Photodynamic antibacterial process double response surface optimization and antibacterial effect evaluation method of 4-BrPA / beta-CD supramolecular photosensitizer

Abstract

The invention discloses a photodynamic antibacterial process double response surface optimization and antibacterial effect evaluation method of a 4-BrPA / beta-CD supramolecular photosensitizer, and belongs to the technical field of photodynamic antibacterial. According to the method, a 4-BrPA / beta-CD supramolecular photosensitizer is used as a photosensitive material, 450 nm visible light is used as an excitation light source, the material concentration, the optical power density and the illumination time are used as investigation factors, and the net bacteriostasis rate measured by a plate colony counting method is used as a response value for Box-Behnken test design; through parallel detection of real-time sampling after illumination and delayed sampling after dark culture, respectively establishing real-time sterilization and delayed sterilization double-response surface models; after background toxicity verification and unified deduction, comparing the coefficient change of interaction items of the double models, and analyzing the differentiated influence of the delay effect on different interaction effects; and determining an optimal process interval by taking the delayed sterilization model as a final basis. Meanwhile, five quantitative evaluation indexes including DGI, CPAI, PTCI, CTRI and HTI are provided. The invention provides a complete method from process optimization to effect evaluation for the photosensitizer.

Description

Technical Field

[0001] This invention relates to the field of photodynamic antibacterial technology and process optimization, specifically to a dual response surface optimization method and antibacterial effect evaluation method for photodynamic antibacterial processes using 4-BrPA / β-CD supramolecular photosensitizer. Background Technology

[0002] Photodynamic therapy (aPDT), which utilizes photosensitizers to generate reactive oxygen species to kill pathogenic microorganisms under light excitation at specific wavelengths, has become an important alternative strategy to address the bacterial resistance crisis. Cyclodextrin-based supramolecular photosensitizers have attracted much attention due to their good biocompatibility and host-guest recognition capabilities. However, to advance these novel photosensitizers from basic laboratory research to industrial applications, key technical challenges need to be addressed, including optimizing process parameters, analyzing the interactions of multiple factors, and systematically evaluating their antibacterial effects.

[0003] On the same day, the applicant filed another invention patent application (invention title: A 4-BrPA / β-CD supramolecular photosensitizer, its preparation method and application), which discloses a method for preparing a 4-BrPA / β-CD supramolecular photosensitizer formed by non-covalent self-assembly of 4-bromophthalic anhydride (4-BrPA) and β-cyclodextrin (β-CD), and confirms that it has good photophysical properties and basic photodynamic activity under visible light excitation. However, this application does not address the key industrialization technical issues such as process parameter optimization and effect evaluation of this photosensitizer in practical photodynamic antibacterial applications.

[0004] Currently, the optimization methods for photodynamic antibacterial processes using novel photosensitizers (including 4-BrPA / β-CD) have the following shortcomings: First, the evaluation time point is singular, failing to consider the delayed killing effect. Photodynamic damage includes two stages: immediate killing during illumination and delayed killing due to the cumulative expression of irreversible damage after illumination ends. However, existing technologies generally use the antibacterial rate detected immediately after illumination as the sole endpoint, ignoring the contribution of the delayed effect to the actual bactericidal effect. When only immediate killing is used as the optimization target, it may lead to bias in the evaluation of the benefits of high light intensity and long-term parameter combinations, thus affecting the accuracy of the process optimization direction.

[0005] Second, the accuracy of endpoint detection methods is insufficient. In existing photodynamic antibacterial studies, the evaluation of antibacterial effects widely employs the OD absorbance method based on an ELISA reader. This method cannot distinguish between live and dead bacteria and suffers from systematic errors due to the inherent color of the photosensitive material, light scattering interference, or bacterial aggregation. Plate count is the internationally recognized gold standard for microbial quantification, but due to its cumbersome operation and low throughput, it has not yet been integrated into methods requiring batch detection, such as response surface methodology.

[0006] Third, the background effect is not effectively separated. The total antibacterial effect of photodynamic antibacterial agents is composed of three coupled parts: material dark toxicity, non-specific phototoxicity, and net photodynamic effect. Existing technologies either completely ignore background subtraction or use a fixed value for uniform subtraction without verifying the fluctuation of background toxicity under different conditions, which may lead to systematic errors in the input data of the response surface model.

[0007] Fourth, the differences in the interaction between the immediate and delayed phases have not been considered. There are complex interactions among material concentration, optical power density, and illumination time, and these interactions may exhibit different or even opposite trends in the immediate and delayed killing phases. However, current research has not yet established a methodology capable of simultaneously capturing and comparing the interaction patterns of the two phases, nor does it possess the technical means to use the temporal evolution characteristics of the interaction as a basis for evaluating photosensitizer properties and making process decisions.

[0008] Furthermore, as a novel photosensitive material, the optimal material concentration, optical power density, illumination time, and interaction mechanism of 4-BrPA / β-CD supramolecular photosensitizer under visible light excitation have not yet been reported, and a systematic method for process optimization and effect evaluation is urgently needed. Summary of the Invention

[0009] This invention aims to overcome the aforementioned deficiencies of existing technologies. For the novel photosensitizer 4-BrPA / β-CD supramolecular photosensitizer, using 450 nm visible light as the excitation source, it provides a dual-response surface optimization method for photodynamic antibacterial processes and a method for evaluating visible light-excited antibacterial properties. Specific objectives include: (1) Establish a response surface optimization process with plate colony counting as the endpoint detection method to solve the problem that the OD value method is not able to distinguish between live and dead bacteria in the photodynamic system; (2) Establish a parallel comparison method of immediate sterilization and delayed sterilization dual response surfaces, and use the delayed effect as the endpoint index to correct the traditional optimization standard, filling the gap in the study of the delayed killing effect of 4-BrPA / β-CD photosensitizer; (3) Establish a unified background toxicity subtraction method based on representative condition point verification to achieve accurate quantification of the net photodynamic effect of 4-BrPA / β-CD photosensitizer; (4) Based on the comparative analysis of the regression coefficients of the dual model, the “selective regulation” law of the delayed effect of 4-BrPA / β-CD photosensitizer under 450 nm excitation on the interaction of different factors is revealed: the material concentration-light power density antagonism is greatly amplified, the light power density-light time antagonism is created, and the material concentration-light time antagonism is significantly alleviated. (5) The Delayed Effect Gain Index (DGI), Antagonistic Amplification Index (CPAI), Antagonistic Generation Index (PTCI), Antagonistic Relief Index (CTRI), and High Intensity Tolerance Index (HTI) were proposed to establish a quantitative evaluation index combination system for the delayed killing ability of 4-BrPA / β-CD supramolecular photosensitizer. (6) Based on the delayed sterilization model, the optimal process range of 4-BrPA / β-CD photosensitizer under 450 nm visible light excitation was determined.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for optimizing the photodynamic antibacterial process of 4-BrPA / β-CD supramolecular photosensitizer using a dual response surface methodology and evaluating its antibacterial effect, comprising the following steps: (1) Establishment of the optimization object and excitation light source: The optimized object of this invention is the 4-BrPA / β-CD supramolecular photosensitizer disclosed in another invention patent filed by the applicant on the same day (invention title: A 4-BrPA / β-CD supramolecular photosensitizer, its preparation method and application). This photosensitizer is formed by the self-assembly of 4-bromophthalic anhydride and β-cyclodextrin. Its maximum absorption wavelength is 234 nm, and it has at least 7 luminescent centers in the visible light range of 440-520 nm, even in the infrared region. Its maximum excitation wavelength is about 460 ± 10 nm, and its maximum emission wavelength is about 620 ± 10 nm, confirming that it has good photophysical properties and basic photodynamic activity under visible light excitation at 460 ± 10 nm.

