CN-121994790-A - Photodynamic antibacterial process double-response-surface optimization and antibacterial effect evaluation method of 4-BrPA/beta-CD supermolecule photosensitizer

CN121994790ACN 121994790 ACN121994790 ACN 121994790ACN-121994790-A

Abstract

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

Inventors

LI CHAOXIA
JIANG BO
XIAO RONG
WANG TIE
CUI ENTIAN
Cao Liurui
WANG JIN
WEI CHANG

Assignees

盐城工学院
盐城工学院技术转移中心有限公司

Dates

Publication Date: 20260508
Application Date: 20260224

Claims (10)

1. The photodynamic antibacterial process double-response surface optimization method of the 4-BrPA/beta-CD supermolecule photosensitizer is characterized in that the optimization method takes the 4-BrPA/beta-CD supermolecule photosensitizer as a photosensitive material and takes 450nm xenon lamp visible light as an excitation light source, and comprises the following steps: (1) The response surface test design is that the concentration A, the optical power density B and the illumination time C of the 4-BrPA/beta-CD supermolecule photosensitizer are taken as investigation factors, and the net light excitation bacteriostasis rate measured by a plate colony counting method is taken as a response value, and three-factor three-level Box-Behnken response surface test design is carried out; (2) Two sets of parallel and independent detection are carried out on the same group of test schemes, wherein the first set is the instant sterilization detection, the sample is sampled immediately after the illumination treatment is finished to count the plate colony, the second set is the delayed sterilization detection, and the sample is continuously subjected to dark culture at 35-39 ℃ for 16-24 hours after the illumination treatment is finished to count the plate colony; (3) Calculating a net bacteriostasis rate, namely respectively calculating net light excitation bacteriostasis rates of immediate sterilization and delayed sterilization by taking colony forming units CFU/mL as quantitative indexes, wherein a calculation formula is that the net bacteriostasis rate (%) = [1- (CFU exp /CFU blank ) ]multipliedby 100% -D-L, wherein CFU exp is an experimental group colony count, CFU blank is a blank control group colony count, D is a dark toxicity bacteriostasis rate, and L is a phototoxicity bacteriostasis rate; (4) The dual model construction, namely respectively taking the instant sterilization net bacteriostasis rate and the delayed sterilization net bacteriostasis rate as response values, respectively establishing a quadratic polynomial regression model comprising a primary term, an interactive term and a quadratic term, and performing variance analysis and significance test to respectively obtain an instant sterilization model and a delayed sterilization model; (5) The dual-model interaction comparison analysis comprises the steps of respectively comparing material concentration-optical power density interaction term coefficients beta A×B and beta A×B ', optical power density-illumination time interaction term coefficients beta B×C and beta B×C ', and material concentration-illumination time interaction term coefficients beta A×C and beta A×C ' in the instant sterilization model and the delayed sterilization model, and analyzing the change direction, change amplitude and significance evolution of each interaction term coefficient between the instant sterilization model and the delayed sterilization model; (6) And (3) process optimization based on a delayed sterilization model, namely, taking the delayed sterilization model as a final process optimization basis, solving a comprehensive regression equation and analyzing a response surface, and determining an optimal process interval of the 4-BrPA/beta-CD supermolecule photosensitizer under the excitation light source.
2. The optimization method according to claim 1, wherein the three-factor three-level setting range in the step (1) is that the concentration A of the 4-BrPA/beta-CD supermolecule photosensitizer is 2-8 mg/L, the optical power density B is 400-600W/m 2, and the illumination time C is 20-40 min; Preferably, the three levels are set to be that the concentration A of the 4-BrPA/beta-CD supermolecule photosensitizer is 2 mg/L, 5 mg/L, 8 mg/L, the optical power density B is 400W/m 2, 500W/m 2, 600W/m 2, and the illumination time C is 20min, 30 min, 40 min.
3. The optimizing method according to claim 1, wherein the step (2) further comprises a dark incubation step of incubating the mixture containing the 4-BrPA/beta-CD supermolecule photosensitizer and bacteria at 35-39 ℃ in a dark condition for 20-40 min to allow the 4-BrPA/beta-CD photosensitizer to bind to the bacteria sufficiently.
4. The optimization method according to claim 1, wherein the calculation of the net antibacterial rate in step (3) further comprises the steps of background toxicity verification and unified subtraction: selecting 5 condition points representing the center and boundary of a response surface design space, wherein the center point is the material concentration of 5 mg/L, the optical power density of 500W/m < 2 >, the illumination time of 30min, the high-light-intensity/long-time point is the material concentration of 5 mg/L, the optical power density of 600W/m < 2 >, the illumination time of 40 min, the low-light-intensity/short-time point is the material concentration of 5 mg/L, the optical power density of 400W/m < 2 >, the illumination time of 20min, the low-concentration point is the material concentration of 2 mg/L, the optical power density of 500W/m < 2 >, the illumination time of 30min, and the high-concentration point is the material concentration of 8 mg/L, the optical power density of 500W/m < 2 > and the illumination time of 30 min; Respectively and independently measuring the dark toxicity bacteriostasis rate D and the phototoxicity bacteriostasis rate L of each condition point in two sets of systems of instant sterilization detection and delayed sterilization detection, when the range of the dark toxicity bacteriostasis rate and the range of the phototoxicity bacteriostasis rate of each condition point are less than or equal to 3%, respectively adopting the arithmetic average value D mean of the D value of each condition point and the arithmetic average value L mean of the L value of each condition point as unified background deduction values of all test groups of a response surface, otherwise, respectively and independently deducting corresponding control values for each test group of the response surface; the two models of instant sterilization and delay sterilization are respectively and independently measured and the background mean value is independently calculated.
