감염성 미생물의 효율적인 2광자 들뜬 광역학적 비활성화를 위한 수산기 의존성을 가진 수용성 풀러레놀

결과 및 토론

수용성 풀러레놀의 특성

수용성 C₆₀ (OH)₄₆ (fullerenol)은 원형 및 단분산으로 결정되었으며 이전 연구에 따라 합성되었습니다 [27]. 풀러레놀의 평균 측면 크기는 저배율(그림 2a)과 HR-TEM 이미지(그림 2b)를 사용하여 측정한 바와 같이 약 1.13 ± 0.04 nm였습니다. 또한, 풀러레놀은 d -풀러레놀 {1\( \overline{1} \)00} 격자 무늬의 간격. 그러나 이러한 입자는 pH 7.0의 수용액에서 수소 결합을 통해 응집체를 형성할 수 있습니다. 형성된 응집체의 평균 크기는 DLS 분석에 의해 밝혀진 바와 같이 약 130 nm였습니다. 또한, 응집체는 pH 7.0 수용액, 1x 인산염 완충 식염수 및 배양 배지와 같은 다양한 생리적 환경에서 3 개월 동안 매우 안정적으로 유지되었습니다(추가 파일 1:표 S1). 풀러레놀의 UV-vis 흡수 스펙트럼에서 흡광도 피크는 약 216 및 309 nm에서 관찰되었으며 이러한 피크는 π –π * 방향족 C=C 결합의 전이 및 n –π * 각각 C=O 숄더의 전환. π -수성 분산액에서 일반적으로 관찰되는 바와 같이 풀러레놀의 전자 전이는 산소를 함유하여(그림 2c), 풀러레놀의 존재를 확인합니다. 준비된 재료의 특성을 확인하기 위해 FTIR, XPS 및 질량 분석기를 사용하여 추가 특성화를 수행했습니다. FTIR을 사용하여 준비된 재료의 노출된 작용기를 분석했습니다. 분석 결과 다음과 같은 특성 재료 밴드가 나타났습니다. 약 1109 cm ⁻¹ 에서 C–O 신축 밴드 (밴드 1), 약 1271 cm ⁻¹ 에서 페놀릭 C–OH 스트레칭 밴드 (밴드 2), 약 1422 cm ⁻¹ 에서 3차 알코올 C=O 스트레칭 밴드 (밴드 3), 약 1674 cm ⁻¹ 에서 C=C 신축 밴드 (밴드 4), 약 1721 cm ⁻¹ 에서 C=O 스트레칭 밴드 (밴드 5), 약 3318 cm ^-1 에서 CH 분자간 수소 결합 및 카르복실레이트 O-H 스트레칭 밴드 (밴드 6). 또한 CO₂ 밴드 간섭이 관찰되었습니다. 이 밴드는 노출된 하이드록실 및 카보닐 그룹과 방향족 C=C 결합을 나타냅니다(그림 2d). 일반적으로 탄소 원자를 지배적으로 포함하는 풀러레놀의 표면 화학을 조사하기 위해 XPS를 수행했습니다. 풀러레놀의 deconvoluted C(1s) 스펙트럼은 비산소화 고리(C–C/C=C, 286.1 eV), C–O 결합(286.9 eV) 및 C=O 결합(288.0 eV)을 나타냅니다. 또한, O(1s)/C(1s) 비율은 약 35.8%였습니다(그림 2e). 풀러레놀의 분자량은 또한 FD 질량 분석기를 사용하여 결정되었습니다(추가 파일 1:그림 S2). the number of hydroxyl groups (C–OH) was confirmed to be 46, which was consistent with the atomic ratios and bonding compositions of the fullerenol summarized in Fig. 2. These characterization results confirm the successful synthesis of fullerenol.

