Enhanced Antimicrobial Photodynamic Therapy against Escherichia coli Using MXene-Based Silver–Curcumin 2D Nanocomposite Photosensitizers

Enhanced Antimicrobial Photodynamic Therapy against Escherichia coli Using MXene-Based Silver–Curcumin 2D Nanocomposite Photosensitizers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhanced Antimicrobial Photodynamic Therapy against Escherichia coli Using MXene-Based Silver–Curcumin 2D Nanocomposite Photosensitizers Saliha noor, Dr.Shahzad Anwar, Dr.Rafaqat Ali Khan, Sawera Malik, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9225233/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract The increasing prevalence of antibiotic-resistant bacteria poses a significant threat to public health. Addressing this challenge requires the development of innovative antimicrobial materials. In this context, MXene-based nanomaterials have emerged as promising candidates for advanced healthcare applications. The photodynamic therapy (aPDT) was evaluated to investigate the antibacterial potential and efficacy of MXene–silver–curcumin (NC) nanocomposites. Nanocomposites are novel photoactive nanomaterials. The synthesis of composites includes a chemical synthesis route and green synthesis and followed by complete characterization of the materials to evaluate their morphological, optical, and cell viability against rhabdomyosarcoma tumor (RD) cell lines. The layered MXene nanosheets depicted in the SEM images were loaded with silver nanoparticles and curcumin. The nanoscale structure and dispersibility in aqueous media were evaluated through dynamic light scattering (DLS), UV–Visible spectroscopy, and fluorescence studies, indicating an approximate hydrodynamic diameter of 220 nm. The bonding among MXene, silver, and curcumin were confirmed by Raman spectroscopy. In antibacterial activity, a strong inhibition zone of 15.01 mm was observed against Escherichia . Cytotoxicity data from the MTT experiment demonstrate that MXene-silver-curcumin NC exhibited limited cell death and minimal toxicity, particularly at lower dosages (31µg/mL) 92%, when compared to the control. On the exposure of 418 nm light on composites, the concentration and light dependent aPDT effects were observed, where the bacterial killing was significantly higher at concentration of 500 ug/mL, and this was explained by the combined effects of MXene conductivity, silver plasmonic enhancement, and curcumin photosensitization. Fluorescence spectroscopy antibacterial photodynamic therapy Raman spectroscopy photosensitizer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Wound infections due to a range of bacteria generally develop during the healing of skin and tissues damaged by external causes, producing serious inflammation and a variety of disorders [ 1 ]. Since prolonged use of antibiotics results in drug resistance, which lessens the effectiveness of treatment, a variety of therapeutic approaches have been developed to increase the effectiveness of wound healing and lower the incidence of infection. These include therapeutic antimicrobials, peptides, hydrogels, nanofibrous dressings, and stem cell therapy [ 2 ]. The necessity for other treatments against infectious diseases is suggested by the appearance of bacterial strains that are resistant to antibiotic treatment. The application of antimicrobial photodynamic therapy (aPDT) represents a strategy that may lead to more effective antimicrobial treatment [ 3 , 4 ] .aPDT uses reactive oxygen, visible light, and a chemical photosensitizer or nontoxic photoactivatable dye. The treatment relies on the energy being transferred to the oxygen molecules, resulting in highly reactive mediators like superoxide and singlet oxygen that are harmful to the cells [ 5 ]. The photo responsive nanomaterials' limited light absorption ability and rapid photogenerated charge recombination usually result in subpar photocatalytic performance. Heterojunction formation [ 6 ] and defect introduction, particularly the creation of oxygen vacancies (OVs) [ 7 ], are generally thought to be efficient methods for improving the photocatalytic properties of nanomaterials. MXenes, a class of 2D materials derived from MAX phases, are promising for biomedical applications due to their biocompatibility. They can be modified to improve their antibacterial performance. The scientific community is actively investigating MXenes for biochemical detection, drug encapsulation, and tissue regeneration[ 7 , 8 , 9 ]. MXene have optical and electrical performance to achieve better results in various fields[ 11 ]. Due to exceptional electrical conductivity, high absorption capacity and conversion efficiencies for near-infrared light, and localized surface plasmon resonance (LSPR), Ti 3 C 2 T x MXenes, an appealing photosensitive material, have recently been shown to have improved photocatalytic properties by increasing the photoinduced charge transfer[ 12 ].Ti 3 C 2 T x MXene good biocompatibility and high absorption capacity make it a promising candidate for a variety of biomedical and biosensing applications, particularly in wound healing [ 13 , 14 ].But Ti 3 C 2 T x MXene frequently has a high conversion temperature (over65°C), which can harm the tissues around[ 13 ]. Due to the short-term pulse impact of light irradiation, the remaining bacteria can also reproduce quickly when light exposure is stopped. MXene-based heterojunctions (Bi 2 S 3 /Ti 3 C 2 T x ) can improve electron transport to produce more ROS and improve biocompatibility when used as photoexcited antibacterial agents [ 16 ].The poor stability, low dispersibility of MXene is resolved by combining it with polymer materials, metal as well as ceramic compounds. Improvements in stability together with enhanced biocompatibility and functional properties enable the composite material to become suitable for biomedical applications [ 17 , 18 ]. Their surfaces can be modified to increase specificity by attaching medications, targeting compounds, or other substances. MXenes also release drugs in a regulated and targeted manner in response to stimuli like temperature, pH, or certain enzymes in the tumor microenvironment.[ 19 ]. Over the past few decades, noble metallic nanoparticles have gained a lot of interest as antibacterial materials because of their possible physicochemical properties [ 20 ]. Coinage metals, particularly Silver (Ag) [ 21 ] and Gold (Au) [ 22 ], exhibit excellent biocompatibility, antimicrobial properties, and corrosion resistance, making them valuable materials for various biomedical applications, [ 23 ]. Silver can effectively mitigate both the oxidation and stacking of MXene nanosheets, which are major limitations to its long-term stability and performance beyond this it also increasing antimicrobial efficiency [ 24 , 25 ].Silver nanoparticles toxicity at a biological platform is a serious concern. Coordination of silver nanoparticles with certain combinational materials is necessary to decrease toxicity and increase antibacterial agent efficacy. AgNPs' size, shape and modification by other materials are presented in order to assess the combined impact on their antibacterial activity [ 26 , 27 ] . Turmeric stands as Curcuma longa containing curcumin as its main active compound that demonstrates three major therapeutic effects: antibacterial along with anti-diabetic and anti-cancer properties. However, curcumin's low bioavailability and solubility limit its effectiveness. Current research involves nanotechnology-based curcumin formulations that improve its performance by increasing delivery efficiency while enabling large-scale manufacturing and local distribution of the substance [ 28 , 29 ]. The nanohybrid system of MXene functionalized with nanoparticles may load and distribute curcumin, enabling regulated release at particular sites.[ 30 ]. In order to load and transport curcumin, a variety of drug delivery methods have been investigated, including inorganic nanomaterials [ 31 ], polymeric nanoparticles, covalent organic frameworks[ 32 ], MOFs [ 33 ], and MXene NSs [ 34 ]. The synergistic/additive effects of co-encapsulating curcumin and silver nanoparticles in various forms have recently been studied [ 35 ]. Combination of curcumin with silver reduce the silver toxicity and enhance antimicrobial activity [ 36 ]. In present work, a promising nanoplatform for wound healing is presented by building MXene nanosheets connected with silver nanoparticles, which have a high photothermal conversion efficiency and an exceptional curcumin-loading capacity. This method combines improved drug delivery with photothermal therapy to provide a non-invasive, dual-modality solution. It is anticipated that combining these elements will result in comprehensive ,extremely successful antimicrobial therapy and wound healing, given the exceptional photothermal and photodynamic performance of Ti 3 C 2 T x NSs and the long-term therapeutic effect of silver and curcumin. Materials and methods Materials Hydrochloric acid (HCL), Lithium fluoride (LiF), pH paper, MAX powder, Deionized water, curcumin, silver nitrate (AgNO3) ≥ 98% are purchased from Sigma Aldrich, black tea from local market. Nutrient Agar, Nutrient Broth, glass Petri dishes (catalog no. D35-10–1-N) were procured from Cellvis. Bacterial strains (ATCC 8739,) were sourced from the NIBGE. Cell Culture Assembly (NCCC) and RD cell lines from NIH, correspondingly,96-well plates, phosphate-buffered saline (PBS), and dimethyl sulfoxide (DMSO) were also used. Methods Synthesis of MXene-silver-curcumin NC With a previously discussed modified least intensive layer delamination (MILD) method, Ti 3 C 2 T x MXene NSs were prepared by chemically etching Al atoms selectively from the Ti 3 AlC 2 MAX phase [ 37 ]. 