[0011] The optimization method of this invention takes the 4-BrPA / β-CD supramolecular photosensitizer as the research object and uses 450 nm xenon lamp visible light as the excitation source.

[0012] (2) Determination of factors and levels in the 4-BrPA / β-CD photodynamic antibacterial response surface methodology: Using 4-BrPA / β-CD supramolecular photosensitizer as the research object and its characteristic absorption peak at 450 nm as the excitation wavelength, the three factors and three levels for response surface optimization were determined based on the results of single-factor experiments as follows: 4-BrPA / β-CD supramolecular photosensitizer concentration (A): 2, 5, 8 mg / L; optical power density (B): 400, 500, 600 W / m²; and illumination time (C): 20, 30, 40 min.

[0013] (3) Parallel experimental design of 4-BrPA / β-CD photodynamic antibacterial dual response surface: The Box-Behnken design was used to generate 17 test sites, including 5 central replicates. All test sites were randomized within the same batch. Each test set included the following four types of experimental units: (1) Experimental group: 4-BrPA / β-CD supramolecular photosensitizer + bacterial solution + light treatment; (2) Dark toxicity control group: 4-BrPA / β-CD supramolecular photosensitizer + bacterial solution + complete light avoidance; (3) Phototoxicity control group: bacterial solution + light treatment (without 4-BrPA / β-CD supramolecular photosensitizer); (4) Growth blank control group: bacterial solution + complete light avoidance (without 4-BrPA / β-CD supramolecular photosensitizer).

[0014] The antibacterial rate was determined at two different evaluation time points: (1) Immediate sterilization model: 3 ml of each of the four types of samples were placed in sterile cuvettes with lids, the lids were tightened, and the samples were completely wrapped with aluminum foil (to avoid light). The samples were then incubated at 37 °C for 30 min and then incubated in the dark (or pre-cultured). After the dark incubation treatment, the dark toxicity control group and the growth blank control group completely wrapped with aluminum foil, as well as the test group and the phototoxicity control group without aluminum foil, were placed under a 450 nm xenon lamp light source and the power density and light time corresponding to the 17 groups of tests. Immediately after the light treatment, samples were taken, diluted appropriately, and spread on solid culture medium. The samples were incubated at 37 °C for 20 h and then plated for colony counting. (2) Delayed sterilization model: The only difference from the immediate sterilization model is that after the light treatment, all samples were incubated in the dark at 37 °C for 20 h, and then samples were taken, diluted, spread, and cultured for counting.

[0015] (4) Calculation of net inhibition rate based on plate colony count: Bacterial viability in each experimental and control group was expressed as colony forming units (CFU / mL). Net light-induced inhibition rate was calculated using the following formula: Net antibacterial rate (%) = [1 - (CFU)] exp / CFU blank )] × 100% - D - L Among them: CFU exp : Colony count in the experimental group; CFU blank : Colony count in the blank control group; D: Dark virulence inhibition rate, calculated as D = [1 - (CFU)] dark / CFU blank )] × 100%; L: phototoxicity inhibition rate, calculated as L = [1 - (CFU)] × 100%; light / CFU blank )] × 100% Calculation.

[0016] (5) Background toxicity verification and uniform deduction: Five representative condition points representing the center and boundary of the design space were selected: center point (5 mg / L, 500 W / m², 30 min); high light intensity / long time point (5 mg / L, 600 W / m², 40 min); low light intensity / short time point (5 mg / L, 400 W / m², 20 min); low concentration point (2 mg / L, 500 W / m², 30 min); and high concentration point (8 mg / L, 500 W / m², 30 min).

[0017] The dark toxicity inhibition rate D and phototoxicity inhibition rate L were calculated at each point to verify their stability within the design space. When the range of D and L values ​​at each point is ≤3%, the arithmetic mean of 5 points was used as the uniform background subtraction value for the 17 experimental groups of the response surface. Otherwise, the corresponding blank control needs to be subtracted separately for each experimental group of the response surface.

[0018] Let the dark toxicity background mean of the instant sterilization model be D. mean The mean background value of phototoxicity is L mean The dark toxicity background mean of the delayed sterilization model is D'. mean The mean background value of phototoxicity is L' mean .

[0019] (6) Construction of a 4-BrPA / β-CD photodynamic antibacterial dual response surface model: Using the immediate sterilization net bacteriostatic rate and the delayed sterilization net bacteriostatic rate as response values ​​Y, a quadratic polynomial regression model was established: Y (immediate sterilization) = β0 + β1A + β2B + β3C + β A×B AB + β A×C AC + β B×C BC + β A×A A² + β B×B B² + β C×C C² and Y' (delayed sterilization) = β'0 + β'1A + β'2B + β'3C + β' A×B AB + β' A×C AC + β' B×C BC+ β' A×A A² + β' B×B B² + β' C×C C².

[0020] The significance of the model (p<0.05), the lack of fit, and the signal-to-noise ratio (Adeq Precision >4) were tested using analysis of variance. The goodness of fit and predictive ability of the model were comprehensively evaluated using the coefficient of determination R², the corrected coefficient of determination Adj R², and the predictive coefficient of determination Pred R², with the requirement that the difference between Pred R² and Adj R² be <0.2.

[0021] (7) Dual-model comparative analysis and evaluation of the delayed killing ability of 4-BrPA / β-CD photosensitizer: A systematic comparative analysis of the regression coefficients, three-dimensional morphology of the response surface, and contour projection characteristics of the immediate sterilization model and the delayed sterilization model reveals the "selective regulation" law of the delayed effect on the interaction of different factors: ① Comparison of constant terms: Calculate the delay effect gain index DGI = β0'-β0, which quantifies the baseline gain of the delay effect of 4-BrPA / / β-CD photosensitizer under 450 nm excitation; a DGI ≥ 5 percentage points is considered to indicate that the photosensitizer has a significant delay enhancement effect; ② Comparison of quadratic coefficients: Compare the changing trends of coefficients A², B², and C². The larger the absolute value of the quadratic coefficient, the smaller the degree of narrowing / widening of the optimal window for that factor in the delay stage, and vice versa. ③ Comparison of interaction terms and intensities: Analyze the changes in the coefficients of the material concentration-light power density interaction term (A×B), the material concentration-illuminance time interaction term (A×C), and the light power density-illuminance time interaction term (B×C). The specific judgment rules are as follows: The criterion for judging delayed-amplification antagonism is: the coefficient β of the interaction term between material concentration and optical power density in the instantaneous sterilization model. A×B In the delayed sterilization model, the coefficient ≤ -3.0 and p < 0.05 indicates that β A×B For coefficients ≤ -8.0 and p < 0.05, the change Δ A×B =β A×B '-β A×B ≤-4.0; Preferred, instantaneous model β A×B The coefficient is -4.69 and p < 0.05, β in the delay model A×B The coefficient is -10.35 and p < 0.05, Δ A×B =-5.66; The criteria for judging delayed creation antagonism are divided into three levels: (1) Significant creation: β in the instant model B×C For coefficients p ≥ 0.05, β' in the delay model B×C The coefficient p < 0.05; (2) Potential creation: β in the instant model B×CFor coefficients p ≥ 0.05, β' in the delay model B×C The coefficient p < 0.10 and Δ B×C =β B×C '-β B×C ≤ -3.0; (3) Trend creation: β in the instant model B×C For coefficients p ≥ 0.05, β' in the delay model B×C The coefficient p ≥ 0.10 and Δ B×C =β B×C '-β B×C ≤ -4.0; Preferred, instantaneous model β B×C The coefficient is -0.85, p>0.05, and β in the delay model B×C The coefficient is -4.97, p < 0.1, Δ B×C =-4.12; The criterion for significantly delaying the relief of antagonism is: the interaction term β between material concentration and light exposure time in the instantaneous sterilization model. A×C For coefficients ≤ -3.0 and p < 0.05, β in the delayed sterilization model A×C 'When the coefficient is ≥ -3.0, its change Δ A×C =β A×C '-β A×C ≥+3.0; Preferred, instantaneous model β A×C With a coefficient of -5.26 and p < 0.05, β in the delay model A×C The coefficient is -2.1, Δ A×C =+3.16.

