Donor Rotation and Charge Enhancement Functionalized Nile Red Derivatives-Mediated Efficient Phototherapy for Drug-Resistant Bacteria Elimination and Monkeypox Virus Inactivation

Donor Rotation and Charge Enhancement Functionalized Nile Red Derivatives-Mediated Efficient Phototherapy for Drug-Resistant Bacteria Elimination and Monkeypox Virus Inactivation | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Aggregate This is a preprint and has not been peer reviewed. Data may be preliminary. 1 May 2025 V1 Latest version Share on Donor Rotation and Charge Enhancement Functionalized Nile Red Derivatives-Mediated Efficient Phototherapy for Drug-Resistant Bacteria Elimination and Monkeypox Virus Inactivation Authors : Laiping Fang , Wei Wang , Jianan Dai , Yike Tu , Shufang Li , Kuo He , Siya Tong , Yuhui Liao 0000-0003-4702-9516 , Ping’an Ma 0000-0003-4198-5240 [email protected] , and Guihua Jiang Authors Info & Affiliations https://doi.org/10.22541/au.174605970.08433930/v1 354 views 229 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The escalating threats of antimicrobial resistance and monkeypox virus infections to public health necessitate innovative therapeutic approaches. Developing materials with balanced photodynamic and photothermal effects for broad-spectrum drug-resistant bacteria elimination and monkeypox virus inactivation remains challenging. Herein, we prepared a series of Nile Red derivatives by a donor rotation and charge enhancement strategy, identifying 5-(dicyanomethylene)-9-[4-(bis(4-methoxyphenyl)amino)phenyl]-7a,12a-dihydro-5H-benzo[a]phenoxazine (TPAOMCN)-featuring alkoxy-triphenylamine and malononitrile, as the optimal candidate. TPAOMCN demonstrated extended near-infrared absorption, enhanced intersystem crossing (ISC) efficiency, and intense molecular motions, enabling dual-modal phototherapy. Electrospun TPAOMCN nanofibers (NFs) with submicron-scale diameter achieved >50°C temperature elevation and 30-fold reactive oxygen species (ROS) generation under irradiation, yielding >99.9% eradication of multidrug-resistant pathogens and virus. In methicillin-resistant S. aureus (MRSA)-induced wound infection and Vaccinia virus-mediated tail-scarred models, TPAOMCN NFs effectively eliminated MRSA colonies and reduced viral load through physical disruption of pathogen membranes, thermal denaturation of viral capsids, and ROS-mediated biomolecule oxidation, while suppressing inflammation and accelerating angiogenesis-mediated tissue repair. This study not only established a molecular engineering strategy for Nile Red to achieve prime PDT-PTT performance but also provided a paradigm for advancing dual-functional phototherapeutic platforms against emerging antimicrobial threats and monkeypox virus infections. Donor Rotation and Charge Enhancement Functionalized Nile Red Derivatives-Mediated Efficient Phototherapy for Drug-Resistant Bacteria Elimination and Monkeypox Virus Inactivation Laiping Fang, ‡,a,b Wei Wang, ‡,c Jianan Dai, ‡,d Yike Tu, b Shufang Li, b Kuo He, e Siya Tong, b Yuhui Liao,* ,c Ping’an Ma,* ,e and Guihua Jiang* ,b Dr. L. P. Fang, Guangdong Second Provincial General Hospital, School of Medicine, Jinan University Guangzhou 518037, P. R. China The Department of Medical Imaging, The Affiliated Guangdong Second Provincial General Hospital of Jinan University, Guangzhou 518037, P. R. China Mrs. Y. K. Tu, Mrs. S. F. Li, Mrs. S. Y. Tong, Prof. G. H. Jiang The Department of Medical Imaging, The Affiliated Guangdong Second Provincial General Hospital of Jinan University, Guangzhou 518037, P. R. China E-mail: [email protected] Dr. W. Wang, Prof. Y. H. Liao Institute for Engineering Medicine, Kunming Medical University Kunming 650500, P. R. China E-mail: [email protected] Dr. J. N. Dai College of Information Technology, Jilin Engineering Research Center of Optoelectronic Materials and Devices, Jilin Normal University, Siping 136000, P. R. China Mr. K. He, Prof. P. A. Ma State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130012, P. R. China E-mail: [email protected] Keywords: drug-resistant bacteria, monkeypox virus, nile red derivatives, photodynamic therapy, photothermal therapy The escalating threats of antimicrobial resistance and monkeypox virus infections to public health necessitate innovative therapeutic approaches. Developing materials with balanced photodynamic and photothermal effects for broad-spectrum drug-resistant bacteria elimination and monkeypox virus inactivation remains challenging. Herein, we prepared a series of Nile Red derivatives by a donor rotation and charge enhancement strategy, identifying 5-(dicyanomethylene)-9-[4-(bis(4-methoxyphenyl)amino)phenyl]-7a,12a-dihydro-5H-benzo[a]phenoxazine (TPAOMCN)-featuring alkoxy-triphenylamine and malononitrile, as the optimal candidate. TPAOMCN demonstrated extended near-infrared absorption, enhanced intersystem crossing (ISC) efficiency, and intense molecular motions, enabling dual-modal phototherapy. Electrospun TPAOMCN nanofibers (NFs) with submicron-scale diameter achieved >50°C temperature elevation and 30-fold reactive oxygen species (ROS) generation under irradiation, yielding >99.9% eradication of multidrug-resistant pathogens and virus. In methicillin-resistant S. aureus (MRSA)-induced wound infection and Vaccinia virus-mediated tail-scarred models, TPAOMCN NFs effectively eliminated MRSA colonies and reduced viral load through physical disruption of pathogen membranes, thermal denaturation of viral capsids, and ROS-mediated biomolecule oxidation, while suppressing inflammation and accelerating angiogenesis-mediated tissue repair. This study not only established a molecular engineering strategy for Nile Red to achieve prime PDT-PTT performance but also provided a paradigm for advancing dual-functional phototherapeutic platforms against emerging antimicrobial threats and monkeypox virus infections. 