Enhancing second harmonic generation-mediated photodynamic therapy via external electric field modulation
Manu Kumar, Avinash Jukanti, Rivka Cahan, Dima Cheskis, Refael Minnes
TL;DR
This study shows that applying an electric field can boost the effectiveness of a light-based therapy for killing bacteria by enhancing the light conversion process.
Contribution
The first study to demonstrate electric field modulation enhancing SHG in photodynamic therapy.
Findings
EEF significantly increased SHG intensity, improving photodynamic activity against bacteria.
BFO-PPIX conjugates reduced bacterial survival to 35.8% under EEF, compared to 48.1% without EEF.
BT-PPIX conjugates showed 57.1% bacterial survival with EEF, versus 78.4% without EEF.
Abstract
Photodynamic therapy (PDT) utilizes light-activated photosensitizers (PS) to produce reactive oxygen species (ROS) for targeted bacterial destruction; however, its efficacy is often limited by inadequate light penetration, necessitating novel enhancements, such as the integration of second harmonic generation (SHG) through harmonic nanoparticles (HNPs) that convert two photons into one of higher frequency, thereby advancing the approach of SHG-based PDT for improved bacterial eradication. Our novel technique explores the impact of an e (EEF) on SHG intensity to augment PDT efficacy against Staphylococcus aureus (S. aureus). We investigated a novel conjugate, Bismuth Ferrite (BFO) in conjunction with protoporphyrin IX (PPIX), and compared it to the Barium Titanate (BT)-PPIX conjugate, under EEF of 0 V, 10 V, and 20 V for a duration of 5 minutes. The experiments utilized a near-infrared…
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TopicsPhotodynamic Therapy Research Studies · Nanoplatforms for cancer theranostics · Cancer Research and Treatments
1. Introduction
Photodynamic therapy (PDT) is a cutting-edge treatment that utilizes a combination of light, photosensitizing chemical agents and molecular oxygen to generate highly cytotoxic reactive oxygen species (ROS). This process efficiently eradicates target cells, including cancerous and bacterial cells [1]. PDT provides significant benefits compared to chemotherapy, surgery, and radiation therapy due to its straightforward approach, noninvasive nature, resistance-free mechanism, safety profile, and minimal side effects. As a result, it stands out as a promising option for both antibacterial and anticancer treatment [2]. PDT offers an innovative and sustainable alternative to traditional antibiotics, bypassing conventional resistance mechanisms [3], and combating multidrug-resistant Staphylococcus aureus (S. aureus) infections [4]. One of the key applications of PDT is in the treatment of non-systemic infections caused by drug-resistant S. aureus. By utilizing photosensitizing agents to generate free radicals that damage bacterial structures, PDT effectively complements antimicrobial therapy and offers a novel solution to the growing challenge of antimicrobial resistance (AMR) [5].
AMR arises naturally due to the selective pressure exerted by antibiotics. However, the widespread misuse and overuse of antibiotics across multiple sectors have significantly accelerated the emergence of resistant microorganisms, posing a major global challenge to healthcare systems [6]. As a result, AMR has become an existential challenge for modern medicine and public health, with implications extending beyond healthcare to food security and agricultural sustainability [7]. Pathogenic bacteria, such as S. aureus, utilize various resistance mechanisms, including target modification and horizontal gene transfer to survive antimicrobial treatments [8]. Additionally, bacteria employ mechanisms, including metabolic pathway adjustments, to resist AMR, highlighting the need for further research to defeat these resistance mechanisms [9]. In this context, PDT represents a powerful tool in the fight against AMR, offering a novel resistance-free approach to bacterial infections and reinforcing the need for innovative solutions to address the escalating global crisis of antibacterial resistance.