[0022] ④ High Intensity Tolerance Index Calculation: HTI = β B×C '- β B×C The unit is the absolute difference of the encoded regression coefficients. HTI > 0: The BC interaction is enhanced in the delayed stage compared to the immediate stage (antagonism weakens or turns into synergy), indicating that the photosensitizer has a delayed compensation effect; HTI < 0: The BC interaction is weakened in the delayed stage compared to the immediate stage (antagonism is enhanced), indicating that the photosensitizer has a delayed amplification effect; The magnitude of |HTI| reflects the intensity of the delayed effect on the regulation of the BC interaction, and the larger the |HTI|, the more significant the regulation; ⑤ Evaluation Index System: Based on the DGI and HTI mentioned above, the following indicators are also calculated: Material concentration-optical power density antagonistic amplification index CPAI = |β A×B ' / β A×B When CPAI ≥ 2.0, it is determined to have significant delayed amplification antagonism capability; Optical power density-time antagonistic generation index PTCI = |β B×C ' - β B×C |, when PTCI ≥ 3.0, it is judged as having significant delayed antagonistic ability; Material concentration-light duration antagonistic mitigation index CTRI = |β A×C ' - β A×C | When CTRI ≥ 3.0, it is considered to have significant delayed relief antagonistic ability; DGI, CPAI, PTCI, CTRI, and HTI were used as a combination of quantitative evaluation indicators for the delayed killing ability of the 4-BrPA / β-CD supramolecular photosensitizer.

[0023] (8) Determination of optimal conditions for photodynamic antibacterial process using 4-BrPA / β-CD photosensitizer: The optimal process conditions predicted by the instantaneous model using response surface methodology are: material concentration 5.96 mg / L and optical power density 487.6 W / m². 2 With an illumination time of 32.99 min, the maximum net antibacterial rate (%) was 84.30%. The predicted optimal process conditions for the delayed model were: material concentration 6.24 mg / L and light power density 479.17 W / m². 2 With an illumination time of 33.04 min, the maximum net antibacterial rate (%) was 87.18%. Based on the optimal process predicted by both models, a material concentration of 6.2 mg / L and a light power density of 480 W / m² were selected. 2 Further process verification was conducted by irradiating the sample for 33 minutes. The experiment was repeated three times. The results showed that the actual average net antibacterial rate (%) of the immediate model and the delayed model was 85.2% and 87.6%, respectively, which was consistent with the model prediction results, proving the accuracy of the model in predicting process conditions.

[0024] (9) Application scenarios: The above optimization methods can be applied to the following scenarios: screening and optimization of photodynamic antibacterial process parameters for 4-BrPA / β-CD photosensitizers; evaluation of the consistency of photodynamic activity of different batches of 4-BrPA / β-CD photosensitizers; and monitoring of the storage stability of 4-BrPA / β-CD photosensitizers.

[0025] The above evaluation method can be applied to the following scenarios: structure-activity relationship study of 4-BrPA / β-CD photosensitizers; analysis of the photodynamic damage mechanism of 4-BrPA / β-CD photosensitizers; comparison of photodynamic efficacy among different preparation processes of 4-BrPA / β-CD photosensitizers.

[0026] The beneficial effects of this invention are: (1) It fills the complete gap in the research on the optimization of photodynamic antibacterial process and delayed effect of 4-BrPA / β-CD supramolecular photosensitizer.

[0027] The 4-BrPA / β-CD supramolecular photosensitizer is a novel photosensitizing material synthesized and disclosed for the first time by the inventors. Its optimal material concentration, optimal optical power density, optimal illumination time, and interaction mechanisms under 450 nm visible light excitation have not been previously reported. This invention systematically and completely discloses for the first time the optimization method and optimal process parameters for the photodynamic antibacterial process of this photosensitizer, laying a key technological foundation for its industrial application.

[0028] (2) Methodological Innovation: The invention pioneered a dual-response surface parallel comparison model, revealing a novel law of the "selective regulation" interaction of the delayed effect. This invention is the first to incorporate the "delayed effect" into the process evaluation system of 4-BrPA / β-CD photosensitizers, establishing a dual-response surface parallel comparison model of immediate and delayed sterilization. Existing technologies all take immediate killing at the end of illumination as the sole optimization endpoint, and completely fail to understand the differentiated impact of the delayed effect on different interactions. Through a dual-model system comparison, this invention discovered and named the "selective regulation" law of the delayed effect—drastically amplifying the material concentration-light power density antagonism, creating the light power density-illumination time antagonism, and significantly alleviating the material concentration-illumination time antagonism. The revelation of this law fundamentally corrects the technical bias that "the delayed effect is a unified effect in a single direction," opening up a new research paradigm of "selective regulation of phase-interaction" for photodynamic process optimization.

[0029] (3) Innovation in detection methods: Integrating plate colony counting into the response surface optimization process.

[0030] This invention overcomes the technical inertia in the field where "response surface optimization generally relies on high-throughput OD value detection," successfully integrating the internationally recognized gold standard for microbial quantification—plate colony counting—into a three-factor, three-level response surface methodology for large-scale experiments targeting 4-BrPA / β-CD photosensitizers. Through ingenious experimental design and rigorous randomization, the data throughput required for model construction was achieved while ensuring detection accuracy. Compared to the OD value method, the plate colony counting method completely eliminates interference from dead bacteria, material color, and light scattering, resulting in higher accuracy and biological authenticity in calculating net inhibition rate.

[0031] (4) Scientific discovery and innovation: For the first time, the interaction phase evolution law of 4-BrPA / β-CD photosensitizer under 450 nm excitation was revealed.