1. Introduction Pathogenic microorganisms, including drug-resistant bacteria and monkeypox virus, pose significant threats to human and public health security. [1-2] While antibiotics remain clinical mainstays against bacterial infections, their overuse accelerates the evolution of untreatable superbugs. [3-5] By 2050, it is projected that 10 million people worldwide will die from drug-resistant infections each year. [6] Simultaneously, the zoonotic monkeypox virus persists without approved targeted therapies, as repurposed smallpox antivirals (cidofovir, brincidofovir, tecovirimat) carry unproven efficacy and safety profiles in large-scale applications. [7-11] These converging crises underscore the critical need for innovative therapeutic platforms capable of simultaneously addressing multidrug-resistant pathogens and enveloped viruses through non-antibiotic mechanisms, circumventing current limitations in conventional treatment paradigms. Scheme 1. (a) Structure design of BCN, TPACN, and TPAOMCN by a donor rotation and charge enhancement strategy. (b) The mechanism of photothermal conversion and ROS generation. (c) Applications of TPAOMCN NFs-mediated PTT and PDT for anti-drug-resistant bacteria infections and monkeypox virus inactivation. Phototherapy, encompassing photothermal therapy (PTT) and photodynamic therapy (PDT), exhibits promising potential for the treatment of pathogenic microorganisms due to their high spatiotemporal controllability, low toxicity, and resistance to drug resistance development. [13-16] Conventional photothermal eradication requires bactericidal temperatures exceeding 70°C that risk collateral tissue damage, [17-18] while subtherapeutic hyperthermia preserves healthy structures but permits bacterial survival. Considering that bacterial proteins, nucleic acids, and lipids are all sensitive to oxidation, combining PDT, which generates ROS that cause irreversible oxidative damage to bacterial structures, can further effectively eradicate bacteria. [19] Combined phototherapy may also be a potential candidate for virus treatment. On the one hand, the lipid envelopes of viruses are particularly sensitive to ROS and prone to oxidation. [20-22] Chen et al. designed a photosensitizer (DTTPB) with a hydrophilic head and two hydrophobic tails to mimic the phospholipid structure of biological membranes, which could completely inactivate human coronaviruses after 20 minutes of irradiation under white light, with minimal cytotoxicity and excellent biocompatibility. [23] Not only that, antiviral PTT is also starting to emerge. [24] A positively charged photothermal agent (N + TT- m CB) could interact with the rabies virus envelope through charge attraction. The combined thermal effects and photothermal-induced inflammatory responses suppress localized viral activity, significantly enhancing survival rates in rabies virus-infected mice. [25] Therefore, the combined therapy of PDT and PTT offers a potential, more rational, safe, and efficient treatment strategy. Current photodynamic therapies face intrinsic limitations in hypoxic microenvironments due to the oxygen-dependent type-II photochemical mechanism that predominantly generates singlet oxygen (¹O₂), [25] while conventional photothermal agents based on inorganic nanomaterials (graphene derivatives, Au/Ag nanoparticles) confront persistent biosafety concerns limiting their further clinical applications despite their high photothermal conversion performance. [26-29] Organic phototheranostic agents offering distinct advantages of inherent biocompatibility, structural adaptability, and programmable metabolic pathways have emerged as compelling alternatives. Nile Red, a clinically translatable fluorescent scaffold, distinguishing itself through a unique π-expanded xanthene architecture that permits precise functional group engineering, may be the optimal candidate. However, its planar structure paradoxically suffers from aggregation-caused ROS quenching effect and faces the fundamental challenge of balancing inherently competitive energy dissipation pathways between photodynamic and photothermal processes. [30-32] Moreover, there are hardly any reports on the synergistic PDT-PTT for monkeypox virus inactivation. In summary, modifying Nile Red for prominent PDT and PTT performance against broad-spectrum drug-resistant bacteria and monkeypox virus holds significant scientific research value and broad application prospects. In this article, we meticulously designed and synthesized three Nile Red derivatives called 2-(9-phenyl-7a,12a-dihydro-5H-benzo[a]phenoxazin-5-ylidene)malononitrile (BCN), 2-(9-(4-(diphenylamino)phenyl)-7a,12a-dihydro-5H-benzo[a]phenoxazin-5-ylidene)malononitrile (TPACN), and TPAOMCN