Method for optimizing parameters of composite nano-photosensitizer for photodynamic microneedle preparation

Abstract

This invention discloses a method for optimizing the parameters of composite nano-photosensitizers used in the preparation of photodynamic microneedles, relating to the field of biomedical technology. The method includes: preparing octahedral Cu₂O nanoparticles and recording synthesis parameters; in-situ loading of sheet-like CuTCPP to obtain CuTCPP@Cu₂O composite nanoparticles and recording loading parameters; mixing the composite particles with a first matrix and a natural small molecule with a second matrix, followed by layered casting and vacuum-assisted filling to prepare a bilayer microneedle test sample; evaluating performance and adjusting synthesis and / or loading parameters accordingly; and preparing the composite nano-photosensitizer with the final parameters after achieving the desired performance. This invention solves the technical problem that existing composite nano-photosensitizers lack an iterative optimization mechanism based on microneedle sample performance feedback in parameter determination, making it difficult to ensure that the final product consistently meets the expected functional standards. It achieves precise locking of photosensitizer synthesis and loading parameters, ensuring that the final product possesses the preset performance characteristics.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for optimizing the parameters of composite nanophotosensitizers used in the preparation of photodynamic microneedles. Background Technology

[0002] The clinical need for treating deep subcutaneous infected wounds is urgent. The performance of composite nano-photosensitizers used in photodynamic microneedles directly affects the antibacterial and repair effects of wounds, making parameter optimization and precise preparation crucial. Current technologies mostly employ a single photosensitizer paired with conventional microneedles, relying on traditional synthesis processes to prepare the photosensitizer, without establishing a parameter optimization system suitable for photodynamic microneedles. While these technologies can be initially applied in closed, stable systems, they exhibit significant limitations when facing the complex microenvironment of wounds. Existing technologies cannot precisely control the synthesis and loading parameters of photosensitizers, making it difficult to ensure the generation of multiple reactive oxygen species and meet the mechanical and drug release properties of the microneedles. The obtained performance data is incomplete and unreliable, failing to meet the needs for precise assessment and efficient management of photodynamic therapy for subcutaneous infected wounds. Summary of the Invention

[0003] This application addresses the technical problem that existing composite nanophotosensitizers lack an iterative optimization mechanism based on microneedle sample performance feedback in parameter determination, making it difficult to ensure that the final product consistently meets the expected functional standards.

[0004] To address the aforementioned technical problems, this application proposes a method for optimizing the parameters of a composite nano-photosensitizer used in the preparation of photodynamic microneedles. The method includes: preparing octahedral Cu₂O nanoparticles and recording the synthesis parameters; in-situ loading of sheet-like CuTCPP onto the octahedral Cu₂O nanoparticles to prepare CuTCPP@Cu₂O composite nanoparticles and recording the in-situ loading parameters; mixing the obtained CuTCPP@Cu₂O composite nanoparticles with a first matrix material and simultaneously mixing natural small molecules with a second matrix material; and preparing a photodynamic microneedle test sample with a bilayer structure through a microneedle forming process using layered casting and vacuum-assisted filling; evaluating the performance of the obtained photodynamic microneedle test sample; and adjusting the synthesis parameters and / or in-situ loading parameters according to the performance evaluation results to achieve a preset target, thereby preparing the composite nano-photosensitizer using the synthesis parameters and in-situ loading parameters that meet the preset target.

[0005] This application proposes one or more technical solutions, which have at least the following technical effects: This application involves the stepwise preparation of inorganic nanoparticles and in-situ loading of organic photosensitizing components. A double-layer microneedle is then prepared via layered casting and vacuum-assisted molding. The preparation parameters are iteratively adjusted based on multi-dimensional performance testing results to stably obtain a composite nano-photosensitizer that meets performance requirements. This results in more stable and compliant reactive oxygen generation, mechanical strength, and drug release performance of the photodynamic microneedle. The precise locking of photosensitizer synthesis and loading parameters ensures that the final product exhibits the predetermined performance characteristics. Attached Figure Description

[0006] 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.

[0007] Figure 1 This is a flowchart illustrating the method for optimizing the parameters of composite nanophotosensitizers used in the preparation of photodynamic microneedles, as provided in the embodiments of this application.

[0008] Figure 2 This is a flowchart illustrating the preset target in the method for optimizing the parameters of composite nanophotosensitive agents used in the preparation of photodynamic microneedles provided in the embodiments of this application. Detailed Implementation

[0009] This application provides a method for optimizing the parameters of composite nanophotosensitive agents used in the preparation of photodynamic microneedles. This method solves the technical problem that the determination of parameters of existing composite nanophotosensitive agents lacks an iterative optimization mechanism based on the performance feedback of microneedle samples, making it difficult to ensure that the final product stably meets the expected functional standards.

[0010] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0011] It should be noted that any variation of the terms "comprising" and "having" is intended to cover non-exclusive inclusion, for example, a process, method, system, product, or server that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or modules that are not explicitly listed or that are inherent to such processes, methods, products, or devices.

[0012] Example 1, as Figure 1As shown, a method for optimizing the parameters of composite nanophotosensitive agents used in the preparation of photodynamic microneedles, wherein the method includes: Octahedral Cu₂O nanoparticles were prepared, and the synthesis parameters were recorded.

[0013] In this embodiment, the octahedral Cu2O nanoparticles are cuprous oxide nanocrystals with a regular octahedral morphology, which can be used as an inorganic carrier to load photosensitizers and construct antibacterial materials with both photodynamic and Fenton-like activities.