[0032] This invention, through comparative analysis of immediate and delayed sterilization models, first discovered and reported the "selective regulation" type of the interaction between different factors of the 4-BrPA / β-CD supramolecular photosensitizer under 450 nm visible light excitation: the material concentration-light power density interaction shows delayed and drastic amplification of antagonism, the light power density-light exposure time interaction shows delayed creation of antagonism, and the material concentration-light exposure time interaction shows delayed and significant relief of antagonism. This pattern reveals that the delayed effect on the interaction between different factors in photodynamic antibacterial processes is not a uniform enhancement, but rather presents three distinct selective regulation types: "drastic amplification of antagonism, creation of antagonism, and significant relief of antagonism." This discovery elucidates the time-dependent nature of the light dose-material dose interaction at the mechanistic level.

[0033] (5) Evaluation index innovation: Establish a five-in-one quantitative index evaluation system of DGI, CPAI, PTCI, CTRI and HTI.

[0034] This invention is the first to achieve a systematic deconstruction and precise quantification of the delayed effect characteristics in photodynamic antibacterial processes. Among them, the Delayed Effect Gain Index (DGI = β0' - β0) characterizes the absolute increase in the antibacterial rate of the delayed sterilization model compared to the immediate sterilization model at the center point of the design space. When DGI ≥ 5 percentage points, the photosensitizer is considered to have a significant delayed gain effect. The Concentration-Power Antagonistic Amplification Index (CPAI = |AB′ / AB|) quantifies the amplification of the concentration-light power antagonism effect during the delayed stage. When CPAI ≥ 2.0, it indicates that the delayed stage amplifies the antagonism severely. The Power-Time Antagonistic Creation Index (PTCI = |BC′ - BC|) measures the creation intensity of the power-time interaction during the delayed stage. When PTCI ≥ 3.0, it indicates that the delayed stage creates significant antagonism. The Concentration-Time Antagonistic Relief Index (CTRI = |AC′ - AC|) characterizes the degree of relief of the concentration-time antagonism during the delayed stage. When CTRI ≥ 3.0, it indicates that the delayed stage significantly relieves the antagonism. The High Intensity Tolerance Index (HTI = BC′ - BC) determines the delayed compensation effect (HTI > 0.0) through its sign. 0) or delayed amplification effect (HTI < 0). This five-in-one evaluation system expands the traditional single-dimensional evaluation of "antibacterial rate" to include five independent dimensions: gain amplitude, antagonistic amplification, antagonistic creation, antagonistic mitigation, and regulation direction. For the first time, it has achieved a complete characterization of the "selective regulation" characteristics of the delayed effect, providing a brand-new methodological tool for the process optimization, mechanism analysis, and quality evaluation of photosensitizers.

[0035] (6) Significant process optimization results.

[0036] The process optimization of the 4-BrPA / β-CD supramolecular photosensitizer was carried out using the method of this invention. The results showed that the optimal process conditions predicted by the immediate sterilization model were a material concentration of 5.96 mg / L, a light power density of 487.6 W / m², and an illumination time of 32.99 min, with a maximum net antibacterial rate of 84.30%. The optimal process conditions predicted by the delayed sterilization model were a material concentration of 6.24 mg / L, a light power density of 479.17 W / m², and an illumination time of 33.04 min, with a maximum net antibacterial rate of 87.18%. Combining the prediction results of the two models, the comprehensive optimal process conditions were determined to be a material concentration of 6.2 mg / L, a light power density of 480 W / m², and an illumination time of 33 min. After three independent and repeated verification experiments, the measured average net antibacterial rate of immediate sterilization under these conditions was 85.2% (with a relative error of <1.1% compared to the predicted value of 84.30%), and the measured average net antibacterial rate of delayed sterilization was 87.6% (with a relative error of <0.5% compared to the predicted value of 87.18%). The verification results are in high agreement with the model predictions, which fully demonstrates that the dual response surface model established in this invention has excellent prediction accuracy, and that the determined process conditions are reliable and repeatable, providing a solid experimental basis for the precise application of 4-BrPA / β-CD supramolecular photosensitizers.

[0037] (7) Form a complete technology chain with material patents.

[0038] This invention, along with another invention patent filed on the same day by the applicant (synthesis of a β-cyclodextrin supramolecular photosensitizer and its visible light response characteristics), seamlessly connects and supports each other, together forming a complete technical chain from "material preparation → structural characterization → photoactivity verification → process optimization → mechanism analysis → effect evaluation". The two patents form a dual intellectual property barrier, providing a solid guarantee for the subsequent industrialization development of 4-BrPA / β-CD supramolecular photosensitizers. Attached Figure Description

[0039] The accompanying drawings are provided to further explain the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 The diagram shows the three-dimensional response surface and contour plot of the instant sterilization model of this invention. Figure 1 A: A three-dimensional graph of material concentration versus optical power density on net antibacterial rate; Figure 1 B: Contour plot of material concentration versus optical power density as a function of net antibacterial rate; Figure 1 C: Three-dimensional graph of material concentration versus light exposure time on net antibacterial rate; Figure 1 D: Contour plot of material concentration versus light exposure time on net antibacterial rate; Figure 1 E: A three-dimensional graph of the effect of light power density - illumination time on net antibacterial rate; Figure 1 F: Contour plot of light power density versus illumination time on net antibacterial rate; Figure 2 The diagram shows the three-dimensional response surface and contour plot of the delayed sterilization model of this invention. Figure 2 A: A three-dimensional graph of material concentration versus optical power density on net antibacterial rate; Figure 2 B: Contour plot of material concentration versus optical power density as a function of net antibacterial rate; Figure 2 C: Three-dimensional graph of material concentration versus light exposure time on net antibacterial rate; Figure 2 D: Contour plot of material concentration versus light exposure time on net antibacterial rate; Figure 2 E: A three-dimensional graph of the effect of light power density - illumination time on net antibacterial rate; Figure 2 F: Contour plot of light power density versus illumination time on net antibacterial rate; Figure 3 This is a comparison chart of the maximum net antibacterial rate under the center point conditions, predicted optimal conditions, and actual verification conditions of the instantaneous and delayed sterilization models of this invention. Figure 4 This is a schematic diagram illustrating the coefficient transformation relationship between the instantaneous and delayed sterilization models of the present invention and the "selective regulation" law of the three sets of interactions; Figure 4 A: A diagram showing the transformation relationship of all coefficients in the instant and delayed sterilization models; Figure 4 B: Schematic diagram of the "selective regulation" law of the interaction of the three groups of instant and delayed sterilization models. Detailed Implementation

[0040] 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 embodiments. This embodiment is a specific implementation example of the optimization and effect evaluation of the photodynamic antibacterial process of 4-BrPA / β-CD supramolecular photosensitizer under 450 nm visible light excitation, but the scope of protection of this invention is not limited to this embodiment.

[0041] Example 1: Optimization of dual response surface methodology for photodynamic antibacterial process using 4-BrPA / β-CD supramolecular photosensitizer.

[0042] 1. Experimental materials: 1.1 4-BrPA / β-CD supramolecular photosensitizer: The preparation method, structural characterization, and visible light response characteristics of the 4-BrPA / β-CD supramolecular photosensitizer used in this embodiment are detailed in Example 1 of another invention patent filed by the applicant on the same day (Invention title: A 4-BrPA / β-CD supramolecular photosensitizer, its preparation method and application). This embodiment will not repeat the synthesis process; the 4-BrPA / β-CD supramolecular photosensitizer powder prepared by this patent is directly used to prepare stock solutions of 2 mg / L, 5 mg / L, and 8 mg / L with sterile 1×PBS + 0.1% peptone solution (hereinafter referred to as PBS solution), and stored at 4°C in the dark for later use.