by a donor rotation and charge enhancement strategy (Scheme 1a). (1) By incorporating triphenylamine (TPA) with rich rotors, TPAOMCN exhibited effective non-radiative transition (NRT) and intense molecular rotation, achieving superior photothermal conversion capabilities. (2) The strong intramolecular charge transfer of TPAOMCN weakened the radiative transition (RT), facilitating the ISC ability, narrowing the single-triplet gap (∆ S-T ), and demonstrating the optimal ROS production (Scheme 1b). (3) In vitro experiments revealed that TPAOMCN NFs exhibited over 99.9% inhibition rate against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (PA), and MRSA, as well as effectively eliminating the monkeypox virus. (4) In both MRSA-infected wound and vaccinia virus-challenged tail scarification models, photoactivated TPAOMCN NFs exerted dual therapeutic-regenerative functions through pathogen eradication and inflammation modulation, achieving 90% accelerated wound healing in bacterial infections and 82% viral load reduction (Scheme 1c). Overall, this study introduced TPAOMCN with exceptional PDT and PTT efficacy, and comprehensively explored its safe and effective broad-spectrum anti-drug-resistant bacteria and antivirus activities. These findings offered valuable insights and served as a reference for the development of next-generation clinical photothepeutic materials against evolving microbial threats. 2. Results and Discussion 2.1. Design, Synthesis, and Characterization As depicted in Figure 1a and Scheme S1, Nile Red derivatives, including BCN, TPACN, and TPAOMCN, were designed and synthesized by a donor rotation and charge enhancement strategy. In brief, the Suzuki-Miyaura couplings of 2-(9-bromo-5H-benzo[a]phenoxazin-5-ylidene)malononitrile with phenylboronic acid, (4-(diphenylamino)phenyl)boronic acid, and (4-(bis(4-methoxyphenyl)amino)phenyl)boronic acid furnished BCN, TPACN, and TPAOMCN, respectively. The experimental steps and characterization data, including 1 H NMR, 13 C NMR, and high-resolution mass spectrometry (HRMS), for the compounds can be found in Figures S1-S9. The fundamental mechanism of photothermal conversion hinges on molecular rotation, prompting the adoption of a donor rotation strategy. Compared to a single benzene ring, the classic molecular rotor-TPA served as a freely rotating electron donor, thereby bolstering the photothermal performance. Furthermore, the superior electron-donating capability of TPA also facilitated an extended absorption range for TPACN. In addition to concurrently optimizing the molecule’s photodynamic properties, it is imperative to augment its electron-donating capability further. To this end, methoxy-modified TPA, which possesses an even greater electron-donating capacity than TPA, has been introduced into the molecular backbone of TPAOMCN to achieve better ROS yield. To demonstrate the rationality and feasibility of the aforementioned design strategy, their ground state (S 0 ) structures were initially optimized. As illustrated in Figure 1b, the dihedral angles formed between 2-(9-bromo-5H-benzo[a]phenoxazin-5-ylidene)malononitrile and the electron donors were measured at -35.72° for BCN, 32.99° for TPACN, and 27.61° for TPAOMCN. Notably, the incorporation of TPA as a terminal moiety induced substantial distortion in the molecular framework. Specifically, the dihedral angles between neighboring benzene rings within the TPA structure reached -31.39° in TPACN and -30.25° in TPAOMCN, highlighting the efficacy of our donor rotation approach. Subsequently, we calculated the energy gap (∆ L-H ) between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) for each molecule (Figure 1c). The ∆ L-H values progressively diminished from BCN (2.46 eV) to TPACN (1.96 eV) and TPAOMCN (1.77 eV). This gradually decreasing energy gap reflected the enhancement of the molecule’s electron-donating ability, which was consistent with our design principles. Additionally, BCN exhibited a relatively uniform electron cloud distribution across both HOMO and LUMO, while TPAOMCN demonstrated a pronounced localization of the electron cloud in the LUMO on the electron-accepting group and in the HOMO on the electron-donating group. TPACN also displayed a comparable trend, confirming that the introduction of TPA markedly facilitated intramolecular charge transfer (ICT). Next, we delved into the photophysical properties of these molecules. Figures 1d-1e revealed their maximum molar extinction coefficients (ɛ), which were 3.2×10 4 , 2.7×10 4 , and 2.2×10 4 L mol -1 cm -1 for BCN, TPACN, and TPAOMCN, respectively. BCN, with its most planar molecular structure, exhibited enhanced light-capturing ability, resulting in the highest absorbance. Correspondingly, the absorption peaks for BCN, TPACN, and TPAOMCN in dimethyl sulfoxide (DMSO) solutions were observed at 531 nm, 600 nm, and 635 nm, respectively, demonstrating a red shift in absorption with increasing electron donor-acceptor strength, as anticipated. The photoluminescence (PL) properties of these molecules in DMSO solutions were also investigated (Figure 1f). BCN displayed distinct red fluorescence with a peak at 655 nm. Surprisingly, neither TPACN nor TPAOMCN exhibited any emission, showcasing a decayed radiative transition (RT) behavior. To elucidate the underlying