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One pot synthesis of water-soluble quercetin derived multifunctional nanoparticles with photothermal and antioxidation capabilities
Colloids and Surfaces B: Biointerfaces 183 (2019) 110429
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One pot synthesis of water-soluble quercetin derived multifunctional nanoparticles with photothermal and antioxidation capabilities Shu-Hua Tanga, Rong Lia, Jin Tana, Ying Wanga, Zi-Tao Jianga,b, a b
T
⁎
Tianjin Key Laboratory of Food Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China School of Food Engineering, Tianjin Tianshi College, Tianjin 301700,China
A R T I C LE I N FO
A B S T R A C T
Keywords: Quercetin Water-soluble Antioxidation Photothermal Multifunctional
As a member of flavonoids, the application of quercetin has been mainly focused on antioxidation study. Fabrication of multifunctional nanoplatforms with quercetin is limited. In the present study, water-soluble quercetin derived nanoparticles (QFNPs) were fabricated through the one pot synthesis strategy with Fe3+, quercetin and poly (vinyl pyrrolidone) (PVP). The raw materials were dissolved in absolute ethanol and the mixed together. After stirring at room temperature for 6 h, the QFNPs could be simply harvested by centrifugation without the need of time-consuming dialysis procedure. Due to the protective effect of PVP, the synthesized nanoparticles could be well dispersed in water with the hydrodynamic size about 23 nm. DPPH free radical scavenging capacity assay showed QFNPs could act as efficient antioxidant. Besides antioxidation activity, the QFNPs also exhibited good photothermal capacity. Temperature stability result suggested the good stability of QFNPs between 35 and 95 °C. MTT and hemolysis assay showed the good biocompatibility of QFNPs. What’s more, the QFNPs showed good cellular antioxidation activity and efficient photothermal killing effect to cancer cells (4T1 cells). The QFNPs could be promising nanoplatform for biomedical application.
1. Introduction Nanoparticles with the capacity of integrating different functions into one platforms have drawn tremendous attention in biomedical field. Great efforts have been devoted to the synthesis and application of multifunctional nanoplatforms for bioimaging and therapy [1,2]. For these biomedical applications, the biosafety of the nanoparticles is one of the main concerns [3]. Multifunctional nanoparticles with good biocompatibility are highly appreciated. Different strategies have been developed for the fabrication of biocompatible nanoparticles. Modification of the nanoparticles with biocompatible macromolecules is one effective approach to promote their biocompatibility. Various kinds of polymers, such as chitosan, dextran,polyethylene glycol (PEG), poly (vinyl pyrrolidone) (PVP), and poly(vinyl alcohol) (PVA), have been applied to fabricate nanoparticle with high biocompatibility and stability [4]. The functionalization of proteins could also render the nanoparticles with high biocompatibility [5]. In addition to surface modification, the synthesis of nanoparticles with biocompatible molecules is another strategy to fabricate biocompatible nanoplatforms. For example, the growth of Au nanoparticles on natural melanin extracted from cuttlefish could generate multifunctional nanoagent with high
biocompatibility [6]. Similarly, small biomolecules, such as dextran [7] and glutathione [8], could also be used as ligands to fabricate highly biocompatible nanoparticles for antioxidation study or bioimaging. Besides these biomolecules, phenolic compounds extracted from plants could also be used for the construction of biocompatible nanoparticles with excellent biological activity [9,10]. Phenolic compounds in plants often contain benzene ring and hydroxyl groups. Their unique chemical structure characteristics render them capacity to defend various diseases [11,12]. Flavonoids primarily derived from benzo-γ-pyrone (phenylchromone) are a large group of phenolic compounds [13]. According to difference in the chemical structure, flavonoids could be classified to be flavonols, flavanones, flavans and anthocyanidins, proanthocyanidins, isoflavones, flavones and neoflavonoids [11]. These flavonoids usually contain multiple phenolic hydroxyl groups, and show excellent antioxidation capacity [14,15]. During which, quercetin with anti-inflammation, anti-viral, anti-tumour, antioxidation and antibacterial activities have attracted great attention [16]. As categorized by chemical structure, quercetin is a type of flavonol. Due to the presence of hydroxyl groups, quercetin shows excellent antioxidation activity [17]. It could obviously inhibit the proliferation
⁎ Corresponding author at: Tianjin Key Laboratory of Food Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China. E-mail addresses: [email protected], [email protected] (Z.-T. Jiang).
https://doi.org/10.1016/j.colsurfb.2019.110429 Received 12 April 2019; Received in revised form 18 July 2019; Accepted 6 August 2019 Available online 11 August 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
Colloids and Surfaces B: Biointerfaces 183 (2019) 110429
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and PVP. After that, 162 mg of FeCl3∙6H2O in 10 mL of absolute ethanol was then added. The solution was further stirred for 6 h in the dark. Greenish black precipitates were collected after centrifugation at 10,000 rpm for 5 min. The precipitates were washed thrice by absolute ethanol and then dispersed in water. Solid QFNPs were obtained by freeze-drying and stored in the refrigerator for further use.
of hepatic cancer cells [18] and neuroblastom cells [16]. Researches suggest that biological activities of quercetin might be attributed to the influence to enzyme activities and signaling pathway of cells [19]. Despite the outstanding bioactivities, application of quercetin is largely restricted by its poor water solubility. Various nano-carriers [20–22] were designed to improve the bioavailability of the quercetin. Although these exploration for antioxidation and anticancer study, direct fabrication of multifunctional nanoplatforms with quercetin is limited. Combining with nanotechnology, construction of such nanoplatforms would provide new opportunities for the application of quercetin. Due to the coordination interaction of phenolic hydroxyl with metal ions, phenolic compound often shows strong binding capacity with these metal ions. Especially, complexes of some phenolic compound such as gallic acid [23] and tannic acid [24] with Fe3+ show strong absorption in the NIR field (700–950 nm), rendering them capacity for photothermal therapy (PTT). Besides the bioactivities, quercetin also shows strong binding capacity with metal ions, such as Cu2+, Mg2+ and Fe3+ [25–27]. However, the potential of complex derived from quercetin and Fe3+ for PTT has not been explored. In the present study, the derived quercetin multifunctional nanoparticles (QFNPs) were fabricated with quercetin, PVP and Fe3+ through a one-pot synthesis strategy. QFNPs showed good water solubility. Upon irradiation by 808 nm laser, 4T1 cells (Mouse breast cancer cells) could be efficiently killed by the photothermal effect of QFNPs, indicating QFNPs could serve as efficient photothermal agents. Besides PTT, QFNPs also showed good antioxidation activity.
2.4. Drug release study Drug release behavior of the QFNPs in PBS (pH 7.4) was investigated. 28.8 mg of QFNPs in 2 mL of PBS was added into the dialysis bag (molecular weight cutoff: 8–14 kDa) with the two ends sealed with clips. The dialysis bag was placed in a preheated release medium (50 mL) at 37 °C. 5 mL of the dissolution medium was withdrawn at selected time points (0.5, 1, 2, 4, 6 and 8 h) and an equal volume of fresh PBS was added then. Content of the released flavonoid was measured by the aluminium chloride colorimetric method [28] with quercetin as standard. Cumulated drug release of the phenolic compound was calculated by the following formula: Cumulated release n−1 degree (%) = [(50Cn+5 ∑i = 1 Ci )/weight of the phenolic compound]×100%, where Cn is the concentration of flavonoid in the dissolution medium at the time point of n [29]. 2.5. Photothermal measurement of QFNPs in vitro Photothermal property of QFNPs was measured under 808 nm laser at power density of 3.0 W cm−2. For the measurement, a range of QFNPs solutions with concentrations of 0.2, 0.5, 1.0 and 2.0 mg mL-1 were treated with laser for 10 min, respectively. Temperature increments of the solutions along time were recorded by a thermocouple probe inserted into the solutions.
