Article Cite This: Langmuir 2018, 34, 9516−9524
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Development of Multifunctional Polydopamine Nanoparticles As a Theranostic Nanoplatform against Cancer Cells Jingjing Wang,† Yuan Guo,‡ Jie Hu,† Wenchao Li,† Yuejun Kang,†,§ Yang Cao,*,‡ and Hui Liu*,†,§,∥
Langmuir 2018.34:9516-9524. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 08/15/18. For personal use only.
†
Institute for Clean Energy and Advanced Materials, Faculty of Materials and Energy, Southwest University, Chongqing 400715, China ‡ Chongqing Key Laboratory of Ultrasound Molecular Imaging, Institute of Ultrasound Imaging, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China § Chongqing Engineering Research Center for Micro-Nano Biomedical Materials and Devices, Chongqing 400715, China ∥ State Key Laboratory of Molecular Engineering of Polymers (Fudan University), Shanghai 200433, China S Supporting Information *
ABSTRACT: Although demanding, the development of multifunctional theranostic nanoplatforms is attracting considerable worldwide interest. Herein, a theranostic nanoplatform with multifunctions based on polydopamine (PDA) nanoparticles (NPs) was developed, owning dual-imaging and dual-therapy functions for cancer theranostic applications. PDA NPs were generated using a facile polymerization method under alkaline conditions, followed by poly(ethylene glycol) (PEG) modification. Then, the obtained NPs were loaded with IR820 and Fe3+ ions to produce the final PEGylated PDA/IR820/Fe3+ (PPIF) NPs. The PPIF NPs thus generated displayed increasingly brighter photoacoustic and magnetic resonance signals with increasing NP concentration and were demonstrated to be cytocompatible and effectively taken up and internalized into HeLa cells. Under near-infrared light irradiation, PPIF NPs can produce heat and reactive oxygen species for photothermal/photodynamic combined cancer therapy. In this study, the versatility of PDA NPs was demonstrated to be promising as a multifunctional nanoplatform for potential cancer theranostic applications.
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INTRODUCTION
Among these NIR-based theranostic nanoplatforms, polydopamine (PDA) nanoparticles (NPs) currently attract particular interest.34,35 PDA NPs with tunable size and desirable stability and biocompatibility can be generated using facile self-polymerization methodology. The desirable NIR-responsive properties of these NPs make them promising candidates for PA imaging-guided PTT theranostic nanoplatforms.36−38 The imaging characteristics of these platforms can be improved by exploiting their inherent chelation properties in combination with other kinds of imaging modalities, such as magnetic resonance (MR) imaging,39 whereas their therapeutic functions can be enhanced by exploiting their strong drugloading properties.40,41 In addition, by loading other types of NIR-responsive agents, their PA and PT properties can be enhanced simultaneously.39,42 Nevertheless, in spite of the great progress that has been achieved when developing PDA NP-based theranostic nanoplatforms, their potential applicability in terms of dual-modal imaging-guided combined therapy still warrants further exploration.
Given the considerable threat to human health posed by cancer, it is of paramount importance to develop effective and powerful anticancer tools.1−7 In this regard, theranostic nanoplatforms, which consist of dual diagnostic and therapeutic components, are proven to have shown appreciable anticancer potential.8−12 These designed theranostic nanoplatforms facilitate the precise diagnosis and therapy of cancer, as well as enable real-time monitoring of the outcomes of therapy. Till now, different types of theranostic nanomaterials have been developed against cancer, including, but not limited to, noble metal-based nanomaterials,13−15 transition metal-based nanostructures, 16−19 copper chalcogenide semiconductors,20−22 and carbon-based nanostructures.23,24 Among these, theranostic nanoplatforms with near-infrared (NIR) laser-responsive properties are particularly attractive because of the therapeutic advantages of NIR light, such as relatively low absorption/scattering, rare nonspecific activation, and deeper tissue penetration.25 These developed nanoplatforms can function effectively in photoacoustic (PA) imaging-guided photothermal therapy (PTT) and/or photodynamic therapy (PDT) for cancer.26−33 © 2018 American Chemical Society
Received: May 28, 2018 Revised: July 21, 2018 Published: July 24, 2018 9516
DOI: 10.1021/acs.langmuir.8b01769 Langmuir 2018, 34, 9516−9524
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mixed at a weight ratio of 0.04:1 under vigorous stirring. After 4 h of reaction, the mixture was dialyzed to remove free Fe3+. The amount of Fe3+ loaded onto the NPs was analyzed by inductively coupled plasma optical emission spectrometry. The NPs thus generated were denoted as PPIF NPs. Characterization Techniques. The size and morphology of the generated NPs were determined by field emission scanning electron microscopy (FESEM, JSM-7800F). Proton NMR (1H NMR) spectra of the NPs were acquired via a Bruker AV 300 NMR (Karlsruhe, Germany) spectrometer in D2O solvent, and their UV−vis−NIR absorption spectra were obtained using a UV spectrophotometer (UV-1800, Shimadzu). The dynamic diameters and ζ-potentials of the NPs were determined by dynamic light scattering (Nano ZS90, Malvern Instruments, U.K.). The stability of IR820 loaded on the NPs was investigated by a