[0014] Specifically, add 80 mL of pure water to a 100 mL round-bottom flask, then add 0.8456 g of copper nitrate trihydrate and 5 g of polyvinylpyrrolidone sequentially. Place the round-bottom flask in an oil bath and heat to 55 °C, stirring for 15 min until the solids are completely dissolved and the solution is completely clear. Dissolve 2.8 g of sodium hydroxide in 10 mL of pure water to obtain an aqueous sodium hydroxide solution. Add the sodium hydroxide solution dropwise to the round-bottom flask using a dropper, maintaining the temperature at 55 °C and stirring for another 30 min. Dissolve 3.6985 g of ascorbic acid in 10 mL of pure water to obtain an aqueous ascorbic acid solution. Add the ascorbic acid solution dropwise to the round-bottom flask using a dropper, maintaining the temperature at 55 °C, and reflux with stirring for 3 h.

[0015] After the reaction was completed, the precipitate was separated by centrifugation at 5000 rpm for 5 min using a high-speed centrifuge, and then washed three times with pure water. The washed precipitate was placed in a drying device and dried at 60℃ for 24 h to obtain octahedral cuprous oxide nanoparticle powder. Throughout the preparation process, all relevant synthesis parameters were fully recorded, including the molar ratio of Cu(NO3)2·3H2O, NaOH and ascorbic acid, reaction temperature, and reaction time.

[0016] CuTCPP@Cu2O composite nanoparticles were prepared by in-situ loading of sheet-like CuTCPP onto the octahedral Cu2O nanoparticles, and the in-situ loading parameters were recorded.

[0017] In this embodiment, the CuTCPP@Cu2O composite nanoparticles are composite photosensitizers obtained by in-situ loading copper carboxyphenyl porphyrin onto octahedral cuprous oxide as a carrier. They possess both photodynamic and Fenton-like antibacterial activities and can be used to prepare photodynamic antibacterial microneedles.

[0018] Optionally, octahedral Cu2O nanoparticles are ultrasonically dispersed in ethanol / pure water, and then a DMF solution of racemic tetra(4-carboxyphenyl)porphyrin is added dropwise and heated to react. Finally, CuTCPP@Cu2O composite nanoparticles are prepared in situ by centrifugation, washing with anhydrous ethanol and drying. The specific implementation process of this step will be described in detail below.

[0019] Next, the in-situ load parameters will be recorded. The specific steps are as follows: Weigh 0.05 g of octahedral Cu₂O nanoparticles and calculate their molar number based on the molar mass of Cu₂O. Weigh 0.004 g of meso-tetra(4-carboxyphenyl)porphyrin and calculate its molar number based on its molar mass. Compare the molar numbers of the two to obtain the molar ratio of Cu₂O to CuTCPP, and record this feed ratio parameter.

[0020] Add Cu2O to a mixture of 18 mL anhydrous ethanol and 12 mL pure water. Turn on the ultrasonic machine, set the power to 800 W and the frequency to 40 kHz, and sonicate for 30 minutes. Record the ultrasonic power, ultrasonic frequency, and ultrasonic time parameters.

[0021] A DMF solution containing 0.004 g of racemic tetra(4-carboxyphenyl)porphyrin was added dropwise to a round-bottom flask containing a Cu2O dispersion. The round-bottom flask was placed in an oil bath and heated to 90°C. The mixture was stirred under reflux for 6 hours, and the reaction temperature and time parameters were recorded.

[0022] After the reaction was complete, the reaction product was transferred to a centrifuge tube. A high-speed centrifuge was set to 5000 rpm and centrifuged for 5 minutes. The precipitate at the bottom was collected, and the centrifugation speed and time parameters were recorded. The precipitate was washed three times with anhydrous ethanol, and the washing solvent and number of washes parameters were recorded.

[0023] The washed precipitate was placed in a drying device, the drying temperature was set to 60℃ and the drying time was 24 hours, and the drying temperature and drying time parameters were recorded.

[0024] Through the above steps, all key parameters during the in-situ loading process are acquired and recorded, providing traceable experimental evidence for subsequent parameter optimization of composite nanophotosensitizers.

[0025] The obtained CuTCPP@Cu2O composite nanoparticles were mixed with the first matrix material, and natural small molecules were mixed with the second matrix material. The photodynamic microneedle test sample with a double-layer structure was prepared by a microneedle forming process of layered casting and vacuum-assisted filling.

[0026] In this embodiment, the photodynamic microneedle test sample is a microneedle-shaped sample with a double-layer structure, loaded with photosensitive composite particles and curcumin, which can be used to detect the photodynamic effect.

[0027] In one embodiment of this application, hyaluronic acid with a molecular weight in the range of 200 to 400 kDa was weighed and added to deionized water. The mixture was stirred at a rate of 250 rpm until the material was completely dissolved, thus obtaining a 5% (w / v) hyaluronic acid solution. Curcumin was added at a ratio of 0.2% of the solution mass, and the mixture was stirred for 2 hours in a completely dark environment to ensure uniform dispersion of the natural small molecules, thereby obtaining a drug-loaded needle tip layer mixture.

[0028] Gelatin with a molecular weight between 50 and 100 kDa was weighed and added to deionized water. The mixture was heated to 55°C and stirred continuously at 300 rpm until the solid material was completely dissolved, resulting in a 20% (w / v) gelatin solution. CuTCPP@Cu2O composite nanoparticles were added at a ratio of 0.5% (w / v) of the solution, and the mixture was stirred continuously at the same speed for 30 minutes to allow the particles to be evenly dispersed in the liquid phase system, thus obtaining an antibacterial base layer mixture.