2. Materials and methods 2.1. Materials 4T1 Cells were obtained from Cell Bank of Shanghai, Chinese Academy of Sciences (Shanghai, China). Ferric chloride hexahydrate, 2, 2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), and PVP (M≈ 58,000) were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Quercetin (95%) was bought from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Absolute ethanol and 2,6di-tert-butyl-4-methylphenol (BHT) were purchased from Guangfu Fine Chemical Research Institute (Tianjin, China). 2′, 7′Dichlorodihydrofluorescein diacetate (DCFH-DA) and 2,2-diphenyl-1picrylhydrazyl (DPPH) were purchased from Sigma Aldrich (St. Louis, MO, USA). Sodium carbonate was bought from Tianjin Yingda Rare Chemical Reagents Factory (Tianjin, China). All chemicals were at least analytical grade and used without further purification. Deionized water was used throughout all the experiments.
2.6. Temperature stability study To investigate the temperature stability of the QFNPs, 100 mL of aqueous solution of QFNPs with concentration of 0.1 mg mL−1 was added into a 250 mL round bottom flask and heated with thermostat water bath. Temperature of the solution was maintained for 10 min at different temperatures (35, 50, 65, 80 and 95 °C) in the heating process. At selected time points (0, 5 and 10 min), absorbance of the solution at 292 nm was monitored during the isothermal periods. While 0 min was the time when temperature of the solution reached the selected temperatures. 2.7. Antioxidation capacity assay of QFNPs The antioxidation capacity of QFNPs was determined by DPPH free radical scavenging activity assay. For the assay, the solutions of QFNPs with different concentrations were mixed with DPPH ethanol solution (50 μg mL−1) at room temperature. After left static in the dark for 1 h, absorbance at 524 nm of the mixed solutions was measured. The scavenging rate (S) of DPPH calculated by S(%) = (1-(A-A1)/ A0)×100. Where A in the equation was the absorbance of the reaction mixture of samples with DPPH solution, A1 was the absorbance of the solutions of samples without DPPH and A0 was the initial absorbance of DPPH without the samples.
2.2. Instrumentation and characterization X-ray diffraction (XRD) analysis was performed on a Utima IV diffractometer (Rigaku, Japan) using Cu Kα radiation (λ = 1.5418 Å). Transmission electron microscopy (TEM) measurement was carried out on a HT7700 transmission electron microscope (Hitachi, Japan). The fourier transform infrared (FT-IR) spectra were recorded by a Nicolet IR iS10 spectrometer (Nicolet, USA). Dynamic light scattering (DLS) was carried out on a Malvern Zetasizer (Nano series ZS, UK). The UV–vis absorption spectra were obtained from a Lambda 25 UV/VIS spectrometer (PerkinElmer, USA). The content of iron ions was measured with inductively coupled plasma mass spectrometer (ICP-MS, Thermo Elemental X7, UK). Element analysis was conducted on vario EL cube elemental analyzer (Elementar, Germany).
2.8. Cytotoxicity assay in vitro Cytotoxicity assay of QFNPs toward 4T1 cells was carried out using the MTT method. Firstly, 4T1 cells were seeded into a 96-well plate (104 cells per well). After incubation for 24 h at 37 °C under 5% CO2, the stale culture medium was replaced by the fresh one. QFNPs were then added. The cells were further incubated with various concentrations of QFNPs for 24 h. After the incubation, the cells were washed by PBS and treated by MTT (10 μL, 5 mg mL−1) for 4 h. After this, 120 μL
2.3. Synthesis of QFNPs For the synthesis of QFNPs, 30 mg of quercetin and 513 mg of PVP were added into a 250 mL flask, followed by 40 mL of absolute ethanol. The mixture was stirred at room temperature to dissolve the quercetin 2
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from the previously reported procedure, no water was used during the synthesis process. Then precipitates would be formed, which is favorable for the separation and purification of the product. The QFNPs could be simply harvested by centrifugation, without the need of timeconsuming dialysis process, which usually takes 24 h or more time. To the best of our knowledge, this process have not be reported for other drugs or natural regents. PVP could serve as protecting ligands to stabilize the nanoparticles, rendering QFNPs high hydrophilicity. To confirm the protective effect of PVP, 100 μL of ethanol solution of Fe3+, quercetin and PVP with the same ratios to the nanoparticle synthesis procedure was added into a 20 mL vial. For control, 100 μL of ethanol solution with equal amount of Fe3+ and quercetin was added into another vial, without the addition of PVP. After natural evaporation of ethanol, same amount of water was added into the two vials. As shown in Fig. S1, the aggregates were formed for the solution without PVP, while a transparent solution was obtained with the addition of PVP. These results verified the protect effect of PVP. After the synthesis, absorption maxima of quercetin at 257 and 374 nm disappeared in the UV–vis absorption spectrum of QFNPs, while a new band at 292 nm appeared (Fig. S2). These might be attributed to the partial oxidation of quercetin by Fe3+ [33]. To confirm that, X-ray photoelectron spectroscopy (XPS) of QFNPs was investigated. Wide scan XPS spectrum of QFNPs indicated the presence of C, O, N and Fe (Fig. S3). Fe 2p3/2 peak position at 709.8 eV and Fe 2p1/2 peak position at 723.2 eV suggested the presence of Fe2+ in QFNPs (Fig. S4), confirming the oxidation of quercetin by Fe3+. Partial oxidation of the quercetin through the loss of two H yields a quinone [33]. The content of Fe and the quinone in QFNPs were also investigated. ICP-MS analysis indicated there was 57 mg Fe in one gram of QFNPs. While the C and N content in the QFNPs were determined to be 45.85% and 7.69% by element analysis. The C is from PVP and quercetin. While the N is from PVP. Thus content of the quinone was calculated to be 105 mg in one gram of QFNPs. Release profile of the from QFNPs in pH 7.4 PBS buffer suggested 41% quinone was released after 8 h (Fig. S5). Morphology of QFNPs was observed by TEM. The synthesized QFNPs are spherical like nanoparticles with an average size of 21 nm (Fig. 1). Micrograph with larger magnification suggested the internal structure of nanoparticles is not compact (Fig. S6). The hydrodynamic size of QFNPs was measured to be 23 nm in pure water, which was well accorded with the results by TEM. While hydrodynamic sizes of QFNPs in pH 7.4 buffer and pH 5 buffer were measured to be 62 nm and 270 nm, suggesting the aggregation trend of QFNPs in lower pH (Fig. S7). Zeta potential of QFNPs was near neutral in the pure water and pH 5 buffer (Fig. S8), indicating the capping of nonionic hydrophilic PVP