mechanisms, we further analyzed their Huang-Rhys (HR) factors, which are closely related to the non-radiative transition (NRT) process. HR factors quantify the change in vibrational quanta during electronic transitions, with more significant HR factors promoting non-radiative decay rates. [33-34] Figure 1g compared the HR factors of BCN, TPACN, and TPAOMCN. Notably, the HR factor of BCN was lower than that of TPACN and significantly lower than that of TPAOMCN. These results suggested that the primary reason for the lack of emission in TPACN and TPAOMCN is the dominant non-radiative decay of excited molecules back to the ground state, involving processes such as ISC and molecular motions. This indicated that TPACN and TPAOMCN may possess potential photodynamic and photothermal properties, laying the foundation for further exploration of their phototherapeutic capabilities. backend=biber, style=alphabetic, sorting=ynt ]biblatex Figure 1. (a) Molecular structures of BCN, TPACN, and TPAOMCN. (b) Optimized S0 geometries. (c) The electronic distribution in LUMO and HOMO and their energy gap determined at the B3LYP/6-311G (d,p) level. (d-e) Non-normalized and normalized absorption spectra of molecule solutions in DMSO. (f) PL spectra of molecule solutions in DMSO. (g) HR factors of molecules. (h) Images and SEM images of TPAOMCN NFs, scale bar: 5.00 μm. 2.2. Photothermal Conversion and ROS Generation By employing the nanoprecipitation method and electrospinning techniques, we successfully prepared the corresponding nanoparticles (NPs) and NFs. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses revealed that the particle size of TPAOMCN NPs was approximately 121.6 nm (Figure S10). Scanning electron microscopy (SEM) showed that the diameters of PVDF NFs (Fs) and TPAOMCN NFs ranged from 0.5 to 1.0 μm (Figures 1h-1i and Figure S11). Subsequently, we evaluated the stability of both TPAOMCN NPs and TPAOMCN NFs. Over a 14-day observation period, the particle size of TPAOMCN NPs remained consistently within 125 nm (Figure S12). Furthermore, PVDF NFs and TPAOMCN NFs maintained structural stability, whether washed with water or 75% ethanol (Figure S13). Then, we first evaluated the photothermal conversion capabilities of these NPs under 660 nm laser irradiation. Notably, TPACN NPs and TPAOMCN NPs exhibited superior photothermal performance compared to water or BCN NPs. Specifically, when exposed to a 660 nm laser at a power density of 0.3 W cm -2 , the temperatures of water, BCN NPs, TPACN NPs, and TPAOMCN NPs were recorded as 26.6°C, 33.8°C, 59.9°C, and 65.5°C, respectively backend=biber, style=alphabetic, sorting=ynt ]biblatex (Figure 2a). Then, we investigated the temperature increase in response to varying concentrations of TPAOMCN NPs under 660 nm laser illumination. As depicted in Figure 2b, a concentration-dependent temperature increase was observed at a concentration of 100 μg mL -1 . Furthermore, we analyzed the impact of different laser power densities on the heating efficiency of TPAOMCN NPs, revealing a clear positive correlation between increased laser power density and the resultant temperature rise (Figure 2c). TPAOMCN NFs could also reach a high temperature above 75°C within only 10 seconds at a laser power of 0.1 W cm -2 (Figure 2d). To assess the photothermal stability of TPAOMCN NFs, we subjected them to three heating and cooling cycles under consistent 660 nm laser irradiation (Figure 2e). Our findings Figure 2. (a) Photothermal heating curves of water and NPs. (b) Concentration dependence of TPAOMCN NPs for photothermal conversion (0.3 W cm -2 ). (c) Laser power dependence of TPAOMCN NPs for photothermal conversion (100 μg mL -1 ). (d) Photothermal heating curves of TPAOMCN NFs or undoping PVDF NFs (0.1 W cm -2 ). (e) The photothermal stability of TPAOMCN NFs. (f-h) Calculated reorganization energy of BCN, TPACN, and TPAOMCN at different wavenumbers, inset: contribution of bond length, bond angle, and dihedral angle to the total reorganization energy. (i) The PL intensity of DCFH with TPAOMCN NFs under laser irradiation (0.3 W cm -2 ). (j-l) The identification of ROS type by DHR123, HPF, and DPBF. (m-o) The ESR signal intensity of different ROS in dark or irradiation (0.3 W cm -2 ) with TPAOMCN NFs. (p) Singlet-triplet energy levels. backend=biber, style=alphabetic, sorting=ynt ]biblatex indicated that the maximum temperature of TPAOMCN NFs remained relatively stable, demonstrating good photothermal stability. To gain deeper insights into the differences in non-radiative transitions among these NPs, we calculated the total reorganization energy (λ). The λ value for BCN (344.59 MeV) was lower than that for TPACN (739.16 MeV) and TPAOMCN (2133.44 MeV), suggesting a more rigid structure of BCN (Figure 2f-2h). Moreover, we examined the contributions of various intramolecular motion modes to non-radiative decay. While the photothermal conversion of BCN primarily stemmed from bond length and bond angle vibrations (contributing 96.5%), the rotation of dihedral angles was the main contributor to the photothermal conversion of TPACN and TPAOMCN, accounting for 58.5% and 84.0%, respectively. This explains, in part, the excellent photothermal conversion properties of TPAOMCN. Subsequently, we simulated their optimal structures in