[0029] Place the microneedle mold stably on a horizontal platform. Slowly inject the drug-loaded needle tip mixture into the mold's needle tip cavity, ensuring the volume just fills the cavity. Place the container holding the mold into a vacuum drying oven and perform three repeated vacuuming operations. Each time, reduce the oven pressure to -0.095 MPa, let it stand for 1 minute, and then restore it to normal pressure. The negative pressure helps expel air bubbles from the solution and promotes full adhesion of the solution to the cavity wall for complete filling. After processing, scrape off any excess hyaluronic acid solution from the mold surface to ensure the needle tip layer is neat.

[0030] Next, slowly pour the antibacterial base layer mixture into the mold after the needle tip layer has been filled, ensuring the liquid level evenly covers the lower needle tip structure. No further vacuuming is required. Maintain a constant internal temperature of 35°C in the forced-air drying oven and place the mold inside for continuous drying for 12 hours, allowing the two layers of different liquid components to solidify layer by layer. After the curing process is complete, gently peel off the mold and remove the finished product to obtain a photodynamic microneedle test sample with a dual-layer structure.

[0031] By combining layered casting with vacuum defoaming molding, and by precisely controlling the material ratio and molding conditions, a double-layered photodynamic microneedle sample with a regular layered structure and uniform distribution of active substances was prepared.

[0032] The performance of the obtained photodynamic microneedle test samples was evaluated. Based on the performance evaluation results, the synthesis parameters and / or in-situ loading parameters were adjusted to achieve the preset target. The composite nano photosensitizer was then prepared by using the synthesis parameters and in-situ loading parameters that met the preset target.

[0033] Specifically, the first step is to evaluate the performance of the obtained photodynamic microneedle test samples, including the detection of microneedle morphology, mechanical strength, drug release, and photosensitizer reactive oxygen generation capacity. The specific implementation process of this step will be described in detail below.

[0034] Next, based on the measured data obtained from the four performance evaluations, i.e., the performance evaluation results, the synthesis parameters and in-situ loading parameters were iteratively adjusted against the preset reactive oxygen species (ROS) generation targets. Using the generation effects of singlet oxygen, superoxide anion radicals, and hydroxyl radicals as the core evaluation criteria, the ROS signal performance corresponding to different parameters was statistically analyzed. Combined with the performance change patterns resulting from parameter variations, the correction direction and adjustment range of each parameter were determined.

[0035] Gradient screening experiments were conducted on the synthesis parameters of octahedral cuprous oxide nanoparticles. Five gradient levels were set for the molar ratio of raw materials, with a difference of 0.5 between adjacent levels; four gradient levels were set for the reaction temperature, with a temperature difference of 5 degrees Celsius between adjacent levels; and four gradient levels were set for the reaction time, with a time difference of 0.5 hours between adjacent levels. Copper nitrate trihydrate, sodium hydroxide, and ascorbic acid were selected as reactants. The reactants were added in the following order: copper nitrate trihydrate first, followed by sodium hydroxide, and finally ascorbic acid. The mixture was stirred at 400 rpm throughout the process, and parallel preparations were carried out with different reaction temperatures and times. By comparing the microstructure and number of surface active sites in each group of products, the synthesis conditions of a raw material molar ratio of 1:20:6, a reaction temperature of 55 degrees Celsius, and a reaction duration of 3 hours were selected. This parameter system can stably form a regular octahedral structure, retain sufficient monovalent copper active sites, and meet the structural basis conditions for the generation of hydroxyl radicals in the Fenton reaction.

[0036] In-situ loading parameter gradient screening experiments were conducted on plate-like porphyrins. Five gradient levels of molar ratio were set, with a difference of 0.2 between adjacent levels; four gradient levels of reaction temperature were set, with a temperature difference of 5 degrees Celsius between adjacent levels; and four gradient levels of reaction time were set, with a time difference of 0.5 hours between adjacent levels. The uniformity of surface coating and the ability to generate reactive oxygen species under light were observed in each group of samples. The final loading parameters determined to be a molar ratio of 1:70, a reaction temperature of 90 degrees Celsius, and a reaction time of 6 hours. These parameters ensure uniform adhesion of the porphyrin material to the particle surface, stable production of singlet oxygen and superoxide anion radicals, and do not damage the original active sites of the substrate particles.

[0037] The composite nano-photosensitizer was prepared according to the optimal parameters determined through screening. Cuprous oxide was added to an ethanol-water mixed solvent system and ultrasonically treated at 800 watts and 40 kHz for 30 minutes to achieve complete particle dispersion. A dimethylformamide solution containing porphyrin was added dropwise at a constant rate of 0.2 mL / s, with a stirring speed of 350 rpm throughout the process. The mixture was then refluxed at 90°C for 6 hours to complete the in-situ loading process. After the reaction, the precipitate was collected by centrifugation at 5000 rpm for 5 minutes using a high-speed centrifuge. The precipitate was washed with 8 mL of anhydrous ethanol for 3 minutes each time, for a total of 3 washes. The precipitate was then dried at 60°C for 24 hours to obtain the final composite nano-photosensitizer sample.

[0038] Prepare a 0.1 mol / L hydrogen peroxide solution. Take 2 mL of this solution and mix it evenly with an equal volume of photosensitizer test solution. Control the reaction temperature at 25°C and allow the reaction to stand for 2 minutes before starting the detection process. A 50 mW / cm² LED light source with a wavelength range of 400 nm to 800 nm is used for detection. Singlet oxygen and superoxide anion free radical signals under pure light conditions, as well as hydroxyl free radical signals in the hydrogen peroxide coexistence system, are detected. A relative signal intensity of at least 35% for the reactive oxygen species characteristic peak is set as the pass / fail threshold. All sample test results meet the set numerical standard, confirming that all performance parameters meet the preset technical objectives.

