[0008] The sensing region includes a reactive oxygen species (ROS) responsive ratiometric SERS nanoprobe, which is composed of gold nanoparticles and Raman signal molecules modified on their surface. The Raman signal molecules include:

[0009] Reactive oxygen species responsive groups, which have Raman characteristic peaks whose intensity decreases with increasing reactive oxygen species concentration;

[0010] Anchoring groups, which are stably bound to the surface of gold nanoparticles through covalent or coordination bonds;

[0011] The drug-loading region is an annular region surrounding the sensing region, and includes a coaxial electrospun membrane composed of coaxial fibers. The coaxial fibers have the following characteristics:

[0012] The shell is composed of a near-infrared light-responsive photothermal melting material, which can absorb light energy and convert it into heat energy under near-infrared light irradiation, causing the shell to melt and crack.

[0013] The core layer comprises a water-soluble biodegradable matrix and an active oxygen scavenger dispersed therein. After the shell is ruptured, the core layer releases the active oxygen scavenger through the dissolution of the water-soluble matrix.

[0014] In conjunction with the first aspect of the present invention, in some embodiments, the gold nanoparticles have a particle size of 30-60 nm; and / or,

[0015] The reactive oxygen species responsive group is one of the following: borate group, borate ester group, carboxyl group, and alkynyl group; and / or,

[0016] The anchoring group is one or more of thiol, diazo group, and selenol.

[0017] In conjunction with the first aspect of the present invention, in some embodiments, the near-infrared light-responsive photothermal melting material comprises:

[0018] The substrate is a biocompatible flexible polymer material;

[0019] Photothermal conversion components can absorb near-infrared light and convert it into heat energy;

[0020] Phase change components can melt after absorbing thermal energy;

[0021] The photothermal conversion component has a mass percentage of 0.5% to 2.0%, and the phase change component has a mass percentage of 1.5% to 5.5%.

[0022] In conjunction with the first aspect of the present invention, in some embodiments, the substrate is one or more selected from polyurethane, polyvinylidene fluoride, polyacrylonitrile, polyether-block-amide, styrene-butadiene-styrene block copolymer, polycarbonate-based polyurethane, non-isocyanate polyurethane, polyhydroxyalkanoate, polyvinyl alcohol, polyetheretherketone, and polyethersulfone; and / or,

[0023] The photothermal conversion component is one or more of the following: polydopamine, graphene / graphene oxide, carbon black, polypyrrole, gold nanorods, and iron(III) oxide@tannic acid nanoparticles; and / or,

[0024] The phase change component is one or more of the following: lauric acid, decanoic acid, myristic acid, palmitic acid, stearic acid, paraffin, polyethylene glycol, n-octadecane, hexadecane, soybean wax, palm wax, erythritol, mannitol, sodium sulfate decahydrate or its hydrated salt, and calcium chloride hexahydrate or its hydrated salt.

[0025] In conjunction with the first aspect of the present invention, in some embodiments, the water-soluble biodegradable matrix is ​​one or more selected from polyvinylpyrrolidone, polyvinyl alcohol, polyethylene oxide, hydroxypropyl methylcellulose, gelatin, chitosan, polyethylene glycol, and copovidone; and / or,

[0026] The active oxygen scavenger is one or more of the following: 1,3-dimethylthiourea, curcumin, epigallocatechin gallate, tannic acid, glutathione, metformin, superoxide dismutase, catalase, cerium dioxide, and manganese dioxide.

[0027] In conjunction with the first aspect of the present invention, in some embodiments, the diameter of the coaxial fiber is 0.6 to 2.2 micrometers, the diameter of the core layer is 0.1 to 1.6 micrometers, and the thickness of the shell layer is 0.1 to 0.8 micrometers.

[0028] In conjunction with the first aspect of the invention, in some embodiments, the thickness of the wearable SERS sensing / drug-loaded patch is 50-200 micrometers; and / or,

[0029] The size of the sensing area is 2~6mm; and / or,

[0030] The width of the drug-loaded region extending outward from the outer periphery of the sensing region is 4-8 mm.

[0031] Secondly, the present invention provides a sensing and treatment system for diabetic foot ulcer wounds, comprising:

[0032] The Raman spectroscopy detection unit is used to detect the Raman spectral signal of the sensing area of ​​the wearable SERS sensor / drug-loaded patch, wherein the Raman spectral signal is the intensity of the Raman characteristic peak whose intensity decreases with increasing reactive oxygen species concentration and the intensity of the internal standard peak.

[0033] The calculation unit is used to calculate the near-infrared light irradiation time based on the Raman spectral signal;

[0034] A control unit is configured to generate and send near-infrared light irradiation control commands based on the irradiation time output by the calculation unit, the commands including the irradiation duration;

[0035] The near-infrared light irradiation unit is used to receive control commands from the control unit and target the drug-loaded area of ​​the wearable SERS sensing / drug-loaded patch with near-infrared light irradiation.

[0036] The Raman spectroscopy detection unit, calculation unit, control unit, and near-infrared light irradiation unit are connected wirelessly and / or via wired means.

[0037] In conjunction with the second aspect of the present invention, in some embodiments, calculating the near-infrared light irradiation time based on the Raman spectral signal includes:

[0038] Calculate the intensity ratio of Raman characteristic peaks whose intensity decreases with increasing reactive oxygen species concentration to that of the internal standard peak;

[0039] The current reactive oxygen species concentration of the diabetic foot ulcer wound is calculated based on the intensity ratio and the preset standard curve.

[0040] Calculate the required release amount of reactive oxygen species scavenger based on the difference between the current reactive oxygen species concentration and the target reactive oxygen species concentration;

[0041] The required irradiation time is determined based on the release amount.

[0042] In conjunction with the second aspect of the present invention, in some embodiments, the irradiation wavelength of the near-infrared irradiation unit is 800~1000 nm.

[0043] Compared with the prior art, the present invention has at least the following beneficial effects:

[0044] This invention achieves quantitative detection of reactive oxygen species (ROS) by detecting the change in the intensity ratio of Raman characteristic peaks before and after ROS binding to a ratio-response SERS nanoprobe in the sensing area. When the ROS concentration in a diabetic foot ulcer exceeds a threshold (100 μM), near-infrared light irradiation is initiated to quantitatively melt the shell of the coaxial electrospun membrane, releasing the required amount of ROS scavenger, thereby controlling the ROS concentration within the range of 20~100 μM and accelerating wound healing. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 This is a route diagram for synthesizing the Raman signal molecule (compound 2) in Example 1 of the present invention.

[0047] Figure 2 The above is the 1H NMR spectrum of compound 1 prepared in Example 1 of this invention.

[0048] Figure 3The nuclear magnetic resonance hydrogen spectrum of compound 2 prepared in Example 1 of this invention.

[0049] Figure 4 This is a scanning electron microscope image of the reactive oxygen species-responsive ratio SERS nanoprobe prepared in Example 2 of the present invention.

[0050] Figure 5 The images show the zeta potential, UV absorption spectrum, and hydration particle size distribution of the reactive oxygen species (ROS) responsive ratio SERS nanoprobe prepared in Example 2 of this invention before and after modification, where: a is the zeta potential, b is the UV absorption spectrum, and c is the hydration particle size distribution of the ROS responsive ratio SERS nanoprobe.

[0051] Figure 6 The mechanical properties of polyurethane nanofiber membranes of different thicknesses prepared in Example 3 are shown in Figures a through d, which are tensile stress-strain cycle diagrams of polyurethane nanofiber membranes with electrospinning times of 2h, 4h, 6h, and 8h, respectively. Figure e is the force-deformation curve of polyurethane nanofiber membranes prepared with different electrospinning times.