PDT relies on photochemical and photobiological reactions triggered by photosensitizers (PS) like curcumin, porphyrins, and methylene blue, which are activated by light within specific absorbance bands [10]. This modern technique operates through a unique mechanism in which a PS, activated by light of specific wavelength, generates ROS that effectively destroy pathogenic microorganisms [11]. Its effectiveness is influenced by various factors, such as the uptake of the PS within cells, the nature of the photochemical reactions triggered, and the presence of sufficient oxygen [12]. Even so, the effectiveness of PDT is hindered by limited light penetration ability within biological tissues, particularly in the visible spectrum (λ ~ 400–700 nm), which poses challenges in restricting its utility in treating deeper-seated infections [13]. Additionally, PDT is limited by poor light penetration, reducing PS activation and effectiveness against deeper bacterial infections [14].
Despite its potential, PDT faces challenges such as limited light penetration and PS efficiency. The development of novel molecular chimeras, such as CM358 for metastatic melanoma, demonstrates the potential of targeted molecular strategies to enhance therapeutic efficacy [15]. Recent advances in PS design have led to reduced dark toxicity and improved therapeutic effects. These engineered PSs feature aggregation-induced emission properties, which enhance their ability to generate ROS, making them more effective for PDT [16]. Many PSs, including porphyrins such as Protoporphyrin IX (PPIX), primarily induce cellular damage through a Type II mechanism. Their efficacy in PDT is heavily influenced by their intracellular localization, as reactive species generated during the process have a diffusion range of less than 20 nm [17,18]. PPIX is an essential PS in PDT due to its strong absorption in the UV-visible spectrum and its efficient ROS generation. Recent advancements in antiviral therapies, such as non-ionizing radiation treatment for SARS-CoV-2, underscore the expanding potential of photodynamic methods in infectious disease management [19]. However, limitations such as shallow light penetration in tissues and non-specific activation hinder its overall antimicrobial effectiveness [20]. To address these challenges, harmonic nanoparticles (HNPs) can be utilized to induce second harmonic generation (SHG), a nonlinear optical process in which two photons of identical frequency combine to produce a new photon with double the frequency and half the wavelength of incident photons. The longer wavelength of the incident light improves its penetration into deeper tissue layers, where SHG converts the light into shorter wavelengths that align with the absorption properties of the PPIX [21,22]. SHG like other emerging optical techniques, such as fiber-optic evanescent wave spectroscopy (FEWS), may enhance diagnostic and treatment accuracy in related therapeutic applications [23]. SHG has the potential to enhance light penetration within biological tissues by using HNPs. When combined with PDT, SHG can significantly boost antibacterial effects while minimizing damage to surrounding healthy tissues [24].
Following above, HNPs such as Barium Titanate (BT) and Bismuth Ferrite (BFO), both non-centrosymmetric materials with high non-linear optical efficiency and excellent biocompatibility, have been investigated for SHG emission and their application in PDT [25,26]. BT exhibits large normalized hyperpolarizability, making it suitable for SHG applications. BT conjugation with Rose Bengal has shown the ability to produce ROS in HeLa cells and cause cell death under the emission of SHG-mediated PDT [27]. On the other hand, BFO harmonic nanoparticles have shown significant applications in SHG imaging for various biological targets. This has shown efficient use in monitoring pulmonary macrophage mice and tracking nanoparticle-loaded cells with high resolution in thick tissue [28]. Additionally, HNPs have been conjugated with PS to form novel HNP-PS complexes, which can generate photons via SHG when exposed to intense light. This conjugate improves therapeutic efficacy by converting visible light-reactive PSs into molecules that respond to near-infrared (NIR) light. NIR light enables deeper tissue penetration, addressing many of the limitations typically encountered in traditional PDT [29]. This demonstrates that the application of nanotechnology in PDT offers a comprehensive approach to effectively managing bacterial infections. Such advancements hold great promise for addressing the challenges of AMR by enabling innovative diagnostic and therapeutic strategies [30]. Moreover, functionalized second harmonic nanoprobes enable the targeting of various biological processes. Their versatility makes them valuable tools for both monitoring and treating bacterial infections [31].
To enhance SHG-based PDT effectiveness, the external electric field (EEF) has proven to be pivotal in modulating the optical properties of HNPs, influencing phenomena like nonlinear optical rectification, SHG, and third harmonic generation (THG) [32,33]. The application of an EEF has served to align the dipoles of nanoparticles within the host matrix, thereby boosting SHG intensity [34]. Additionally, investigation on quantum analysis has further revealed that the SHG dynamics can transition from Poisson statistics to chaotic phenomena as electric field intensities escalate, which in turn modifies the spectral statistics of SHG resonance [35].