[0039] Clearly define the adjustment rules for parameters that fail to meet standards. When the reactive oxygen species (ROS) signal intensity is below the acceptable threshold, slightly increase the porphyrin loading ratio or extend the loading reaction time. If structural sites are damaged, lower the reaction temperature and re-produce raw materials. Once the finalized parameters are implemented in the batch production stage, a set of samples is randomly selected every 20 batches for comprehensive ROS testing to verify the consistency of product performance and ensure that multiple batches of finished products consistently meet the standards.

[0040] By iteratively optimizing process parameters through performance data feedback, and by implementing standardized verification and judgment rules and batch quality control measures, a qualified composite nanophotosensitizer capable of simultaneously generating three types of target reactive oxygen species was finally obtained.

[0041] Furthermore, the method provided in this application embodiment includes: The synthesis parameters include: the molar ratio of Cu(NO3)2·3H2O, NaOH and ascorbic acid, reaction temperature, and reaction time.

[0042] Optionally, calculate the molar number of each reactant. The molar mass of Cu(NO3)2·3H2O is 241.55 g / mol, and the amount added is 0.8456 g, so the molar number is 0.8456 g ÷ 241.55 g / mol ≈ 0.0035 mol. The molar mass of NaOH is 40 g / mol, and the amount added is 2.8 g, so the molar number is 2.8 g ÷ 40 g / mol = 0.07 mol. The molar mass of ascorbic acid is 176 g / mol, and the amount added is 3.6985 g, so the molar number is 3.6985 g ÷ 176 g / mol ≈ 0.021 mol. Therefore, the molar ratio of Cu(NO3)2·3H2O, NaOH, and ascorbic acid is 1:20:6. Place the round-bottom flask in an oil bath, set the oil bath temperature to 55℃, and monitor and maintain the reaction system temperature at 55℃ throughout the process. This determines the reaction temperature parameter as 55℃. After the NaOH aqueous solution was added dropwise, the mixture was stirred at 55°C for 30 min, and the reaction time for precursor formation was recorded as 30 min. After the ascorbic acid aqueous solution was added dropwise, the mixture was refluxed and stirred at 55°C for 3 h, and the reduction reaction time was recorded as 3 h. Based on this, the key reaction time parameters for the synthesis process were determined to be 30 min and 3 h.

[0043] The molar ratio of Cu(NO3)2·3H2O, NaOH, and ascorbic acid is 1:20:6, with NaOH being 20 times the molar amount of Cu(NO3)2·3H2O. This provides a strongly alkaline reaction environment, ensuring complete conversion of copper ions into the copper hydroxide precursor and preventing copper ion residue. The molar amount of ascorbic acid is 6 times that of Cu(NO3)2·3H2O, providing sufficient reducing power to reduce copper ions in the copper hydroxide precursor to cuprous ions, generating Cu2O. Simultaneously, the reduction reaction rate is controlled to avoid over-reduction and the formation of elemental copper, ensuring the product is pure-phase Cu2O and providing a foundation for the subsequent formation of an octahedral morphology.

[0044] The reaction temperature of 55℃ can regulate the kinetics of precursor formation and crystal growth. Too low a temperature will result in a slow reaction rate, incomplete precursor formation, and insufficient crystal growth. Too high a temperature will result in an excessively fast crystal growth rate, particle agglomeration, and uneven morphology. 55℃ can ensure uniform precursor formation and promote the growth of Cu2O crystals along specific crystal planes to form a regular octahedral structure.

[0045] A 30-minute precursor formation time ensures that copper ions react fully with NaOH to completely convert into copper hydroxide precursor, providing a homogeneous reaction system for the subsequent reduction reaction. A 3-hour reduction reaction time ensures that ascorbic acid fully reduces the precursor to Cu2O, while allowing sufficient time for the crystals to grow into a complete octahedral structure. This avoids incomplete reduction and impurities in the product due to too short a reaction time, or particle agglomeration and uneven morphology due to too long a reaction time.

[0046] Furthermore, the method provided in this application embodiment includes: Cu2O was added to ethanol / pure water and sonicated for a predetermined time until the nanoparticles were uniformly dispersed to obtain a first intermediate. A DMF solution of racemic tetra(4-carboxyphenyl)porphyrin was added dropwise to the first intermediate, and the mixture was heated to a predetermined temperature and reacted for a predetermined time to obtain a second intermediate. The second intermediate was separated and precipitated using a high-speed centrifuge, washed with anhydrous ethanol, and dried to obtain CuTCPP@Cu2O powder.

[0047] Specifically, 18 mL of anhydrous ethanol and 12 mL of pure water were added to a 50 mL round-bottom flask, along with 0.05 g of octahedral Cu₂O nanoparticles. An ultrasonic machine was turned on, set to 800 W power and 40 kHz frequency, and ultrasonic treatment was performed for 30 minutes to ensure complete and uniform dispersion of the Cu₂O nanoparticles in the ethanol and pure water mixture, yielding the first intermediate.

[0048] Weigh 0.004 g of racemic tetra(4-carboxyphenyl)porphyrin and dissolve it in 1 mL of DMF. Stir until completely dissolved to obtain a DMF solution of racemic tetra(4-carboxyphenyl)porphyrin. Add this solution dropwise to a round-bottom flask containing the first intermediate using a dropper. Then place the round-bottom flask in an oil bath and heat to 90 °C. Stir continuously for 6 hours under reflux to allow CuTCPP to be loaded in situ onto the surface of Cu2O nanoparticles, thus obtaining the second intermediate.