[0052] Figure 7 The results of the unidirectional moisture conduction test of polyurethane nanofiber membranes of different thicknesses prepared in Example 3 (flowing from the inside to the outside) are shown in a~d. The graphs are the changes in water content of polyurethane nanofiber membranes with electrospinning time of 2h, 4h, 6h and 8h respectively. The graph is the quantitative graph of unidirectional moisture conduction of polyurethane nanofiber membranes prepared with different electrospinning time.

[0053] Figure 8 The image shows the Raman spectra of the sensor patches prepared for different electro-spraying times in Example 4.

[0054] Figure 9 This is a scanning electron microscope image of the sensing patch prepared in Example 4 of the present invention.

[0055] Figure 10 The figures shown are Raman spectra, SERS intensity versus logarithmic concentration of hydrogen peroxide ratio, and specificity test results of the sensing patch prepared in Example 5 of this invention after responding to different concentrations of hydrogen peroxide. Figure a shows the Raman spectra of the sensing patch after responding to different concentrations of hydrogen peroxide, and figure b shows the results of I... 999 / I 1081 The curve showing the relationship between the concentration of hydrogen peroxide solution and the logarithmic concentration of the solution, with c representing the specificity test plot.

[0056] Figure 11 Figure 1 shows the characterization of the therapeutic patch prepared in Example 6. Figure 2a is a scanning electron microscope image of the drug-loaded patch, Figure 3b is a histogram of fiber diameter distribution of the drug-loaded patch, and Figure 4c is a transmission electron microscope image of the coaxial fibers of the drug-loaded patch.

[0057] Figure 12The images show the relationship between 808 nm laser irradiation time and temperature and the relationship between 808 nm laser irradiation time and drug release for the treatment patch in Example 7. Image a shows the relationship between irradiation time and temperature for a laser with an excitation wavelength of 808 nm, and image b shows the relationship between irradiation time and cumulative drug release for a laser with an excitation wavelength of 808 nm.

[0058] Figure 13 The images shown are from Example 7, which depict intracellular antioxidant assays and cytotoxicity assays of the sensing / therapeutic patch. Image a shows the intracellular antioxidant assay, and image b shows the cytotoxicity assay.

[0059] Figure 14 This is a wound healing diagram of the sensor / therapeutic patch applied to the foot ulcer lesions of diabetic mice in Example 8.

[0060] Figure 15 This is a Raman spectral image taken on day 7 after applying the sensing / treatment patch to the paw ulcers of diabetic mice in Example 8. a) Sensing / treatment patch + near-infrared light group; b) 1,3-dimethylthiourea group; c) Sensing / treatment patch group; d) Day 7 after different treatments. 999 / I 1081 The changes (due to the high initial hydrogen peroxide concentration, the sensing / treatment patch was replaced at 0 and 6 hours, and two near-infrared irradiations were performed).

[0061] Figure 16 This section presents a histological analysis of skin tissue collected from paw ulcer wounds in diabetic mice after different treatments using a sensing / treatment patch, as described in Example 8. a) Representative HE staining results of wound tissue sections from different groups on days 7 and 13 post-injury; b) Quantitative epithelial thickness graph; c) Quantitative granulation tissue thickness graph (the lengths of the green and blue lines represent epithelial and granulation tissue thickness, and the yellow triangular arrows represent hair follicles); d) Representative Masson staining results from different groups on days 7 and 13; e) Quantitative results of the corresponding collagen volume fraction. Data are expressed as mean ± SD.

[0062] Figure 17 The images shown are of the heart, liver, spleen, lungs, kidneys, and other organs of diabetic mice euthanized on day 21 after the sensor / treatment patch was applied to the foot ulcer wound in Example 8.

[0063] Figure 18The images shown are immunofluorescence images of the sensor / treatment patch applied to the foot ulcers of diabetic mice in Example 8. a is DAPI (4,6-diamidinyl-2-phenylindole dihydrochloride) / hydrogen peroxide staining, c is DAPI / TNF-α (tumor necrosis factor-α) staining, e is DAPI / VEGF (vascular endothelial growth factor) staining, b is the quantitative statistical result of the percentage of hydrogen peroxide, d is the quantitative statistical result of tumor necrosis factor-α, and f is the quantitative statistical result of the percentage of vascular endothelial growth factor. Detailed Implementation

[0064] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0065] Although it is known that low levels of reactive oxygen species (ROS) in DFU wounds are detrimental to healing, there are still some challenges in the on-site analysis and treatment of DFU. For example, in terms of DFU analysis, the preparation process of electrochemical methods is complex and the signal is prone to drift; in terms of fluorescence spectroscopy, it is difficult to completely remove excess fluorescent tag molecules; and in terms of colorimetry, it is easily interfered with by endogenous colors such as blood (red). In terms of treatment, burst release of ROS scavenging components can lead to overtreatment, while sustained release can result in insufficient efficacy.

[0066] To address the above problems, this invention provides a wearable SERS sensing / drug-loaded patch for diabetic foot ulcers, comprising a flexible transparent substrate, wherein a sensing area and a drug-loaded area are disposed on one side surface of the flexible transparent substrate; wherein:

[0067] The sensing region includes a reactive oxygen species (ROS) responsive ratiometric SERS nanoprobe, which is composed of gold nanoparticles and Raman signal molecules modified on their surface. The Raman signal molecules include:

[0068] Reactive oxygen species responsive groups, which have Raman characteristic peaks whose intensity decreases with increasing reactive oxygen species concentration;

[0069] Anchoring groups, which are stably bound to the surface of gold nanoparticles through covalent or coordination bonds;

[0070] The drug-loading region is an annular region surrounding the sensing region, and includes a coaxial electrospun membrane composed of coaxial fibers. The coaxial fibers have the following characteristics:

[0071] The shell is composed of a near-infrared light-responsive photothermal melting material, which can absorb light energy and convert it into heat energy under near-infrared light irradiation, causing the shell to melt and crack.

[0072] The core layer comprises a water-soluble biodegradable matrix and an active oxygen scavenger dispersed therein. After the shell is ruptured, the core layer releases the active oxygen scavenger through the dissolution of the water-soluble matrix.

[0073] The sensing region of this patch contains a reactive oxygen species (ROS)-responsive ratiometric SERS nanoprobe with ROS-responsive groups exhibiting Raman characteristic peaks whose intensity decreases with increasing ROS concentration. The drug-loaded region comprises a coaxial electrospun membrane. The shell of this membrane is composed of a near-infrared light-responsive photothermal melting material, which absorbs light energy and converts it into heat energy under near-infrared light irradiation, causing the shell to melt and rupture. The core layer contains a water-soluble, biodegradable matrix and ROS scavengers dispersed within it. When the ROS concentration in a diabetic foot ulcer exceeds a threshold, near-infrared light irradiation is initiated to quantitatively melt the shell of the coaxial electrospun membrane, releasing the required amount of ROS scavengers, thereby controlling the ROS concentration within the range of 20–100 μmol / L. Using this patch can solve the problem of excessively low ROS concentration caused by the sudden release of ROS scavengers, leading to slow healing of DFU wounds.

[0074] This patch uses a flexible, transparent substrate. The transparency of the substrate allows the excitation light from the Raman spectroscopy detection unit to penetrate directly to the reactive oxygen species (ROS) ratiometric SERS nanoprobe in the sensing area, avoiding interference from absorption or scattering of the light signal by the substrate material. Furthermore, it facilitates clinical observation of wound healing. The flexibility of the substrate allows the sensing area to fit snugly against irregular foot ulcers, ensuring full contact between the ROS ratiometric SERS nanoprobe and wound exudate, avoiding false-negative test results caused by gaps.