100 mL polypropylene plastic vial was filled with an etchant solution of 20 mL HCl (9 M) and 1.33 g LiF while being constantly stirred on a hot plate. To avoid overheating placed in water bath and 1 g of Ti 3 AlC 2 MAX powder was subsequently mixed to the mixture gradually and agitated for 24 hours at 35°C. To eliminate unetched MAX powder, the obtained mixture was centrifuged and washed using DI water. The delaminated Ti 3 C2T x MXene flakes stock solution was made by applying a washing cycle at 5000 rpm four to five times until the solution's pH reached ≥ 6. A green synthesis process mediated by plant extract was used to create silver nanoparticles. 50 ml of boiling deionized water were mixed with 5g of black tea to create black tea extract. 0.34 grams of silver nitrate (AgNO₃) were dissolved in 100 ml of deionized water to create a 1 molar solution. 1 M AgNO 3 solution was added in 1:1 volumetric proportion with the prepared black tea extract and then subjected to a water bath at temperature of 84ºC for 20 minutes to allow reduction of silver ions to occur. The centrifugation of the solution was done at 10000 rpm for 10 minutes to isolate the silver nanoparticles. The supernatant was poured off and remaining material was harvested. For the composite preparation 1 gram of MXene, 0.5 grams of silver nanoparticles (AgNPs), and 0.5g of curcumin were added to 10 milliliters of deionized (DI) water. The mixture was then mixed on a hot plate under controlled temperature of 45ºC to 50ºC and stirred for 1 hour. Following this heating and mix phase, the mixture that resulted was put in an oven to dry and composite material has been synthesized as illustrated in Fig. 1 . Optical characterization of MXene-silver-curcumin NC Dynamic light scattering study (DLS) DLS determines the hydrodynamic size of a particle in an aqueous environment. This was used to determine the stability and divisions of size when the synthesized nanoparticles were suspended in a media. MXene-silver-curcumin NC suspensions were put in a cuvette containing two milliliters of each of the self-assembled particle samples in the experimental setup. The cuvette was subsequently subjected to a dynamic light scattering instrument (Microtrac Nanotrac Wave II). Particle motion patterns and solution stability were affirmed by the interaction of the incoming light with the mobile particles, which were moving randomly within the solution. Scanning electron microscopy (SEM) analysis of MXene-silver-curcumin NC SEM (FESEM, MAIA3 TESCAN) analysis was used to examine MXene-silver-curcumin NC morphology. Samples were positioned on stubs and morphological examination was assessed. UV and fluorescence spectroscopy With a fluorescence spectroscopy (FluoroMax-4, Horiba Scientific: USA), fluorescence spectroscopic measurements were obtained to assess the intrinsic fluorescence properties of material. For the antibacterial investigation MXene-silver-curcumin NC interacted with E. Coli bacteria at varying concentration and times. MXene-silver-curcumin NC synthesis was confirmed by using a (Shi Madzu UV-VIS 2101PC) UV-vis absorption spectrophotometer. Culture medium preparation Nutrient broth was made by dissolving 16 g of powder of nutrient broth in 1000 ml of distilled water. The suspension was autoclaved at 121°C for an hour. In the same way, 1000 ml of distilled water and 33 g of nutrient agar powder were combined, autoclaved, and allowed to set in a petri dish at 4°C. For the preparation of fresh bacterial inoculums, one colony of E. coli was transferred from an agar plate to nutrient broth. Bacteria were cultured for four hours at 37°C for bacterial growth. Antibacterial Activity of MXene-silver-curcumin nanocomposite The antibacterial efficacy of various nanoparticles (NPs) against monostreak E. coli was performed by using agar diffusion method. Using a cotton swab, broth cultures were equally distributed across the nutrient agar plates. Wells were dug on the agar plates using autoclaved micropipette tips and loaded with 50 µl of suspensions of each NPs. A digital vernier caliper was used to measure the zones of inhibition in millimeters after the plates were incubated for the entire night at 37°C. For MXene-silver-curcumin nanocomposite, each experiment was carried out three times in order to get mean values. The agar well diffusion method, which was previously published in the literature with various modifications, was used to test the antibacterial activity of the produced nanocomposites [ 38 ] Evaluation of MXene-silver-curcumin nanocomposite for enhanced antibacterial photodynamic therapy (aPDT) For four hours, the E. Coli-type culture collection of bacterial strains was cultured in nutrient broth at 37°C till growth. For the photobacterial assay, a 40µl of the cultured E. coli suspension was introduced into 40ml of deionized water, followed by the addition of 500 ml of nanocomposites (NCs) into a petri dish. This solution was then exposed to light of a specific wavelength for 60 minutes. At 10-minute intervals throughout the 60-minute exposure, 20µl samples of the solution were spread onto nutrient agar plates and subsequently incubated overnight to quantify bacterial viability. The colony counting method was used to calculate the bacterial growth inhibition. Analysis of Data For data analysis and plot processing, we have utilized Origin Pro 8.5 software. Surface morphology and size distribution of MXene-silver-curcumin NC These NC shape and size distribution were described using DLS and scanning electron microscopy, respectively. Scanning electron microscopy was used to examine the morphology of MXene and MXene-silver-curcumin NC, as seen in Fig .2(a) and Fig .2(c) which confirm synthesis of MXene from MAX phase and presence of MXene nanosheets along with silver and curcumin particles loaded on the surface. The monodisperse MXene and MXene-silver-curcumin NCs solution with a hydrodynamic size of 200nm [ 39 ]and 220 nm is displayed by the DLS measurement in Fig. 2 (b) and 2(d). Among the many advantageous characteristics that make MXene-silver-curcumin NCs potentially of significant interest in a wide range of practical applications are excellent conductivity, chemical stability pre catalytic activity, and antibacterial activity. UV visible spectroscopic evaluations of MXene-Silver-Curcumin NC The formation of silver nanoparticles was confirmed by the fact that the UV-Vis absorption peak of black-tea-mediated AgNPs was observed at 350 nm, which agrees with the surface plasma resonance peak of the synthesized AgNPs of Thymus vulgaris [ 40 ], the Ti 3 C 2 T x MXene nanosheets had large absorption band ranging 300–420 nm, which is similar to the 200–420 nm recorded range of Ti 3 C 2 T x MXenes .The minor change can be attributed to variation in surface terminations and synthesis parameter[ 41 ]. The UV-Vis absorption spectra of curcumin show distinct peaks at approximately 427 nm as also in previous study [ 42 ] respectively, all these parameters conforming particle formation as shown in Fig. 3 (a-c). The UV-Vis spectrum of the synthesized composite exhibited two distinct maximum absorbance peaks at 272 nm and 354 nm Fig. 3 (d), suggesting its potential for biosensing applications. Fluorescence based spectroscopic analysis of MXene-silver-curcumin NC The distilled water was used to examine MXene-Silver-Curcumin wavelengths in quartz cuvettes. A shift found at excitation wavelengths of 390nm respectively, demonstrating the fluorescent nature of MXene-silver-curcumin NC as shown in figure below. In Fig .4(a, b, c) it is clear that MXene Fluorescence intensity peak is at 455nm[ 43 ] that of Silver is at 394nm [ 38 ] and Fluorescence intensity peak of curcumin is at 534nm [ 44 ]. These findings demonstrated that MXene-silver-curcumin NC are fluorescent nanomaterials that can be tuned for use in biomedical applications, as is the case in this investigation against the bacterial strain E. coli . The fluorescence of the MXene–silver–curcumin composite shows a notable blue shift, with the emission peak moving from the wide range of the individual components to a sharp peak at 390 nm shown in Fig. 4 (d) This phenomenon is associated to structural effects within the composite, electron redistribution, and non-radiative energy transfer which are in line with observations made on the interaction of Cu-MOF@Rhodamine B with analytes [ 45 ]. Analysis of MXene-silver-curcumin NCs using confocal microscopy Evaluations of MXene-silver-curcumin NC using confocal microscopy were conducted at an excitation wavelength of 488 nm. MXene-silver-curcumin NC confocal microscopy analysis is shown in Fig. 5 (a-f) based on temporal interactions at 500 µg/ml concentration. A single optical section of the sample, imaged using confocal fluorescence microscopy, is illustrated in Fig. 5 (a) The image shows the fluorescent behavior of MXene-silver-Curcumin NC, which are visible as fluorescently-labeled structures with varied morphology, including both isolated particles and larger fluorescent sheets in Fig. 5 (b). As presented in Fig. 5 (c) fluorescence histogram of pure tunable MXene-silver-curcumin NC, verified greatest intensity peaks in the green region correspond to the fluorescence distribution of the nanoparticles in that area, while Fig. 5 (e) show 2D slice of the fluorescent sheets of MXene-silver-curcumin NC with variations in intensity representing the distribution of the material within the focal plane. Raman spectroscopic analysis of MXene-Silver-curcumin NC The MXene-silver-curcumin NC vibrational characteristics are revealed by the Raman spectrum, which is shown in Fig. 6 . Different peaks at 303 cm⁻¹, 420 cm⁻¹, 797 cm⁻¹, and 1273 cm⁻¹ are indication of Ti–C vibrations and surface terminations (–O, –OH, –F). Curcumin showed notable peaks at 1258, which were connected to C–O stretching vibrations and C–O–C stretching, respectively [ 46 ].More Raman bands at greater shifts indicate the possibility of surface vibrational modes, phonon interactions, or