[0049] After the reaction was complete, the second intermediate was transferred to a centrifuge tube and centrifuged at 5000 rpm for 5 minutes using a high-speed centrifuge. The precipitate at the bottom was collected. The precipitate was washed three times with anhydrous ethanol to remove unreacted impurities. The washed precipitate was placed in a drying apparatus and dried at 60°C for 24 hours to obtain CuTCPP@Cu2O composite nanoparticle powder.

[0050] By solvent dispersion, in-situ loading reaction and post-processing, uniform loading of sheet-like CuTCPP on the surface of octahedral Cu2O nanoparticles was achieved, and a high-purity and stable CuTCPP@Cu2O composite nanophotosensitizer was prepared.

[0051] Furthermore, the method provided in this application embodiment includes: The first matrix material is gelatin with a molecular weight of 50-100 kDa; the second matrix material is hyaluronic acid with a molecular weight of 200-400 kDa; and the natural small molecule is curcumin.

[0052] Optionally, gelatin with a molecular weight of 50-100 kDa is selected as the first matrix material. This material has excellent biocompatibility and can be firmly bound to CuTCPP@Cu2O composite nanoparticles, thus stably confining the photosensitive active material to the microneedle tip area. At the same time, gelatin has suitable mechanical strength after molding, which can ensure that the microneedle tip has the ability to puncture and penetrate, meeting the requirements for skin puncture. It can also improve the regularity of the molded structure by relying on its own colloidal properties, reducing the loss of active ingredients during the preparation process.

[0053] Hyaluronic acid with a molecular weight of 200-400 kDa was selected as the second matrix material. This material exhibits excellent hydrophilicity and can form a support structure for the microneedle base, thus mitigating skin irritation caused by needle puncture. Hyaluronic acid can slowly release active ingredients upon contact with the skin, prolonging the duration of action. At the same time, it can connect the needle tip layer structure, improving the overall fit and structural stability of the double-layer microneedle, further optimizing the compatibility of microneedle use.

[0054] Curcumin, a natural small molecule, is added to the substrate matrix and possesses natural antibacterial and anti-inflammatory properties. It can form a synergistic effect with the CuTCPP@Cu2O composite nano photosensitizer. This substance has high biosafety and will not cause adverse skin irritation. It can help enhance the antibacterial and repairing effects under photodynamic therapy, enrich the overall performance of microneedling, and improve the efficacy of the composite nano photosensitizer.

[0055] Furthermore, the method provided in this application embodiment includes: Vacuum-assisted filling involves repeatedly evacuating the solution added to the mold three times to remove air bubbles.

[0056] Specifically, a process of repeated vacuuming three times is used to remove air bubbles trapped in the solution inside the mold. The solution poured into the mold cavity easily traps air, and tiny air bubbles cannot dissipate on their own by natural settling. Multiple negative pressure suctions can cause air bubbles of all sizes inside the solution to gradually precipitate out and be discharged from the cavity, thoroughly cleaning the gas remaining deep inside the cavity and on the surface, avoiding the retention of trace air bubble impurities after a single treatment.

[0057] Retained air bubbles can cause voids and defects after microneedle molding, damaging the delicate shape and structure of the needle tip. Vacuum debubbling treatment ensures the integrity and regularity of the overall microneedle contour, maintaining consistency between the molded morphology and the mold cavity structure. Cavities formed by air bubbles reduce the density and mechanical properties of the microneedle body, making it prone to breakage and damage during use. Removing air bubbles results in a compact and uniform internal structure of the matrix, effectively improving the structural stability during microneedle puncture operations.

[0058] Bubbles can also interfere with the dispersion of active substances in the matrix, causing local component imbalance. After the bubble removal operation is completed, it can be ensured that the composite nanoparticles and natural small molecules are uniformly dispersed in the matrix, so that the performance of microneedles remains consistent and can stably exert photodynamic effects.

[0059] Furthermore, the method provided in this application embodiment includes: The parameters of the microneedle forming process include: the solution concentration of the second matrix material, the solution concentration of the first matrix material, the drying temperature, and the drying time.

[0060] Specifically, the solution concentration corresponding to the first matrix material was obtained through a gradient mixing experiment. Gelatin with molecular weights ranging from 50 to 100 kDa was weighed and dissolved in an equal volume of deionized water to prepare solutions of various concentrations. CuTCPP@Cu2O composite nanoparticles were then incorporated into solutions of different concentrations. The uniformity of particle dispersion and solution flow characteristics were observed. Combined with the microneedle tip formation morphology and puncture mechanics, 20% was ultimately determined to be the suitable solution concentration. At this concentration, the gelatin solution exhibits moderate fluidity, allowing it to smoothly fill the fine cavities of the mold. This not only securely encapsulates the composite photosensitizer, preventing the release and loss of active components, but also imparts sufficient mechanical strength to the substrate after molding, ensuring the overall stability of the microneedle structure.

[0061] The concentration of the second matrix material solution was determined using a gradient preparation experiment. Hyaluronic acid with a molecular weight of 200-400 kDa was used, and multiple solutions were prepared according to different dissolution ratios. After incorporating curcumin, the dispersion of small molecules was observed, and the formation effect of the solution on the needle tip material was tested. A suitable solution concentration of 5% was selected. This concentration of hyaluronic acid solution has a reasonable viscosity, forms a regular shape when filling the needle tip, can stably load curcumin, and can quickly dissolve and decompose the drug in the wound microenvironment.