[0075] The sensing region of this invention includes a reactive oxygen species (ROS)-responsive ratiometric SERS nanoprobe composed of gold nanoparticles and Raman signal molecules modified on their surface. The concentration of ROS in diabetic foot ulcer wounds is very low. Using gold nanoparticles as the SERS substrate, their surface plasmon resonance effect can amplify the Raman spectral signal intensity by more than a million times, enabling the detection of weak molecular vibrational signals and thus ensuring high sensitivity. The Raman signal molecules employ a dual-group design: the anchoring group (such as a thiol group) can stably bind to the surface of the gold nanoparticles through covalent bonds (such as Au-S bonds), ensuring detection stability; while the ROS-responsive group can specifically react with ROS in the wound microenvironment (such as H2O2, ·OH, and other inflammatory markers). As the concentration of ROS increases, the intensity of the Raman characteristic peak of the ROS-responsive group decreases; this Raman characteristic peak is named the ROS response peak. The Raman signal molecules also possess a Raman characteristic peak whose intensity does not change with the ROS concentration; this Raman characteristic peak is named the internal standard peak. When using this patch, the intensity of the reactive oxygen species response peak and the internal standard peak are detected simultaneously, and the concentration of reactive oxygen species can be calculated based on the intensity ratio of the two.

[0076] The coaxial fiber used in the drug-carrying region of this invention mainly consists of a shell layer and a core layer. The mechanism by which it releases reactive oxygen species (ROS) scavengers is closely related to the characteristics of each layer: the shell layer is composed of a near-infrared photoresponsive photothermal melting material, which is solid at room temperature and can effectively encapsulate the core layer, preventing premature leakage of the ROS scavengers. The core layer is composed of a water-soluble biodegradable matrix and ROS scavengers. The main function of the water-soluble biodegradable matrix is ​​to uniformly disperse the ROS scavenger molecules, preventing crystallization or aggregation, while its biodegradability avoids secondary damage to the body. When preparing the core layer using coaxial electrospinning, the water-soluble biodegradable matrix also provides spinnability and mechanical strength. The water solubility and degradability of the water-soluble biodegradable matrix prevent foreign body reactions with non-degradable materials. In some embodiments, the water-soluble biodegradable matrix is ​​one or more of polyvinylpyrrolidone, polyvinyl alcohol, polyethylene oxide, hydroxypropyl methylcellulose, gelatin, chitosan, polyethylene glycol, and copovidone.

[0077] When near-infrared light irradiates the drug-loaded area, the photothermal melting material of the shell absorbs the light energy and converts it into heat energy, raising the temperature above the melting point of the near-infrared light-responsive photothermal melting material, causing the shell to melt and rupture. As the shell melts and ruptures, the core layer dissolves upon contact with wound exudate, releasing reactive oxygen species scavengers that directly act on the ulcer wound. The longer the irradiation time, the larger the melting area of ​​the shell, and the greater the release of reactive oxygen species scavengers, thus achieving on-demand control of drug release.

[0078] In some embodiments of the present invention, the sensing region further includes a micro / nanofiber membrane for loading a reactive oxygen species (ROS) responsive ratio type (SERS) nanoprobe. In some embodiments of the present invention, a micro / nanofiber membrane is adhered to a flexible transparent substrate, and the ROS responsive ratio type SERS nanoprobe is loaded onto the micro / nanofiber membrane. Specifically, the ROS responsive ratio type SERS nanoprobe is sprayed onto the surface of the micro / nanofiber membrane using an electrostatic spraying method, and the micro / nanofiber membrane is then spread and adhered to the flexible transparent substrate. The micro / nanofiber membrane is preferably a polyurethane nanofiber membrane, and the polyurethane nanofiber membrane is preferably prepared by electrospinning.

[0079] In some embodiments of the present invention, the gold nanoparticles have a particle size of 30-60 nm. The surface plasmon resonance (SPR) effect of gold nanoparticles is the core mechanism for SERS signal enhancement, and the SPR peak position is directly related to the particle size. The SPR peak position of gold nanoparticles in this particle size range is typically located at 520-650 nm. Figure 5 b. The ultraviolet absorption spectrum shows a strong absorption peak at 520 nm, which is close to the excitation wavelength. The Raman spectral signal can be amplified by 10 nm through localized surface plasmon resonance (LSPR). 6 ~10 8 This ensures a high sensitivity response to low concentrations of reactive oxygen species (10 μM H2O2, Example 5).

[0080] In some embodiments, the reactive oxygen species (ROS) responding group is one of a borate group (-B(OH)2), a borate ester group, a carboxyl group, or an alkynyl group; the anchoring group is one or more of a thiol group, a diazo group, or a selenyl group. Preferably, the ROS responding group is a borate group or a borate ester group, and the anchoring group is a thiol group. When the anchoring group is a thiol group, the Raman signal molecule can not only stably bind to gold nanoparticles, but the intensity of the Raman characteristic peak of -CS- in the Raman signal molecule does not change with the concentration of ROS, and can be used as an internal standard peak. The Raman characteristic peak of -BO- has a high degree of separation from the Raman characteristic peak of -CS-, and the coexistence of the two in one Raman signal molecule can avoid spectral overlap and ensure ratiometric detection. In addition, the hydrolysis products of the borate group or the borate ester group are non-toxic small molecules that can be metabolized by the body. Further, the borate ester group is a pinacol borate ester group or a pinacol selenoborate ester group.

[0081] In some embodiments, the gold nanoparticles are one or more of the following: gold nanospheres, gold nanostars, gold nanorods, gold nanocubes, gold nanowires, and gold nanocages.

[0082] In some embodiments, the Raman signal molecule is one or more of 4-((2-mercaptoethyl)carbamoyl)phenylboronic acid, 4-mercaptophenylboronic acid, 4-mercaptophenylboronic acid pinacol ester, selenophenylboronic acid pinacol ester, 4-mercaptobenzoic acid, and 4-diazobenzyne.

[0083] In some embodiments of the present invention, 4-((2-mercaptoethyl)carbamoyl)phenylboronic acid is used as the Raman signal molecule, and gold nanospheres (AuNPs) with an average particle size of 50 nm are used as gold nanoparticles. The thiol groups are bonded to AuNPs by -S-Au- bonds, and the reactive oxygen species response peak is located at 999 cm⁻¹. -1 At this location, the Raman characteristic peak belongs to the symmetrical stretching of the -BO- bond, and its intensity gradually decreases with increasing reactive oxygen species concentration; the internal standard peak is located at 1081 cm⁻¹. -1 At this point, a Raman characteristic peak representing -CS- bond stretching is observed, and its intensity remains essentially constant with increasing reactive oxygen species concentration. Based on the intensity ratio I... 999 / I 1081 The concentration of reactive oxygen species in the wound can be calculated.

[0084] In some embodiments, the near-infrared light-responsive photothermal melting material comprises:

[0085] The substrate is a biocompatible flexible polymer material;

[0086] Photothermal conversion components can absorb near-infrared light and convert it into heat energy;

[0087] Phase change components can melt after absorbing thermal energy;

[0088] The photothermal conversion component has a mass percentage of 0.5% to 2.0%, and the phase change component has a mass percentage of 1.5% to 5.5%.

[0089] Near-infrared responsive photothermal melting materials are synergistically composed of a substrate, a photothermal conversion component, and a phase change component. The substrate is made of a biocompatible flexible polymer material, ensuring the safety of the shell in a biological environment while providing good flexibility to adapt to wound application requirements. The photothermal conversion component specifically absorbs near-infrared light and efficiently converts light energy into heat energy; its mass percentage is 0.5%–2.0%, a proportion that ensures effective absorption of near-infrared light while avoiding excessive content that could negatively impact the overall performance of the shell. The phase change component, after absorbing the heat energy transferred from the photothermal conversion component, heats up to its phase change temperature and melts; its mass percentage is 1.5%–5.5%, ensuring controllable melting of the shell under appropriate heat input. The synergistic effect of these components enables the shell to achieve responsive melting in response to near-infrared light through a chain reaction under near-infrared irradiation.