structural flaws originating from the nanostructures (Wang et al., 2017). Antibacterial assessment of MXene-silver-curcumin NC against E. coli The E. Coli culture collection of bacterial strains was incubated for 4 hours at 37°C in nutrient broth. For the MXene-silver-curcumin NC, the concentration range was 500–31 µg/ml. Every sample was examined three times. MXene and curcumin with no zone, silver with zone of 14.98 mm, and MXene-silver-curcumin with 15.01 mm zone of inhibition are shown in Fig. 7 (a), while graphical representation of these zone is shown in Fig. 7 (b) and in supplementary data S1while Fig. 7 (c) show zone of inhibition at various concentration due to interactions between MXene-silver-curcumin and E-coli . The MXene-silver-curcumin zone of inhibition at varying concentrations is graphically shown in Fig. 7 (d). The effects of varying concentrations of MXene-silver-curcumin NC on E. coli bacteria revealed a linear relationship between bacterial mortality and particle concentrations. Bacterial disintegration peaked at higher concentrations and was negligible at lower ones shown in supplementary data S2 aPDT Analysis of MXene-silver-curcumin NC Against E. coli As depicted in Fig. 8 (a), the presence of both the nanocomposites and light resulted in significant bacterial killing, with the number of possible bacterial colonies progressively decreasing over the 60-minute interaction period, reaching a very low count after 60 minutes. In contrast, Fig. 8 (b) demonstrates that in the absence of light, the nanocomposites exhibited negligible bactericidal activity, leading to very limited bacterial reduction despite interaction. Furthermore, Fig. 8 (c) confirms that light exposure alone did not effectively kill the bacteria. These findings collectively indicate that a material acting as a photosensitizer is essential to generate reactive oxygen species (ROS) in the presence of light, thereby facilitating effective bacterial killing. aPDT analysis of MXene-silver-curcumin NC against E. coli using conventional fluorescence spectrometer Conventional fluorescence spectrometer was used to assess the interactions of MXene-silver-curcumin NC with E. coli based on time. MXene-silver-curcumin NC were cultured with E. Coli for 10 to 60 minutes, with a 10-minute break between each measurement. NC operate as photosensitizers when exposed to particular wavelengths of light, generating reactive oxidative species such singlet oxygen. These ROS cause bacterial membrane, protein, and DNA oxidative damage, which results in cell death. Figure 9 (a) indicates the declining pattern in fluorescence intensity peaks with time interactions, which shows how well MXene-silver-curcumin NC kill bacteria. MXene-silver-curcumin NC incubation resulted in a considerable drop in the fluorescence intensity of bacterial cells, suggesting a gradual decline in bacterial growth. Figure 9 (b)show the time interaction of composite with bacterial in the absence of light indicating very small decreasing pattern of fluorescence intensity peak. Figure 9 (c) show interaction of bacteria with light which show negligible decrease in fluorescence intensity peak which indicate that bacteria were not effect by light only. Colony forming units of MXene-silver-curcumin NC at different concentrations Particles were inoculated with E. coli bacterial inoculums to assess the Colony Forming Unit calculation as in previous study [47].The growth of pure bacterial colonies was then studied using various serial dilutions dispersed on an agar plate. CFU/mL at various doses is shown in Fig. 10 (a-e) and graphical analysis in Fig. 8 (f) verified that the minimum CFU/ml was 500 µg/ml. Figure 10 (e) indicates at least 10 colonies were observed at peak concentrations of 500 µg/ml, while Fig. 10 (a) shows a maximum of 268 colonies were counted at low values of 31 µg/ml in 20 minutes while at 500 µg/ml concentration bacterial colonies reduce to 1 at 30 min and at concentration of 31 µg/ml bacterial colonies were 25. Data was gathered in triplicate and plotted against each value (± SD) to determine the average and standard deviation as observed in Fig. 10 (f) colony formation unit based on both concentration (31–500 µg/ml) and time interaction (10–60 min). From Fig. 10 (g) it can be seen that E.coli bacteria and particles treated with varying doses of MXene-silver-curcumin exhibited a linear relationship between bacterial death and particle concentrations. Bacterial disintegration peaked at higher MXene-silver-curcumin NCs concentrations and was negligible at lower concentrations. Agar plates were utilized to calculate the average CFU/mL values which are included in the Supplementary data S3, after E. Coli inoculums were incubated with MXene-Ag-Curcumin NCs for 24 hours. Concentration and time based interaction of MXene-silver-curcumin NC with E. coli using conventional fluorescence spectrometer Conventional fluorescence spectrometer was used to assess the interactions of MXene-silver-curcumin NC with E. coli based on concentrations and time as studied earlier [ 38 ]. Illustration in Fig. 11 (a-e) shows a declining trend in fluorescence intensity peaks with time interactions, which indicates how well MXene-silver-curcumin NC kill bacteria at various concentrations. Demonstrate that two factors concentration and time interactions have an impact on killing efficiency. The maximum 500 µg/ml concentrations of MXene-silver-curcumin NC (10–60 min) possessed the maximum killing efficiency, as per Flores' concentration-time interaction-based analysis. MXene-silver-curcumin NC antibacterial properties are revealed through their interaction with bacterial cells. A notable reduction in fluorescence intensity was noted during incubation of bacterial cells with MXene-silver-curcumin NC suggesting a gradual slowdown in bacterial development. According to this result, MXene-silver-curcumin NC successfully limit bacterial growth, which is in line with other antibacterial testing. The bacteria's outer membrane is where the nanoparticles first bind, changing its shape and eventually damaging its cells[ 25 ]. Flavins and NADPH, two naturally occurring fluorescent chemicals that cause bacterial cells to auto-fluoresce, are probably released as a result of this damage. MXene-silver-curcumin NC strong antibacterial qualities are demonstrated by the immediate decrease in fluorescence intensity that results from this procedure. In vitro toxicity of MXene-silver-curcumin NC-treated RD cells As illustrated in Fig. 12 , cytotoxicity data from the MTT experiment demonstrate that MXene-silver-curcumin NC exhibited limited cell death and minimal toxicity, particularly at lower dosages (62 and 31µg/mL), when compared to the untreated control shown in supplementary data S4. These results imply that MXene-silver-curcumin NC have a significant potential as a drug delivery vehicle for biological applications due to their low toxicity which were found similar to earlier study of ZnO nanoflower. Conclusion MXene-silver-curcumin NC which have a size of 220 nm, were successfully produced by chemical and green synthesis method indicating their potential for use in a range of biomedical applications. UV-visible absorption and fluorescence spectroscopy were used to confirm their optical and chemical characteristics. MXene nanosheets structures with silver and curcumin loaded on the surface were discovered by scanning electron microscopy (SEM). MXene-silver-curcumin NC antibacterial properties were tested against E .coli by using ager well diffusion method and RD cell lines were used to assess their cytotoxicity. Confocal microscopy, fluorescence investigations and antimicrobial photodynamic treatment (aPDT) provided insights into the interaction between MXene-silver-curcumin NC and bacterial cells, suggesting potential as enzyme-driven biochemical processes. Together, these results indicate that MXene–silver–curcumin NC are promising multifunctional photosensitizers for aPDT and fluorescence imaging. Consequently, more in vivo studies are conducted to confirm MXene-silver-curcumin NC biocompatibility with a wider range of pathogens and to conduct a concentration-effect analysis.These results simply that composites (NC) can be used as effective light-responsive nanoplatforms, combining bioimaging and therapeutic functionalities, and have the potential to be used as alternative antimicrobials in the future. Declarations Ethical Approval Not Applicable Conflict of Interest The authors declare no conflict of interest. Funding This study received no any funding from external source. Author Contribution Saliha noor, contributed in Writing, Experiments,AnalysisShahzad Anwar, Supervision,methdology,Rafaqat Ali Khan*, writing ,evaluation and experimentationSawera Malik, analysisHina Ali, Evaluation, methodology, Farwa Nurjis, analysis and methdologyShaista Taimur , evaluations and experimentaionMuhammad Iftakhar, co supervision,methdologyMuhammad Saleem,analysis and proof readingBabar Manzoor Atta, Analysis and methdology Acknowledgement The authors acknowledge Miss Fatima Batool for her cooperation during lab work. Data Availability No data was used in this study. References Moeini A, Pedram P, Makvandi P, Malinconico M, Gomez d’Ayala G (2020) Wound healing and antimicrobial effect of active secondary metabolites in chitosan-based wound dressings: A review. 