DMSO was added into the wells. The absorbance of the solution in each well was recorded by a multimode microplate reader. Each treatment group was repeated by five times. 2.9. Hemolysis assay Hemolysis assay was carried out according to the literature [30] with slight modifications. Blood of Kunming mouse obtained from Tianjin Medical University was collected in anticoagulant tube. After centrifugation at 3000 rpm for 5 min, supernatant was discarded. The erythrocytes were washed three times with physiological saline. The final red blood cells pellet was re-suspended with 1.5 mL of physiological saline. 120 μL of the diluted red blood cells suspension was mixed with 0.88 mL of QFNPs solutions at varied concentrations. For the positive and negative controls, 120 μL of the diluted red blood cells suspension was mixed with 0.88 mL of deionized water and physiological saline. After incubation at 37 °C for 3 h, the samples were centrifuged and the supernatants were transferred to a 96-well plate (100 μL/well). The plate was then placed in the multimode microplate reader. Absorbance of the supernatants at 540 nm was recorded. The hemolytic activity of red blood cells was calculated with the following formula: Percent hemolysis (%) = (sample absorbance-negative control absorbance)/ (positive control absorbance-negative control absorbance) × 100. 2.10. Cellular antioxidation activity assays The cellular antioxidation activity assay was performed according to literature with some modifications [31]. For the study, 4T1 cells were seeded in 96-well plates. After the incubation for 24 h, the culture medium was removed and the cells were washed with PBS. The cells were further treated with 10 μM DCFH-DA in treated medium for 1 h. Subsequently, the treated medium was discarded and the cells were washed twice with PBS. After that, the experimental groups were treated with 20 μL of QFNPs with different concentrations and 60 μL of HBSS. The blank and control group were treated with 20 μL of H2O and 60 μL of HBSS. After further incubation at 37 °C for 10 min, all the cells except the blank group were treated with 20 μL of 0.6 mM AAPH in HBSS. The blank group was treated with 20 μL of HBSS. Then fluorescence of the wells was monitored by the plate reader. Increment of fluorescence at 525 nm with the excitation wavelength of 488 nm was monitored by a microplate reader every 5 min for one hour. All samples and controls were repeated at least three times. 2.11. Cellular photothermal therapy assessment The photothermal therapy effect of QFNPs toward 4T1 cells was also studied. For the assay, 4T1 cells were cultured in a 96-well culture plate with the density of 104 cell per well for 24 h. The cells were washed with PBS and treated with QFNPs (800 μg mL−1, diluted in cell culture medium) for 1 h. Cells treated with cell culture medium without QFNPs were used as control. Subsequently, all the cells were irradiated by 808 nm laser under the power densities of 7 W cm-2 for 5 min. MTT assays were performed to evaluate the cell viability. All the cells were stained with calcein acetoxymethyl ester (calcein AM) and propidium iodide (PI). Then, luminescence imaging of the cells was carried out on the confocal microscopy. 3. Results and discussion Water soluble QFNPs were simply synthesized by mixing Fe3+, quercetin and PVP at room temperature. Ethanol was used as solvent during the synthesis. Firstly, quercetin and PVP were mixed and dissolved in ethanol. Both of the phenolic hydroxyl groups and the amide moieties of PVP could coordinate to Fe3+ [32]. Coordination polymer nanoparticles would be formed after the introduction of Fe3+. Different
Fig. 1. TEM micrograph of QFNPs. 3
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Fig. 2. DPPH radical scavenging capacity of QFNPs.
Fig. 4. Cellular antioxidation assay of 4T1 cells treated with various concentrations of QFNPs.
Fig. 5. Cell viability of 4T1 cells treated with different concentration of QFNPs through MTT assay.
when DPPH concentration decreased, it caused a decrease in absorbance at 524 nm. As shown in Fig. 2, the scavenging rate of DPPH increased as the concentration of QFNPs increased, suggesting the efficient radical scavenging capacity of QFNPs. Due to the good water solubility, QFNPs could provide a powerful tool for antioxidation study. Before the investigation of the potential of QFNPs for PTT, UV–vis absorption spectra of QFNPs were measured. As shown in Fig. S11, obvious NIR absorption around 808 nm was observed, and the absorption increased when the concentration of QFNPs was raised, indicating the possibility of QFNPs for PTT. After that, QFNPs solutions with different concentrations were irradiated by a 808 nm laser with the power of 3 W cm−2 for 10 min. Temperature changes of the solutions were then recorded. As shown in Fig. 3a, obvious temperature increments were observed. The temperature increment increased as the concentration of QFNPs increased. Temperature of QFNPs solution with concentration of 2.0 mg mL-1 increased by nearly 40 °C after 10 min irradiation, while temperature increment of pure water was only about 5 °C under the same condition (Fig. 3a). The thermal imaging of QFNPs was recorded (Fig. S12). The color of solutions turned from yellow to nearly white as the concentration of the solutions and irradiation time increased. Besides photothermal property, temperature stability of the QFNPs was also studied. As shown in Fig. S13, absorbance of the solution at 292 nm slightly changed at different temperatures, indicating the good temperature stability of the QFNPs between 35 and 95 °C. These results indicated QFNPs could be used as efficient photothermal agent. Photo-stability of QFNPs was also investigated. QFNPs solution was irradiated by 808 nm laser at 3 W cm−2 for 5-cycle laser off/on. During
Fig. 3. (a) Temperature elevation of QFNPs solutions with different mass concentration. (b) The photothermal response of QFNPs solution with the concentration of 1 mg mL−1 for 5-cycle laser off/on.