the singlet excited (S 1 ) state. As shown in Figure S14, compared to the ground (S 0 ) state structure, the dihedral angles 1 and 2 in TPAOMCN changed by 59.29° and 56.24°, respectively, which were greater than the changes observed in TPACN (33.52° and 33.88°). This indicated that the transition from the S 0 to S 1 state in TPAOMCN involves a larger molecular rotation angle change upon photoexcitation. Consequently, when returning to the S 0 state, TPAOMCN underwent more intense molecular motion, leading to enhanced photothermal conversion performance. To evaluate the photosensitivity of TPAOMCN NFs, we first assessed their overall ROS generation efficiency using 2’,7’-dichlorodihydrofluorescein (DCFH) as a fluorescent indicator. As depicted in Figure 2i and Figure S15, DCFH exhibited minimal emission when exposed to a 660 nm laser alone. However, the fluorescence signal of DCFH at 525 nm markedly increased with prolonged 660 nm laser exposure in the presence of TPAOMCN NFs. After 5 minutes of laser irradiation, TPAOMCN NFs exhibited a striking 30-fold increase in DCFH emission intensity, highlighting their exceptional ROS production capabilities. To further verify the types of ROS, we employed specific indicators: 1,3-diphenylisobenzofuran (DPBF) for singlet oxygen ( 1 O 2 ), dihydrorhodamine (DHR123) for superoxide radical (O 2 -• ), and hydroxyphenyl fluorescein (HPF) for hydroxyl radical (•OH). Notably, no significant fluorescence increase was observed in the DHR123-only or HPF-only groups (Figure S16). The fluorescence intensities of DHR123 and HPF increased dramatically by 51.0% and 387.0%, respectively, when combined with TPAOMCN NFs, indicating efficient generation of O 2 -• and •OH (Figure 2j-2k). Additionally, the absorbance of DPBF solutions decreased by 20.5% after 5 minutes of irradiation, suggesting that TPAOMCN NFs could also produce 1 O 2 (Figure 2l). Furthermore, we conducted electron-spin resonance (ESR) spectroscopy using 2,2,6,6-tetramethylpiperidine (TEMP) as the 1 O 2 indicator and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) as the spin-trapping reagent for radicals. As illustrated in Figure 2m-2o, characteristic ROS signals were observed for TEMP and BMPO when mixed with TPAOMCN NFs under irradiation, confirming the generation of 1 O 2 , O 2 -• , and •OH. To gain deeper insights about our experimental findings, theoretical calculations were performed. It is well-established that a smaller energy gap of ΔE S-T favors ISC, thereby enhancing ROS generation. Accordingly, we computed the ΔE S1-T2 and ΔE S1-T1 values for the molecules. As shown in Figure 2p, ΔE S1-T2 values for all three molecules were relatively small. TPAOMCN exhibited the smallest ΔE S1-T1 value of 0.58 eV compared to TPACN (0.74 eV). BCN displayed the largest ΔE S1-T1 , indicating its inferior ROS production capability. In summary, TPAOMCN NFs demonstrated outstanding ROS generation and photothermal conversion properties, which are beneficial for PDT and PTT. 2.3. In Vitro Antibacterial and Antiviral Ability Motivated by their excellent ROS generation and photothermal properties, the antibacterial activity of TPAOMCN NFs was assessed against four bacterial strains: E. coli, PA, S. aureus, and MRSA. Using SYTO9/PI staining, it was observed that bacteria remained viable after treatment with PBS or NFs alone, but NFs-treated bacteria exposed to laser exhibited significant red PI fluorescence, indicating bacterial death (Figure 3a). This was further confirmed by colony-forming unit (CFU) counts, which showed that laser-activated NFs nearly eliminated all E. coli, with inhibition rates exceeding 99.9%, and similarly high efficacy against PA, S. aureus, and MRSA (Figures 3b-3f). SEM images provided visual evidence of bacterial membrane damage caused by the NFs plus laser treatment, with bacteria displaying wrinkled and collapsed surfaces compared to smooth, intact controls (Figure 3g). Crystal violet staining of biofilms revealed that laser-activated NFs reduced biofilm biomass by over 75%, as indicated by lighter staining compared to controls (Figures 3h-3i). backend=biber, style=alphabetic, sorting=ynt ]biblatex Figure 3. (a) SYTO9/PI fluorescence staining of four bacterial strains (E. coli, P. aeruginosa, S. aureus, and MRSA) with different treatments (SYTO9: green fluorescence; PI: red fluorescence), scale bar: 50 μm. (b) Photographs and (c-f) CFU statistical results of different bactericidal efficacies with different treatments. (g) SEM images of the four bacteria with treatment of PBS or TPAOMCN NFs with laser, scale bar: 1 μm. (h) Crystalline violet staining and (i) Statistical results of biofilms of MRSA after different treatments, scale bar: 0.5 cm. (j) Photographs and (k) cell viability analysis of BHK-21 cells when Vaccinia virus was pretreated by different treatments. (l) Confocal images and (m) Fluorescence intensity of GFP-vaccinia virus with TPAOMCN NFs after coincubation for 1 h with or without 660 nm laser irradiation (0.3 W cm -2 ) for 10 min. (n) Detection of the vaccinia virus load after vaccinia virus coincubating with PBS, or TPAOMCN NFs for 1 h with or without 660 nm laser illumination (0.3 W cm -2 ) for 10 min. (o-q) The expression levels of IL-1β, IL-6, and TNF-α of RAW264.7 cells dealt with different treatments. Data