[0090] In some embodiments, the substrate is one or more of polyurethane, polyvinylidene fluoride, polyacrylonitrile, polyether-block-amide, styrene-butadiene-styrene block copolymer, polycarbonate-based polyurethane, non-isocyanate polyurethane, polyhydroxyalkanoate, polyvinyl alcohol, polyetheretherketone, and polyethersulfone. These substrates ensure the safety of the shell in a biological environment while providing good flexibility to meet the needs of wound applications.

[0091] In some embodiments, the photothermal conversion component is one or more selected from polydopamine, graphene / graphene oxide, carbon black, polypyrrole, gold nanorods, and iron(III) oxide@tannic acid nanoparticles. These photothermal conversion components have photothermal conversion functions, and most are biosafety-free.

[0092] In some embodiments, the phase change component is one or more of lauric acid, decanoic acid, myristic acid, palmitic acid, stearic acid, paraffin wax, polyethylene glycol, n-octadecane, hexadecane, soybean wax, palm wax, erythritol, mannitol, sodium sulfate decahydrate or its hydrated salt, and calcium chloride hexahydrate or its hydrated salt. These phase change components possess phase change functionality; upon absorbing heat, they undergo a solid-to-liquid phase change, and upon cessation of near-infrared light irradiation, as heat dissipates, they undergo a liquid-to-solid phase change. This allows for brief melting of the shell layer during near-infrared light irradiation, releasing active oxygen scavengers from the core layer.

[0093] In some embodiments, the water-soluble biodegradable matrix is ​​one or more of polyvinylpyrrolidone, polyvinyl alcohol, polyethylene oxide, hydroxypropyl methylcellulose, gelatin, chitosan, polyethylene glycol, and copovidone.

[0094] In some embodiments, the reactive oxygen species (ROS) scavenger is one or more selected from 1,3-dimethylthiourea, curcumin, epigallocatechin gallate, tannic acid, glutathione, metformin, superoxide dismutase, catalase, cerium dioxide, and manganese dioxide. These ROS scavengers inhibit inflammatory responses by scavenging reactive oxygen free radicals.

[0095] In some embodiments, the flexible transparent substrate is a transparent wound dressing with a thickness of ≤100 micrometers. This thickness avoids light loss caused by excessive thickness. In embodiments of the present invention, the flexible transparent substrate is a medical transparent dressing.

[0096] In some embodiments, the thickness of the wearable SERS sensing / drug-loaded patch is 50 to 200 micrometers; this thickness can simultaneously meet the requirements of structural stability and flexible fit.

[0097] In some embodiments, the size of the sensing area is 2-6 mm; since the distribution of reactive oxygen species in the DFU wound is uneven, a circular area of ​​2-6 mm can focus on the core area of ​​inflammation and avoid interference from healthy tissue signals.

[0098] In some embodiments, the drug-loaded region extends outward from the outer periphery of the sensing region by a width of 4-8 mm. Since the diameter of a DFU wound is typically 5-15 mm, the 4-8 mm extension width allows the total diameter of the drug-loaded region to reach 10-14 mm.

[0099] In some embodiments, the coaxial fiber has a diameter of 0.6–2.2 micrometers, the core layer has a diameter of 0.1–1.6 micrometers, and the shell layer thickness is 0.1–0.8 micrometers. Fiber membranes within this diameter range exhibit high porosity (Example 3 shows good moisture transfer capacity in unidirectional moisture conduction tests). Figure 7 This ensures rapid absorption of wound exudate while providing sufficient surface area for drug release. The core diameter determines the loading capacity of the reactive oxygen species scavenger; a diameter of 0.1–1.6 micrometers can accommodate different drug concentration requirements. The shell thickness of 0.1–0.8 micrometers ensures photothermal response sensitivity—excessive thickness (>0.8 micrometers) requires a longer laser irradiation time. Figure 12 (a shows that the shell completely ruptured after 6 minutes of 808nm laser irradiation). If it is too thin (<0.1 micrometers), it may degrade prematurely during storage.

[0100] The present invention also provides a sensing and treatment system for diabetic foot ulcer wounds, comprising:

[0101] The Raman spectroscopy detection unit is used to detect the Raman spectral signal of the sensing area of ​​the wearable SERS sensor / drug-loaded patch, wherein the Raman spectral signal is the intensity of the Raman characteristic peak whose intensity decreases with increasing reactive oxygen species concentration and the intensity of the internal standard peak.

[0102] The calculation unit is used to calculate the near-infrared light irradiation time based on the Raman spectral signal;

[0103] A control unit is configured to generate and send near-infrared light irradiation control commands based on the irradiation time output by the calculation unit, the commands including the irradiation duration;

[0104] The near-infrared light irradiation unit is used to receive control commands from the control unit and target the drug-loaded area of ​​the wearable SERS sensing / drug-loaded patch with near-infrared light irradiation.

[0105] The Raman spectroscopy detection unit, calculation unit, control unit, and near-infrared light irradiation unit are connected wirelessly and / or via wired means.

[0106] The diabetic foot ulcer wound sensing and treatment system is composed of a Raman spectroscopy detection unit, a calculation unit, a control unit, and a near-infrared light irradiation unit working in coordination. Each unit is connected by wireless and / or wired means to achieve efficient linkage. Its working process starts with the Raman spectroscopy detection unit, which is responsible for detecting the signal intensities of the reactive oxygen species response peak and the internal standard peak in the sensing area of the wearable SERS sensing / drug-loading patch. Subsequently, the calculation unit receives and processes the detection results, and accurately calculates the required near-infrared light irradiation time based on the Raman spectroscopy signal. The control unit generates and outputs a near-infrared light irradiation control instruction according to the irradiation time output by the calculation unit. Finally, the near-infrared light irradiation unit responds to the control instruction and conducts targeted near-infrared light irradiation on the drug-loading area of the wearable SERS sensing / drug-loading patch, thereby realizing intelligent sensing monitoring and precise light-controlled treatment of the diabetic foot ulcer wound.

[0107] In some embodiments, calculating the near-infrared light irradiation time based on the Raman spectroscopy signal includes:

[0108] Calculating the intensity ratio of the Raman characteristic peak whose intensity decreases as the reactive oxygen species concentration increases and the internal standard peak;

[0109] Calculating the current reactive oxygen species concentration in the diabetic foot ulcer wound according to the intensity ratio and the preset standard curve;

[0110] Based on the difference between the current reactive oxygen species concentration and the target reactive oxygen species concentration, calculating the release amount of the required reactive oxygen species scavenger;

[0111] According to the release amount and the melting characteristics of the coaxial electrospun membrane shell layer, calculating the area of the shell layer that needs to be melted; according to the melted area and the photothermal conversion efficiency of the near-infrared light irradiation unit, calculating the required irradiation time and / or intensity.

[0112] The reactive oxygen species response peak decreases as the reactive oxygen species concentration increases, and the intensity of the internal standard peak remains unchanged due to the stable chemical environment of the anchoring group. By calculating the intensity ratio of the two, interference factors such as probe aggregation, light fluctuation, and substrate scattering can be eliminated, accurately reflecting the reactive oxygen species concentration, and avoiding errors caused by single peak intensity detection.

[0113] The preset standard curve is the standard curve of the intensity ratio of the reactive oxygen species response peak to the internal standard peak and the reactive oxygen species concentration. The calculation unit can convert the intensity ratio of the two into a specific reactive oxygen species concentration according to this preset standard curve. Taking 4-((2-mercaptoethyl)carbamoyl)phenylboronic acid as the Raman signal molecule as an example, as Figure 10 shown in b, the preset standard curve is y = -5.90x + 20.41; x = logC, where C is the current reactive oxygen species concentration; y = I 999 / I<0​​​​The intensity of the Raman characteristic peak at that location, i.e., the intensity of the reactive oxygen species response peak, I 1081 Located at 1081cm -1 The intensity of the Raman characteristic peak at the point is the intensity of the internal standard peak. When using other Raman signal molecules, a standard curve can be pre-established for the ratio of the intensity of the reactive oxygen species response peak to the internal standard peak and the reactive oxygen species concentration (10~200 μM).