Functional characterization of the synthesized water-soluble C₆₀ (OH)₄₆ fullerenol. 아 Low-magnified TEM image and b HR-TEM image of a water-soluble fullerenol illustrating the materials {1\( \overline{1} \)00} lattice planes and the mean size of 1.11 ± 0.03 nm with a d -spacing of 0.213 nm. ㄷ UV–vis and d FTIR spectra of nanomaterial. 이 Deconvoluted C(1s) XPS spectra and fitted peaks obtained using Gaussian function:nonoxygenated ring (C–C/C=C), C–O bond, and C=O bond, respectively. The atomic ratio and bonding composition of fullerenol are shown as summarized in the table. The O(1s)/C(1s) atomic ratio is 35.8%

ROS Generation of Water-Soluble Fullerenol Under TPE

A PS absorbs and transfers light energy to other nonabsorbing molecules to generate ROS, which kill targeted cells, damage tumor vasculature, and activate an antitumor immune response. PSs have a particular arrangement of electrons in their molecular orbitals. Similar to nearly all molecules, at ground (singlet) state, PSs have couples of electrons with opposite spins in low-energy molecular orbitals. The absorption of light at an appropriate wavelength lifts an electron to a high-energy orbital without changing its spin. This is a short-lived (nanoseconds) excited singlet (S₁ ) state, and the PS can lose its energy and return to the ground state by emitting light (fluorescence) or heat. Alternatively, intersystem crossing, wherein the spin of the excited electron is inverted, can occur in the S₁ 상태. This electron spin inversion is responsible for the relatively long life (lasting microseconds) of the excited triplet (T₁ ) state. Radiative triplet-to-singlet transitions are inhibited because they require a change in electron spin, which is a slow process. From the T₁ state, the PS can return to the ground state by emitting light (phosphorescence) or transferring energy to another molecule. It can also lose energy through internal conversion or radiationless transitions when colliding with other molecules. The longer the life of the PS in the T₁ state is, the higher are its chances of colliding with another molecule, resulting in ROS production [57,58,59]. The photosensitization of water-soluble fullerenols results in their transition to a long-lived T₁ state and subsequent energy or electron transfer to molecular oxygen, yielding ROS such as ¹ O₂ 및 O₂ ^{. -} , which have major roles in PDT. Therefore, ¹ O₂ 및 O₂ ^{. -} produced by water-soluble C₆₀ (OH)₄₆ must be detected directly using laser irradiation. To detect ¹ O₂ 및 O₂ ^{. -} formation during PDT, in this study, PDT was initiated by combining excited the triplet water-soluble C₆₀ (OH)₄₆ , oxygen, and light configured to a suitable wavelength and energy as well as by introducing SOSG, t -MVP, XTT, and GSH reagents [33, 34, 49,50,51]. To exploit the potential bactericidal capability of the materials, a wavelength of approximately 760 nm was determined to be the most efficient for deriving the relative maximum TPA ratio of the water-soluble C₆₀ (OH)₄₆ under TPE (Fig. 3a); this is attributable to the interband transitions involved [52]. This wavelength was used in subsequent experiments in this study. The water-soluble C₆₀ (OH)₄₆ was photoexcited through TPE at a power of 211.2 nJ pixel ⁻¹ with 800 scans (Ex, 760 nm; total effective exposure time, ~ 3.2621 s) and delivered dose of 3 or 6 μg mL ⁻¹ (Additional file 1:Table S2). Furthermore, to confirm the involvement of ROS in the PDT effects of the water-soluble C₆₀ (OH)₄₆ , α -tocopherol was used for ROS neutralization [49, 53]. The quantity of generated ROS was reduced after the addition of α -tocopherol, but the observed bacterial viability increased as expected. Additionally, the quantity of generated ROS depended on the delivered dose. To prevent ¹ O₂ 및 O₂ ^{. -} production possibly engendered by inadvertent exposure of water-soluble C₆₀ (OH)₄₆ to white light—which could have compromised the experiments in this study [60]—subsequent PDT experiments were conducted in the dark. This study focused on the quantities of generated ¹ O₂ 및 O₂ ^{. -} . The water-soluble C₆₀ (OH)₄₆ exhibited considerable antibacterial effects, demonstrating its potential for application in PDT. Notably, after the same experiment, the water-soluble C₆₀ (OH)₂₁ (Additional file 1:Figs. S3, S4; Fig. 3a) was less effective in forming ¹ O₂ 및 O₂ ^{. -} when compared with the water-soluble C₆₀ (OH)₄₆ (Additional file 1:Table S2). The water-soluble C₆₀ (OH)₄₆ generated more ¹ O₂ 및 O₂ ^{. -} than did the water-soluble C₆₀ (OH)₂₁; additionally, the water-soluble C₆₀ (OH)₄₆ and water-soluble C₆₀ (OH)₂₁ had Φ _Δ values of approximately 0.93 and 0.85, respectively (for reference, Φ _Δ =0.64 is the QY of TSPP dissolved in D₂ O [29, 30]).