[0043] 1.2 Culture medium: LB broth medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.2-7.4, autoclaved at 121℃ for 20 min; LB agar medium: Add 1.5% agar powder to LB broth and autoclave at 121℃ for 20 min.

[0044] 1.3 Preparation of test strains and bacterial stock solutions: After rapidly thawing the Escherichia coli (E. coli, ATCC 25922) culture tubes stored at -80℃ in the laboratory at approximately 37℃, 100 μl of each tube was serially diluted 10-fold, starting from 10... 6 200 μl of the diluted solution was inoculated onto LB agar plates using the spread plate method and incubated at 37°C for 12–24 h to complete the first activation. Single colonies were picked from the first activation plates and 60 plates were activated from each plate. All cells from the second activation were washed with sterile PBS to prepare a 25 ml bacterial stock solution, which was stored at 4°C for later use. Cell pellet was then obtained by centrifugation (8000 r / min, 5 min at 4°C) and washed three times with sterile PBS. Finally, the bacteria were resuspended in sterile PBS to prepare a cell concentration of approximately 1 × 10⁻⁶ cells / mL. 7 CFU / ml bacterial stock solution.

[0045] 1.4 Preparation of high-concentration 4-BrPA / β-CD material stock solution: Dissolve 4-BrPA / β-CD material in PBS solution to prepare a stock solution of 2560 mg / L. Place the stock solution in a 10 ml plastic centrifuge tube with high transparency. Irradiate the tube with a 365 nm UV lamp at a distance of 10 cm for 1-2 h. The tube can be used only after being checked for sterility by plate count method.

[0046] 1.5 Excitation source calibration: The excitation source used in this embodiment is a xenon lamp with a center wavelength of 450 nm, an irradiation area of ​​10 cm × 10 cm, and an adjustable optical power density range of 0-2000 W / m². Before each experiment, the optical power density is calibrated at the well surface of the culture plate using an optical power meter to ensure that the actual irradiation intensity deviates from the design value by ≤5%.

[0047] 2. Test methods: 2.1 Response surface methodology: Box-Behnken design was performed using Design-Expert 13.0 software, with material concentration (A), 450 nm light power density (B), and illumination time (C) as the three factors. The net light-induced inhibition rate, determined by plate colony counting, was used as the response value, generating 17 test sites (including 5 central point replicates). The randomization order of the test sites and the combination of factor levels are shown in Table 1.

[0048] Table 1 Box-Behnken Design (BBD) Experimental Design Scheme 2.2 Sampling and Grouping: For the 17 groups of experiments in Table 1, four types of experimental units were set up as shown in Table 2. For each group of experiments, four sterile, stoppered quartz cuvettes were used. Using aseptic technique, 2.7 ml of sterile material solution and 0.3 ml of diluted bacterial stock solution (~1×10⁻⁶) were added to the experimental group and the dark toxicity control group cuvettes. 7 (CFU / ml), at this point the concentration of the material in the cuvette remained almost unchanged, while the bacterial content was ~1×10⁻⁶. 6 CFU / ml; Add 2.7 ml of sterile PBS solution and 0.3 ml of diluted bacterial stock solution (~1×10⁻⁶) to the phototoxicity control group and the growth blank control group. 7 If the concentration is CFU / ml, then the bacterial content is also ~1×10⁻⁶. 6 CFU / ml. Each experiment was performed in triplicate, and the results were averaged.

[0049] Table 2. Four types of experimental unit designs for each group of BBD trials. 2.3 Dark incubation: All four types of experimental units were placed in a 37°C constant temperature incubator, wrapped in aluminum foil and incubated in the dark for 30 minutes to allow the 4-BrPA / β-CD photosensitizer to fully bind with the bacteria.

[0050] 2.4 450 nm Xenon lamp illumination treatment: Perform lighting treatment according to the parameters in Table 1: Experimental group and phototoxicity control group: Irradiated with 450 nm xenon lamps according to the set light power density (400 / 500 / 600 W / m²) and irradiation time (20 / 30 / 40 min); Dark toxicity control group and growth blank control group: They were tightly wrapped in aluminum foil, kept in the dark throughout the process, and placed in the same environment as the experimental group and phototoxicity control group for 450 nm xenon lamp irradiation.

[0051] 2.5 Two-time-point sampling and plate colony counting: (1) Instant sterilization model: Immediately after the 450 nm light treatment, take 100 μL of bacterial culture from each cuvette, dilute it 5-fold with sterile PBS, and then perform serial 10-fold dilutions (10... -1 10 -2 10 -3 ), take 200 μL of each and spread it on LB agar plates, incubate at 37℃ for 20 h, select an appropriate dilution (expected colony count 30-300 CFU) for plate colony counting. Spread each dilution on 3 parallel plates, and take the average of the results.

[0052] (2) Delayed sterilization model: After the 450 nm light treatment, all cuvettes were placed in a 37℃ constant temperature incubator for 20 h of dark incubation. After the dark incubation, the same as the instant sterilization model was performed, including sampling, dilution, plating, incubation, counting, and averaging.

[0053] 2.6 Background toxicity verification and determination of uniform deduction values: According to step (5) of the technical solution, five representative condition points were selected, and their dark toxicity inhibition rate and phototoxicity inhibition rate were measured in two evaluation systems: instant sterilization and delayed sterilization. Three independent repeated experiments were set up for each condition point, and the average value of the results was taken.

[0054] 2.7 Calculation of net inhibition rate at each test site: As described in step (4) of the technical solution, the net inhibition rate based on plate colony count is calculated. The net light-induced inhibition rate at each test point is calculated using the following formula: Net antibacterial rate (%) = [1 - (CFU)] exp / CFU blank )] × 100% - D - L Among them: CFU exp : Colony count in the experimental group; CFU blank : Colony count in the blank control group; D: Dark virulence inhibition rate, calculated as D = [1 - (CFU)] dark / CFU blank)] × 100%; L: phototoxicity inhibition rate, calculated as L = [1 - (CFU)] × 100%; light / CFU blank )] × 100% Calculation.

[0055] When the range of both the dark toxicity inhibition rate and the phototoxicity inhibition rate at each point is ≤3%, the arithmetic mean of 5 points is used as the uniform background subtraction value for the 17 test groups of the response surface. At this time, the net photoexcitation inhibition rate at each test point is calculated according to the following formula: Net antibacterial rate (%) = [1 - (CFU)] exp / CFU blank )] × 100% - D mean - L mean Among them: CFU exp Mean colony count of the experimental group; CFU blank Mean colony count in the blank control group; D mean : The dark toxicity background mean of the corresponding model; L mean : The mean background phototoxicity value of the corresponding model.

[0056] 3. Results Analysis: 3.1 Five validation results of the immediate sterilization model and the delayed sterilization model: According to method 2.6, the representative 5-point dark toxicity inhibition rate D and phototoxicity inhibition rate L in the immediate sterilization model and the delayed sterilization model were obtained respectively, and the results are shown in Table 3.