[0114] The required release amount of reactive oxygen species (ROS) scavenger is calculated based on the difference (ΔC) between the current ROS concentration C and the target ROS concentration C0, where ΔC = C - C0. For example, if the current ROS concentration C is 180 μM and the target ROS concentration C0 is 80 μM, sufficient ROS scavenger to scavenger 100 μM needs to be released, i.e., ΔC = 100 μM, Q = λΔC, where Q represents the drug release amount and λ is the conversion coefficient between Q and ΔC. The shell of the coaxial fiber in the drug-loaded region is composed of a near-infrared light-responsive photothermal melting material, which requires the absorption of a certain amount of energy to melt and rupture. The irradiation time / intensity must satisfy: effective energy = irradiation intensity × irradiation time × irradiation area × η, where η is the photothermal conversion efficiency of the photothermal conversion component, which is a fixed value. Q = k × effective energy, where k is an inherent constant of the material. It can be deduced that irradiation time = Q / (k × irradiation intensity × irradiation area × η). Example 7 demonstrates that the cumulative release amount of the ROS scavenger is related to the near-infrared light irradiation time. Figure 12 (b) By curve fitting, a quantitative functional relationship can be established between the cumulative release of reactive oxygen species (ROS) scavengers and the near-infrared light irradiation time. Based on this, the required irradiation time can be calculated from the target release amount. After determining the required drug release amount by combining the current ROS concentration C of the system, the near-infrared light irradiation time corresponding to achieving that drug release amount can be accurately calculated.

[0115] In some embodiments, the irradiation wavelength of the near-infrared irradiation unit is 800~1000 nm.

[0116] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0117] The thermoplastic polyurethane 1185A used in Example 3 has a weight-average molecular weight of 100,000 and was purchased from BASF AG.

[0118] The polyvinylpyrrolidone used in Example 3 had a weight-average molecular weight of 1,300,000 and was purchased from Aladdin Reagent (Shanghai) Co., Ltd.

[0119] The medical transparent skin patch used in Example 6 was purchased from 3M Company, USA.

[0120] The electrospinning machine, model SR-100, was purchased from Shandong Nafibo Technology Development Co., Ltd.

[0121] The needle specifications are universal.

[0122] Example 1: Preparation of Raman signal molecules

[0123] The route for synthesizing the Raman signaling molecule (compound 2) in this embodiment is as follows: Figure 1 As shown, the specific steps are as follows:

[0124] S1. N,N-diisopropylethylamine (DIPEA, 1 mL) and cystamine dihydrochloride (225 mg) were mixed in 4 mL of N,N-dimethylformamide (DMF) for 5 minutes in an ice-water bath. Then, 4-carboxyphenylboronic acid (498 mg) and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU, 802 mg) were added to the mixture. The reaction mixture was stirred at 0 °C for 30 minutes, and then reacted at room temperature for 12 hours. Ethyl acetate was added to the reaction mixture until no white flocculent precipitate formed. The mixture was washed three times with 50 mL of deionized water and filtered to obtain compound 1, the structural formula of which is shown below. Figure 1 The proton NMR spectrum is shown below. Figure 2 .

[0125] S2. Compound 1 (200 mg) was dissolved in 10 mL of ethanol at 70 °C, and then a 10 mL ethanol solution containing 4 equivalents of sodium borohydride (67.53 mg) was added dropwise. The mixture was heated at 80 °C for 1 hour. After cooling to 25 °C, the solution was added to 30 mL of ice water, and 1 M hydrochloric acid was added to adjust the pH to 3. After evaporating the ethanol, the solution was extracted three times with ethyl acetate and washed three times with deionized water. The organic layer was dried over anhydrous sulfuric acid, and the solvent was evaporated. The residue was rapidly purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to obtain the Raman signal molecule—compound 2, the structure of which is shown in [image missing]. Figure 1 The proton NMR spectrum is shown below. Figure 3 .

[0126] Example 2: Preparation of reactive oxygen species-responsive ratio SERS nanoprobes (hereinafter referred to as probes)

[0127] S1. AuNPs with an average particle size of 50 nm were prepared according to the classic citrate reduction reaction.

[0128] S1. At room temperature, 60 μL of freshly prepared compound 2 (0.1 mM) was added to 10 mL of AuNPs (76 pM / L), and the mixture was stirred for 12 hours. Excess compound 2 was removed by centrifugation (3000 rpm, 15 min), and the precipitate was resuspended in deionized water. The above steps were repeated 3 times, with the volume of deionized water reduced by a factor of three on the last attempt, to obtain a probe suspension (hereinafter referred to as probe suspension). The probe suspension was stored at 4°C for later use.

[0129] Scanning electron microscope image of the probe as follows Figure 4 As shown, the probe particle size is 50 nm.

[0130] The zeta potential and UV absorption spectra before and after probe modification are as follows: Figure 5 As shown, the probe potential increases after modification, indicating that the negatively charged citrate ions on the surface of the gold nanospheres are replaced by responsive Raman molecules, indicating that the molecular modification is successful.

[0131] Example 3: Preparation and Optimization of Polyurethane Nanofiber Membranes

[0132] The polyurethane nanofiber membrane provided in this embodiment is prepared by electrospinning. The preparation methods below are divided into four groups according to the electrospinning time to optimize the thickness of the polyurethane nanofiber membrane, so as to achieve the best mechanical properties and water permeability of the polyurethane nanofiber membrane.

[0133] The method for preparing polyurethane nanofiber membranes by electrospinning specifically includes the following steps:

[0134] S1. Dissolve polyurethane (TPU) in a mixture of DMF / THF (1:1, v:v) and stir magnetically for 6 to 12 hours to obtain a uniform 18% (w / v) TPU spinning solution.

[0135] S2. Transfer the resulting solution to a 10 mL syringe and degas it before electrospinning.

[0136] S3. Under an applied voltage of 8.5 kV, a syringe advance speed of 1.0 mL / h, and a distance of 15 cm between the needle and the collector, polyurethane nanofiber membranes of different thicknesses were obtained by electrospinning for 2, 4, 6, and 8 h, for a total of four groups.

[0137] S4. After vacuum drying at room temperature for 1 day, the polyurethane nanofiber membrane was hydrophilically modified using a plasma cleaner with the same parameters, and then tested using a computer-controlled tensile testing instrument and a moisture management instrument.

[0138] like Figure 6 , Figure 7As shown, as the thickness of the polyurethane nanofiber membrane increases, its mechanical properties increase, while its water permeability decreases. In this invention, the polyurethane nanofiber membrane with the most suitable mechanical properties and water permeability, namely the polyurethane nanofiber membrane with an electrospinning time of 4 hours and a thickness of 155 micrometers, was selected for subsequent experiments.

[0139] Example 4: Preparation and Optimization of Sensing Region

[0140] This embodiment uses electrostatic spraying to prepare the sensing area of ​​the wearable SERS sensing / drug-loaded patch:

[0141] The polyurethane nanofiber membrane prepared in Example 3 with an electrospun time of 4 hours was fixed onto a metal collecting plate, and its position was carefully aligned to ensure accurate collection during the electrostatic spraying process. 1 mL of the probe suspension prepared in Example 2 was loaded into a 10 mL syringe, which was then attached to a syringe pump. A 23G needle was selected, and the positive voltage was adjusted to 6.5 kV and the negative voltage to -1.5 kV. The needle was 15 cm away from the metal collecting plate, and the syringe injection rate was 1.0 mL / h. The spraying process lasted for 1 hour. After the probe was sprayed, another 1 mL was added, and this process was repeated three times to obtain samples sprayed for 1 hour, 2 hours, and 3 hours, respectively. Finally, the entire sample was removed from the collecting plate, vacuum dried at room temperature, and cut into 4 mm diameter discs to obtain a patch containing only the sensing area (hereinafter referred to as the sensing patch). Its scanning electron microscope image is shown below. Figure 9 .