To significantly enhance PDT treatment through EEF for increasing SHG intensity, we devised an innovative experimental apparatus aimed at augmenting the local electric field around HNPs. This approach significantly increased SHG intensity for both BT and BFO materials, making these systems highly effective for PDT applications. To the best of our knowledge, this method has not been previously explored in the context of EEF modulated SHG intensity in application of PDT. In our experiments, applying an EEF to HNPs resulted in a notable improvement in SHG intensity compared to controls without EEF exposure. Furthermore, SHG enhancement was further amplified after conjugating the HNPs with PPIX, with the effect being even more pronounced under EEF application. This improved light conversion efficiency between the HNPs, and the PS led to enhanced antimicrobial activity, consistent with increased SHG-mediated activation. The increased SHG intensity induced by the EEF demonstrated significant efficacy against antibiotic-resistant bacteria, such as S. aureus, highlighting the pronounced enhancement of photodynamic effects when EEF is applied.
2. Materials and methods
2.1 Synthesis and size characterization of HNPs-PPIX conjugate
A colloidal suspension of HNPs-conjugate was prepared by mixing and sonicating on ice. This process involved combining 6 mg each of Bismuth Ferrite (BFO, CAS No: 12010-42-3, LTS Research Laboratories, Inc, USA) or Barium Titanate (BT, 467634, Sigma-Aldrich, USA) with 6 mg of the photosensitizing agent, Protoporphyrin IX (PPIX, P8293-1G, Sigma-Aldrich, USA) in a 15 ml solution consisting of 6% solvent (14.1 ml double-distilled water and 0.9 ml diethylene glycol, Cat. No: 121160010, Thermo-Scientific, Netherlands). The HNPs and PPIX were combined at a weight-to-weight (w/w) ratio of 1:1, resulting in HNP-PPIX conjugates at a final concentration of 800 μg/ml. After synthesis, the samples were filtered through a 0.22 μm PVDF membrane (FPV-203–030, Jet Bio-fil, China) before being used in experiments.
The filtered BFO and BFO–PPIX conjugate dispersions were vortexed immediately prior to analysis and transferred into polystyrene disposable square cuvettes (10 × 10 × 45 mm; 1.5 mL; two optical windows). Particle size was then measured using Dynamic Light Scattering (Zetasizer Ultra, MADLS®, Malvern), and the results are reported as size-by-number distributions (number vs. hydrodynamic diameter, nm). Moreover, scanning transmission electron microscopy (STEM) was employed to image BT and BT-PPIX conjugates.
The optimal diameter of BFO was found to be 68.7 ± 1.0 nm suitable for SHG intensity studies, while the size of BFO-PPIX conjugates measured 79.7 ± 2.2 nm [36]. The size of BT-PPIX conjugates was optimized at 53 ± 9 nm according to Ref. [22], which was suitable for SHG intensity studies throughout the experiments.
2.2 Effect of external electric field on the HNPs-PPIX conjugates
To investigate the effect of EEF on HNPs and HNPs-PPIX conjugate only, 3 ml of each sample solution was placed in a quartz cuvette positioned between two stainless steel electrodes. The electrodes, measuring 46 mm × 6 mm × 0.01 mm and spaced 8 mm apart, were connected to the positive and negative terminals of a DC voltage source (TENMA®, 72–10495 digital control DC power supply, 0-30V). Approximately 30 mm of each electrode’s length was submerged in the solution (Fig 1). The samples were exposed to electric field strength: 0, 1.25 and 2.5 V/mm for applied voltages of 0 V, 10 V, and 20 V for 5 minutes, after which the HNPs and HNP-PPIX conjugates were immediately assessed for studying SHG intensity and PDT efficacy. While performing the experiments, only minimal bubble formation was observed, indicating negligible electrochemical activity, solution heating, and electrode discoloration that did not affect the HNPs and HNPs-PPIX conjugate samples. Therefore, 5-minutes exposure was chosen, balancing effective conditioning with no significant alteration of sample and ensuring reliable results.