[0062] The drying temperature was selected through multi-level temperature control comparison experiments. Multiple temperature values ​​were set within a range for curing and molding operations. The magnitude of matrix dehydration shrinkage, the probability of structural cracking, and the physicochemical stability of active substances were observed at different temperatures. 35℃ was determined to be the suitable drying temperature. This temperature will not cause rapid dehydration and deformation damage to the matrix, nor will it delay the curing process due to excessively low temperatures. It ensures the simultaneous shaping of the double-layer structure while avoiding the damage of high temperatures to the photosensitizer and natural small molecule activity.

[0063] The drying time is determined based on the curing state. Different drying times are sequentially set at a fixed drying temperature, and the removal of moisture and the compactness of the solidified structure are observed layer by layer until the material completely loses its fluidity and its shape no longer changes. A drying time of 12 hours is then determined based on this. This time allows for complete removal of moisture from the solution, ensuring complete curing of both matrix layers. This effectively avoids deformation and softening issues during later use, guaranteeing long-term stability of the microneedle's morphology and performance.

[0064] Furthermore, the method provided in this application embodiment includes: The performance of the obtained photodynamic microneedle test samples was evaluated, including: microneedle morphology evaluation, mechanical strength testing, drug release characteristic testing, and reactive oxygen generation capacity detection of the composite nano photosensitizer.

[0065] Specifically, the photodynamic microneedle test sample was bonded and fixed to the surface of the scanning electron microscope (SEM) stage using conductive adhesive. Gold sputtering was performed using a gold target with a sputtering current of 10 mA and a sputtering time of 90 seconds. The processed sample was then placed inside the SEM, with an accelerating voltage of 8 kV. Microscopic morphology was observed at 500x and 2000x magnification. Simultaneously, an optical camera was used to capture the macroscopic morphology of the microneedle array under natural light at a distance of 10 cm. The acceptable criteria were set as an array row / column deviation of no more than 5 μm and a needle tip taper angle maintained between 15 and 30 degrees. The array arrangement regularity, needle tip morphology, and uniformity of active ingredient distribution were compared with standard morphology maps, and all morphology observation data were recorded simultaneously.

[0066] Mechanical strength tests were conducted under constant conditions of 25°C and 50% humidity. Photodynamic microneedles, all with a uniform length of 600 micrometers and a base diameter of 300 micrometers, were securely fixed in the loading slot of a universal materials testing platform using a specialized fixture. The device was set to a compression travel rate of 2 mm / min, with an initial preload of 0.02 N and a maximum compression stroke of 1 mm. Pressure and displacement data were collected by slowly pressing down along the direction perpendicular to the needle tip, and force-displacement curves were generated simultaneously. Each sample was tested five times. Data deviating more than 10% from the group average were considered outliers and discarded. The average of the remaining valid values ​​was calculated to analyze the microneedle's load-bearing limit. The maximum pressure a single microneedle could withstand was determined to be 0.27 N, thus determining the sample's puncture capability.

[0067] A phosphate buffer solution with a pH of 7.4 and a molar concentration of 0.01 mol / L was prepared as the release medium. 20 mL of the buffer solution was placed in a thermostatic container, and the internal temperature was maintained at 37°C. Uniformly sized microneedles were completely immersed in the buffer solution. 2 mL samples were extracted at 1 minute, 3 minutes, and 6 minutes. After each sampling, 2 mL of fresh buffer solution was added to maintain a stable system volume. The extracted samples were placed in a microplate reader, and the absorbance was read at a detection wavelength of 420 nm. The cumulative drug release ratio at different time points was calculated, and a drug release curve was plotted to evaluate the drug release rate and total drug release.

[0068] A 0.1 mg / mL composite nano-photosensitizer test solution was prepared. 0.1 mol / L 5,5-dimethyl-1-pyrroline-N-oxide reagent and 0.05 mol / L 2,2,6,6-tetramethylpiperidine reagent were mixed with the test solution at a volume ratio of 1:1, ensuring a total detection system volume of 5 mL. After mixing and stirring, the mixture was allowed to stand for 30 seconds, then irradiated for 1 minute using an LED light source with a power of 50 mW / cm² and a wavelength range of 400 nm to 800 nm, maintaining a light distance of 5 cm. Immediately after irradiation, the mixture was fed into an electron paramagnetic spectrometer. Signal acquisition was performed at a microwave frequency of 9.4 GHz and a scan width of 1000 Gauss. The type and level of reactive oxygen species generated were determined based on the characteristic peak shapes observed.

[0069] Four performance tests were completed using complete quantitative detection parameters to comprehensively evaluate the appearance, mechanical properties, drug release capacity, and photosensitivity of microneedles, providing reliable experimental basis for optimizing the preparation parameters of composite nanophotosensitive agents.

[0070] Furthermore, such as Figure 2 As shown, the method provided in this application embodiment includes: Composite photosensitizers are prepared by using synthesis parameters and in-situ loading parameters that meet preset objectives. The preset objectives include enabling the composite photosensitizers to generate singlet oxygen and superoxide anion radicals under light irradiation and to generate hydroxyl radicals in the presence of hydrogen peroxide.

[0071] In one embodiment, the composite nanophotosensitizer prepared with the specified parameters has the ability to generate multiple types of reactive oxygen species. Different reactive oxygen species can play different functional roles, jointly supporting the photodynamic microneedles to achieve wound repair and antibacterial effects.