[0142] By controlling the spraying time to increase the probe loading amount, the SERS signal of the sensing patch gradually increased with the increase in probe loading amount, indicating that more probes were successfully modified on the surface of the polyurethane nanofiber film. Its Raman spectrum is shown below. Figure 8 The sensor patch obtained by spraying the probe suspension for 3 hours showed good SERS signal-to-noise ratio, and the polyurethane nanofiber membrane provided good coverage of the probe's characteristic wavelength band (999 cm⁻¹). -1 and 1081 cm -1 There was no interference, which could meet the subsequent sensing requirements. Subsequent experiments used a sensing patch obtained by spraying a probe suspension for 3 hours.

[0143] Example 5: Sensing and Anti-interference Test of Sensor Patch

[0144] This embodiment tests the sensing and anti-interference capabilities of the sensor patch obtained by spraying probe suspension for 3 hours in Example 4. The specific test steps are as follows:

[0145] S1. Using a pipette, transfer 5 μL of freshly prepared hydrogen peroxide solutions of different concentrations (10 μM, 25 μM, 50 μM, 100 μM, 200 μM) onto a clean glass slide. Then, place a sensor patch over the hydrogen peroxide droplet. Observe that the droplet significantly penetrates to the probe-containing side of the sensor patch. After incubating at room temperature for 1 hour, perform SERS testing with the transparent skin patch side facing up. For each concentration, collect at least 9 spectra (9 distinct points), and repeat the experiment three times using three different sensor patches to verify reproducibility.

[0146] S2. Various relevant interfering organisms and ROS were tested using the following solutions: PBS, magnesium sulfate, copper sulfate, potassium chloride, sodium carbonate, sodium sulfate, sodium sulfide, calcium chloride, zinc sulfate, sodium bisulfite, glutathione, L-cysteine, sodium hypochlorite, hydroxyl radical, superoxide anion, peroxynitrite anion, and hydrogen peroxide (all at 200 μM). Testing was performed using a sensor patch, similar to the method used in S1.

[0147] Preparation method of hydroxyl radical solution: Mix equal volumes of 1 mM hydrogen peroxide aqueous solution and 10 mM ferrous chloride aqueous solution, and dilute to the required concentration to obtain hydroxyl radical solution.

[0148] Preparation method of superoxide anion solution: Quickly weigh 0.0142 g potassium superoxide (0.2 mM), dissolve it in 10 mL of ultra-dry dimethyl sulfoxide, and weigh 1.2 equivalents of 18-crown ether-6 to aid dissolution. Sonicate for 5 min to completely dissolve it, and then dilute the solution to the required concentration to obtain superoxide anion solution.

[0149] Preparation method of peroxynitrite anion solution: Weigh 0.5 g of sodium hydroxide and dilute with pure water to prepare a 10 mL solution. Pipette 0.3 mL of 30% hydrogen peroxide solution and 0.08 mL of 96% sulfuric acid and dilute with pure water to prepare a 5 mL solution. Weigh 0.2 g of sodium nitrite and dilute with pure water to prepare a 5 mL solution. Quickly mix the latter two solutions and immediately pour them into the first solution. Add 0.08 g of manganese dioxide, filter, and seal the filtrate in a -18℃ refrigerator to obtain the peroxynitrite anion solution. The concentration of peroxynitrite anion is detected by UV-Vis spectroscopy at 302 nm. The accurate concentration should be determined using a UV spectrometer before each use.

[0150] like Figure 10 As shown, the sensor patch exhibits a linear response to hydrogen peroxide and excellent specificity.

[0151] Example 6: Preparation of a sensing / drug-loaded patch

[0152] This embodiment provides a method for preparing a sensing / drug-loaded patch, the specific steps of which are as follows:

[0153] S1. Slowly add 2 mL of dopamine hydrochloride solution (20 mg / mL) dropwise to 18 mL of Tris-HCl buffer (50 mM, pH=8), and stir continuously at 1500 rpm for 24 hours at room temperature. The solution gradually changes from colorless to black to obtain polydopamine. After the reaction is complete, centrifuge to collect the precipitate, wash three times with deionized water / ethanol alternately, and dry under vacuum at 60 °C overnight to obtain black polydopamine powder.

[0154] S2. Preparation of coaxial electrospun membrane:

[0155] S21. The preparation process of the shell solution is as follows. First, a 26% w / v TPU solution was prepared by mixing 1.3 g of polyurethane (TPU) in 5 mL of a mixed solvent system of N,N-dimethylformamide / tetrahydrofuran (1:1, v / v) and stirring at 80°C until clear and transparent. Then, 30% lauric acid (LA) was added to the TPU solution, and after stirring at a constant temperature for 6 h, 1% polydopamine (PDA) was added, and stirring was continued at 80°C for 1 h to obtain the shell solution.

[0156] S22. A core solution was prepared by mixing 0.15 g of 1,3-dimethylthiourea (DMTU) and 0.6 g of polyvinylpyrrolidone (PVP) in 5 mL of ethanol for 12 hours at room temperature using magnetic stirring.

[0157] S23. The core solution and shell solution were separately loaded into 10 mL syringes and connected to coaxial spinnerets (size: 17 for the core solution, 22 for the shell solution). Next, an aluminum foil roller collector was covered and collected using a rotating drum collector at 3 r / s. The distance between the spinneret and the collector was set to 15 cm. Two syringe pumps were used to provide a continuous feed rate of 0.12 mL / h for the core solution and 1.0 mL / h for the shell solution. The electric field strength was set to 12.5 kV. After collection, the samples were vacuum dried at room temperature for 1 day to remove residual trace solvents. Finally, they were cut into rings with an inner diameter of 4 mm and an outer diameter of 1 cm to obtain a therapeutic patch with hydrogen peroxide scavenging function.

[0158] S3. Place the sensor patch inside the ring of the treatment patch to form concentric circles. At the same time, place the side of the sensor patch with the probe facing outwards and attach the back side with the adhesive side of 3M medical transparent skin to obtain the sensor / treatment patch.

[0159] Example 7: Photothermal properties and in vitro drug release test of the therapeutic patch

[0160] This embodiment tests the photothermal properties and in vitro drug release of the therapeutic patch prepared in step S2 of Example 6.

[0161] Photothermal performance testing method: The treatment patch was placed in a 35 mm cell culture dish, and 2 mL of PBS buffer solution was added. An 808 nm NIR laser was used, with the laser power density set to 0.442 W / cm². 2 The treatment patch was irradiated for 6 minutes, with the NIR laser positioned 5 cm above the sample. The temperature was recorded every 30 seconds using a thermal imager.

[0162] In vitro drug release assay: First, place the treatment patch in a cell culture dish and add 2 mL of PBS buffer. Then, use an 808 nm NIR (near-infrared) laser at 0.442 W / cm². 2 Each sample was irradiated with a power density of 5 cm at a distance of 1, 2, 3, 4, 5, and 6 min for a total of six groups. After irradiation, the solution in the petri dishes was collected, and 1 mL of fresh PBS solution was added. The samples were then placed in a 37°C incubator and shaken for 15 minutes. After shaking, a total of 3 mL of solution was collected. Each group was repeated three times. Finally, the amount of DMTU was determined using high-performance liquid chromatography (HPLC). The experiment was repeated three times.