The schematic representation depicts the setup with two stainless steel electrodes inserted into a 4 ml quartz cuvette.The cuvette was filled with 3 ml of HNPs or HNPs-PPIX conjugate and connected to the negative and positive terminals of a DC power supply. The samples were exposed to external electric fields (EEF),0, 1.25 and 2.5 V/mm for applied voltages from 0 V to 10 V and 20 V for duration of 5 minutes.
2.3 Laser setup and experiments
A near-infrared (NIR) femtosecond pulsed laser (Astrella, Coherent, USA) was employed for the experiments. The laser operated at a wavelength of 798 nm with a pulse width of 35 fs and a repetition rate of 71.4 MHz. The beam alignment was achieved using a broadband dielectric mirror (BB1-E03, Thorlabs, Inc.) and an adjustable iris diaphragm (SM2D25 - SM2 Lever-Actuated Iris Diaphragm (Ø1.4 - Ø25.0 mm), Thorlabs, Inc.). The real-time power reading was recorded using a power detector (Coherent PowerMax-USB PM10). The direct high-power laser beam with a mean power of 282.5 ± 0.5 mW was used for direct measurements of SHG emissions. However, for laser irradiations of bacterial samples in 96-well plates, the laser beam was arranged to be normally incident on the samples from above (Fig 2). Also, by adjusting the iris diaphragm, the laser power incident on the samples was reduced to 51.5 ± 2.1 mW, which was an appropriate level for these experiments. The beam diameter of 5 mm was large enough to uniformly cover the full area of each sample. Spectral emission following laser excitation of BFO and BFO-PPIX conjugates, as well as BT and BT-PPIX conjugates, were recorded using an Ocean Optics QE Pro Spectrometer (Ocean Insight, USA). Further, spectral analysis was conducted using Origin Pro, 2018 (Northampton, USA).
The schematic representation depicts a 3-D setup of photodynamic therapy (PDT) treatment in bacteria using NIR femtosecond pulse laser at 798 nm, mirrors, iris and bacteria.The laser beam originates from the source and is directed towards the first mirror, which reflects it towards the iris. The beam then passes through the iris, where its diameter can be adjusted to target the bacterial surface effectively. The beam is subsequently directed to a second mirror, which reflects it perpendicularly downward onto the bacterial surface, ensuring precise coverage for the treatment.
2.4 Bacterial culture
S. aureus (ATCC 25923) was cultured on Brain Heart Agar (BHA, Himedia, India) plates for 24 hours. Subsequently, the cells were inoculated into Brain Heart Infusion (BHI) broth (Himedia, India) and incubated at 37 ± 1˚C with constant shaking at 170 rpm until the optical density at 600 nm reached 1.0 ± 0.003, corresponding to a cell concentration of 10^8^ cells/ml. The culture was then diluted in sterile 0.9% saline solution to achieve the final concentration of 10^4^ cells/ml, suitable for use in photodynamic treatment.
2.5 Dark toxicity
A 200 μl bacterial suspension (10^4^ cells/ml) in 0.9% sterile saline solution was incubated in the dark with BFO and BFO- PPIX drug concentrations of 4 μg/200 μl, 8 μg/200 μl, 12 μg/200 μl, and 16 μg/200 μl while shaking for 30 minutes. Following incubation, 100 μl of treated bacterial suspension was evenly spread onto BHA plates with a spreader and incubated at 37˚C for 24 hours to allow colony-forming units (CFUs). The CFUs were then counted using a colony counter Scan 500 (Interscience, Saint-Nom-la-Bretèche, France) [36].
Dark toxicity for BT and BT-PPIX conjugates was considered according to Ref. [22].
2.6 Light toxicity
A bacterial suspension of 200 μl (10^4^ cells/ml) in 0.9% sterile saline solution was exposed to a femtosecond laser for 4, 8, and 12 minutes. After treatment, 100 μl of irradiated bacterial suspension was evenly spread onto BHA plates using a spreader and incubated at 37°C for 24 hours. The CFUs were quantified using a colony counter Scan 500 [36].