5. The optimization method according to claim 1, wherein the model evaluation criteria in the step (4) includes comprehensively evaluating the model fitting goodness and predictive ability with a decision coefficient R2, a correction decision coefficient Adj R2, and a prediction decision coefficient Pred R2, requiring a difference between Pred R2 and Adj R2 of <0.2; When the mismatch item shows statistical significance p <0.05, if the repeated actual measurement value of the central point is extremely poor <2% and the difference value of Pred R2 and Adj R2 is less than 0.2, the mismatch significance is judged to be caused by too small pure error, and the effectiveness and the prediction capability of the model are not affected.
6. The optimization method according to claim 1, wherein in the step (5), the "selective regulation" type of interaction of the delayed effect on different factors of the 4-BrPA/β -CD supermolecule photosensitizer is determined according to the comparison result: When the material concentration-optical power density interaction term coefficient beta A×B in the instant sterilization model is a significant negative value and the beta A×B ' negative direction in the delayed sterilization model is further enhanced, the variation delta A×B =β A×B '-β A×B is less than or equal to-4.0, judging that the delay severe amplification antagonism is performed; When the coefficient beta B×C of the optical power density-illumination time interaction term in the instant sterilization model is not obvious, p is more than or equal to 0.05, the coefficient beta B×C ' in the delay sterilization model is weak and obvious, p is less than 0.1, and the variation delta B×C =β B×C '-β B×C is less than or equal to-3.0, judging that the potential creation level in the delay creation antagonism is realized; When the material concentration-illumination time interaction term coefficient beta A×C in the instant sterilization model is a significant negative value and beta A×C ' in the delayed sterilization model is reduced negatively, and the variation delta A×C =β A×C '-β A×C is more than or equal to +3.0, determining that the antagonism is significantly relieved; The comparison analysis of the step (5) also comprises the comparison of the three-dimensional shape and the contour projection characteristics of the response surface of the instant sterilization model and the delay sterilization model, and concretely comprises the steps of A-B interaction, namely, comparing the diagonal distortion degree, the falling edge abruptness and the contour ellipse length-width ratio of the three-dimensional curved surface; B-C interaction, namely comparing whether the three-dimensional curved surface has obvious diagonal distortion and the transformation degree of the contour line from circle to ellipse, and A-C interaction, namely comparing the distortion weakening degree of the three-dimensional curved surface and the transformation degree of the contour line from ellipse to circle.
7. The optimization method according to any one of claims 1 to 6, wherein the optimal process interval determined in the step (6) is 4-BrPA/beta-CD supermolecule photosensitizer concentration 5.96-6.24 mg/L, optical power density 479.17-487.6W/m2, and illumination time 32.99-33.04 min.
8. The method for evaluating the visible light excitation antibacterial property of the 4-BrPA/beta-CD supermolecule photosensitizer is characterized by comprising the following steps of: (I) According to the method of steps (1) to (4) in claim 1, an instant sterilization model and a delayed sterilization model are respectively obtained, wherein the constant term of the instant sterilization model is beta 0 , the coefficient of the interaction term is beta A×B 、β A×C 、β B×C , the constant term of the delayed sterilization model is beta 0 ', and the coefficient of the interaction term is beta A×B '、β A×C '、β B×C '; (II) calculating the following evaluation index: Delay effect gain index dgi=β 0 '-β 0 in percent of the bacteriostatic rate. When DGI is more than or equal to 5 percent, judging that the photosensitizer has obvious delay synergistic effect; the material concentration-optical power density antagonistic amplification index CPAI= |beta A×B '/β A×B |, and the CPAI is judged to have remarkable delay antagonistic ability when being more than or equal to 2.0; The optical power density-illumination time antagonism generation index PTCI = |beta B×C ' - β B×C |, and the PTCI is more than or equal to 3.0, and the antagonism is judged to have obvious delay generation; The material concentration-illumination time antagonism relieving index CTRI= |beta A×C ' - β A×C |, and the CTRI is more than or equal to 3.0, the material concentration-illumination time antagonism relieving index CTRI is judged to have obvious delayed release antagonism capability; High intensity tolerance index hti=β B×C ' - β B×C in absolute difference of coded regression coefficients. HTI > 0, wherein the BC interaction in the delay stage is enhanced compared with that in the immediate stage, antagonism is weakened or is converted into synergy, and the photosensitizer is judged to have a delay compensation effect; HTI < 0, namely the BC interaction in the delay stage is weakened and antagonism is enhanced compared with that in the immediate stage, and the photosensitizer is judged to have a delay amplification effect; (III) DGI, CPAI, PTCI, CTRI and HTI are used as a quantitative evaluation index combination system of the delayed killing capability of the 4-BrPA/beta-CD supermolecule photosensitizer.
9. Use of the optimization method of any one of claims 1 to 7 in any one of the following scenarios: screening and optimizing photodynamic antibacterial process parameters of the 4-BrPA/beta-CD photosensitizer; 4-BrPA/beta-CD photosensitizer is subjected to consistency evaluation of photodynamic activity of different preparation batches; storage stability monitoring of 4-BrPA/beta-CD photosensitizer.
10. Use of the assessment method of claim 8 in any of the following scenarios: structure-activity relationship research of 4-BrPA/beta-CD photosensitizer; Photodynamic damage mechanism analysis of the 4-BrPA/beta-CD photosensitizer; Photodynamic efficacy comparison between different preparation processes of 4-BrPA/beta-CD photosensitizer.