polymers. More negative zeta potential of QFNPs was observed in the pH 7.4 buffer. The XRD pattern indicated QFNPs are amorphous materials (Fig. S9). FT-IR spectra of quercetin, PVP and QFNPs are shown in Fig. S10. Observation of the band at 1289 cm−1 in the spectrum of QFNPs, representing the stretching vibration of C–N, suggested the presence of PVP in QFNPs. The C]O stretching vibration of PVP at 1670 cm−1 in PVP was shifted to 1653 cm−1 in QFNPs, due to the interaction between iron ions and PVP. Observation of the band at 1211 cm−1 for the stretching vibration of C–O in phenols and 3420 cm−1 for the stretching vibration of OeH in the spectrum of QFNPs indicated the presence of phenolic hydroxyl, which rendered QFNPs antioxidation capacity. DPPH free radical scavenging capacity assay was carried out to evaluate the antioxidation activity of QFNPs. DPPH was a very stable free radical. It showed strong absorbance around 524 nm. Therefore,
4
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Fig. 6. Bright field and fluorescence images of calcein AM (green, live cells) and PI (red, dead cells) stained 4T1 cells: (a) treated without QFNPs and laser; (b) treated with laser only; (c) treated with QFNPs only; (d) treated with QFNPs and laser. Scar bar is 100 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
each heating cycle, the solution was firstly irradiated by 808 nm laser for 5 min. The laser was subsequently turned off, left the solution untreated for another 5 min. Temperature oscillations of the solution during the heating cycles were recorded. As shown in Fig. 3b, the maximal temperature of QFNPs solution could reach 42.1 °C during the first heating cycle, which could still reach as high as 43.5 °C in the fifth heating cycle, indicating the good photothermal stability of QFNPs. The results suggest the excellent potential of QFNPs for PTT. Encouraged by the good free radical scavenging capacity, cellular antioxidation study of QFNPs toward 4T1 cells was carried out. DCFHDA was used to monitor the cellular oxidation conditions. After incubation with cells, the acetate groups of DCFH-DA could be removed by cellular esterases. DCFH-DA or DCFH in cell are nonfluorescent in reduction state. They would be fluorescent after the oxidation by oxidants or free radicals. Thus intensity of the fluorescence could be used as indicators of the oxidation status of the cells. The decrease of the fluorescence indicates the increase of antioxidation effect. AAPH was used as free radical generation source for the oxidation of DCFH-DA or DCFH in this study. For the study, 4T1 cells were firstly incubated with DCFH-DA. After the incubation, extracellular DCFH-DA was washed away. Then QFNPs and AAPH were added. Fluorescence at 525 nm of the cells treated with different concentration of QFNPs was monitored during a period of 1 h. The cells treated without AAPH and QFNPs were set as blank group and those treated without QFNPs as control. Integrated intensities of the cell groups during 1 h were shown in Fig. 4. Fluorescence signal was observed in the blank group, indicating the DCFH-DA could penetrate the cell membrane and be trapped intracellular. Fluorescence signal of the control group was much higher than the blank group, suggesting the effective oxidation of DFCH by AAPH. Fluorescence signal of the cells treated with QFNPs decreased with the increase of its concentration, indicating scavenging of the free radical generated from AAPH by QFNPs. All these results suggested that QFNPs could be efficient cellular antioxidants. Before the investigation of PTT effect of QFNPs for cancer cells,
cytotoxicity of QFNPs was studied. MTT assay was performed to assess the cytotoxicity of QFNPs toward 4T1 cells. As shown in Fig. 5, slight increment in the cell viability was observed after incubation for 24 h, when the concentration of QFNPs was below 0.5 mg mL−1. Cell viability was as high as 90% even though the concentration of QFNPs reached 0.8 mg mL−1, suggesting the low cytotoxicity of QFNPs. Besides cytotoxicity assay, hemocompatibility of QFNPs was investigated by erythrocyte hemolysis assay. As shown in Fig. S14, the percent hemolysis gradually increased with increasing concentration of QFNPs. The percent hemolysis was 1.7% when the concentration of the QFNPs was 250 μg mL−1, suggesting good hemocompatibility of QFNPs. After confirming the biocompatibility of QFNPs, PTT efficiency of QFNPs to 4T1 cells was further evaluated through MTT assay. An aliquot of 0.8 mg mL−1 of QFNPs was adopted to study the photothermal killing effect. 4T1 cells incubated with or without QFNPs were treated with 808 laser at 7 W cm-2 for 5 min. After the irradiation, the viability was only 15.3% for the cells incubated with QFNPs (Fig. S15), while 83.5% cells still survived for the groups incubated without QFNPs, suggesting the good PTT efficiency of QFNPs. The photothermal cell killing effect was further confirmed by the cell staining experiments. After the laser irradiation, the cells were stained with calcein AM and PI. The calcein AM could easily penetrate the cell membrane. The AM group of calcein AM could be removed by the esterase of live cells, resulting intense green fluorescence. It was widely used for the staining of live cells. While PI couldn’t pass through the cell membrane of live cell. But it could pass the disordered regions of dead cell and went into the nucleus. After that, PI would be embedded in the DNA double helix, generating red fluorescence. Thus PI was widely used for staining of dead cells. As shown in Fig. 6, obvious green fluorescence was observed for the cells treated without QFNPs (Fig. 6a and 6b) and the cells treated with QFNPs only (Fig. 6c), while no obvious red fluorescence signal was detected for those cells, indicating most of those cells survived after the treatment. However, obvious red fluorescence was observed for the cells treated with both of laser and QFNPs (Fig. 6d), while little green fluorescence was observed 5
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in the cells, suggesting almost all of the cells died after the treatment. These results further the good photothermal efficiency of QFNPs.
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4. Conclusions In summary, a simple one-pot synthesis strategy was developed for the fabrication of QFNPs with Fe3+, quercetin and PVP. The fabricated QFNPs showed good potential for PTT, along with efficient antioxidation capacity. Furthermore, low toxicity of Fe3+, quercetin and PVP renders QFNPs excellent biocompatibility. The synthesized QFNPs could be powerful tools for the application in biomedical field. This study will provide a new idea for exploration of the application of flavonoids. Acknowledgements This work was supported by the Tianjin Natural Science Foundation of China (17JCQNJC02400 and 16JCYBJC43300). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110429. References [1] S.M. Janib, A.S. Moses, J.A. MacKay, Imaging and drug delivery using theranostic nanoparticles, Adv. Drug Deliv. Rev. 62 (11) (2010) 1052–1063. [2] J. Xie, S. Lee, X. Chen, Nanoparticle-based theranostic agents, Adv. Drug Deliv. Rev. 62 (11) (2010) 1064–1079. [3] S. Naahidi, M. Jafari, F. Edalat, K. Raymond, A. Khademhosseini, P. Chen, Biocompatibility of engineered nanoparticles for drug delivery, J. Control. Release 166 (2) (2013) 182–194. [4] M. Muthiah, I.K. Park, C.S. Cho, Surface modification of iron oxide nanoparticles by biocompatible polymers for tissue imaging and targeting, Biotechnol. Adv. 31 (8) (2013) 1224–1236. [5] M.F. Zhang, M. Yudasaka, K. Ajima, A. Miyawaki, S. Iijima, Light-assisted oxidation of single-wall carbon nanohorns for abundant creation of oxygenated groups that enable chemical modifications with proteins to enhance biocompatibility, ACS Nano 1 (4) (2007) 265–272. [6] Z. Wang, N. Yu, W. Yu, H. Xu, X. Li, M. Li, C. Peng, Q. Wang, M. Zhu, Z. Chen, In situ growth of Au nanoparticles on natural melanin as biocompatible and multifunctional nanoagent for efficient tumor theranostics, J. Mater. Chem. B 7 (1) (2019) 133–142. [7] J.M. Perez, A. Asati, S. Nath, C. Kaittanis, Synthesis of biocompatible dextrancoated nanoceria with pH-dependent antioxidant properties, Small 4 (5) (2008) 552–556. [8] H.F. Qian, C.Q. Dong, J.F. Weng, J.C. Ren, Facile one-pot synthesis of luminescent, water-soluble, and biocompatible glutathione-coated CdTe nanocrystals, Small 2 (6) (2006) 747–751. [9] A.M. Abdellah, M.A. Sliem, M. Bakr, R.M. Amin, Green synthesis and biological activity of silver–curcumin nanoconjugates, Future Med. Chem. 10 (22) (2018) 2577–2588. [10] M.C. Moulton, L.K. Braydich-Stolle, M.N. Nadagouda, S. Kunzelman, S.M. Hussain, R.S. Varma, Synthesis, characterization and biocompatibility of "green" synthesized silver nanoparticles using tea polyphenols, Nanoscale 2 (5) (2010) 763–770. [11] C. Kanadaswami, L.-T. Lee, P.-P.H. Lee, J.-J. Hwang, F.-C. Ke, Y.-T. Huang, M.T. Lee, The antitumor activities of flavonoids, In Vivo 19 (5) (2005) 895–909.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
One pot synthesis of water-soluble quercetin derived multifunctional nanoparticles with photothermal and antioxidation capabilities Shu-Hua Tanga, Rong Lia, Jin Tana, Ying Wanga, Zi-Tao Jianga,b, a b
T
⁎
Tianjin Key Laboratory of Food Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China School of Food Engineering, Tianjin Tianshi College, Tianjin 301700,China
A R T I C LE I N FO
A B S T R A C T
Keywords: Quercetin Water-soluble Antioxidation Photothermal Multifunctional
As a member of flavonoids, the application of quercetin has been mainly focused on antioxidation study. Fabrication of multifunctional nanoplatforms with quercetin is limited. In the present study, water-soluble quercetin derived nanoparticles (QFNPs) were fabricated through the one pot synthesis strategy with Fe3+, quercetin and poly (vinyl pyrrolidone) (PVP). The raw materials were dissolved in absolute ethanol and the mixed together. After stirring at room temperature for 6 h, the QFNPs could be simply harvested by centrifugation without the need of time-consuming dialysis procedure. Due to the protective effect of PVP, the synthesized nanoparticles could be well dispersed in water with the hydrodynamic size about 23 nm. DPPH free radical scavenging capacity assay showed QFNPs could act as efficient antioxidant. Besides antioxidation activity, the QFNPs also exhibited good photothermal capacity. Temperature stability result suggested the good stability of QFNPs between 35 and 95 °C. MTT and hemolysis assay showed the good biocompatibility of QFNPs. What’s more, the QFNPs showed good cellular antioxidation activity and efficient photothermal killing effect to cancer cells (4T1 cells). The QFNPs could be promising nanoplatform for biomedical application.