[0072] Singlet oxygen and superoxide anion free radicals generated under light have strong oxidizing properties. These two types of substances can directly act on harmful microorganisms at the skin wound surface, destroying the cell membrane structure of microorganisms, blocking the internal nutrient transport and metabolic processes of bacteria, and effectively inhibiting the continuous reproduction and spread of pathogens. At the same time, they can oxidize and remove inflammatory metabolic substances accumulated on the wound surface, relieving local redness and inflammation symptoms, and initially achieving an antibacterial and soothing effect on the wound.

[0073] The hydroxyl radicals generated in the hydrogen peroxide system have higher oxidative activity, enabling deep oxidation and degradation of stubborn bacterial debris and wound impurities that are difficult to decompose with ordinary reactive oxygen species. Human skin naturally produces trace amounts of hydrogen peroxide at damaged sites. Photosensitizers can trigger reactions based on the wound's own endogenous substances, continuously and stably producing hydroxyl radicals, extending the effective duration of action, and further enhancing wound cleaning and repair.

[0074] The three reactive oxygen species (ROS) have non-overlapping mechanisms of action, and their target sites are complementary. Their combined action can construct a multi-layered protection and repair system, avoiding the problems of insufficient action of a single ROS and the development of pathogen tolerance. By controlling the synthesis and in-situ loading parameters according to this preset target, the composite nano-photosensitizer can be guaranteed to possess both excellent photosensitivity and Fenton reaction activity, with stable and balanced oxygen production performance.

[0075] When the composite nano photosensitizer with this functional property is incorporated into the photodynamic microneedle, it allows the microneedle to exert multiple effects simultaneously after penetrating the skin, comprehensively improving the antibacterial, anti-inflammatory, and repair effects of the wound. It also ensures that the various performance characteristics of the finished microneedle product are stable and meet the actual medical repair needs.

[0076] Example 2: This application also provides a photodynamic microneedle patch, comprising: An antibacterial base layer, made of gelatin, containing the composite nano photosensitizer; and a drug-loaded needle tip layer, made of hyaluronic acid, containing natural small molecules.

[0077] Optionally, the photodynamic microneedle patch employs a dual-layer composite structure, consisting of a tightly bonded antibacterial base layer and a drug-loaded needle tip layer. The antibacterial base layer uses gelatin with a molecular weight of 50 to 100 kDa as the matrix material, loaded with CuTCPP@Cu2O composite nanophotosensitizer prepared using a parameter optimization method. The drug-loaded needle tip layer uses hyaluronic acid with a molecular weight of 200 to 400 kDa as the matrix material, loaded with curcumin, a natural small molecule. The two layers have clearly defined functions and work synergistically to achieve the dual core functions of subcutaneous antibacterial and anti-inflammatory repair. High-purity deionized water with a resistivity greater than 18.2 megohmcm is used uniformly in the preparation solution to ensure stable colloidal dissolution.

[0078] In preparing the drug-loaded needle tip layer, hyaluronic acid of a predetermined molecular weight was added to high-purity deionized water and stirred continuously at 250 rpm until the material was completely dissolved, resulting in a 5% (w / v) hyaluronic acid solution. Curcumin was then added at a ratio of 0.2% (w / v) of the solution mass. The mixture was stirred for 2 hours in a dark environment to ensure uniform dispersion of the curcumin and form a drug-loaded mixture. The drug-loaded mixture was slowly injected into the needle tip cavity of the PDMS microneedle negative mold. Three repeated vacuuming operations were performed, each time reducing the pressure to -0.095 MPa and holding it for 1 minute before restoring it to normal pressure. The negative pressure was used to expel air bubbles from the solution, ensuring the solution fully filled the needle tip cavity. Excess solution outside the cavity was removed. After debubbling, the mixture was allowed to stand for two minutes to stabilize the liquid surface. No pre-drying was required before proceeding to the next step. The finished microneedle was designed with a height of 600 micrometers and a needle tip base diameter of 300 micrometers.

[0079] In preparing the antibacterial substrate layer, gelatin with a predetermined molecular weight is added to high-purity deionized water. The mixture is heated to 55 degrees Celsius and continuously stirred at 300 rpm until completely dissolved, resulting in a 20% (w / v) gelatin solution. A CuTCPP@Cu2O composite nano-photosensitizer, obtained through parameter optimization, is added at a ratio of 0.5% of the solution mass. The mixture is stirred at the same rate for 30 minutes to ensure uniform dispersion of the composite nano-photosensitizer, forming an antibacterial mixture. This antibacterial mixture is then slowly poured onto a mold that has already been filled with the needle tip layer. The substrate thickness is uniformly controlled at 200 micrometers, forming a complete substrate cover layer without the need for further vacuuming.

[0080] After the double-layer solution casting is completed, the microneedle mold is placed in a constant temperature environment of 35 degrees Celsius for continuous drying for 12 hours, allowing the hyaluronic acid drug-loaded needle tip layer and the gelatin antibacterial base layer to solidify completely layer by layer. Demolding is then performed under conditions of 25 degrees Celsius and 50% humidity. The mold is held steadily by both hands along its sides, and the mold cavity is peeled off smoothly and evenly upwards, avoiding pulling or bending to prevent needle tip breakage and layer separation. Once demolding is complete, a photodynamic microneedle patch with a well-defined structure and uniform component distribution is obtained.