Description

Photodynamic antibacterial process double-response-surface optimization and antibacterial effect evaluation method of 4-BrPA/beta-CD supermolecule photosensitizer Technical Field The invention relates to the field of photodynamic antibiosis technology and technology optimization, in particular to a photodynamic antibiosis technology double-response-surface optimization and bacteriostasis effect evaluation method of a 4-BrPA/beta-CD supermolecule photosensitizer. Background Photodynamic antimicrobial therapy (aPDT) has become an important alternative strategy to deal with the crisis of bacterial resistance by utilizing photosensitizers to generate active oxygen under the excitation of light of specific wavelengths to kill pathogenic microorganisms. Cyclodextrin-based supermolecule photosensitizers are paid attention to because of good biocompatibility and host-guest recognition capability, but the novel photosensitizers are pushed to industrial application from laboratory basic research, and key technical problems such as process parameter optimization, multi-factor interaction analysis, bacteriostasis effect system evaluation and the like need to be solved. The inventor submits another patent application (the application name: a 4-BrPA/beta-CD supermolecule photosensitizer, a preparation method and application thereof) on the same day, and the application discloses a preparation method of the 4-BrPA/beta-CD supermolecule photosensitizer formed by non-covalent self-assembly of 4-bromophthalic anhydride (4-BrPA) and beta-cyclodextrin (beta-CD), and the preparation method proves that the 4-BrPA/beta-CD supermolecule photosensitizer has good photophysical property and basic photodynamic activity under the excitation of visible light. However, the application does not relate to the industrial key technical problems of process parameter optimization, effect evaluation and the like of the photosensitizer in practical photodynamic antibacterial application. At present, the photodynamic antibacterial process optimization method aiming at novel photosensitizers (comprising 4-BrPA/beta-CD) has the following defects: First, the evaluation time is single and the delay killing effect is not considered. Photodynamic damage includes both immediate killing during illumination and delayed killing due to irreversible damage accumulation expression after illumination. However, the prior art generally uses the bacteriostasis rate detected immediately after the illumination is finished as a unique endpoint index, and ignores the contribution of the delay effect to the actual sterilization effect. When only instant killing is used as an optimization target, deviation can occur in the evaluation of the high-light-intensity and long-time parameter combination benefit, and the accuracy of the process optimization direction is further affected. Second, the end point detection method is not accurate enough. In the existing photodynamic antibacterial research, an OD value absorbance method based on an enzyme-labeled instrument is widely adopted for antibacterial effect evaluation, living bacteria and dead bacteria cannot be distinguished by the method, and systematic errors exist in the colored light scattering interference or bacteria aggregation state of the photosensitive material. The plate colony counting method is an internationally accepted microorganism quantitative gold standard, but is not integrated into a method system requiring batch detection such as response surface optimization at present due to complex operation and low flux. Third, the background effects are not effectively separated. The total antibacterial effect of photodynamic antibiosis is formed by coupling three parts of material dark toxicity, nonspecific phototoxicity and net photodynamic effect. The prior art either ignores background subtraction entirely or uses fixed value unified subtraction without verifiable assessment of background toxicity fluctuations under different conditions, resulting in possible systematic errors in the input data of the response surface model. Fourth, the difference in interaction between the real-time and delay phases is not of concern. There are complex interactions between the three factors of material concentration, optical power density and illumination time, and these interactions may exhibit different or even opposite trends in the immediate killing phase versus the delayed killing phase. However, the existing research has not established a method system capable of capturing and comparing interaction rules of two time phases at the same time, and also lacks a technical means for taking the time phase evolution characteristics of the interaction as the basis of photosensitizer characteristic evaluation and process decision. In addition, the 4-BrPA/beta-CD supermolecule photosensitizer is used as a novel photosensitive material, and the optimal material concentration, optical power density, illumination time and interactio