1. Introduction Nanoparticles with the capacity of integrating different functions into one platforms have drawn tremendous attention in biomedical field. Great efforts have been devoted to the synthesis and application of multifunctional nanoplatforms for bioimaging and therapy [1,2]. For these biomedical applications, the biosafety of the nanoparticles is one of the main concerns [3]. Multifunctional nanoparticles with good biocompatibility are highly appreciated. Different strategies have been developed for the fabrication of biocompatible nanoparticles. Modification of the nanoparticles with biocompatible macromolecules is one effective approach to promote their biocompatibility. Various kinds of polymers, such as chitosan, dextran,polyethylene glycol (PEG), poly (vinyl pyrrolidone) (PVP), and poly(vinyl alcohol) (PVA), have been applied to fabricate nanoparticle with high biocompatibility and stability [4]. The functionalization of proteins could also render the nanoparticles with high biocompatibility [5]. In addition to surface modification, the synthesis of nanoparticles with biocompatible molecules is another strategy to fabricate biocompatible nanoplatforms. For example, the growth of Au nanoparticles on natural melanin extracted from cuttlefish could generate multifunctional nanoagent with high
biocompatibility [6]. Similarly, small biomolecules, such as dextran [7] and glutathione [8], could also be used as ligands to fabricate highly biocompatible nanoparticles for antioxidation study or bioimaging. Besides these biomolecules, phenolic compounds extracted from plants could also be used for the construction of biocompatible nanoparticles with excellent biological activity [9,10]. Phenolic compounds in plants often contain benzene ring and hydroxyl groups. Their unique chemical structure characteristics render them capacity to defend various diseases [11,12]. Flavonoids primarily derived from benzo-γ-pyrone (phenylchromone) are a large group of phenolic compounds [13]. According to difference in the chemical structure, flavonoids could be classified to be flavonols, flavanones, flavans and anthocyanidins, proanthocyanidins, isoflavones, flavones and neoflavonoids [11]. These flavonoids usually contain multiple phenolic hydroxyl groups, and show excellent antioxidation capacity [14,15]. During which, quercetin with anti-inflammation, anti-viral, anti-tumour, antioxidation and antibacterial activities have attracted great attention [16]. As categorized by chemical structure, quercetin is a type of flavonol. Due to the presence of hydroxyl groups, quercetin shows excellent antioxidation activity [17]. It could obviously inhibit the proliferation
⁎ Corresponding author at: Tianjin Key Laboratory of Food Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China. E-mail addresses: [email protected], [email protected] (Z.-T. Jiang).
https://doi.org/10.1016/j.colsurfb.2019.110429 Received 12 April 2019; Received in revised form 18 July 2019; Accepted 6 August 2019 Available online 11 August 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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and PVP. After that, 162 mg of FeCl3∙6H2O in 10 mL of absolute ethanol was then added. The solution was further stirred for 6 h in the dark. Greenish black precipitates were collected after centrifugation at 10,000 rpm for 5 min. The precipitates were washed thrice by absolute ethanol and then dispersed in water. Solid QFNPs were obtained by freeze-drying and stored in the refrigerator for further use.
of hepatic cancer cells [18] and neuroblastom cells [16]. Researches suggest that biological activities of quercetin might be attributed to the influence to enzyme activities and signaling pathway of cells [19]. Despite the outstanding bioactivities, application of quercetin is largely restricted by its poor water solubility. Various nano-carriers [20–22] were designed to improve the bioavailability of the quercetin. Although these exploration for antioxidation and anticancer study, direct fabrication of multifunctional nanoplatforms with quercetin is limited. Combining with nanotechnology, construction of such nanoplatforms would provide new opportunities for the application of quercetin. Due to the coordination interaction of phenolic hydroxyl with metal ions, phenolic compound often shows strong binding capacity with these metal ions. Especially, complexes of some phenolic compound such as gallic acid [23] and tannic acid [24] with Fe3+ show strong absorption in the NIR field (700–950 nm), rendering them capacity for photothermal therapy (PTT). Besides the bioactivities, quercetin also shows strong binding capacity with metal ions, such as Cu2+, Mg2+ and Fe3+ [25–27]. However, the potential of complex derived from quercetin and Fe3+ for PTT has not been explored. In the present study, the derived quercetin multifunctional nanoparticles (QFNPs) were fabricated with quercetin, PVP and Fe3+ through a one-pot synthesis strategy. QFNPs showed good water solubility. Upon irradiation by 808 nm laser, 4T1 cells (Mouse breast cancer cells) could be efficiently killed by the photothermal effect of QFNPs, indicating QFNPs could serve as efficient photothermal agents. Besides PTT, QFNPs also showed good antioxidation activity.
2.4. Drug release study Drug release behavior of the QFNPs in PBS (pH 7.4) was investigated. 28.8 mg of QFNPs in 2 mL of PBS was added into the dialysis bag (molecular weight cutoff: 8–14 kDa) with the two ends sealed with clips. The dialysis bag was placed in a preheated release medium (50 mL) at 37 °C. 5 mL of the dissolution medium was withdrawn at selected time points (0.5, 1, 2, 4, 6 and 8 h) and an equal volume of fresh PBS was added then. Content of the released flavonoid was measured by the aluminium chloride colorimetric method [28] with quercetin as standard. Cumulated drug release of the phenolic compound was calculated by the following formula: Cumulated release n−1 degree (%) = [(50Cn+5 ∑i = 1 Ci )/weight of the phenolic compound]×100%, where Cn is the concentration of flavonoid in the dissolution medium at the time point of n [29]. 2.5. Photothermal measurement of QFNPs in vitro Photothermal property of QFNPs was measured under 808 nm laser at power density of 3.0 W cm−2. For the measurement, a range of QFNPs solutions with concentrations of 0.2, 0.5, 1.0 and 2.0 mg mL-1 were treated with laser for 10 min, respectively. Temperature increments of the solutions along time were recorded by a thermocouple probe inserted into the solutions.