2.7 PDT experiments
Suspensions of cultured S. aureus bacteria (200 μl at 10^4^ cells/ml) in a 0.9% sterile saline solution were incubated in the dark for 30 minutes with 16 μg of BFO, PPIX, or BFO-PPIX conjugates. Additionally, to investigate the influence of an EEF on PDT efficacy, 8 μg of BT, PPIX, or BT-PPIX conjugates were employed in distinct experiments. In both scenarios, the drug was exposed to an EEF for 5 minutes before treating the bacteria with the drug to test PDT efficiency. These distinct experimental setups facilitated a comparative assessment of the PDT effects on S. aureus under EEF conditions between the BFO and BFO-PPIX conjugates and the BT and BT-PPIX conjugates. Following the incubation, the samples were further exposed to a femtosecond laser beam for 8 minutes. The temperature of samples was monitored using an infrared thermometer (−50°C – 380°C), indicating a temperature increase from 20.3 ± 0.1°C before to 20.8 ± 0.1°C after irradiation. To determine PDT efficacy post-irradiation, 100 μl of treated bacterial suspension was spread over BHA plates with a spreader and incubated at 37˚C for 24 hours. After 24 hours, the CFUs were counted using a colony counter Scan 500.
3. Results
3.1 Effects of EEF on HNPs and HNPs-PPIX conjugates for SHG intensity
BFO and BT were subjected independently to an external electric field with voltages of 0 V, 10 V, and 20 V for a duration of 5 minutes. Subsequently, the samples were irradiated using a NIR femtosecond pulse laser. We saw a clear increase in the SHG intensity at 20 V compared to 10 V and 0 V for both BT and BFO (Fig 3).
(A) BFO and (B) BT HNPs were exposed to an external electric field (EEF) ranging from 0 V to 10 V and 20 V for a duration of 5-minutes.Following this, the samples were tested for second harmonic generation (SHG) intensity enhancement using an NIR femtosecond pulse laser. An enhancement in SHG intensity was observed under the influence of EEF. These results validate the proportional relationship between EEF and SHG intensity.
Further, BFO and BT conjugates with PPIX were exposed to varying EEF levels (0 V, 10 V, and 20 V) for a duration of 5 minutes. In the absence of an applied electric field (0 V), the SHG intensity reflects the intrinsic nonlinear optical properties of the conjugates that provide their baseline response before external modulation. Following the application of 10 V, there was an argumentation in SHG intensity. However, at 20 V, the SHG intensity peaks showed a clear increase compared to those at 0 V and 10 V in the BFO–PPIX conjugates. This enhancement was further confirmed by the BT–PPIX conjugates, which also exhibited stronger SHG intensity under the same applied electric field. (Figs 4A and 4B). Additionally, a noticeable enhancement in emission intensity was observed for BFO-PPIX and BT-PPIX conjugates, which reflect fluorescence emissions wavelength. There was an increase in emission intensity at 619.6 nm compared to 0 V and 10 V. A 20 V electric field exposure produced the highest enhancement in emission intensity, highlighting the effect of stronger electric fields on the emission properties of the BFO-PPIX and BT-PPIX conjugates (Figs 4C and 4D).
Demonstrates the Harmonic nanoparticles-Protoporphyrin IX (HNPs-PPIX conjugate): (A) Bismuth Ferrite (BFO) and (B) Barium Titanate (BT), both are conjugates with PPIX, highlights the effect of exposure to an external Electric Field (EEF) ranging from 0V to 10 V and 20V over 5 minutes.This exposure results in enhanced second harmonic generation (SHG) intensity efficiency as the electric field strength increases on conjugates. (C) and (D) show the emission spectra, with fluorescence peak providing evidence of the conjugate’s properties after EEF exposure and under excitation by a pulse laser, confirming the improved SHG efficiency.