[0163] like Figure 12 As shown, with the increase of laser irradiation time, the temperature of the sensing / drug-loaded patch increases and a phase transition gradually occurs, resulting in an increase in drug release. This indicates that the drug release can be controlled by controlling the laser irradiation time.

[0164] Example 7: Cytotoxicity and Intracellular Antioxidant Assays of Sensing / Therapeutic Patches

[0165] This embodiment performs cytotoxicity and intracellular antioxidant assays on the sensing / therapeutic patch prepared in step S3 of Example 6. The specific methods are as follows:

[0166] S1. To evaluate the scavenging effect of the sensing / therapeutic patch on intracellular reactive oxygen species (ROS), this experiment used a 2',7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe to detect hydrogen peroxide levels in cells. In the experiment, mouse fibroblasts (L929 cells) were first seeded into confocal culture dishes and incubated for 24 hours to ensure sufficient cell adhesion to the culture surface. After cell adhesion, the original culture medium was removed, and the prescribed dose of the DCFH-DA fluorescent probe was added according to the reagent application instructions. Incubation continued for 15 minutes to allow the probe to fully penetrate the cells. Subsequently, the cells were washed three times with PBS to remove any residual probe that had not penetrated the cells. After entering the cells, DCFH-DA was hydrolyzed by intracellular esterases into non-fluorescent DCFH, which can be oxidized by intracellular ROS (such as hydrogen peroxide) to form green fluorescent DCF. Therefore, the fluorescence intensity directly reflects the intracellular ROS level.

[0167] To compare the reactive oxygen species scavenging effects under different conditions, the experiment set up multiple treatment groups: a blank control group using only phosphate buffer solution (hereinafter referred to as the phosphate buffer solution group), a control group with only hydrogen peroxide (H2O2) added (hereinafter referred to as the hydrogen peroxide group), a group treated with H2O2 and the antioxidant DMTU (hereinafter referred to as the hydrogen peroxide + 1,3-dimethylthiourea group), and experimental groups with H2O2 and sensing / treatment patches combined with near-infrared irradiation for different durations (irradiation for 0 minutes, referred to as the hydrogen peroxide + sensing / treatment patch group; irradiation for 3 minutes, referred to as the hydrogen peroxide + sensing / treatment patch group + near-infrared light - 3 minutes; irradiation for 4 minutes, referred to as the hydrogen peroxide + sensing / treatment patch group + near-infrared light - 4 minutes; irradiation for 4 minutes, referred to as the hydrogen peroxide + sensing / treatment patch group + near-infrared light - 5 minutes). Cells in each group were incubated for 2 hours under corresponding conditions to simulate the cellular microenvironment during wound repair. After incubation, cell morphology and fluorescence distribution were observed using a fluorescence confocal microscope to qualitatively assess the differences in reactive oxygen species (ROS) levels among the groups. Simultaneously, to further quantify the ROS scavenging effect, a fluorescence microplate analyzer was used for detection: L929 cells were seeded into 96-well plates and treated according to the same DCFH-DA fluorescence staining steps described above, i.e., adding probes, incubation, and washing. The fluorescence intensity of each group of cells was then quantitatively detected using a microplate analyzer to objectively compare the scavenging capacity of different treatments for intracellular hydrogen peroxide.

[0168] S2. Cytotoxicity assay for sensing / therapeutic patches

[0169] The cytotoxicity of the sensing / treatment patch to L929 cells was evaluated using CCK-8 and live / dead cell staining assays. Briefly, L929 cells were seeded into 96-well plates (10,000 cells / well) and incubated overnight until they were fully adhered to the plate. Then, the sensing / drug-loaded patch was added to the corresponding wells of the plate and incubated for different times. Each group contained six replicate wells. Finally, CCK-8 reagent was added to the wells and co-incubated for 2 hours, and absorbance was measured at 450 nm using a microplate reader.

[0170] To visualize the cytotoxicity of the sensing / drug-loaded patch, similar to the CCK-8 assay, L929 cells were attached to a confocal culture dish, the sensing / drug-loaded patch was added, and the cells were cultured for different time periods. The culture medium was then removed, and the cells were washed three times with PBS. Calcein-AM (2 μM) and PI (4 μM) were added to the cell suspension, and the cells were incubated in a cell culture incubator for 20 minutes. Live cells (yellow-green fluorescence, 492 nm) and dead cells (red fluorescence, 545 nm) were distinguished using fluorescence microscopy.

[0171] like Figure 13 As shown in Figure a, the hydrogen peroxide group exhibited strong green fluorescence, while the fluorescence intensity of the hydrogen peroxide + 1,3-dimethylthiourea group was significantly reduced, indicating that the antioxidant 1,3-dimethylthiourea can effectively remove hydrogen peroxide from cells. After 3 minutes of near-infrared irradiation, the fluorescence intensity of the hydrogen peroxide + sensing / treatment patch + near-infrared light - 3 minutes group was significantly reduced, proving that the prepared sensing / treatment patch releases DMTU to eradicate hydrogen peroxide under photothermal conditions. With increasing irradiation time, the fluorescence intensity gradually weakened, indicating that the sensing / treatment patch, combined with near-infrared light irradiation time, has the function of regulating intracellular hydrogen peroxide content. Figure 13 As shown in b, both the sensing / therapeutic patch group and L929 mouse fibroblasts showed strong green fluorescence (survival) after incubation, while almost no red fluorescence signal (death) was observed, indicating that the sensing / therapeutic patch has high biocompatibility and is conducive to cell growth.

[0172] Example 8: Application Case

[0173] This embodiment aims to verify the effect of the sensing / therapeutic patch prepared in step S3 of Example 6 on hydrogen peroxide detection and treatment in foot ulcers of diabetic mice.

[0174] The experiment first established a type 2 diabetic mouse model: 52 eight-week-old DB mice, weighing approximately 44 grams, were selected and acclimatized for 7 days to stabilize their physiological state. Random blood glucose monitoring confirmed that the mice's blood glucose level exceeded 16.7 mmol / L. -1 This indicates that the type 2 diabetes model has been successfully constructed.

[0175] Subsequently, a diabetic foot ulcer wound model was constructed: all diabetic mice were first fasted for 4 hours, and after anesthesia, a circular skin wound with a diameter of 4 mm was created on the dorsum of their feet using a puncture biopsy needle. To compare the effects of different intervention methods, the mice were randomly divided into four groups, with the following specific treatments: phosphate buffer solution group: 5 μL of 1.0× sterile PBS solution was directly applied to the wound; sensor / treatment patch group: after applying 5 μL of 1.0× sterile PBS solution, only the sensor / treatment patch was used to monitor the reactive oxygen species concentration in the wound, without near-infrared light irradiation; 1,3-dimethylthiourea group: 5 μL of DMTU solution was applied to the wound; sensor / treatment patch + near-infrared light group: 1 hour after applying 5 μL of 1.0× sterile PBS solution, the hydrogen peroxide concentration was detected using the sensor / treatment patch to determine the required near-infrared irradiation time, followed by near-infrared light irradiation to trigger drug release.

[0176] Dynamic monitoring of hydrogen peroxide levels in the wound was conducted on days 1, 3, 5, and 7 of the experiment (every 6 hours). The results are as follows: Figure 15 As shown; since wound dryness may affect the accuracy of detection, 5 μL of PBS was dripped onto the wound before each monitoring, and then the sensing / treatment patch was used for hydrogen peroxide detection.

[0177] To further evaluate the ability of the sensing / treatment patch to clear hydrogen peroxide overexpressing in wounds under near-infrared light irradiation, a hydrogen peroxide solution (200 μM) was added to the wound on day 6 to simulate the therapeutic effect under a high reactive oxygen species environment. When the hydrogen peroxide concentration was detected to be higher than 100 μM, near-infrared light (808 nm and 0.44 W·cm⁻¹) was used. -2 The duration of irradiation was controlled to regulate the release of DMTU. Finally, the wound healing process was observed and photographed on days 0, 3, 7, 13, and 21. Figure 14 As shown, the sensing / therapeutic patch prepared in step S3 of Example 6 can effectively relieve the symptoms.