2. Materials and methods 2.1. Materials 4T1 Cells were obtained from Cell Bank of Shanghai, Chinese Academy of Sciences (Shanghai, China). Ferric chloride hexahydrate, 2, 2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), and PVP (M≈ 58,000) were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Quercetin (95%) was bought from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Absolute ethanol and 2,6di-tert-butyl-4-methylphenol (BHT) were purchased from Guangfu Fine Chemical Research Institute (Tianjin, China). 2′, 7′Dichlorodihydrofluorescein diacetate (DCFH-DA) and 2,2-diphenyl-1picrylhydrazyl (DPPH) were purchased from Sigma Aldrich (St. Louis, MO, USA). Sodium carbonate was bought from Tianjin Yingda Rare Chemical Reagents Factory (Tianjin, China). All chemicals were at least analytical grade and used without further purification. Deionized water was used throughout all the experiments.
2.6. Temperature stability study To investigate the temperature stability of the QFNPs, 100 mL of aqueous solution of QFNPs with concentration of 0.1 mg mL−1 was added into a 250 mL round bottom flask and heated with thermostat water bath. Temperature of the solution was maintained for 10 min at different temperatures (35, 50, 65, 80 and 95 °C) in the heating process. At selected time points (0, 5 and 10 min), absorbance of the solution at 292 nm was monitored during the isothermal periods. While 0 min was the time when temperature of the solution reached the selected temperatures. 2.7. Antioxidation capacity assay of QFNPs The antioxidation capacity of QFNPs was determined by DPPH free radical scavenging activity assay. For the assay, the solutions of QFNPs with different concentrations were mixed with DPPH ethanol solution (50 μg mL−1) at room temperature. After left static in the dark for 1 h, absorbance at 524 nm of the mixed solutions was measured. The scavenging rate (S) of DPPH calculated by S(%) = (1-(A-A1)/ A0)×100. Where A in the equation was the absorbance of the reaction mixture of samples with DPPH solution, A1 was the absorbance of the solutions of samples without DPPH and A0 was the initial absorbance of DPPH without the samples.
2.2. Instrumentation and characterization X-ray diffraction (XRD) analysis was performed on a Utima IV diffractometer (Rigaku, Japan) using Cu Kα radiation (λ = 1.5418 Å). Transmission electron microscopy (TEM) measurement was carried out on a HT7700 transmission electron microscope (Hitachi, Japan). The fourier transform infrared (FT-IR) spectra were recorded by a Nicolet IR iS10 spectrometer (Nicolet, USA). Dynamic light scattering (DLS) was carried out on a Malvern Zetasizer (Nano series ZS, UK). The UV–vis absorption spectra were obtained from a Lambda 25 UV/VIS spectrometer (PerkinElmer, USA). The content of iron ions was measured with inductively coupled plasma mass spectrometer (ICP-MS, Thermo Elemental X7, UK). Element analysis was conducted on vario EL cube elemental analyzer (Elementar, Germany).
2.8. Cytotoxicity assay in vitro Cytotoxicity assay of QFNPs toward 4T1 cells was carried out using the MTT method. Firstly, 4T1 cells were seeded into a 96-well plate (104 cells per well). After incubation for 24 h at 37 °C under 5% CO2, the stale culture medium was replaced by the fresh one. QFNPs were then added. The cells were further incubated with various concentrations of QFNPs for 24 h. After the incubation, the cells were washed by PBS and treated by MTT (10 μL, 5 mg mL−1) for 4 h. After this, 120 μL
2.3. Synthesis of QFNPs For the synthesis of QFNPs, 30 mg of quercetin and 513 mg of PVP were added into a 250 mL flask, followed by 40 mL of absolute ethanol. The mixture was stirred at room temperature to dissolve the quercetin 2
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from the previously reported procedure, no water was used during the synthesis process. Then precipitates would be formed, which is favorable for the separation and purification of the product. The QFNPs could be simply harvested by centrifugation, without the need of timeconsuming dialysis process, which usually takes 24 h or more time. To the best of our knowledge, this process have not be reported for other drugs or natural regents. PVP could serve as protecting ligands to stabilize the nanoparticles, rendering QFNPs high hydrophilicity. To confirm the protective effect of PVP, 100 μL of ethanol solution of Fe3+, quercetin and PVP with the same ratios to the nanoparticle synthesis procedure was added into a 20 mL vial. For control, 100 μL of ethanol solution with equal amount of Fe3+ and quercetin was added into another vial, without the addition of PVP. After natural evaporation of ethanol, same amount of water was added into the two vials. As shown in Fig. S1, the aggregates were formed for the solution without PVP, while a transparent solution was obtained with the addition of PVP. These results verified the protect effect of PVP. After the synthesis, absorption maxima of quercetin at 257 and 374 nm disappeared in the UV–vis absorption spectrum of QFNPs, while a new band at 292 nm appeared (Fig. S2). These might be attributed to the partial oxidation of quercetin by Fe3+ [33]. To confirm that, X-ray photoelectron spectroscopy (XPS) of QFNPs was investigated. Wide scan XPS spectrum of QFNPs indicated the presence of C, O, N and Fe (Fig. S3). Fe 2p3/2 peak position at 709.8 eV and Fe 2p1/2 peak position at 723.2 eV suggested the presence of Fe2+ in QFNPs (Fig. S4), confirming the oxidation of quercetin by Fe3+. Partial oxidation of the quercetin through the loss of two H yields a quinone [33]. The content of Fe and the quinone in QFNPs were also investigated. ICP-MS analysis indicated there was 57 mg Fe in one gram of QFNPs. While the C and N content in the QFNPs were determined to be 45.85% and 7.69% by element analysis. The C is from PVP and quercetin. While the N is from PVP. Thus content of the quinone was calculated to be 105 mg in one gram of QFNPs. Release profile of the from QFNPs in pH 7.4 PBS buffer suggested 41% quinone was released after 8 h (Fig. S5). Morphology of QFNPs was observed by TEM. The synthesized QFNPs are spherical like nanoparticles with an average size of 21 nm (Fig. 1). Micrograph with larger magnification suggested the internal structure of nanoparticles is not compact (Fig. S6). The hydrodynamic size of QFNPs was measured to be 23 nm in pure water, which was well accorded with the results by TEM. While hydrodynamic sizes of QFNPs in pH 7.4 buffer and pH 5 buffer were measured to be 62 nm and 270 nm, suggesting the aggregation trend of QFNPs in lower pH (Fig. S7). Zeta potential of QFNPs was near neutral in the pure water and pH 5 buffer (Fig. S8), indicating the capping of nonionic hydrophilic PVP