3.2 NIR photodynamic effects after EEF exposure on HNPs-PPIX conjugates in S. aureus
We evaluated the photodynamic effects in 200 μl of S. aureus with 16 μg of BFO-PPIX conjugates and compared the results under EEF exposure with excitation from a pulsed laser beam. EEF was first applied to the conjugates alone. These EEF-exposed conjugates were then tested on bacteria to observe their response under laser irradiation. Our goal was to examine how the EEF influences the HNPs and their conjugation with PPIX to boost SHG intensity. After confirming this enhancement, we treated the bacteria and found that the EEF-treated conjugates led to a much greater level of bacterial destruction under pulse laser irradiation.
The control group (bacteria without drug and laser treatment) exhibited a survival rate of 100%, signifying the baseline viability of S. aureus. Laser treatment without drug resulted in a bacterial survival rate of 91.6 ± 1.5%, whereas BFO-PPIX treatment with laser significantly decreased bacterial survival rate to 48.1 ± 3.2%. The impact of EEF exposure on BFO-PPIX conjugates led to a further decrease in bacterial survival rate to 35.8 ± 3.0%, compared to laser and BFO-PPIX conjugates alone (Fig 5A). Moreover, we prepared a separate experiment to test the effect of EEF on BT-PPIX conjugates with 8 μg in S. aureus. The laser treatment demonstrated bacterial survival rate of 94.9 ± 2.9%. BT-PPIX conjugates with laser demonstrated a bacterial survival rate of 78.4 ± 3.7%. However, EEF-exposed BT-PPIX conjugates demonstrated bacterial survival rate of 57.1 ± 1.0% (Fig 5B).
Viability of S. aureus (200 µl) after photodynamic treatment under various conditions: (A) Laser only; BFO-PPIX conjugates (16 µg); (B) Laser only; BT-PPIX conjugates (8 µg); and exposure of these conjugates to an external electric field (EEF) for duration of 5 minutes in both cases.The experiments were conducted using an NIR femtoseconds pulsed laser at 798 nm, with total treatment duration lasting 8 minutes. Photodynamic effects were compared with the control (without drug and laser treatment). The comparative results validate the influence of EEF on both conjugates, confirming the enhancement of second harmonic generation (SHG) intensity and its impact on photodynamic effects. The error bars represent the standard errors from three consecutive experiments. Each drug was tested using a consistent cell density of 104 cells/ml.
4. Discussions
To enhance the efficacy of SHG-based PDT, we introduced an innovative experimental approach focusing on the effect of an EEF on HNPs, termed EEF-modulated SHG intensity. This applied field amplified the local electric field surrounding the HNPs, increasing SHG intensity when excited by an NIR femtosecond pulse laser at 798 nm. This enhancement significantly improved the antibacterial performance of the system as demonstrated by the reduced S. aureus survival following treatment with EEF-exposed conjugates under pulse laser irradiation.
The measured sizes of BFO and BT as HNPs, along with their conjugates with PPIX, and their UV-Vis absorption and fluorescence emissions were consistent with observations reported in our previous published study [36,37]. Furthermore, our latest findings detected a significant SHG intensity peak at 399 nm under varying EEF conditions (0 V, 10 V, and 20 V) over 5 minutes exposure. These experiments, conducted using an NIR femtosecond pulse laser, provided exciting insights into SHG intensity behavior.
In the absence of EEF, the pulse laser induced SHG in HNPs due to their intrinsic nonlinear characteristics. Under these conditions, SHG efficiency was moderate and primarily influenced by the nanoparticles’ natural alignment of dipole moments [38,39]. The SHG intensity of BFO increased steadily as the applied electric field was raised from 0 V to 10 V and then to 20 V. A similar enhancement trend was also observed in BT, confirming the consistency of this effect. A 10 V electric field effectively aligned the dipole moments of the HNPs, improving SHG intensity relative to 0 V. At 20 V, the electric field alignment was maximized. The combination of optimized dipole alignment and the high intensity of the femtosecond pulse laser produced a significantly elevated SHG intensity peak compared to 0 V and 10 V. Femtosecond pulse lasers offer high temporal and spatial coherence, which was crucial for efficient SHG [40].