[0057] Table 3. Five-point validation results of the instant sterilization model and the delayed sterilization model. Table 3 shows that the range of background values ​​at 5 representative points in both the immediate and delayed sterilization models is <3%, indicating that the 4-BrPA / β-CD supramolecular photosensitizer exhibits good stability in background toxicity within the design space. Therefore, the arithmetic mean of the 5 points can be used as a uniform background subtraction value. Instant sterilization model: D mean (%) = 3.63, L mean (%) = 0.88; Delayed sterilization model: D' mean (%) = 8.81, L' mean (%) = 0.77; Instant sterilization model: Net antibacterial rate (%) = [1 - (CFU)] exp / CFU blank )] ×100% - 3.63-0.88; Delayed sterilization model: Net inhibition rate (%) = [1 - (CFU')] exp / CFU' blank )] ×100% -8.81-0.77.

[0058] 3.2 Calculation and analysis of net inhibition rate of the immediate sterilization model and the delayed sterilization model: The net antibacterial rates of the 17 test sites for the immediate sterilization model and the delayed sterilization model, calculated according to the results in 3.1, are listed in Table 4.

[0059] Table 4. Net antibacterial rate results of response surface methodology for 4-BrPA / β-CD supramolecular photosensitizers Using Design-Expert 13.0 software, quadratic polynomial regression was performed on the response value Y with the immediate bacteriostatic rate and the delayed bacteriostatic rate, respectively, to obtain the regression equation and the results of the analysis of variance.

[0060] (1) Immediate sterilization model (450 nm excitation): Y=81.76+7.59A-4.21B+7.15C-4.69AB-5.26AC-0.85BC-10.36A 2 -24.10B 2 -9.33C 2 .

[0061] Model evaluation metrics: R² = 0.9923; Adj R² = 0.9824; Pred R² = 0.8814; AdeqPrecision = 25.952; Lack of fit p = 0.0028 (Note: The repeated measured values ​​of the 5 center points are highly consistent, the pure error is extremely small, the difference between Pred R² and Adj R² is <0.2, the model is effective).

[0062] (2) Delayed sterilization model (450 nm excitation): Y'=83.58+10.18A-6.24B+5.60C-10.35AB-2.10AC-4.97BC-14.16A 2 -28.96B 2 -9.46C 2 .

[0063] Model evaluation metrics: R² = 0.9772; Adj R² = 0.9478; Pred R² = 0.6358; AdeqPrecision = 15.38; Lack of fit p < 0.0001 (Note: Repeated measured values ​​at the 5 center points are highly consistent, and the pure error is extremely small); It is worth noting that the prediction determination coefficient of the delayed sterilization model (Pred R² = 0.6358) is lower than that of the immediate model, and the difference between Adj R² and Pred R² exceeds 0.2.

[0064] This result indicates that the dose-response relationship of the 4-BrPA / β-CD supramolecular photosensitizer is more complex in the delayed-kill phase than in the immediate phase, potentially involving biological mechanisms that quadratic polynomial models fail to fully capture, such as threshold effects, nonlinear expression of damage accumulation, or dynamic repair of sublethal bacterial populations. Nevertheless, the model can still effectively identify the main effects and interaction trends of key influencing factors (signal-to-noise ratio 15.38 > 4), and its qualitative guidance value for optimal process conditions remains unaffected.

[0065] Click on "Net Removal Rate" in the "Analysis" option of the software, select the "ANOVA" tab for analysis of variance, and the regression analysis of the immediate sterilization model and the delayed sterilization model shown in Tables 5 and 6 will appear.

[0066] Table 5 Regression Analysis Model of the Instant Sterilization Model Table 6 Regression Analysis Model of Delayed Sterilization Model The 3D response surface plots and contour plots of the instant sterilization model and the delayed sterilization model are as follows: Figure 1 and Figure 2 As shown. Figure 1 The results show that in the instant sterilization model, both contour lines AB and AC are slightly elliptical, with a slight elongation along the main diagonal. Furthermore, the p-values ​​for the interaction terms AB and AC in Table 5 are 0.0046 and 0.0025, respectively, both <0.05. These findings corroborate each other, indicating a strong antagonistic interaction between material concentration and light power density, and between material concentration and illumination time. However, contour line BC is circular, suggesting that the antagonistic interaction between factors B and C is not significant. Figure 2The results show that in the delayed sterilization model, only the AB contour line is slightly elliptical and elongated along the main diagonal. The p-value of the interaction term AB in Table 6 is 0.0038 (<0.05), which corroborates the observation that there is a strong antagonistic interaction between material concentration and light power density. However, the AC and BC contour lines are circular, indicating that the antagonistic interaction between illumination time and material concentration and light power density is not significant.

[0067] Figure 1 and Figure 2 The surface morphology reflects the process sensitivity. The three-dimensional response surface plots of AB, AC, and BC exhibit a significantly convex (downward-curving) bell-shaped profile, and the quadratic coefficients are negative and highly significant (p<0.0001) or relatively significant (p<0.05), confirming that the material concentration, optical power density, and illumination time all have theoretical maximum values ​​within the experimental range, and the response surface methodology can accurately locate their optimal process levels. The slope of the three-dimensional response surface along the B-axis (optical power) and along the A-axis (concentration) and C-axis (time) shows a significant and steep decrease along the B-axis, which coincides with the extremely high negative coefficients of the B² term (-24.1 and -28.96), indicating that optical power density is a key sensitive parameter of this process system, and its optimal window width is significantly narrower than that of material concentration and illumination time, requiring precise control. In contrast, the process windows for material concentration and illumination time are wider and exhibit better robustness.

[0068] The experimental results of the validation of the optimal process conditions predicted by the delayed sterilization model are as follows: Figure 3 The optimal process conditions predicted by the immediate sterilization model were a material concentration of 5.96 mg / L, a light power density of 487.6 W / m², and an illumination time of 32.99 min, achieving a maximum net antibacterial rate of 84.30%. The optimal process conditions predicted by the delayed sterilization model were a material concentration of 6.24 mg / L, a light power density of 479.17 W / m², and an illumination time of 33.04 min, achieving a maximum net antibacterial rate of 87.18%. Based on the combined predictions of both models, the optimal process conditions were determined to be a material concentration of 6.2 mg / L, a light power density of 480 W / m², and an illumination time of 33 min. After three independent and repeated verification experiments, the measured average net antibacterial rate of immediate sterilization under these conditions was 85.2% (with a relative error of <1.1% compared to the predicted value of 84.30%), and the measured average net antibacterial rate of delayed sterilization was 87.6% (with a relative error of <0.5% compared to the predicted value of 87.18%). The verification results are in high agreement with the model predictions, which fully demonstrates that the dual response surface model established in this invention has excellent prediction accuracy and that the determined process conditions are reliable and repeatable.