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Carbohydr Polym Technol Appl 10(April):100795. https://doi.org/10.1016/j.carpta.2025.100795 Yang, J., Wang, C., Liu, X., Yin, Y., Ma, Y. H., Gao, Y., … Song, Y. (2020). Gallium–Carbenicillin Framework Coated Defect-Rich Hollow TiO2 as a Photocatalyzed Oxidative Stress Amplifier against Complex Infections. Advanced Functional Materials , 30 (43), 1–13. https://doi.org/10.1002/adfm.202004861 Faghani, A., Gholami, M. F., Trunk, M., Müller, J., Pachfule, P., Vogl, S., … Adeli,M. (2020). Metal-Assisted and Solvent-Mediated Synthesis of Two-Dimensional Triazine Structures on Gram Scale. Journal of the American Chemical Society , 142 (30), 12976–12986. https://doi.org/10.1021/jacs.0c02399 Huang G, Yan Y, Xu D, Wu J, Xu C, Fu L, Lin B (2021) Curcumin-loaded nanoMOFs@CMFP: A biological preserving paste with antibacterial properties and long-acting, controllable release. Food Chemistry , 337 (August 2020), 127987. https://doi.org/10.1016/j.foodchem.2020.127987 Pandey, R. P., Rasheed, P. 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Int J Biol Macromol 75:306–315. https://doi.org/10.1016/j.ijbiomac.2015.01.050 Iftikhar M, Shahzad F, Iqbal A, Mumtaz M, Ahmad I, Hassan T, Koo CM (2024) Synergistic terahertz shielding effects of electrically conductive MXene and shape-controlled magnetic nickel in polyvinylidene fluoride (PVDF) composites. J Alloys Compd 989(March):174306. https://doi.org/10.1016/j.jallcom.2024.174306 Khan RA, Anwar S, Ali H, Aziz U, Khanam B, Zakria M, Raffi M (2024) Antibacterial Efficacy of Tryptophan Coordinated Silver Nanoparticles Against E. coli: Spectroscopic and Microscopic Evaluation of Bacterial Cell Death. J Fluoresc. https://doi.org/10.1007/s10895-024-03987-0 Rozmysłowska-wojciechowska A, Mitrzak J, Szuplewska A (n.d.). Engineering of 2D Ti 3 C 2 MXene Surface Charge and its Influence on Biological Properties, 1–18 Wilson P, Venkateshwari S (2022) Green Synthesis and Characterization of Silver Nanoparticles from. Thymus Vulgaris Leaf Extract. (July) Scalable Synthesis of Ti 3 C 2 T (n.d.), 1–22. https://doi.org/10.1002/adem.201901241 Zebib B, Noirot V (2010) Stabilization of Curcumin by Complexation with Divalent Cations in Glycerol / Water System, 2010 . https://doi.org/10.1155/2010/292760 Rawat, A., Chourasia, N. K., Saini, S. K., Rajput, G., Yadav, A., Chourasia, R. K.,… Kulriya, P. K. (2023). Materials Advances Investigation of charge carrier dynamics in a, 6427–6438. https://doi.org/10.1039/d3ma00429e Wu F, Sun M, Xiang Y, Wu Y, Tong D (2010) Curcumin as a colorimetric and fluorescent chemosensor for selective recognition of fluoride ion. J Lumin 130(2):304–308. https://doi.org/10.1016/j.jlumin.2009.09.007 Kumar T, Bhardwaj VK (2025) Fluorescent Cu-MOF @ Rhodamine-B Nanocomposite for the Selective Sensing of Nitrate in Human Serum Gunasekaran S, Natarajan RK, Natarajan S, Rathikha R (2008) Structural investigation on curcumin. Asian J Chem 20(4):2903–2913 Ghasemi M, Khorsandi K, Kianmehr Z (2021) Photodynamic inactivation with curcumin and silver nanoparticles hinders Pseudomonas aeruginosa planktonic and biofilm formation: evaluation of glutathione peroxidase activity and ROS production. World Journal of Microbiology and Biotechnology , 4 . https://doi.org/10.1007/s11274-021-03104-4 Additional Declarations No competing interests reported. Supplementary Files supplemtrydataffpaper1.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 04 May, 2026 Reviewers agreed at journal 06 Apr, 2026 Reviews received at journal 01 Apr, 2026 Reviewers agreed at journal 30 Mar, 2026 Reviewers invited by journal 30 Mar, 2026 Editor assigned by journal 27 Mar, 2026 Submission checks completed at journal 27 Mar, 2026 First submitted to journal 25 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9225233","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":614870613,"identity":"6be5d7e7-ceb5-4dcf-9c64-af26d28dbaed","order_by":0,"name":"Saliha noor","email":"","orcid":"","institution":"University of Poonch Rawalakot","correspondingAuthor":false,"prefix":"","firstName":"Saliha","middleName":"","lastName":"noor","suffix":""},{"id":614870619,"identity":"11949616-a605-4e3b-989a-273fd9d48d80","order_by":1,"name":"Dr.Shahzad Anwar","email":"","orcid":"","institution":"Pakistan Institute of Engineering and Applied Sciences","correspondingAuthor":false,"prefix":"Dr.","firstName":"Shahzad","middleName":"","lastName":"Anwar","suffix":""},{"id":614870621,"identity":"a26a9a17-4bd3-410b-830a-51a7a413fdf8","order_by":2,"name":"Dr.Rafaqat Ali Khan","email":"data:image/png;base64,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","orcid":"","institution":"Pakistan Institute of Engineering and Applied Sciences, PO 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1","display":"","copyAsset":false,"role":"figure","size":512652,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis methodology of nanostructure\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/933517929986071d642fbe19.png"},{"id":105984855,"identity":"bdd42ad9-ccc2-4b88-bbd8-f1494c4ac31f","added_by":"auto","created_at":"2026-04-02 07:18:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":701148,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological and hydrodynamic study of images of MXene and MXene-silver-curcumin NC (a) SEM Images of MXene (b) hydrodynamic size of MXene (c) SEM Images of MXene-silver-curcumin NC (d) hydrodynamic size of MXene-silver-curcumin NC\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/56c5e36a75be68cd33f904b2.png"},{"id":105984856,"identity":"d0d8e4bc-14fe-403d-92b3-682bf4a93021","added_by":"auto","created_at":"2026-04-02 07:18:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2368065,"visible":true,"origin":"","legend":"\u003cp\u003eSpectroscopic characterization of MXene, silver curcumin and their NC (a)UV–Vis absorption spectrum of silver NPs suspension (b) UV–Vis absorption spectrum of MXene NS (c) UV–Vis absorption spectrum of curcumin (d) UV–Vis absorption spectrum of MXene-silver-curcumin NC.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/f42bccbffe8046b1365e0525.png"},{"id":105984858,"identity":"df2027a6-9b1c-44ed-9632-f35d5cc48976","added_by":"auto","created_at":"2026-04-02 07:18:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2511190,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectroscopic characterization of MXene-silver-curcumin NC (a) fluorescence emission spectrum of silver in suspension (b) Fluorescence emission spectrum of MXene suspension (c) Fluorescence emission spectrum of curcumin in suspension (d) Fluorescence emission spectrum of MXene-silver-curcumin suspension.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/6d3dede8a587deb2aa7f1a85.png"},{"id":106093688,"identity":"2dc22238-6a16-44c3-a744-7b7fd860258a","added_by":"auto","created_at":"2026-04-03 11:38:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":545870,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal microscopy images of MXene-silver-curcumin NC at excitation wavelengths 488 nm (a) optical images showing surface morphology of MXene-silver-curcumin NC (b) fluorescence image depicting distribution of MXene-silver-curcumin NC (c) histogram of MXene-silver-curcumin NCs (d) 3D Fluorescence intensity profile of MXene-silver-curcumin NC\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/af11151e7d8b83b7539c502d.png"},{"id":106094203,"identity":"5befdfe7-1ecc-4b61-84fa-d31f43f13af5","added_by":"auto","created_at":"2026-04-03 11:41:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":163726,"visible":true,"origin":"","legend":"\u003cp\u003eRaman spectrum indicating the vibrational modes of MXene-silver-curcumin NC\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/bd2a4cda1cdee38e03aa1ee9.png"},{"id":105984860,"identity":"91e2efa7-e597-42cf-be3a-cd519acb9ca4","added_by":"auto","created_at":"2026-04-02 07:18:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":713188,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of MXene, silver, curcumin and there NC (a) zone of inhibition MXene, Silver, curcumin and there NCsagainst \u003cem\u003eE. coli\u003c/em\u003e (b) graphical representation of zone of inhibition of MXene, Silver, curcumin and their NCs (c,d) zone of inhabitation of MXene-silver-CurcuminNCs at different concentrations and there graphical representation.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/e21e9bc4acfd3718d136ce44.png"},{"id":105984861,"identity":"83e71bd3-972b-4c91-b3b4-794f2201a110","added_by":"auto","created_at":"2026-04-02 07:18:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":727597,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of antibacterial action of MXene-silver-curcumin NC (a) Time-dependent antibacterial activity of MXene-silver-curcumin NC under 418 nm light exposure (b) Time-dependent antibacterial activity of MXene-silver-curcumin NC without light (c) Time-dependent activity ofonly bacteria under 418 nm light exposure.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/b8cd987264baf0ecc77c54be.png"},{"id":106094044,"identity":"5b5346dd-03a0-4f91-9808-9a294cd287a2","added_by":"auto","created_at":"2026-04-03 11:40:47","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":287824,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence-based evaluation of MXene-silver-curcumin NC against E. coli as a function of exposure time (a) time-dependent fluorescence response (0–60 minutes) under 418nm light (b) time-dependent fluorescence response (0–60 minutes) without light (c) time-dependent fluorescence response (0–60 minutes) only bacteria and light.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/265036e224789df954257f77.png"},{"id":106093712,"identity":"223a17c5-1e0d-4297-8363-be1f33d585b3","added_by":"auto","created_at":"2026-04-03 11:38:46","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":674161,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of antibacterial action of MXene-silver-curcumin NC at varying concentrations (a-e) Concentration and Time-dependent antibacterial activity of MXene-silver-curcumin NCs under 418 nm light (f) graphical representation of colony-forming units per milliliter (CFU/mL) at dissimilar concentrations and time of MXene-silver-curcumin NCs (g) graphical representation of fluorescence intensity of MXene-silver-curcumin NCs at concentrations 500-31 µg/ml.