are expressed as the mean ± SD ( n = 3). Statistical analysis between groups was conducted by One-way ANOVA. P -values are denoted as follows: ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001. Additionally, the antiviral potential of NFs with laser was also explored using vaccinia virus-infected BHK-21 cells. Viruses pretreated with ”no laser” or ”PBS + laser” could lead to cell death upon co-incubation with host cells. Meanwhile, viruses pretreated with laser-irradiated TPAOMCN NPs exhibited good cell viability (Figures 3j-3k). This was corroborated by GFP-labeled virus experiments. As depicted in Figure 3l-3m, after co-incubation with host cells, viruses pretreated with PBS emitted strong green fluorescence, resulting in substantial cell death. Similar outcomes were observed with TPAOMCN NFs in the absence of laser irradiation. However, very faint green fluorescence was observed in the TPAOMCN NFs plus laser group, indicating that 99% of viruses have been cleared. Inactivated viruses were unable to infect host cells, thus maintaining the viability of the host cells and reducing the viral load over 50% (Figure 3n). Furthermore, the anti-inflammatory effects of the NFs were assessed using mouse macrophage RAW264.7 cells stimulated with MHV-A59 virus and various treatments. After 24 hours, Enzyme-Linked Immunosorbent Assay (ELISA) kits were used to measure the expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in cell lysates. The results showed that NFs did not induce overexpression of these cytokines, highlighting their potential for future applications in treating monkeypox virus and other inflammatory conditions (Figures 3o-3q). 2.4. Antibacterial Wound Healing Assessment backend=biber, style=alphabetic, sorting=ynt ]biblatex The efficacy of phototherapy using NFs against MRSA in vivo was assessed. Mice with MRSA-infected wounds on their dorsal skin were divided into six treatment groups: (1) phosphate-buffered saline (PBS); (2) PBS plus laser; (3) NFs alone; (4) NFs with laser; (5) washed NFs; and (6) washed NFs with laser. On day 12, wound healing progress was documented photographically (Figure 4a). Over the 12-day period, groups receiving only NFs or washed NFs without laser showed minimal improvement compared to controls. However, wounds treated with NFs and laser irradiation progressively decreased in size, achieving over 90% healing by day 12, unlike the <50% healing in non-laser-treated groups (Figures 4b-4c). backend=biber, style=alphabetic, sorting=ynt ]biblatex Figure 4. (a) Photographs and (b) Schematic pictures of wounds with different treatments in 12 days, scale bar: 0.5 cm. (c) Relative wound area statistics of mice treated with different treatments. (d) Photographs and (e) CFU statistical results of MRSA colonies at wounds after different treatments for 12 days. (f-g) IL-1β and IL-6 in the serum of mice with different treatments after 12 days. (h) H&E, (i) TNF-α immunohistochemical, (j) CD31 immunofluorescence, and (k) Masson staining of the skin tissues obtained from mice of different groups, scale bar: 400 μm. Data are expressed as the mean ± SD ( n = 3). Statistical analysis between groups was conducted by One-way ANOVA. P -values are denoted as follows: ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001. To further assess antibacterial activity, MRSA colonies in tissue homogenates were counted. Groups treated with NFs (either unwashed or washed) plus laser showed significantly fewer colonies compared to others (Figures 4d-4e). Additionally, inflammatory cytokines IL-1β and IL-6 were measured, revealing high levels in PBS and NFs-only groups, indicative of inflammation in infected wounds. Laser-treated NFs significantly reduced these cytokine levels, suggesting effective wound repair and inflammation control (Figures 4f-4g). Histological evaluations, including H&E staining, CD31 immunofluorescence, TNF-α immunohistochemistry (IHC), and Masson staining further supported these findings. Treatment with NFs and laser resulted in more hair follicles, sebaceous glands, intact epidermal and dermal layers (Figure 4h), reduced inflammatory damage (Figure 4i), increased new blood vessel formation (Figure 4j), and enhanced collagen fiber deposition (Figure 4k) compared to other treatments. Collectively, these results demonstrated that NFs, when combined with laser, significantly accelerated wound healing and inhibited inflammatory response in MRSA-infected wounds. 2.5. Antiviral Wound Healing Performance We established a tail-scarified mouse model infected with Vaccinia virus as a surrogate for monkeypox, aiming to mimic the characteristic pustular lesions of monkeypox. Notably, it has been reported that tail scarification with Vaccinia virus serves as a mouse model for assessing the efficacy of smallpox vaccines. By inoculating Vaccinia virus into the tails of mice using the scarification technique, abundant scabbing was observed at the tail wounds 12 days post-inoculation, indicating sustained viral replication at the site and eliciting a robust inflammatory response. The efficacy of phototherapy using TPAOMCN NFs against the virus in vivo was further assessed. On the one hand, TPAOMCN NFs dressings can adhere closely to wounds, forming a protective layer that minimizes the interference of external