[0069] A bar chart comparing the regression coefficients of the instant sterilization model and the delayed sterilization model is shown below. Figure 4 As shown. Figure 4This indicates that the delayed effect dramatically amplifies the antagonistic interaction between material concentration and optical power density (AC), which is the primary decision-making basis for process optimization. This study reveals for the first time that, under 450 nm visible light excitation, the interaction (AB) between material concentration and optical power density of the 4-BrPA / β-CD supramolecular photosensitizer is dramatically enhanced during the delayed sterilization stage: β... A×B The coefficients increased sharply and negatively from -4.69 (moderate antagonism) in the immediate model to β' in the delayed model. A×B -10.35 (strong antagonism), an increase of 121%. Figure 1 and Figure 2 In the three-dimensional response surface plot, the AB surface drastically transforms from a "slight diagonal distortion" in the immediate stage to a "steep descent cliff" in the delayed stage, and the contour projection is abruptly elongated from an ellipse to a highly flattened limiting ellipse. This finding demonstrates that while high material concentration and high optical power density can coexist in the immediate stage, the combined negative effects (photobleaching, photosensitizer degradation, and bacterial stress) are significantly amplified and accumulated in the delayed stage, ultimately resulting in a strong antagonism. This is a key mechanism feature that traditional "immediate evaluation" methods cannot capture at all, and it is also the primary decision-making basis for optimizing the 4-BrPA / β-CD photodynamic antibacterial process—when aiming for delayed sterilization, the "high concentration + high power" combination strategy must be clearly avoided, and a clear trade-off must be made between material cost and energy cost.

[0070] Figure 4 This study also indicates that the delay effect "creates" an antagonistic relationship between optical power density and illumination time, introducing new constraints for optical dose design. Furthermore, this study found that the interaction between optical power density and illumination time (BC) is essentially insignificant in the immediate phase (β). B×C = -0.85, p>0.05, contour circles), but suddenly exhibits strong antagonism in the delayed phase (β' B×C = -4.97, p = 0.0806 < 0.1, and Δ B×C ≤ -3.0, contour lines are elliptical. Figure 1 and Figure 2 In the three-dimensional response surface plot, the BC surface abruptly changes from a near-circular, undistorted state in the immediate stage to a significant diagonal distortion in the delayed stage. This finding demonstrates that the damaging effects of excessive illumination (high power density + long duration) are not immediately apparent, but are significantly amplified in the delayed stage. This principle provides a new constraint on light dose design—even if the immediate lethality is acceptable, the combination of "high power density + long duration" will be severely "penalized" in the delayed evaluation system.

[0071] Figure 4It also shows that the material concentration-light duration (AC) interaction exhibits a "delayed but significant mitigating antagonism": in the instantaneous sterilization model, the AC response surface shows a significant diagonal distortion, the contour projection is significantly elliptical, and β... A×C The coefficient reached -5.26 (p<0.05), indicating that high concentration and long-term light exposure are mutually exclusive and cannot be achieved simultaneously in the immediate stage; while in the delayed sterilization model, the diagonal distortion of the AC response surface was significantly reduced, the contour lines approached a circle, and β' A×C The coefficient rose to -2.10 (Δ=+3.16, a 60% reduction), intuitively indicating that the mutual interference between the two in the delayed phase was significantly weakened, transforming from an antagonistic relationship of "one gaining at the expense of the other" to a nearly independent positive contributing factor. The scientific implications of this phenomenon are: the concentration-time antagonism in the immediate phase mainly stems from instantaneous competition in the photochemical reaction process (such as photobleaching and instantaneous quenching of reactive oxygen species), which terminates after light exposure; while the bactericidal effect in the delayed phase depends on the degree of accumulated irreversible damage. High concentration means more initial damage sites, and prolonged light exposure means each site experiences longer oxidative stress. The two exhibit an additive effect rather than a competitive effect at the level of damage accumulation, thus significantly mitigating the antagonism. This finding provides important guidance for process optimization: in clinical scenarios targeting delayed killing, a "high concentration + long duration" combination strategy can be confidently adopted. The two not only do not weaken each other but may even achieve synergistic gains through the additive effect of damage accumulation.

[0072] Finally, it should be noted that the above embodiments are merely illustrative of several implementations of the present invention and are not intended to limit the scope of the invention. For those skilled in the art, any modifications, equivalent substitutions, or improvements made without departing from the concept of the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A dual response surface methodology for optimizing the photodynamic antibacterial process of a 4-BrPA / β-CD supramolecular photosensitizer, wherein the 4-BrPA / β-CD supramolecular photosensitizer is formed by non-covalent self-assembly of 4-bromophthalic anhydride and β-cyclodextrin, characterized in that... The optimization method uses the 4-BrPA / β-CD supramolecular photosensitizer as the photosensitizer and a 450nm xenon lamp visible light source as the excitation source, and includes the following steps: (1) Response surface design: The concentration of 4-BrPA / β-CD supramolecular photosensitizer A, light power density B, and illumination time C were investigated as factors, and the net light-excited inhibition rate determined by plate colony counting method was used as the response value. A three-factor, three-level Box-Behnken response surface design was carried out. Each group of experiments was set up with four types of experimental units: experimental group, dark toxicity control group, phototoxicity control group, and growth blank control group. (2) Parallel detection at two time points: Two sets of parallel and independent detections were performed on the same set of test schemes. The first set was the immediate sterilization detection, in which samples were taken immediately after the light treatment and plate colony counting was performed. The second set was the delayed sterilization detection, in which samples were incubated in the dark at 35-39℃ for 16-24 hours after the light treatment and then samples were taken for plate colony counting. (3) Calculation of net inhibition rate: Using colony forming units (CFU / mL) as the quantitative index, the net photoexcitation inhibition rate for immediate sterilization and delayed sterilization was calculated separately. The calculation formula is: Net inhibition rate (%) = [1 - (CFU / mL)] exp / CFU blank )]×100%-DL, where CFU exp For colony counting in the experimental group, CFU blank The colony count is for the blank control group. D represents the dark toxicity inhibition rate, and L represents the phototoxicity inhibition rate. (4) Dual model construction: Using the immediate sterilization net bacteriostatic rate and the delayed sterilization net bacteriostatic rate as response values, a quadratic polynomial regression model containing a first term, an interaction term and a quadratic term was established for each. Variance analysis and significance test were performed to obtain the immediate sterilization model and the delayed sterilization model respectively. (5) Comparative analysis of the interaction between the two models: The coefficient β of the interaction term between material concentration and light power density in the instant sterilization model and the delayed sterilization model was compared. A×B With β A×B 'Optical power density-illuminance time interaction coefficient β' B×C With β B×C ', Material concentration-light duration interaction coefficient β A×C With β A×C 'Compare and contrast the interaction coefficients of each model separately, and analyze the direction, magnitude, and significance of their changes between the instant sterilization model and the delayed sterilization model;' (6) Process optimization based on delayed sterilization model: The delayed sterilization model is used as the basis for final process optimization. By combining regression equation solution and response surface analysis, the optimal process range of the 4-BrPA / β-CD supramolecular photosensitizer under the excitation light source is determined.

2. The optimization method according to claim 1, characterized in that, The three-factor, three-level setting range in step (1) is as follows: the concentration A of 4-BrPA / β-CD supramolecular photosensitizer is 2-8 mg / L, the light power density B is 400-600 W / m², and the illumination time C is 20-40 min; Preferably, the three levels are set as follows: the concentration A of the 4-BrPA / β-CD supramolecular photosensitizer is 2 mg / L, 5 mg / L, and 8 mg / L; the light power density B is 400 W / m², 500 W / m², and 600 W / m²; and the illumination time C is 20 min, 30 min, and 40 min.