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/cbee09157ab147ceb6df19d5.png"},{"id":105984864,"identity":"10c92d3c-c776-428c-9e05-c4bbd636dc25","added_by":"auto","created_at":"2026-04-02 07:18:23","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":591964,"visible":true,"origin":"","legend":"\u003cp\u003e(a-f) Conventional fluorescence spectra of from 0-60 minutes with concentrations of MXene-silver-curcumin NC (31- 500 µg/ml)\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/1737bdd1d7068faca08ded78.png"},{"id":106093325,"identity":"9c9c9bac-a8a6-40d8-9082-50def9cf205d","added_by":"auto","created_at":"2026-04-03 11:36:45","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":176568,"visible":true,"origin":"","legend":"\u003cp\u003eCytoxicity evaluation of RD cells treated with MXene-silver-curcumin NC at different concentrations (31, 62, 125,250 and 500 µg/mL)\u003c/p\u003e","description":"","filename":"image12.png","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/5e10e18d9f22d43b70bf804f.png"},{"id":106401830,"identity":"af5c99d0-9a2f-40f1-849c-af87188c2d5d","added_by":"auto","created_at":"2026-04-08 09:09:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11069734,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/b911208d-39cd-4eb7-a115-45be0cea6dac.pdf"},{"id":105984854,"identity":"1fcbedce-1d0e-4574-93f0-49e37c483803","added_by":"auto","created_at":"2026-04-02 07:18:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":285991,"visible":true,"origin":"","legend":"","description":"","filename":"supplemtrydataffpaper1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9225233/v1/c6c0bf68bae98934df69a1bb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eEnhanced Antimicrobial Photodynamic Therapy against Escherichia coli Using MXene-Based Silver–Curcumin 2D Nanocomposite Photosensitizers\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWound infections due to a range of bacteria generally develop during the healing of skin and tissues damaged by external causes, producing serious inflammation and a variety of disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Since prolonged use of antibiotics results in drug resistance, which lessens the effectiveness of treatment, a variety of therapeutic approaches have been developed to increase the effectiveness of wound healing and lower the incidence of infection. These include therapeutic antimicrobials, peptides, hydrogels, nanofibrous dressings, and stem cell therapy [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The necessity for other treatments against infectious diseases is suggested by the appearance of bacterial strains that are resistant to antibiotic treatment. The application of antimicrobial photodynamic therapy (aPDT) represents a strategy that may lead to more effective antimicrobial treatment [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] .aPDT uses reactive oxygen, visible light, and a chemical photosensitizer or nontoxic photoactivatable dye. The treatment relies on the energy being transferred to the oxygen molecules, resulting in highly reactive mediators like superoxide and singlet oxygen that are harmful to the cells [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The photo responsive nanomaterials' limited light absorption ability and rapid photogenerated charge recombination usually result in subpar photocatalytic performance. Heterojunction formation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and defect introduction, particularly the creation of oxygen vacancies (OVs) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], are generally thought to be efficient methods for improving the photocatalytic properties of nanomaterials.\u003c/p\u003e \u003cp\u003eMXenes, a class of 2D materials derived from MAX phases, are promising for biomedical applications due to their biocompatibility. They can be modified to improve their antibacterial performance. The scientific community is actively investigating MXenes for biochemical detection, drug encapsulation, and tissue regeneration[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. MXene have optical and electrical performance to achieve better results in various fields[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Due to exceptional electrical conductivity, high absorption capacity and conversion efficiencies for near-infrared light, and localized surface plasmon resonance (LSPR), Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXenes, an appealing photosensitive material, have recently been shown to have improved photocatalytic properties by increasing the photoinduced charge transfer[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene good biocompatibility and high absorption capacity make it a promising candidate for a variety of biomedical and biosensing applications, particularly in wound healing [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].But Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene frequently has a high conversion temperature (over65\u0026deg;C), which can harm the tissues around[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Due to the short-term pulse impact of light irradiation, the remaining bacteria can also reproduce quickly when light exposure is stopped. MXene-based heterojunctions (Bi\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e) can improve electron transport to produce more ROS and improve biocompatibility when used as photoexcited antibacterial agents [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].The poor stability, low dispersibility of MXene is resolved by combining it with polymer materials, metal as well as ceramic compounds. Improvements in stability together with enhanced biocompatibility and functional properties enable the composite material to become suitable for biomedical applications [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Their surfaces can be modified to increase specificity by attaching medications, targeting compounds, or other substances. MXenes also release drugs in a regulated and targeted manner in response to stimuli like temperature, pH, or certain enzymes in the tumor microenvironment.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOver the past few decades, noble metallic nanoparticles have gained a lot of interest as antibacterial materials because of their possible physicochemical properties [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Coinage metals, particularly Silver (Ag) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and Gold (Au) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], exhibit excellent biocompatibility, antimicrobial properties, and corrosion resistance, making them valuable materials for various biomedical applications, [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Silver can effectively mitigate both the oxidation and stacking of MXene nanosheets, which are major limitations to its long-term stability and performance beyond this it also increasing antimicrobial efficiency [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].Silver nanoparticles toxicity at a biological platform is a serious concern. Coordination of silver nanoparticles with certain combinational materials is necessary to decrease toxicity and increase antibacterial agent efficacy. AgNPs' size, shape and modification by other materials are presented in order to assess the combined impact on their antibacterial activity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] .\u003c/p\u003e \u003cp\u003eTurmeric stands as Curcuma longa containing curcumin as its main active compound that demonstrates three major therapeutic effects: antibacterial along with anti-diabetic and anti-cancer properties. However, curcumin's low bioavailability and solubility limit its effectiveness. Current research involves nanotechnology-based curcumin formulations that improve its performance by increasing delivery efficiency while enabling large-scale manufacturing and local distribution of the substance [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The nanohybrid system of MXene functionalized with nanoparticles may load and distribute curcumin, enabling regulated release at particular sites.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In order to load and transport curcumin, a variety of drug delivery methods have been investigated, including inorganic nanomaterials [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], polymeric nanoparticles, covalent organic frameworks[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], MOFs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and MXene NSs [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The synergistic/additive effects of co-encapsulating curcumin and silver nanoparticles in various forms have recently been studied [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Combination of curcumin with silver reduce the silver toxicity and enhance antimicrobial activity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn present work, a promising nanoplatform for wound healing is presented by building MXene nanosheets connected with silver nanoparticles, which have a high photothermal conversion efficiency and an exceptional curcumin-loading capacity. This method combines improved drug delivery with photothermal therapy to provide a non-invasive, dual-modality solution. It is anticipated that combining these elements will result in comprehensive ,extremely successful antimicrobial therapy and wound healing, given the exceptional photothermal and photodynamic performance of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e NSs and the long-term therapeutic effect of silver and curcumin.