Similarly, we identified a response in SHG intensity upon applying an electric field to BFO-PPIX. An application of 10 V resulted in an increase in SHG intensity relative to 0 V. At 20 V, the SHG response was amplified. This pronounced intensity was considerably greater compared to both 0 V and 10 V. These observational results were cross verified using BT-PPIX conjugates under EEF exposure. This indicates that improving the local electric field around HNPs and their conjugates with PS can significantly boost the efficiency of SHG. This happens by tuning the nanoparticles to resonate with the laser’s fundamental harmonic wavelength. When the resonance matches, it strengthens the nearby electric field, which enhances the SHG emission [41].
Our results showed a clear proportional relationship between the applied electric field and SHG intensity. As the strength of the electric field increased, the SHG intensity consistently exhibited a noticeable enhancement. The 20 V EEF exhibited the most substantial SHG intensity production relative to 0 V and 10 V. These findings underscore that optimizing the local electric field around HNPs is crucial for enhancing SHG intensity, which can significantly improve PDT by converting NIR light into blue light. This conversion allows for deeper tissue penetration and more effective activation of photosensitizers, leading to better therapeutic outcomes [42,43].
The SHG emission from HNPs-PPIX conjugate was assessed for treatment with 200 μl of S. aureus, following 5 minutes of exposure to an EEF and 8 minutes of irradiation with a pulsed laser. Further, EEF-exposed BFO-PPIX conjugates under pulsed laser treatment led to a significant reduction in the survival rate of S. aureus to 35.8 ± 3.0%, compared to bacteria without EEF exposed conjugates. Likewise, EEF-exposed BT-PPIX conjugates under pulsed laser treatment demonstrated a 57.1 ± 1.0% survival of S. aureus relative to bacteria without EEF exposed conjugate. These findings suggest a synergistic interaction between HNPs-PPIX and EEF under pulsed laser irradiation, leading to a notable enhancement in S. aureus viability reduction.
EEF-exposed HNPs effectively absorbed NIR laser energy, transferring it to the conjugated PS and enhanced antibacterial efficacy by increasing SHG-mediated activation. These phenomena can damage to essential cellular components, including proteins, membranes, and DNA, leading to bacterial cell death [44].Under NIR pulsed laser irradiation, EEF-modulated HNPs-PPIX conjugates exhibited augmented antibacterial activity and diminished S. aureus viability via SHG-based PDT mechanisms through the modulation of SHG intensity, presenting a promising approach for bacterial infection treatment.
Overall, the experimental results suggest that an increasing EEF exposure augments SHG intensity in PDT, while potentially facilitating nanoparticle mobility and PS binding. This enhancement may improve conjugation stability and overall efficiency [45,46]. Additionally, the applied electric field may facilitate conjugating penetration into cells, thereby strengthening intracellular therapeutic efficacy. This strategy could direct conjugates to targeted locations, improving the precision of drug delivery [47]. Improved uptake and delivery efficiency may also allow for lower PS dosages to achieve the desired therapeutic effect by diminishing bacterial viability.
5. Conclusion
This study validated our novel technique for enhancing SHG intensity and its impact on PDT efficacy through an EEF applied to HNPs-PPIX conjugates. A comparative analysis of BFO-PPIX and BT-PPIX conjugates revealed that EEF exposure before treatment with an NIR femtosecond pulsed laser markedly decreased S. aureus survival rates. Specifically, BFO-PPIX conjugates exposed to EEF showed a bacterial survival rate of 35.8 ± 3.0%, compared to 48.1 ± 3.2% without EEF exposure. Similarly, BT-PPIX conjugates exhibited a survival rate of 57.1 ± 1.0% with EEF exposure, in contrast to 78.4 ± 3.7% without it, following 5 minutes of EEF treatment at 20 V. These findings highlight the potential of EEF to significantly enhance SHG intensity in HNPs-PPIX conjugates, thereby improving SHG-based PDT outcomes. To the best of our knowledge, this study represents the first systematic examination of how an externally applied electric field can modulate SHG and explore its potential to improve PDT efficiency.
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