아 Relative TPA spectra of the material. TPE as a function of the wavelength (720–820 nm) at 98.56 nJ pixel ⁻¹ that was used to monitor the signals. Delivered dose, 3 μg mL ⁻¹ water-soluble C₆₀ (OH)₄₆ or C₆₀ (OH)₂₁ fullerenol. The number of surviving b material-treated bacteria was determined by CFU counting assay and is expressed as the percentage (%) for c bacteria that corresponds to the unit of CFU mL ⁻¹ . Delivered dose, OD600 ~ 0.05 of E. 대장균 and 0–9 μg mL ⁻¹ water-soluble fullerenol. d Measurement of phosphorescence spectra at 1270 nm for material. Delivered dose, 3 μg mL ⁻¹ water-soluble fullerenol. Data are means ± SD (n =6)

Antimicrobial Ability Determination Using TPE

Before the execution of antimicrobial experiments, the toxicity of water-soluble fullerenols must be examined to exclude factors that could contribute to bacterial elimination and confound experimental results. In addition, to prevent possible ROS production engendered by the inadvertent exposure of experimental materials to white light, which could confound experimental results [35], PDT experiments must be conducted in the dark. This study applied Gram-negative E. 대장균 as the experimental template. A CFU counting assay was conducted to determine the number of surviving bacteria (expressed herein as a percentage, corresponding to CFU mL ⁻¹ ). The bacteria were treated with two types of the prepared water-soluble fullerenols (dose range, 0 to 9 μg mL ⁻¹ ) and incubated in the dark for 3 h at 37 °C to determine absorbance at 600 nm (OD₆₀₀ ~ 0.05; Additional file 1:Fig. S1). The growth levels of the bacteria treated with the water-soluble fullerenols were first monitored by measuring absorbance at 600 nm. The initial absorbance was 0.05 OD₆₀₀ , and the absorbance associated with both materials reached approximately 0.37 over time. Accordingly, neither material inhibited bacterial proferation. Moreover, the materials engendered a nearly 0 log₁₀ reduction in the number of surviving bacteria (Fig. 3b), corresponding to a viability of approximately 100% (Fig. 3c). Accordingly, the materials were determined to exhibit excellent biocompatibility with the bacteria. Consequently, the materials subjected to 3 h of incubation in the dark at 37 °C were used to conduct experiments. Although the water-soluble fullerenol could generate ROS, interactions between materials and reagents (i.e., SOSG, t -MVP, XTT, and GSH) may result in false-positive ROS signals, thereby confounding PDT results [52]. Therefore, to exclude this possibility, bacteria were introduced and treated with materials in the present study. The amount of ROS generated from the photoexcited material-treated E. 대장균 was observed. Table 2 presents the observed amount of ROS, revealing a similar trend to that in Tables S2–S3 (Additional file 1:materials alone and material-treated-Gram-positive Bacillus subtilis (B. subtilis )); these results were consistent with the ¹ O₂ phosphorescence signal emitted from the materials at 1270 nm (Fig. 3d). PDT against E. 대장균 was performed using irradiation with a low dose of energy (211.2 nJ pixel ⁻¹ with 800 scans, total effective exposure time ~ 3.2621 s; Ex, 760 nm). The effects PDT on the viability of E. 대장균 treated with two-photon photoexcited materials were then determined (Fig. 4). No bactericidal effects were observed on bacteria alone (with or without laser exposure) or on the panel of material-treated bacteria without laser treatment (Fig. 4a). After TPE, bacterial viability was relatively low; specifically, the viability observed for the panel that was treated with the water-soluble C₆₀ (OH)₂₁ was nearly 15%, corresponding to an approximately 0.823 log₁₀ reduction (Fig. 4b). By contrast, the bacterial viability observed for the panel treated with the water-soluble C₆₀ (OH)₄₆ was approximately 0 (100% elimination efficiency, corresponding to a ~ 7.736 log₁₀ reduction). When the dose was increased, complete bactericidal effects were observed for both materials (Fig. 4c, d). However, antimicrobial effects did not differ by bacteria type (Gram-negative E. coli or Gram-positive B. 자막 ) after photoexcitation (Additional file 1:Fig. S5). In addition, regarding the fullerenols that eliminated bacteria, a higher composition of hydroxyl groups increased bactericidal capability when compared with a lower composition under identical treatment conditions.