environmental factors on wound healing. On the other hand, the structure of TPAOMCN NFs can mimic the extracellular matrix, providing a favorable environment for cell growth and promoting wound healing. Thus, we systematically evaluated the antiviral, anti-inflammatory, and wound-healing effects of TPAOMCN NFs. Intravenous injection of PBS or topical application of TPAOMCN NFs without subsequent laser irradiation did not improve the tail lesions in infected mice, with substantial scabbing persisting. However, when treated with TPAOMCN NFs and irradiated with 660 nm laser (0.3 W cm -2 ) for 10 min, a significant acceleration in scab resolution and wound healing was observed (Figures 5a-5b). Compared to other groups, the wound area on the tails of mice in the TPAOMCN NFs plus laser group also gradually decreased as the treatment progressed (Figure 5d). Furthermore, assessment of Vaccinia virus titers in the tail lesion tissues revealed that TPAOMCN NFs treatment combined with 660 nm laser irradiation nearly eradicated the virus compared to other groups (Figure 5e). To evaluate the virus clearance capability of different treatments, tail lesion tissues were collected 12 days later. Hematoxylin and Eosin (H&E) staining suggested that treatment with TPAOMCN NFs and laser resulted in more hair follicles, sebaceous glands, and intact epidermal and dermal layers (Figure 5c). Immunohistochemistry (IHC) staining of Vaccinia virus antigen showed that in the absence of subsequent laser irradiation, all groups exhibited extremely high IHC density for viral antigens. Conversely, when TPAOMCN NFs were subjected to 660 nm laser irradiation, both before and after washing, the IHC density of viral antigens in the mouse skin was markedly reduced (Figure 5f). ELISA was also employed to assess the expression of IL-1β and IL-6 secreted by inflammatory cells. In the PBS or standalone TPAOMCN NFs groups, the levels of pro-inflammatory cytokines in the tail lesion tissues were very high, indicating a strong inflammatory response triggered by Vaccinia virus infection. The introduction of 660 nm laser irradiation led to a substantial decrease in pro-inflammatory cytokine levels in both the pre- and post-washed TPAOMCN NFs treatment groups (Figures 5g-5h). IHC staining for TNF-α supported these findings (Figure 5i). Masson trichrome staining results further demonstrated that the TPAOMCN NFs + laser group exhibited the highest collagen fiber deposition and the best wound healing (Figure 5j). This combination of virus clearance, anti-inflammation, and wound repair fully demonstrates the antiviral and skin-beautifying potential of the ” TPAOMCN NFs + laser” therapeutic strategy for monkeypox. Figure 5. (a) Changes in the appearance of tail wounds before and after different treatments, scale bar: 0.5 cm. (b) Schematic pictures of wounds with different treatments in 12 days. (c) H&E staining of the skin tissues obtained from mice in different groups, scale bar: 400 μm. (d) Quantitative analysis of tail lesions in different treatment groups using ImageJ software. (e) Detection of the vaccinia virus load in tail lesions subjected to different treatments. (f) Immunohistochemical staining for vaccinia virus antigen of staining intensity in tail lesions subjected to different treatments. (g-h) Detection of IL-1β and IL-6 in tail wounds after 12 days. (i) TNF-α immunohistochemical and (j) Masson staining of the tail skin tissues obtained from mice in different groups, scale bar: 400 μm. Data are expressed as the mean ± SD ( n = 3). Statistical analysis between groups was conducted by One-way ANOVA. P -values are denoted as follows: ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001. backend=biber, style=alphabetic, sorting=ynt ]biblatex 2.6. Biosafety Evaluation Biological safety is essential for in vivo applications. To evaluate the biocompatibility of TPAOMCN NPs and TPAOMCN NFs, CCK-8 assays and hemolysis tests were conducted. Normal mouse epithelial-like fibroblast cells (L929) and Raw264.7 cells were exposed to varying concentrations of TPAOMCN NPs for 24 hours, after which cell viability was assessed. Notably, even at a high concentration of 180 μg mL -1 , cell viability remained unaffected, suggesting minimal cytotoxicity of TPAOMCN to normal cells (Figures 6a-6b). Additionally, Figure 6c demonstrated that prolonged exposure to TPAOMCN NPs for 3 hours did not induce hemolysis even at high concentrations. Also, neither PVDF NFs (Fs) nor TPAOMCN NFs caused hemolysis (Figure 6d). To further validate the in vivo biocompatibility, TPAOMCN NFs were applied to the skin of healthy mice for a week. Subsequent analysis of body weight changes, biochemical indexes, and blood routines was conducted. Specifically, in comparison to the PBS-treated control group, the body weight of mice treated with TPAOMCN NFs remained stable without any significant decrease (Figure 6e). Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), urea, creatinine (CR), as well as counts of red blood cells (RBC), white blood cells (WBC), platelets (PLT), and hemoglobin (HGB) all stayed within normal limits in the TPAOMCN NFs group, indicating no liver or kidney dysfunction (Figures 6f-6g). H&E staining also confirmed that TPAOMCN NFs treatment did not induce pathological changes in various organs of the mice (Figure 6h). Moreover, skin appearance remained unaffected by TPAOMCN NFs treatment, as evidenced by H&E and Masson staining (Figure 6i). Collectively, these findings underscore the excellent biocompatibility of TPAOMCN NFs, highlighting their potential for potent antiviral applications. Figure 6. (a) and (b) Assessment of the viability of L929 and Raw264.7 cells incubated with different concentrations of TPAOMCN NPs. (c) Assessment of hemolysis in red blood cells incubated with different concentrations of TPAOMCN NPs. (d) Assessment of hemolysis in red blood cells incubated with PVDF NFs or TPAOMCN NFs. (e-g) Weight changes, biochemical indexes, and blood routines of healthy mice and TPAOMCN NFs-coated mice. (h) H&E staining of the main organs harvested from healthy mice coated with TPAOMCN NFs. (i) H&E and Masson staining of the skin tissues obtained from the healthy mice after dressing of TPAOMCN NFs for 7 days. Data are expressed as the mean ± SD (n = 3). Statistical analysis between groups was conducted by One-way ANOVA. P -values are denoted as follows: ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001. 3. Conclusion In summary, three Nile Red derivatives, namely BCN, TPACN, and TPAOMCN, were meticulously crafted and synthesized through the donor rotation and charge enhancement strategy. Notably, TPAOMCN, featuring backend=biber, style=alphabetic, sorting=ynt ]biblatex alkyl-modified TPA as the electron donor and rotator, exhibited exceptional performance in both PTT and PDT. The introduction of alkoxy-modified TPA into the molecular framework can effectively promote molecular rotation to improve photothermal conversion, while promoting ISC to facilitate the generation of ROS. Specifically, exceeding 80°C photothermal conversion temperature and a remarkable 30-fold increase in ROS production were observed in TPAOMCN-based NFs under 660 nm laser irradiation. In MRSA-induced wound infection and Vaccinia virus-mediated tail-scarred models, TPAOMCN NFs combined with laser irradiation led to a significant reduction in MRSA colonies and viral load within lesions, suppressed inflammatory response, and accelerated tissue repair. This study not only introduced a novel approach to modify Nile Red for enhanced PDT and PTT performance but also advanced the development of phototherapeutic materials for the treatment of drug-resistant bacteria and monkeypox virus. backend=biber, style=alphabetic, sorting=ynt ]biblatex Acknowledgements L. P. Fang, W. Wang, and J. N. Dai contributed equally to this work. This work was supported by National Natural Science Foundation of China (82271948), China Postdoctoral Science Foundation (2024M760593), 2023 Postdoctoral Workstation Project (2023BSGZ011), Lifting Project of Guangdong Second Provincial General Hospital (TJGC-2023001), Guangdong Medical Research Fund of 2025 (B2025545), the Major Research Plan of the National Natural Science Foundation of China (2022YFC2410000), Experts and Youths Project of Guangdong Second Provincial General Hospital (2024B002), Fujian Province Science and Technology Plan (2024D022), and 2024 Smart Imaging New Technology Innovation Fund (NMED2024CX-01-005). Conflict of Interests The authors declare no conflict of interests. backend=biber, style=alphabetic, sorting=ynt ]biblatex Supporting Information Supporting Information is available from the Wiley Online Library or from the author. 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Matsumoto, K. Hosoya, T. Hattori, K. Kaya, J. Am. Chem. Soc. 2007 , 129 , 13626-13632. Supplementary Material File (agg-ms.pdf) Download 8.45 MB Information & Authors Information Version history V1 Version 1 01 May 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Aggregate Keywords drug-resistant bacteria monkeypox virus nile red derivatives photodynamic therapy photothermal therapy Authors Affiliations Laiping Fang Guangdong Second Provincial General Hospital View all articles by this author Wei Wang Kunming Medical University View all articles by this author Jianan Dai Jilin Normal University View all articles by this author Yike Tu Guangdong Second Provincial General Hospital View all articles by this author Shufang Li Guangdong Second Provincial General Hospital View all articles by this author Kuo He Chang Chun Institute of Applied Chemistry Chinese Academy of Sciences View all articles by this author Siya Tong Guangdong Second Provincial General Hospital View all articles by this author Yuhui Liao 0000-0003-4702-9516 Kunming Medical University View all articles by this author Ping’an Ma 0000-0003-4198-5240 [email protected] Chang Chun Institute of Applied Chemistry Chinese Academy of Sciences View all articles by this author Guihua Jiang Guangdong Second Provincial General Hospital View all articles by this author Metrics & Citations Metrics Article Usage 354 views 229 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Laiping Fang, Wei Wang, Jianan Dai, et al. Donor Rotation and Charge Enhancement Functionalized Nile Red Derivatives-Mediated Efficient Phototherapy for Drug-Resistant Bacteria Elimination and Monkeypox Virus Inactivation. Authorea . 01 May 2025. DOI: https://doi.org/10.22541/au.174605970.08433930/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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