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eHydrochloric acid (HCL), Lithium fluoride (LiF), pH paper, MAX powder, Deionized water, curcumin, silver nitrate (AgNO3)\u0026thinsp;\u0026ge;\u0026thinsp;98% are purchased from Sigma Aldrich, black tea from local market. Nutrient Agar, Nutrient Broth, glass Petri dishes (catalog no. D35-10\u0026ndash;1-N) were procured from Cellvis. Bacterial strains (ATCC 8739,) were sourced from the NIBGE. Cell Culture Assembly (NCCC) and RD cell lines from NIH, correspondingly,96-well plates, phosphate-buffered saline (PBS), and dimethyl sulfoxide (DMSO) were also used.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eSynthesis of MXene-silver-curcumin NC\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eWith a previously discussed modified least intensive layer delamination (MILD) method, Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene NSs were prepared by chemically etching Al atoms selectively from the Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e MAX phase [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. 100 mL polypropylene plastic vial was filled with an etchant solution of 20 mL HCl (9 M) and 1.33 g LiF while being constantly stirred on a hot plate. To avoid overheating placed in water bath and 1 g of Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e MAX powder was subsequently mixed to the mixture gradually and agitated for 24 hours at 35\u0026deg;C. To eliminate unetched MAX powder, the obtained mixture was centrifuged and washed using DI water. The delaminated Ti\u003csub\u003e3\u003c/sub\u003eC2T\u003csub\u003ex\u003c/sub\u003e MXene flakes stock solution was made by applying a washing cycle at 5000 rpm four to five times until the solution's pH reached\u0026thinsp;\u0026ge;\u0026thinsp;6.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA green synthesis process mediated by plant extract was used to create silver nanoparticles. 50 ml of boiling deionized water were mixed with 5g of black tea to create black tea extract. 0.34 grams of silver nitrate (AgNO₃) were dissolved in 100 ml of deionized water to create a 1 molar solution. 1 M AgNO\u003csub\u003e3\u003c/sub\u003e solution was added in 1:1 volumetric proportion with the prepared black tea extract and then subjected to a water bath at temperature of 84\u0026ordm;C for 20 minutes to allow reduction of silver ions to occur. The centrifugation of the solution was done at 10000 rpm for 10 minutes to isolate the silver nanoparticles. The supernatant was poured off and remaining material was harvested. For the composite preparation 1 gram of MXene, 0.5 grams of silver nanoparticles (AgNPs), and 0.5g of curcumin were added to 10 milliliters of deionized (DI) water. The mixture was then mixed on a hot plate under controlled temperature of 45\u0026ordm;C to 50\u0026ordm;C and stirred for 1 hour. Following this heating and mix phase, the mixture that resulted was put in an oven to dry and composite material has been synthesized as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOptical characterization of MXene-silver-curcumin NC\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDynamic light scattering study (DLS)\u003c/h2\u003e \u003cp\u003eDLS determines the hydrodynamic size of a particle in an aqueous environment. This was used to determine the stability and divisions of size when the synthesized nanoparticles were suspended in a media. MXene-silver-curcumin NC suspensions were put in a cuvette containing two milliliters of each of the self-assembled particle samples in the experimental setup. The cuvette was subsequently subjected to a dynamic light scattering instrument (Microtrac Nanotrac Wave II). Particle motion patterns and solution stability were affirmed by the interaction of the incoming light with the mobile particles, which were moving randomly within the solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy (SEM) analysis of MXene-silver-curcumin NC\u003c/h2\u003e \u003cp\u003eSEM (FESEM, MAIA3 TESCAN) analysis was used to examine MXene-silver-curcumin NC morphology. Samples were positioned on stubs and morphological examination was assessed.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eUV and fluorescence spectroscopy\u003c/h3\u003e\n\u003cp\u003eWith a fluorescence spectroscopy (FluoroMax-4, Horiba Scientific: USA), fluorescence spectroscopic measurements were obtained to assess the intrinsic fluorescence properties of material. For the antibacterial investigation MXene-silver-curcumin NC interacted with \u003cem\u003eE. Coli\u003c/em\u003e bacteria at varying concentration and times. MXene-silver-curcumin NC synthesis was confirmed by using a (Shi Madzu UV-VIS 2101PC) UV-vis absorption spectrophotometer.\u003c/p\u003e\n\u003ch3\u003eCulture medium preparation\u003c/h3\u003e\n\u003cp\u003eNutrient broth was made by dissolving 16 g of powder of nutrient broth in 1000 ml of distilled water. The suspension was autoclaved at 121\u0026deg;C for an hour. In the same way, 1000 ml of distilled water and 33 g of nutrient agar powder were combined, autoclaved, and allowed to set in a petri dish at 4\u0026deg;C. For the preparation of fresh bacterial inoculums, one colony of \u003cem\u003eE. coli\u003c/em\u003e was transferred from an agar plate to nutrient broth. Bacteria were cultured for four hours at 37\u0026deg;C for bacterial growth.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial Activity of MXene-silver-curcumin nanocomposite\u003c/h2\u003e \u003cp\u003eThe antibacterial efficacy of various nanoparticles (NPs) against monostreak \u003cem\u003eE. coli\u003c/em\u003e was performed by using agar diffusion method. Using a cotton swab, broth cultures were equally distributed across the nutrient agar plates. Wells were dug on the agar plates using autoclaved micropipette tips and loaded with 50 \u0026micro;l of suspensions of each NPs. A digital vernier caliper was used to measure the zones of inhibition in millimeters after the plates were incubated for the entire night at 37\u0026deg;C. For MXene-silver-curcumin nanocomposite, each experiment was carried out three times in order to get mean values. The agar well diffusion method, which was previously published in the literature with various modifications, was used to test the antibacterial activity of the produced nanocomposites [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of MXene-silver-curcumin nanocomposite for enhanced antibacterial photodynamic therapy (aPDT)\u003c/h2\u003e \u003cp\u003eFor four hours, the E. Coli-type culture collection of bacterial strains was cultured in nutrient broth at 37\u0026deg;C till growth. For the photobacterial assay, a 40\u0026micro;l of the cultured E. coli suspension was introduced into 40ml of deionized water, followed by the addition of 500 ml of nanocomposites (NCs) into a petri dish. This solution was then exposed to light of a specific wavelength for 60 minutes. At 10-minute intervals throughout the 60-minute exposure, 20\u0026micro;l samples of the solution were spread onto nutrient agar plates and subsequently incubated overnight to quantify bacterial viability. The colony counting method was used to calculate the bacterial growth inhibition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Data\u003c/h2\u003e \u003cp\u003eFor data analysis and plot processing, we have utilized Origin Pro 8.5 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSurface morphology and size distribution of MXene-silver-curcumin NC\u003c/h2\u003e \u003cp\u003eThese NC shape and size distribution were described using DLS and scanning electron microscopy, respectively. Scanning electron microscopy was used to examine the morphology of MXene and MXene-silver-curcumin NC, as seen in Fig .2(a) and Fig .2(c) which confirm synthesis of MXene from MAX phase and presence of MXene nanosheets along with silver and curcumin particles loaded on the surface. The monodisperse MXene and MXene-silver-curcumin NCs solution with a hydrodynamic size of 200nm [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]and 220 nm is displayed by the DLS measurement in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(b) and 2(d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong the many advantageous characteristics that make MXene-silver-curcumin NCs potentially of significant interest in a wide range of practical applications are excellent conductivity, chemical stability pre catalytic activity, and antibacterial activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eUV visible spectroscopic evaluations of MXene-Silver-Curcumin NC\u003c/h2\u003e \u003cp\u003eThe formation of silver nanoparticles was confirmed by the fact that the UV-Vis absorption peak of black-tea-mediated AgNPs was observed at 350 nm, which agrees with the surface plasma resonance peak of the synthesized AgNPs of Thymus vulgaris [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], the Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene nanosheets had large absorption band ranging 300\u0026ndash;420 nm, which is similar to the 200\u0026ndash;420 nm recorded range of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXenes .The minor change can be attributed to variation in surface terminations and synthesis parameter[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe UV-Vis absorption spectra of curcumin show distinct peaks at approximately 427 nm as also in previous study [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] respectively, all these parameters conforming particle formation as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a-c). The UV-Vis spectrum of the synthesized composite exhibited two distinct maximum absorbance peaks at 272 nm and 354 nm Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(d), suggesting its potential for biosensing applications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence based spectroscopic analysis of MXene-silver-curcumin NC\u003c/h2\u003e \u003cp\u003eThe distilled water was used to examine MXene-Silver-Curcumin wavelengths in quartz cuvettes. A shift found at excitation wavelengths of 390nm respectively, demonstrating the fluorescent nature of MXene-silver-curcumin NC as shown in figure below.