[0178] To investigate the wound healing mechanism of diabetic foot ulcers, mice were euthanized immediately after the experiment by overdose of sodium pentobarbital. Major organs (heart, liver, spleen, lungs, and kidneys) and foot skin were dissected as part of the study on the fixed wound healing mechanism. The excised skin and organs were immersed in 4% paraformaldehyde solution. After 24 hours, the fixed tissue sections were stained with hematoxylin and eosin (H&E), Masson's solution, tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), and ROS (hydrogen peroxide).

[0179] like Figure 16As shown, histological studies indicate that the sensing / treatment patch + near-infrared light combination demonstrates a significant ability to stimulate tissue regeneration and DFU recovery by promoting re-epithelialization and increasing collagen deposition, based on maintaining redox balance.

[0180] like Figure 17 As shown, the sensing / treatment patch + near-infrared light assembly has a good ability to promote wound healing without producing any negative toxicity.

[0181] like Figure 18 As shown, the sensing / treatment patch combined with the near-infrared light group effectively monitors hydrogen peroxide in the wound, and then removes excess hydrogen peroxide as needed to achieve redox homeostasis, thereby inhibiting the inflammatory response, promoting angiogenesis, accelerating collagen deposition, and ultimately achieving compensatory healing of DFU wounds.

[0182] Statistical analysis was performed using one-way ANOVA to compare differences between groups, with p < 0.05 considered statistically significant. Experimental data are expressed as mean ± standard deviation (Mean ± SD). The significance symbols in the graphs are labeled as follows: "*" represents p < 0.05, and "**" represents p < 0.01.

[0183] The results showed that the sensor / treatment patch could accurately observe changes in hydrogen peroxide concentration in diabetic foot ulcers in mice, and that the drug release could be controlled on demand by controlling the near-infrared light irradiation time, thus effectively treating diabetic foot ulcers in mice.

[0184] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A wearable SERS sensing / drug-loaded patch for diabetic foot ulcer wounds, characterized in that, The wearable SERS sensing / drug-loading patch comprises a flexible transparent substrate, a sensing area and a drug-loading area are arranged on one side surface of the flexible transparent substrate; wherein: The sensing area comprises active oxygen response ratio type SERS nanoprobes, the active oxygen response ratio type SERS nanoprobes are composed of gold nanoparticles and Raman signal molecules modified on the surface of the gold nanoparticles, and the Raman signal molecules comprise: An active oxygen response group having a Raman characteristic peak with a decreasing intensity with an increase in the concentration of active oxygen; An anchoring group stably combined with the surface of the gold nanoparticles through a covalent bond or a coordination bond; The drug-loading area is an annular area sleeved on the periphery of the sensing area, and comprises a coaxial electrospun membrane composed of coaxial fibers, the coaxial fibers have: A shell layer composed of a near-infrared light response type photothermal melting material, the near-infrared light response type photothermal melting material can absorb light energy and convert it into heat energy under near-infrared light irradiation, so that the shell layer is melted and broken; A core layer comprising a water-soluble biodegradable matrix and an active oxygen scavenger dispersed in the water-soluble biodegradable matrix, the core layer releases the active oxygen scavenger by dissolving the water-soluble matrix after the shell layer is broken.

2. The wearable SERS sensing / drug carrying patch of claim 1, wherein: The particle size of the gold nanoparticles is 30-60 nm; and / or, The active oxygen response group is one of a boronic acid group, a boronic ester group, a carboxyl group and a diazonium group; and / or, The anchoring group is one or more of a mercapto group, an alkynyl group and a seleno group.

3. The wearable SERS sensing / drug carrying patch of claim 1, wherein: The near-infrared light response type photothermal melting material comprises: A substrate, which is a biocompatible flexible polymer material; A photothermal conversion component, which can absorb near-infrared light and convert it into heat energy; A phase change component, which can melt after absorbing heat energy; The mass percentage of the photothermal conversion component is 0.5%-2.0%, and the mass percentage of the phase change component is 1.5%-5.5%.

4. The wearable SERS sensing / drug-loading patch according to claim 3, wherein: The substrate is one or more of polyurethane, polyvinylidene fluoride, polyacrylonitrile, polyether-block-amide, styrene-butadiene-styrene block copolymer, polycarbonate-based polyurethane, non-isocyanate polyurethane, polyhydroxyalkanoate, polyvinyl alcohol, polyether ether ketone and polyether sulfone; and / or, The photothermal conversion component is one or more of polydopamine, graphene / oxidized graphene, carbon black, polypyrrole, gold nanorod and ferroferric oxide@tannic acid nanoparticles; and / or, The phase change component is one or more of lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, paraffin, polyethylene glycol, n-octadecane, hexadecane, soybean wax, palm wax, erythritol, mannitol, sodium sulfate decahydrate or a hydrated salt thereof, calcium chloride hexahydrate or a hydrated salt thereof.

5. The wearable SERS sensing / drug carrying patch of claim 1, wherein: The water-soluble biodegradable matrix is one or more of polyvinylpyrrolidone, polyvinyl alcohol, polyethylene oxide, hydroxypropyl methyl cellulose, gelatin, chitosan, polyethylene glycol and copolyvione; and / or, The active oxygen scavenger is one or more of 1,3-dimethyl thiourea, curcumin, epigallocatechin gallate, tannic acid, glutathione, metformin, superoxide dismutase, catalase, cerium dioxide and manganese dioxide.

6. The wearable SERS sensing / drug carrying patch of claim 1, wherein: The coaxial fiber has a diameter of 0.6-2.2 microns, the core layer has a diameter of 0.1-1.6 microns, and the shell layer has a thickness of 0.1-0.8 microns.

7. The wearable SERS sensing / drug carrying patch of claim 1, wherein: The wearable SERS sensing / drug-loaded patch has a thickness of 50-200 microns; and / or, The sensing area has a size of 2-6 mm; and / or, The drug-loaded area extends outward from the outer peripheral edge of the sensing area with a width of 4-8 mm.

8. A diabetic foot ulcer wound sensing and treatment system, characterized by, Comprise: a Raman spectrum detection unit for detecting the Raman spectrum signal of the sensing area of the wearable SERS sensing / drug-loaded patch of claim 1, the Raman spectrum signal being an active oxygen response peak and an internal standard peak; a calculation unit for calculating the near-infrared light irradiation time based on the Raman spectrum signal; a control unit for generating and sending a near-infrared light irradiation control instruction according to the irradiation time output by the calculation unit, the instruction containing the irradiation time length; a near-infrared light irradiation unit for receiving the control instruction of the control unit and performing targeted near-infrared light irradiation on the drug-loaded area of the wearable SERS sensing / drug-loaded patch; wherein the Raman spectrum detection unit, the calculation unit, the control unit and the near-infrared light irradiation unit are connected in communication by wireless and / or wired means.

9. The diabetic foot ulcer wound sensing and treatment system of claim 8, characterized in that: calculating the near-infrared light irradiation time based on the Raman spectrum signal comprises: calculating the intensity ratio of the active oxygen response peak and the internal standard peak; calculating the current active oxygen concentration of the diabetic foot ulcer wound according to the intensity ratio and a preset standard curve; calculating the release amount of the required active oxygen scavenger based on the difference between the current active oxygen concentration and the target active oxygen concentration; obtaining the required irradiation time according to the release amount.

10. The diabetic foot ulcer wound sensing and treatment system of claim 8, wherein, The irradiation wavelength of the near-infrared light irradiation unit is 800-1000 nm.

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