[0081] In practical applications, the drug-loaded microneedle patch utilizes the excellent mechanical strength of hyaluronic acid to achieve physical skin penetration. Upon contact with the wound microenvironment, it rapidly dissolves and releases curcumin, eliminating local inflammatory responses and reducing tissue damage caused by inflammatory factors. The antibacterial base layer adheres tightly to the skin surface. Under light irradiation, it drives the composite nano-photosensitizer to generate singlet oxygen and superoxide anion free radicals, disrupting bacterial biofilms and clearing deep subcutaneous bacteria. Simultaneously, it utilizes hydrogen peroxide in the wound microenvironment to generate hydroxyl free radicals through the Fenton reaction, further enhancing the antibacterial effect. This dual-function synergistic effect promotes angiogenesis and tissue repair in infected wounds.

[0082] In summary, the method for optimizing the parameters of composite nanophotosensitive agents for the preparation of photodynamic microneedles provided in this application has the following technical effects: This application utilizes multiple performance test data from photodynamic microneedle samples, iteratively adjusting synthesis and in-situ loading parameters to screen parameters that meet preset standards for the generation of various reactive oxygen species, thus preparing a composite nano-photosensitizer. A microneedle patch is constructed using two types of matrices layered together, with corresponding functional components loaded in each layer for synergistic effects. This results in a stable antibacterial and anti-inflammatory effect, effectively enhancing wound healing capabilities. The photodynamic microneedles demonstrate stable and reliable performance in actual use, achieving precise locking of photosensitizer synthesis and loading parameters, ensuring the final product possesses the preset performance characteristics.

[0083] The above description of the disclosed embodiments enables those skilled in the art to make or use this application. 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 this application. Therefore, this application 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 disclosed herein.

[0084] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of this application and its equivalents, this application also intends to include such modifications and variations.

Claims

1. A method for optimizing the parameters of composite nano-photosensitizers used in the preparation of photodynamic microneedles, characterized in that, include: Octahedral Cu₂O nanoparticles were prepared, and the synthesis parameters were recorded. CuTCPP@Cu2O composite nanoparticles were prepared by in-situ loading sheet-like CuTCPP onto the octahedral Cu2O nanoparticles, and the in-situ loading parameters were recorded. The obtained CuTCPP@Cu2O composite nanoparticles were mixed with the first matrix material, and natural small molecules were mixed with the second matrix material. The photodynamic microneedle test sample with a double-layer structure was prepared by a microneedle forming process of layered casting and vacuum-assisted filling. The performance of the obtained photodynamic microneedle test samples was evaluated. Based on the performance evaluation results, the synthesis parameters and / or in-situ loading parameters were adjusted to achieve the preset target. The composite nano photosensitizer was then prepared by using the synthesis parameters and in-situ loading parameters that met the preset target.

2. The method for optimizing the parameters of composite nanophotosensitive agents for the preparation of photodynamic microneedles according to claim 1, characterized in that, The performance of the obtained photodynamic microneedle test samples was evaluated, including: microneedle morphology evaluation, mechanical strength testing, drug release characteristic testing, and reactive oxygen generation capacity detection of the composite nano photosensitizer.

3. The method for optimizing the parameters of composite nanophotosensitive agents for the preparation of photodynamic microneedles according to claim 1, characterized in that, The first matrix material is gelatin with a molecular weight of 50-100 kDa; the second matrix material is hyaluronic acid with a molecular weight of 200-400 kDa; and the natural small molecule is curcumin.

4. The method for optimizing the parameters of composite nanophotosensitive agents for the preparation of photodynamic microneedles according to claim 1, characterized in that, CuTCPP@Cu2O composite nanoparticles were prepared by in-situ loading sheet-like CuTCPP onto the octahedral Cu2O nanoparticles, comprising: Cu2O was added to ethanol / pure water and sonicated for a predetermined time until the nanoparticles were uniformly dispersed to obtain the first intermediate. A DMF solution of meso-tetra(4-carboxyphenyl)porphyrin was added dropwise to the first intermediate, and the mixture was heated to a predetermined temperature and reacted for a predetermined time to obtain the second intermediate. The second intermediate was separated and precipitated using a high-speed centrifuge, washed with anhydrous ethanol, and dried to obtain CuTCPP@Cu2O powder.

5. The method for optimizing the parameters of composite nanophotosensitive agents for the preparation of photodynamic microneedles according to claim 1, characterized in that, The synthesis parameters include: the molar ratio of Cu(NO3)2·3H2O, NaOH and ascorbic acid, reaction temperature, and reaction time.

6. The method for optimizing the parameters of composite nanophotosensitive agents for the preparation of photodynamic microneedles according to claim 1, characterized in that, The parameters of the microneedle forming process include: the solution concentration of the second matrix material, the solution concentration of the first matrix material, the drying temperature, and the drying time.

7. The method for optimizing the parameters of composite nanophotosensitive agents for the preparation of photodynamic microneedles according to claim 1, characterized in that, Composite photosensitizers are prepared by using synthesis parameters and in-situ loading parameters that meet preset objectives. The preset objectives include enabling the composite photosensitizers to generate singlet oxygen and superoxide anion radicals under light irradiation and to generate hydroxyl radicals in the presence of hydrogen peroxide.

8. The method for optimizing the parameters of composite nanophotosensitive agents for the preparation of photodynamic microneedles according to claim 1, characterized in that, Vacuum-assisted filling involves repeatedly evacuating the solution added to the mold three times to remove air bubbles.

9. A photodynamic microneedle patch, the photodynamic microneedle patch comprising a composite nano-photosensitizer prepared by the composite nano-photosensitizer parameter optimization method for photodynamic microneedle preparation as described in any one of claims 1-8, comprising: An antibacterial base layer, the antibacterial base layer being made of gelatin and containing the composite nano photosensitizer; The drug-loaded needle tip layer is made of hyaluronic acid and contains natural small molecules.

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