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn Fig .4(a, b, c) it is clear that MXene Fluorescence intensity peak is at 455nm[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] that of Silver is at 394nm [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and Fluorescence intensity peak of curcumin is at 534nm [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. These findings demonstrated that MXene-silver-curcumin NC are fluorescent nanomaterials that can be tuned for use in biomedical applications, as is the case in this investigation against the bacterial strain \u003cem\u003eE. coli\u003c/em\u003e. The fluorescence of the MXene\u0026ndash;silver\u0026ndash;curcumin composite shows a notable blue shift, with the emission peak moving from the wide range of the individual components to a sharp peak at 390 nm shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(d) This phenomenon is associated to structural effects within the composite, electron redistribution, and non-radiative energy transfer which are in line with observations made on the interaction of Cu-MOF@Rhodamine B with analytes [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eAnalysis of MXene-silver-curcumin NCs using confocal microscopy\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eEvaluations of MXene-silver-curcumin NC using confocal microscopy were conducted at an excitation wavelength of 488 nm. MXene-silver-curcumin NC confocal microscopy analysis is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(a-f) based on temporal interactions at 500 \u0026micro;g/ml concentration. A single optical section of the sample, imaged using confocal fluorescence microscopy, is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(a)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe image shows the fluorescent behavior of MXene-silver-Curcumin NC, which are visible as fluorescently-labeled structures with varied morphology, including both isolated particles and larger fluorescent sheets in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(b). As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(c) fluorescence histogram of pure tunable MXene-silver-curcumin NC, verified greatest intensity peaks in the green region correspond to the fluorescence distribution of the nanoparticles in that area, while Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(e) show 2D slice of the fluorescent sheets of MXene-silver-curcumin NC with variations in intensity representing the distribution of the material within the focal plane.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRaman spectroscopic analysis of MXene-Silver-curcumin NC\u003c/h2\u003e \u003cp\u003eThe MXene-silver-curcumin NC vibrational characteristics are revealed by the Raman spectrum, which is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Different peaks at 303 cm⁻\u0026sup1;, 420 cm⁻\u0026sup1;, 797 cm⁻\u0026sup1;, and 1273 cm⁻\u0026sup1; are indication of Ti\u0026ndash;C vibrations and surface terminations (\u0026ndash;O, \u0026ndash;OH, \u0026ndash;F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCurcumin showed notable peaks at 1258, which were connected to C\u0026ndash;O stretching vibrations and C\u0026ndash;O\u0026ndash;C stretching, respectively [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].More Raman bands at greater shifts indicate the possibility of surface vibrational modes, phonon interactions, or structural flaws originating from the nanostructures (Wang et al., 2017).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibacterial assessment of MXene-silver-curcumin NC against\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eE. Coli\u003c/em\u003e culture collection of bacterial strains was incubated for 4 hours at 37\u0026deg;C in nutrient broth. For the MXene-silver-curcumin NC, the concentration range was 500\u0026ndash;31 \u0026micro;g/ml. Every sample was examined three times.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMXene and curcumin with no zone, silver with zone of 14.98 mm, and MXene-silver-curcumin with 15.01 mm zone of inhibition are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(a), while graphical representation of these zone is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(b) and in supplementary data S1while Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(c) show zone of inhibition at various concentration due to interactions between MXene-silver-curcumin and \u003cem\u003eE-coli\u003c/em\u003e. The MXene-silver-curcumin zone of inhibition at varying concentrations is graphically shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(d). The effects of varying concentrations of MXene-silver-curcumin NC on \u003cem\u003eE. coli\u003c/em\u003e bacteria revealed a linear relationship between bacterial mortality and particle concentrations. Bacterial disintegration peaked at higher concentrations and was negligible at lower ones shown in supplementary data S2\u003c/p\u003e \u003cp\u003e \u003cb\u003eaPDT Analysis of MXene-silver-curcumin NC Against\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e(a), the presence of both the nanocomposites and light resulted in significant bacterial killing, with the number of possible bacterial colonies progressively decreasing over the 60-minute interaction period, reaching a very low count after 60 minutes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e(b) demonstrates that in the absence of light, the nanocomposites exhibited negligible bactericidal activity, leading to very limited bacterial reduction despite interaction. Furthermore, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e(c) confirms that light exposure alone did not effectively kill the bacteria. These findings collectively indicate that a material acting as a photosensitizer is essential to generate reactive oxygen species (ROS) in the presence of light, thereby facilitating effective bacterial killing.\u003c/p\u003e \u003cp\u003e \u003cb\u003eaPDT analysis of MXene-silver-curcumin NC against\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eusing conventional fluorescence spectrometer\u003c/b\u003e\u003c/p\u003e \u003cp\u003eConventional fluorescence spectrometer was used to assess the interactions of MXene-silver-curcumin NC with \u003cem\u003eE. coli\u003c/em\u003e based on time. MXene-silver-curcumin NC were cultured with E. Coli for 10 to 60 minutes, with a 10-minute break between each measurement. NC operate as photosensitizers when exposed to particular wavelengths of light, generating reactive oxidative species such singlet oxygen. These ROS cause bacterial membrane, protein, and DNA oxidative damage, which results in cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e(a) indicates the declining pattern in fluorescence intensity peaks with time interactions, which shows how well MXene-silver-curcumin NC kill bacteria. MXene-silver-curcumin NC incubation resulted in a considerable drop in the fluorescence intensity of bacterial cells, suggesting a gradual decline in bacterial growth. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e(b)show the time interaction of composite with bacterial in the absence of light indicating very small decreasing pattern of fluorescence intensity peak. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e(c) show interaction of bacteria with light which show negligible decrease in fluorescence intensity peak which indicate that bacteria were not effect by light only.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eColony forming units of MXene-silver-curcumin NC at different concentrations\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eParticles were inoculated with \u003cem\u003eE. coli\u003c/em\u003e bacterial inoculums to assess the Colony Forming Unit calculation as in previous study [47].The growth of pure bacterial colonies was then studied using various serial dilutions dispersed on an agar plate. CFU/mL at various doses is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e(a-e) and graphical analysis in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e(f) verified that the minimum CFU/ml was 500 \u0026micro;g/ml.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e(e) indicates at least 10 colonies were observed at peak concentrations of 500 \u0026micro;g/ml, while Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e(a) shows a maximum of 268 colonies were counted at low values of 31 \u0026micro;g/ml in 20 minutes while at 500 \u0026micro;g/ml concentration bacterial colonies reduce to 1 at 30 min and at concentration of 31 \u0026micro;g/ml bacterial colonies were 25. Data was gathered in triplicate and plotted against each value (\u0026plusmn;\u0026thinsp;SD) to determine the average and standard deviation as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e(f) colony formation unit based on both concentration (31\u0026ndash;500 \u0026micro;g/ml) and time interaction (10\u0026ndash;60 min). From Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e(g) it can be seen that \u003cem\u003eE.coli\u003c/em\u003e bacteria and particles treated with varying doses of MXene-silver-curcumin exhibited a linear relationship between bacterial death and particle concentrations.\u003c/p\u003e \u003cp\u003eBacterial disintegration peaked at higher MXene-silver-curcumin NCs concentrations and was negligible at lower concentrations. Agar plates were utilized to calculate the average CFU/mL values which are included in the Supplementary data S3, after E. Coli inoculums were incubated with MXene-Ag-Curcumin NCs for 24 hours.