Viability (%) was quantified according to the determined viable count of material-treated bacteria through a CFU assay conducted using short excitation with a TPE power of 211.2 nJ pixel ⁻¹ with 800 scans (approximately 3.2621 s of total effective exposure time; Ex, 760 nm) to deliver a dose of a , b 3 or c , d 6 μg mL ⁻¹ . Delivered dose, OD600 0.05 of E. 대장균 . Data are presented as means ± SD (n =6). For C₆₀ (OH)₄₆ - and C₆₀ (OH)₂₁ -treated E. 대장균 with photoexcitation, a 피 <0.001 and p =0.662, b 피 <0.001 and p =0.658, c 피 <0.001 and p <0.001, and d 피 <0.001 and p <0.001. *p value obtained by Student’s t test

Observation of Water-Soluble Fullerenol-Treated E. 대장균 Using TEM and Investigation of Two-Photon Properties

To observe the disruption of material-treated bacteria after photoexcitation, the water-soluble C₆₀ (OH)₄₆ with high PDT efficiency was selected, and bacteria were imaged using TEM. Bare E. 대장균 (Fig. 5a) were incubated with the water-soluble fullerenol for 3 h, resulting in the substantial adsorption of materials on the bacterial surfaces. Nevertheless, no unusual morphologies were observed, indicating normal live bacterial morphology (Fig. 5b). Uptake assay results revealed the adsorption of materials onto the bacterial surface, with the corresponding burst rate being approximately 85% within the first 3 h of incubation (Fig. 5c); the rate reached saturation from the 3rd to the 10th hour. Therefore, the materials were adsorbed and formed an external barrier on the bacterial surface. However, the E. 대장균 exhibited a distorted appearance and severe morphological changes over 3 days of incubation (Fig. 5d), resulting in a 0.940 log₁₀ reduction that corresponded to a nearly 18% viability (Fig.5f; Additional file 1:Fig. S6). Material absorption and coating on the bacterial surface suppressed the absorption of nutrients essential for microbial growth and engendered changes in membrane (wall) permeability, thereby inducing internal osmotic imbalances and inhibiting microbial growth. In other words, the water-soluble fullerenol had antibacterial (bacteriostatic or bactericidal) effects after 3 days of incubation. Furthermore, the photoexcited material-treated bacteria, particularly E. 대장균 , exhibited unique morphologies with severe damage after 3 h of incubation (Fig. 5e, f; Additional file 1:Fig. S6). No heat-generated bubbles formed on the bacterial surface incurred damage, indicating that the water-soluble fullerenol did not have photothermal-mediated heat properties after photoexcitation (Additional file 1:Fig. S7). 의 생존 가능성 E. 대장균 was also determined through fluorescence and quantification (Fig. 6). The green fluorescence indicative of living bacteria in Fig. 6a reveals that the bacteria exposed to laser treatment alone were largely undamaged, which is consistent with the results presented in Fig. 5a. Dead bacteria were detectable after treatment with the materials and laser exposure (red fluorescence in Fig. 6b), a finding that is also consistent with that in Fig. 5e. Bacterial viability was quantified for further antimicrobial testing. Nearly complete elimination of the material-treated bacteria (Fig. 6c) was observed. Viability was also quantified using a CFU assay (Figs. 4a, b and 5f, and Additional file 1:Fig. S6) to demonstrate the antibacterial efficiency of the water-soluble C₆₀ (OH)₄₆ in PDT. According to the results in Figs. 4, 5, and 6; Table 2; and Table S2 (Additional file 1), E. 대장균 treated with the water-soluble C₆₀ (OH)₄₆ was susceptible to photoexcitation, leading to a higher death rate, increased ROS generation, and more severe morphological collapse compared with E. 대장균 treated with the water-soluble C₆₀ (OH)₂₁ . In general, the absolute cross section for TPE makes fluorophores efficient for nonlinear microscopic studies because the ratio of the energy absorbed to the input energy flux to a specimen is high, thereby minimizing possible photodamage to specimens [39, 40]. When two-photon techniques are used to image molecular activities in living biological preparations and turbid tissues, a favorable cross section is desirable [61]. In the present study, the absolute cross section for TPE calculated for the water-soluble C₆₀ (OH)₄₆ was approximately 1037 GM (Goeppert-Mayer unit, with 1 GM =10 ⁻⁵⁰ cm ⁴ s photon ⁻¹ ) at a 760-nm excitation wavelength (fluorescein was the standard reference for the cross section [39, 40]; Fig. 1b and Tables 1 and 3); the absolute cross section calculated for the water-soluble C₆₀ (OH)₂₁ was approximately 1230 GM, which is similar to values obtained in relevant studies [62, 63]. These absolute cross sections could facilitate the two-photon process. Moreover, the fluorescence of the water-soluble C₆₀ (OH)₄₆ was illuminated through a two-photon process (Fig. 1b). The relative fluorescence QY was approximately 0.02 (the QY of Cy5.5 in dimethyl sulfoxide [31] served as a reference:QY_ref =0.28); similarly, the absolute QY [64] was approximately 0.01, and the same QYs were derived for one-photon excitation and TPE [31]. By contrast, the water-soluble C₆₀ (OH)₂₁ had lower relative and absolute QYs (0.06 and 0.05, respectively). In addition, this study investigated the lifetime of the fullerenols. The effects of radiative and nonradiative decay rates on QY and lifetime were calculated. The average lifetime of the water-soluble C₆₀ (OH)₄₆ was approximately 7.797 ns, as calculated from observed lifetimes of 0.149, 1.775, and 19.679 ns; the average lifetime of the water-soluble C₆₀ (OH)₂₁ was approximately 5.251 ns (Fig. 7 and Table 4). Therefore, the ratio of radiative to nonradiative decay rates of the water-soluble C₆₀ (OH)₄₆ was approximately0.020 (derived from rates of approximately 2.565 × 10 ⁶ s ⁻¹ to 1.257 × 10 ⁸ s ⁻¹ ), whereas that of the water-soluble C₆₀ (OH)₂₁ was approximately 0.064 (approximately 1.143 × 10 ⁷ s ⁻¹ and 1.790 × 10 ⁸ s ⁻¹ ; Additional file 1:Table S4). This finding is attributable to the existence of a hydroxyl group on the surface of the water-soluble fullerenol, which induced the nonradiative recombination of electron–hole pairs, leading to the inhibition of intrinsic state emission. However, hydroxylgroups at the edge of the water-soluble fullerenol may have a high occupied molecular orbital. This can be attributed to the strong orbital interaction between hydroxyl groups, which could thus increase the efficiency of intersystem crossing (rather than fluorescence generation) and generate numerous nanomaterial triplets with a high composition of hydroxyl groups; therefore, this would result in a high Φ _Δ value for the water-soluble fullerenol and induce the fullerenol to react with oxygen according to the Jablonski diagram [65]. Consequently, two-photon PDT can be effectively performed using ultralow energy in an extremely short time, thereby providing an alternative approach to killing malignant species.