3. The optimization method according to claim 1, characterized in that, In step (2), before the light treatment, a dark incubation step is also included: the mixture containing 4-BrPA / β-CD supramolecular photosensitizer and bacteria is incubated at 35-39℃ in the dark for 20-40 minutes to allow the 4-BrPA / β-CD photosensitizer to fully bind with the bacteria.

4. The optimization method according to claim 1, characterized in that, Step (3) includes background toxicity verification and uniform deduction steps before calculating the net inhibition rate: Five condition points representing the center and boundary of the response surface design space were selected: Center point: material concentration 5 mg / L, light power density 500 W / m², illumination time 30 min; High light intensity / long time point: material concentration 5 mg / L, light power density 600 W / m², illumination time 40 min; Low light intensity / short time point: material concentration 5 mg / L, light power density 400 W / m², illumination time 20 min; Low concentration point: material concentration 2 mg / L, light power density 500 W / m², illumination time 30 min; High concentration point: material concentration 8 mg / L, light power density 500 W / m², illumination time 30 min. The dark toxicity inhibition rate (D) and phototoxicity inhibition rate (L) were independently determined at each condition point in both the immediate sterilization detection and delayed sterilization detection systems. When the range of both the dark toxicity inhibition rate and the phototoxicity inhibition rate at each condition point was ≤3%, the arithmetic mean D of the D values ​​at each condition point was used. mean The arithmetic mean of L values ​​L mean Use it as a uniform background subtraction value for all experimental groups of the response surface; otherwise, subtract the corresponding control value for each experimental group of the response surface separately. The background mean was measured and calculated independently for both the instant sterilization and delayed sterilization models.

5. The optimization method according to claim 1, characterized in that, The model evaluation criteria in step (4) include: using the coefficient of determination R², the corrected coefficient of determination Adj R², and the predicted coefficient of determination Pred R² to comprehensively evaluate the model's goodness of fit and predictive ability, requiring the difference between Pred R² and Adj R² to be <0.2; When the lack of fit is statistically significant (p < 0.05), if the range of repeated measured values ​​of the center point is < 2% and the difference between Pred R² and Adj R² is < 0.2, then the lack of fit is determined to be due to the small pure error and does not affect the effectiveness and predictive ability of the model.

6. The optimization method according to claim 1, characterized in that, In step (5), based on the comparison results, the type of "selective regulation" of the delayed effect on the interaction of different factors of the 4-BrPA / β-CD supramolecular photosensitizer is determined: When the interaction term coefficient β of material concentration and light power density in the instant sterilization model A×B It is a significantly negative value, and β in the delayed sterilization model A×B The negative effect is further enhanced, and its change Δ A×B =β A×B '-β A×B When the value is ≤-4.0, it is determined to be a delayed and drastic amplification antagonism; When the interaction term coefficient β of light power density-illuminance time in the instant sterilization model B×C Not significant, p≥0.05, while in the delayed sterilization model, β B×C The coefficient is weakly significant, p < 0.1, and its change Δ B×C =β B×C '-β B×C When the value is ≤-3.0, it is determined to be a potential creation level in delayed creation antagonism; When the interaction term coefficient β of material concentration-lighting time in the instant sterilization model A×C The value was significantly negative, while in the delayed sterilization model, β... A×C 'Negative weakening, its change Δ A×C =β A×C '-β A×C A value ≥ +3.0 is considered a significant delay in antagonistic response; The comparative analysis in step (5) also includes a comparison of the three-dimensional morphology of the response surface and the contour projection characteristics of the instant sterilization model and the delayed sterilization model. Specifically, it includes: AB interaction: comparing the degree of diagonal distortion, steepness of the descent edge, and aspect ratio of the contour ellipse of the three-dimensional surface; BC interaction: comparing whether there is significant diagonal distortion of the three-dimensional surface and the degree of transformation of the contour lines from circles to ellipses; AC interaction: comparing the degree of reduction of distortion of the three-dimensional surface and the degree of transformation of the contour lines from ellipses to circles.

7. The optimization method according to any one of claims 1 to 6, characterized in that, The optimal process range determined in step (6) is: 4-BrPA / β-CD supramolecular photosensitizer concentration 5.96-6.24 mg / L, light power density 479.17-487.6 W / m², and illumination time 32.99-33.04 min.

8. A method for evaluating the visible light-excited antibacterial properties of a 4-BrPA / β-CD supramolecular photosensitizer, characterized in that, Includes the following steps: (I) According to the method described in steps (1) to (4) of claim 1, an instant sterilization model and a delayed sterilization model are obtained respectively, wherein the constant term of the instant sterilization model is β0 and the interaction term coefficients are β0 and β0, respectively. A×B β A×C β B×C The constant term of the delayed sterilization model is β0', and the interaction term coefficients are β0' and β0', respectively. A×B '、β A×C '、β B×C '; (II) Calculate the following evaluation indicators: The delayed-effect gain index (DGI) is calculated as β0' - β0, expressed in percentage points of inhibition rate. A DGI ≥ 5 percentage points indicates that the photosensitizer has a significant delayed-effect synergistic effect. Material concentration-optical power density antagonistic amplification index CPAI = |β A×B ' / β A×B When CPAI ≥ 2.0, it is determined to have significant delayed amplification antagonism capability; Optical power density-time antagonistic generation index PTCI = |β B×C ' - β B×C |, when PTCI ≥ 3.0, it is judged as having significant delayed antagonistic ability; Material concentration-light duration antagonistic mitigation index CTRI = |β A×C ' - β A×C | When CTRI ≥ 3.0, it is considered to have significant delayed relief antagonistic ability; High Intensity Tolerance Index (HTI) = β B×C ' - β B×C The unit is the absolute difference of the encoded regression coefficients. HTI > 0: The BC interaction is enhanced in the delayed stage compared to the immediate stage, the antagonism is weakened or turns into synergy, and the photosensitizer is determined to have a delayed compensation effect; HTI < 0: The BC interaction is weaker in the delayed stage than in the immediate stage, and the antagonism is enhanced, indicating that the photosensitizer has a delayed amplification effect; the magnitude of |HTI| reflects the intensity of the delayed effect on the regulation of the BC interaction, and the larger the |HTI|, the more significant the regulation; (III) A combination system of DGI, CPAI, PTCI, CTRI and HTI as quantitative evaluation indicators for the delayed killing ability of the 4-BrPA / β-CD supramolecular photosensitizer.

9. The application of the optimization method according to any one of claims 1 to 7 in any of the following scenarios: Screening and optimization of photodynamic antibacterial process parameters for 4-BrPA / β-CD photosensitizer; Consistency evaluation of photodynamic activity of different batches of 4-BrPA / β-CD photosensitizer; Storage stability monitoring of 4-BrPA / β-CD photosensitizer.

10. The application of the evaluation method according to claim 8 in any of the following scenarios: Structure-activity relationship study of 4-BrPA / β-CD photosensitizer; Elucidation of the photodynamic damage mechanism of 4-BrPA / β-CD photosensitizer; Comparison of photodynamic efficacy among different preparation processes of 4-BrPA / β-CD photosensitizers.

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