DMSO was added into the wells. The absorbance of the solution in each well was recorded by a multimode microplate reader. Each treatment group was repeated by five times. 2.9. Hemolysis assay Hemolysis assay was carried out according to the literature [30] with slight modifications. Blood of Kunming mouse obtained from Tianjin Medical University was collected in anticoagulant tube. After centrifugation at 3000 rpm for 5 min, supernatant was discarded. The erythrocytes were washed three times with physiological saline. The final red blood cells pellet was re-suspended with 1.5 mL of physiological saline. 120 μL of the diluted red blood cells suspension was mixed with 0.88 mL of QFNPs solutions at varied concentrations. For the positive and negative controls, 120 μL of the diluted red blood cells suspension was mixed with 0.88 mL of deionized water and physiological saline. After incubation at 37 °C for 3 h, the samples were centrifuged and the supernatants were transferred to a 96-well plate (100 μL/well). The plate was then placed in the multimode microplate reader. Absorbance of the supernatants at 540 nm was recorded. The hemolytic activity of red blood cells was calculated with the following formula: Percent hemolysis (%) = (sample absorbance-negative control absorbance)/ (positive control absorbance-negative control absorbance) × 100. 2.10. Cellular antioxidation activity assays The cellular antioxidation activity assay was performed according to literature with some modifications [31]. For the study, 4T1 cells were seeded in 96-well plates. After the incubation for 24 h, the culture medium was removed and the cells were washed with PBS. The cells were further treated with 10 μM DCFH-DA in treated medium for 1 h. Subsequently, the treated medium was discarded and the cells were washed twice with PBS. After that, the experimental groups were treated with 20 μL of QFNPs with different concentrations and 60 μL of HBSS. The blank and control group were treated with 20 μL of H2O and 60 μL of HBSS. After further incubation at 37 °C for 10 min, all the cells except the blank group were treated with 20 μL of 0.6 mM AAPH in HBSS. The blank group was treated with 20 μL of HBSS. Then fluorescence of the wells was monitored by the plate reader. Increment of fluorescence at 525 nm with the excitation wavelength of 488 nm was monitored by a microplate reader every 5 min for one hour. All samples and controls were repeated at least three times. 2.11. Cellular photothermal therapy assessment The photothermal therapy effect of QFNPs toward 4T1 cells was also studied. For the assay, 4T1 cells were cultured in a 96-well culture plate with the density of 104 cell per well for 24 h. The cells were washed with PBS and treated with QFNPs (800 μg mL−1, diluted in cell culture medium) for 1 h. Cells treated with cell culture medium without QFNPs were used as control. Subsequently, all the cells were irradiated by 808 nm laser under the power densities of 7 W cm-2 for 5 min. MTT assays were performed to evaluate the cell viability. All the cells were stained with calcein acetoxymethyl ester (calcein AM) and propidium iodide (PI). Then, luminescence imaging of the cells was carried out on the confocal microscopy. 3. Results and discussion Water soluble QFNPs were simply synthesized by mixing Fe3+, quercetin and PVP at room temperature. Ethanol was used as solvent during the synthesis. Firstly, quercetin and PVP were mixed and dissolved in ethanol. Both of the phenolic hydroxyl groups and the amide moieties of PVP could coordinate to Fe3+ [32]. Coordination polymer nanoparticles would be formed after the introduction of Fe3+. Different
Fig. 1. TEM micrograph of QFNPs. 3
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Fig. 2. DPPH radical scavenging capacity of QFNPs.
Fig. 4. Cellular antioxidation assay of 4T1 cells treated with various concentrations of QFNPs.
Fig. 5. Cell viability of 4T1 cells treated with different concentration of QFNPs through MTT assay.
when DPPH concentration decreased, it caused a decrease in absorbance at 524 nm. As shown in Fig. 2, the scavenging rate of DPPH increased as the concentration of QFNPs increased, suggesting the efficient radical scavenging capacity of QFNPs. Due to the good water solubility, QFNPs could provide a powerful tool for antioxidation study. Before the investigation of the potential of QFNPs for PTT, UV–vis absorption spectra of QFNPs were measured. As shown in Fig. S11, obvious NIR absorption around 808 nm was observed, and the absorption increased when the concentration of QFNPs was raised, indicating the possibility of QFNPs for PTT. After that, QFNPs solutions with different concentrations were irradiated by a 808 nm laser with the power of 3 W cm−2 for 10 min. Temperature changes of the solutions were then recorded. As shown in Fig. 3a, obvious temperature increments were observed. The temperature increment increased as the concentration of QFNPs increased. Temperature of QFNPs solution with concentration of 2.0 mg mL-1 increased by nearly 40 °C after 10 min irradiation, while temperature increment of pure water was only about 5 °C under the same condition (Fig. 3a). The thermal imaging of QFNPs was recorded (Fig. S12). The color of solutions turned from yellow to nearly white as the concentration of the solutions and irradiation time increased. Besides photothermal property, temperature stability of the QFNPs was also studied. As shown in Fig. S13, absorbance of the solution at 292 nm slightly changed at different temperatures, indicating the good temperature stability of the QFNPs between 35 and 95 °C. These results indicated QFNPs could be used as efficient photothermal agent. Photo-stability of QFNPs was also investigated. QFNPs solution was irradiated by 808 nm laser at 3 W cm−2 for 5-cycle laser off/on. During
Fig. 3. (a) Temperature elevation of QFNPs solutions with different mass concentration. (b) The photothermal response of QFNPs solution with the concentration of 1 mg mL−1 for 5-cycle laser off/on.