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConcentration and time based interaction of MXene-silver-curcumin NC with\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eusing conventional fluorescence spectrometer\u003c/b\u003e\u003c/p\u003e \u003cp\u003eConventional fluorescence spectrometer was used to assess the interactions of MXene-silver-curcumin NC with \u003cem\u003eE. coli\u003c/em\u003e based on concentrations and time as studied earlier [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Illustration in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e(a-e) shows a declining trend in fluorescence intensity peaks with time interactions, which indicates how well MXene-silver-curcumin NC kill bacteria at various concentrations. Demonstrate that two factors concentration and time interactions have an impact on killing efficiency. The maximum 500 \u0026micro;g/ml concentrations of MXene-silver-curcumin NC (10\u0026ndash;60 min) possessed the maximum killing efficiency, as per Flores' concentration-time interaction-based analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMXene-silver-curcumin NC antibacterial properties are revealed through their interaction with bacterial cells. A notable reduction in fluorescence intensity was noted during incubation of bacterial cells with MXene-silver-curcumin NC suggesting a gradual slowdown in bacterial development. According to this result, MXene-silver-curcumin NC successfully limit bacterial growth, which is in line with other antibacterial testing. The bacteria's outer membrane is where the nanoparticles first bind, changing its shape and eventually damaging its cells[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Flavins and NADPH, two naturally occurring fluorescent chemicals that cause bacterial cells to auto-fluoresce, are probably released as a result of this damage. MXene-silver-curcumin NC strong antibacterial qualities are demonstrated by the immediate decrease in fluorescence intensity that results from this procedure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro toxicity of MXene-silver-curcumin NC-treated RD cells\u003c/h2\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e, cytotoxicity data from the MTT experiment demonstrate that MXene-silver-curcumin NC exhibited limited cell death and minimal toxicity, particularly at lower dosages (62 and 31\u0026micro;g/mL), when compared to the untreated control shown in supplementary data S4.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results imply that MXene-silver-curcumin NC have a significant potential as a drug delivery vehicle for biological applications due to their low toxicity which were found similar to earlier study of ZnO nanoflower.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMXene-silver-curcumin NC which have a size of 220 nm, were successfully produced by chemical and green synthesis method indicating their potential for use in a range of biomedical applications. UV-visible absorption and fluorescence spectroscopy were used to confirm their optical and chemical characteristics. MXene nanosheets structures with silver and curcumin loaded on the surface were discovered by scanning electron microscopy (SEM). MXene-silver-curcumin NC antibacterial properties were tested against \u003cem\u003eE .coli\u003c/em\u003e by using ager well diffusion method and RD cell lines were used to assess their cytotoxicity. Confocal microscopy, fluorescence investigations and antimicrobial photodynamic treatment (aPDT) provided insights into the interaction between MXene-silver-curcumin NC and bacterial cells, suggesting potential as enzyme-driven biochemical processes. Together, these results indicate that MXene\u0026ndash;silver\u0026ndash;curcumin NC are promising multifunctional photosensitizers for aPDT and fluorescence imaging. Consequently, more in vivo studies are conducted to confirm MXene-silver-curcumin NC biocompatibility with a wider range of pathogens and to conduct a concentration-effect analysis.These results simply that composites (NC) can be used as effective light-responsive nanoplatforms, combining bioimaging and therapeutic functionalities, and have the potential to be used as alternative antimicrobials in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthical Approval\u003c/h2\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003ch2\u003eConflict of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study received no any funding from external source.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eSaliha noor, contributed in Writing, Experiments,AnalysisShahzad Anwar, Supervision,methdology,Rafaqat Ali Khan*, writing ,evaluation and experimentationSawera Malik, analysisHina Ali, Evaluation, methodology, Farwa Nurjis, analysis and methdologyShaista Taimur , evaluations and experimentaionMuhammad Iftakhar, co supervision,methdologyMuhammad Saleem,analysis and proof readingBabar Manzoor Atta, Analysis and methdology\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors acknowledge Miss Fatima Batool for her cooperation during lab work.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eNo data was used in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMoeini A, Pedram P, Makvandi P, Malinconico M, Gomez d\u0026rsquo;Ayala G (2020) Wound healing and antimicrobial effect of active secondary metabolites in chitosan-based wound dressings: A review. \u003cem\u003eCarbohydrate Polymers\u003c/em\u003e, \u003cem\u003e233\u003c/em\u003e(November 2019), 115839. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.carbpol.2020.115839\u003c/span\u003e\u003cspan address=\"10.1016/j.carbpol.2020.115839\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaxminarayan R, Matsoso P, Pant S, Brower C, R\u0026oslash;ttingen JA, Klugman K, Davies S (2016) Access to effective antimicrobials: A worldwide challenge. 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Asian J Chem 20(4):2903\u0026ndash;2913\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhasemi M, Khorsandi K, Kianmehr Z (2021) Photodynamic inactivation with curcumin and silver nanoparticles hinders Pseudomonas aeruginosa planktonic and biofilm formation: evaluation of glutathione peroxidase activity and ROS production. \u003cem\u003eWorld Journal of Microbiology and Biotechnology\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11274-021-03104-4\u003c/span\u003e\u003cspan address=\"10.1007/s11274-021-03104-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Fluorescence spectroscopy, antibacterial photodynamic therapy, Raman spectroscopy, photosensitizer","lastPublishedDoi":"10.21203/rs.3.rs-9225233/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9225233/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe increasing prevalence of antibiotic-resistant bacteria poses a significant threat to public health. Addressing this challenge requires the development of innovative antimicrobial materials. In this context, MXene-based nanomaterials have emerged as promising candidates for advanced healthcare applications. The photodynamic therapy (aPDT) was evaluated to investigate the antibacterial potential and efficacy of MXene\u0026ndash;silver\u0026ndash;curcumin (NC) nanocomposites. Nanocomposites are novel photoactive nanomaterials. The synthesis of composites includes a chemical synthesis route and green synthesis and followed by complete characterization of the materials to evaluate their morphological, optical, and cell viability against rhabdomyosarcoma tumor (RD) cell lines. The layered MXene nanosheets depicted in the SEM images were loaded with silver nanoparticles and curcumin. The nanoscale structure and dispersibility in aqueous media were evaluated through dynamic light scattering (DLS), UV\u0026ndash;Visible spectroscopy, and fluorescence studies, indicating an approximate hydrodynamic diameter of 220 nm. The bonding among MXene, silver, and curcumin were confirmed by Raman spectroscopy. In antibacterial activity, a strong inhibition zone of 15.01 mm was observed against \u003cem\u003eEscherichia\u003c/em\u003e. Cytotoxicity data from the MTT experiment demonstrate that MXene-silver-curcumin NC exhibited limited cell death and minimal toxicity, particularly at lower dosages (31\u0026micro;g/mL) 92%, when compared to the control. On the exposure of 418 nm light on composites, the concentration and light dependent aPDT effects were observed, where the bacterial killing was significantly higher at concentration of 500 ug/mL, and this was explained by the combined effects of MXene conductivity, silver plasmonic enhancement, and curcumin photosensitization.\u003c/p\u003e","manuscriptTitle":"Enhanced Antimicrobial Photodynamic Therapy against Escherichia coli Using MXene-Based Silver–Curcumin 2D Nanocomposite Photosensitizers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-02 07:18:17","doi":"10.21203/rs.3.rs-9225233/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-04T16:20:49+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"230151770523945468041461282214426792686","date":"2026-04-06T11:32:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-01T22:38:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2486805352822456704225585937075226918","date":"2026-03-30T22:27:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-30T16:41:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-27T11:02:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-27T11:01:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2026-03-25T15:42:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f48de423-e77a-4c59-ac4d-bee1ed364a71","owner":[],"postedDate":"April 2nd, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-04T16:20:49+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T12:53:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-02 07:18:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9225233","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9225233","identity":"rs-9225233","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}