TEM 이미지. 아 Showing bare bacteria without any treatment. Bacteria treated with material for b 3 h and d 3 days of incubation. 이 The photoexcited material-treated bacteria (3 h of incubation) with a TPE power of 211.2 nJ pixel ⁻¹ with 800 scans (approximately 3.2621 s of total effective exposure time; Ex, 760 nm). ㄷ Uptake assay of bacteria and material at 37 °C. 에 Viability (%) was quantified following the determined viable count of material-treated bacteria via CFU assay by short excitation with the same treatment. Delivered dose OD600 ~ 0.05 of E. 대장균 and 3 μg mL ⁻¹ water-soluble fullerenol C₆₀ (OH)₄₆ . Data are means ± SD (n =6)

Images obtained after laser photoexcitation exposure (211.2 nJ pixel ⁻¹ ) with 800 scans (approximately 3.2621 s of total effective exposure time; Ex, 760 nm) of a , b material-treated bacteria. The Live/Dead kit was used to stain bacteria before images were obtained. Scale bar, 50 μm. ㄷ Viability (%) determination results. Delivered dose, OD600 ~ 0.05 of E. 대장균 and 3 μg mL ⁻¹ water-soluble fullerenol C₆₀ (OH)₄₆ . For the percentages alive and dead, p <0.001. *p value obtained using Student’s t test. Data are presented as mean ± SD (n =6)

Time-resolved room-temperature PL decay profiles of material (98.56 nJ pixel ⁻¹ ). Excitation wavelength, 760 nm. Delivered dose, OD₆₀₀ ~ 0.05 of E. 대장균 and 3 μg mL ⁻¹ 재료. Data are presented as means ± SD (n =6)