polymers. More negative zeta potential of QFNPs was observed in the pH 7.4 buffer. The XRD pattern indicated QFNPs are amorphous materials (Fig. S9). FT-IR spectra of quercetin, PVP and QFNPs are shown in Fig. S10. Observation of the band at 1289 cm−1 in the spectrum of QFNPs, representing the stretching vibration of C–N, suggested the presence of PVP in QFNPs. The C]O stretching vibration of PVP at 1670 cm−1 in PVP was shifted to 1653 cm−1 in QFNPs, due to the interaction between iron ions and PVP. Observation of the band at 1211 cm−1 for the stretching vibration of C–O in phenols and 3420 cm−1 for the stretching vibration of OeH in the spectrum of QFNPs indicated the presence of phenolic hydroxyl, which rendered QFNPs antioxidation capacity. DPPH free radical scavenging capacity assay was carried out to evaluate the antioxidation activity of QFNPs. DPPH was a very stable free radical. It showed strong absorbance around 524 nm. Therefore,
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Fig. 6. Bright field and fluorescence images of calcein AM (green, live cells) and PI (red, dead cells) stained 4T1 cells: (a) treated without QFNPs and laser; (b) treated with laser only; (c) treated with QFNPs only; (d) treated with QFNPs and laser. Scar bar is 100 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
each heating cycle, the solution was firstly irradiated by 808 nm laser for 5 min. The laser was subsequently turned off, left the solution untreated for another 5 min. Temperature oscillations of the solution during the heating cycles were recorded. As shown in Fig. 3b, the maximal temperature of QFNPs solution could reach 42.1 °C during the first heating cycle, which could still reach as high as 43.5 °C in the fifth heating cycle, indicating the good photothermal stability of QFNPs. The results suggest the excellent potential of QFNPs for PTT. Encouraged by the good free radical scavenging capacity, cellular antioxidation study of QFNPs toward 4T1 cells was carried out. DCFHDA was used to monitor the cellular oxidation conditions. After incubation with cells, the acetate groups of DCFH-DA could be removed by cellular esterases. DCFH-DA or DCFH in cell are nonfluorescent in reduction state. They would be fluorescent after the oxidation by oxidants or free radicals. Thus intensity of the fluorescence could be used as indicators of the oxidation status of the cells. The decrease of the fluorescence indicates the increase of antioxidation effect. AAPH was used as free radical generation source for the oxidation of DCFH-DA or DCFH in this study. For the study, 4T1 cells were firstly incubated with DCFH-DA. After the incubation, extracellular DCFH-DA was washed away. Then QFNPs and AAPH were added. Fluorescence at 525 nm of the cells treated with different concentration of QFNPs was monitored during a period of 1 h. The cells treated without AAPH and QFNPs were set as blank group and those treated without QFNPs as control. Integrated intensities of the cell groups during 1 h were shown in Fig. 4. Fluorescence signal was observed in the blank group, indicating the DCFH-DA could penetrate the cell membrane and be trapped intracellular. Fluorescence signal of the control group was much higher than the blank group, suggesting the effective oxidation of DFCH by AAPH. Fluorescence signal of the cells treated with QFNPs decreased with the increase of its concentration, indicating scavenging of the free radical generated from AAPH by QFNPs. All these results suggested that QFNPs could be efficient cellular antioxidants. Before the investigation of PTT effect of QFNPs for cancer cells,
cytotoxicity of QFNPs was studied. MTT assay was performed to assess the cytotoxicity of QFNPs toward 4T1 cells. As shown in Fig. 5, slight increment in the cell viability was observed after incubation for 24 h, when the concentration of QFNPs was below 0.5 mg mL−1. Cell viability was as high as 90% even though the concentration of QFNPs reached 0.8 mg mL−1, suggesting the low cytotoxicity of QFNPs. Besides cytotoxicity assay, hemocompatibility of QFNPs was investigated by erythrocyte hemolysis assay. As shown in Fig. S14, the percent hemolysis gradually increased with increasing concentration of QFNPs. The percent hemolysis was 1.7% when the concentration of the QFNPs was 250 μg mL−1, suggesting good hemocompatibility of QFNPs. After confirming the biocompatibility of QFNPs, PTT efficiency of QFNPs to 4T1 cells was further evaluated through MTT assay. An aliquot of 0.8 mg mL−1 of QFNPs was adopted to study the photothermal killing effect. 4T1 cells incubated with or without QFNPs were treated with 808 laser at 7 W cm-2 for 5 min. After the irradiation, the viability was only 15.3% for the cells incubated with QFNPs (Fig. S15), while 83.5% cells still survived for the groups incubated without QFNPs, suggesting the good PTT efficiency of QFNPs. The photothermal cell killing effect was further confirmed by the cell staining experiments. After the laser irradiation, the cells were stained with calcein AM and PI. The calcein AM could easily penetrate the cell membrane. The AM group of calcein AM could be removed by the esterase of live cells, resulting intense green fluorescence. It was widely used for the staining of live cells. While PI couldn’t pass through the cell membrane of live cell. But it could pass the disordered regions of dead cell and went into the nucleus. After that, PI would be embedded in the DNA double helix, generating red fluorescence. Thus PI was widely used for staining of dead cells. As shown in Fig. 6, obvious green fluorescence was observed for the cells treated without QFNPs (Fig. 6a and 6b) and the cells treated with QFNPs only (Fig. 6c), while no obvious red fluorescence signal was detected for those cells, indicating most of those cells survived after the treatment. However, obvious red fluorescence was observed for the cells treated with both of laser and QFNPs (Fig. 6d), while little green fluorescence was observed 5
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in the cells, suggesting almost all of the cells died after the treatment. These results further the good photothermal efficiency of QFNPs.
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4. Conclusions In summary, a simple one-pot synthesis strategy was developed for the fabrication of QFNPs with Fe3+, quercetin and PVP. The fabricated QFNPs showed good potential for PTT, along with efficient antioxidation capacity. Furthermore, low toxicity of Fe3+, quercetin and PVP renders QFNPs excellent biocompatibility. The synthesized QFNPs could be powerful tools for the application in biomedical field. This study will provide a new idea for exploration of the application of flavonoids. Acknowledgements This work was supported by the Tianjin Natural Science Foundation of China (17JCQNJC02400 and 16JCYBJC43300). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110429. References [1] S.M. Janib, A.S. Moses, J.A. MacKay, Imaging and drug delivery using theranostic nanoparticles, Adv. Drug Deliv. Rev. 62 (11) (2010) 1052–1063. [2] J. Xie, S. Lee, X. Chen, Nanoparticle-based theranostic agents, Adv. Drug Deliv. Rev. 62 (11) (2010) 1064–1079. [3] S. Naahidi, M. Jafari, F. Edalat, K. Raymond, A. Khademhosseini, P. Chen, Biocompatibility of engineered nanoparticles for drug delivery, J. Control. Release 166 (2) (2013) 182–194. [4] M. Muthiah, I.K. Park, C.S. Cho, Surface modification of iron oxide nanoparticles by biocompatible polymers for tissue imaging and targeting, Biotechnol. Adv. 31 (8) (2013) 1224–1236. [5] M.F. Zhang, M. Yudasaka, K. Ajima, A. Miyawaki, S. Iijima, Light-assisted oxidation of single-wall carbon nanohorns for abundant creation of oxygenated groups that enable chemical modifications with proteins to enhance biocompatibility, ACS Nano 1 (4) (2007) 265–272. [6] Z. Wang, N. Yu, W. Yu, H. Xu, X. Li, M. Li, C. Peng, Q. Wang, M. Zhu, Z. Chen, In situ growth of Au nanoparticles on natural melanin as biocompatible and multifunctional nanoagent for efficient tumor theranostics, J. Mater. Chem. B 7 (1) (2019) 133–142. [7] J.M. Perez, A. Asati, S. Nath, C. Kaittanis, Synthesis of biocompatible dextrancoated nanoceria with pH-dependent antioxidant properties, Small 4 (5) (2008) 552–556. [8] H.F. Qian, C.Q. Dong, J.F. Weng, J.C. Ren, Facile one-pot synthesis of luminescent, water-soluble, and biocompatible glutathione-coated CdTe nanocrystals, Small 2 (6) (2006) 747–751. [9] A.M. Abdellah, M.A. Sliem, M. Bakr, R.M. Amin, Green synthesis and biological activity of silver–curcumin nanoconjugates, Future Med. Chem. 10 (22) (2018) 2577–2588. [10] M.C. Moulton, L.K. Braydich-Stolle, M.N. Nadagouda, S. Kunzelman, S.M. Hussain, R.S. Varma, Synthesis, characterization and biocompatibility of "green" synthesized silver nanoparticles using tea polyphenols, Nanoscale 2 (5) (2010) 763–770. [11] C. Kanadaswami, L.-T. Lee, P.-P.H. Lee, J.-J. Hwang, F.-C. Ke, Y.-T. Huang, M.T. Lee, The antitumor activities of flavonoids, In Vivo 19 (5) (2005) 895–909.
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