cumulative release experiment. Briefly, 2 mL of the PPIF NP solution was enclosed in a semipermeable membrane (molecular weight cutoff = 14 000). The membrane was then immersed into an aqueous solution containing 10% FBS (78 mL). The whole system was maintained at a constant temperature of 37 °C in a shaker. The buffer medium (3.0 mL) was withdrawn at predetermined time intervals and the medium volume was maintained constant through replenishing with a comparable volume of buffer. The release profile of IR820 was measured using a UV−vis spectrophotometer. Evaluation of PA and MR Imaging Performance. A PA imaging device (Vevo LAZR, Canada) was employed to evaluate the photoacoustic properties of PPIF NPs. The test was performed using agarose gel molds. For analysis, aqueous solutions of PPIF NPs were injected into the hole. PA images with a depth of 20.00 mm and width of 23.04 mm were acquired under a laser wavelength of 706 nm. For T1-weighted MR imaging, aqueous solutions containing different concentrations of PPIF were transferred to tubes and analyses were performed using MR imaging equipment (Philips Healthcare, MA). All T1-weighted images were obtained using the parameters as follows: echo time = 9.2 ms, repetition time = 30 ms, field of view = 160 mm × 160 mm, and slice thickness = 3 mm. The T1 signal intensities of region of interest for all samples were analyzed. Detection of Reactive Oxygen Species. The ROS generation was measured using DPBF as a probe, which shows reduced absorbance in the presence of singlet oxygen and is commonly used for ROS detection.47−49 Typically, 20 μL of a DPBF solution (1.5 mg/mL in dimethylsulfoxide) was added to 3 mL of a solution containing PP, PPIF, and IR820. The concentration of PPIF was 75 ppm, whereas the concentrations of PP and IR820 were maintained constant with their contents in PPIF NPs. NP-free water was used as a control. The entire solution was irradiated using an 808 nm laser (LWIRL808, LASERWAVE, 0.5 W/cm2), and thereafter the absorbance of DPBF at 410 nm was measured every 2 min. Evaluation of Photothermal Performance. For comparison, IR820, PP, and PPIF NPs were suspended in aqueous solution and irradiated with an 808 nm NIR laser (1.0 W/cm2). The concentration of PPIF was 200 ppm, whereas the concentrations of PP and IR820 were maintained constant with their contents in PPIF NPs. NP-free water was used as a control. The resulting temperature changes were recorded at designated time intervals. Aqueous solutions of PPIF NPs at different concentrations were then irradiated under the same laser conditions to examine the concentration-dependent photothermal properties. The photothermal reproducibility and stability were evaluated by treating a 200 ppm PPIF NP solution using five cycles of heating (10 min) and cooling (20 min). NP-free DI water was used as a blank control. After the five cycles of heating and cooling, the PPIF NPs were characterized and compared with the prepared NPs. Cell Culture. HeLa cells (a human cervical carcinoma cell line) and L929 cells (a mouse fibroblast cell line) were cultured and subcultured in MEM supplemented with 10% FBS and 1% penicillin− streptomycin at 37 °C and 5% CO2 in a humidified incubator. In Vitro Cytotoxicity Assay. The viabilities of cells treated with PPIF NPs of different concentrations were quantified using a CCK-8 colorimetric assay. Briefly, 1.5 × 104 HeLa and L929 cells per well were co-incubated with PPIF NPs of different concentrations. Cells
Herein, we accordingly developed PDA NP-based theranostic nanoplatforms for PA/MR imaging-guided combined PTT/PDT. PDA NPs were fabricated using a well-developed self-polymerization method, followed by poly(ethylene glycol) (PEG) modification. The obtained PEGylated PDA (PP) NPs were subsequently loaded with IR820 and iron ions (Fe3+) to obtain the final PEGylated PDA/IR820/Fe3+ (PPIF) NPs. IR820, an NIR dye with a characteristic absorbance peak at approximately 820 nm, has been reported to show considerable promise with respect to PA imaging and PTT/PDT.43,44 Fe3+ ions can act as contrast agents with desirable biocompatibility for T1-weighted MR imaging.41,45 We hence evaluated the PA and MR imaging abilities of the generated PPIF NPs, as well as their reactive oxygen species (ROS)- and heat-generating abilities under NIR light irradiation. Furthermore, their cytocompatibility was investigated by CCK-8 assay, whereas their cellular uptake behavior was examined by flow cytometry and their intracellular localization and ROS-generating properties was investigated confocal laser scanning microscopy (LSM). Finally, their therapeutic performance against cancer cells was characterized using calcein AM/propidium iodide (PI) staining and the CCK-8 assay.
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MATERIALS AND METHODS
Materials. Dopami ne hydrochloride (9 8%), IR820 (C46H50ClN2NaO6S2, 80%), ammonium hydroxide (NH4OH, 28− 30 wt % aqueous solution), and Iron(III) chloride hexahydrate (FeCl3·6H2O, 97%) were bought from J&K Scientific Ltd. Ethanol (C2H5OH, AR) and 1,3-diphenylisobenzofuran (DPBF) were provided by Shanghai Titan Co., Ltd (Shanghai, China). Poly(ethylene glycol) (mPEG-NH2 MW 2000, 98%) was obtained from Shanghai Yanyi Co., Ltd (Shanghai, China). Minimum essential medium (MEM), penicillin−streptomycin solution, fetal bovine serum (FBS), and trypsin-containing ethylenediaminetetraacetic acid were purchased from Thermo Fisher Scientific (MA). Phosphate buffer saline (PBS), ROS assay kit, solutions of CCK-8, and Hoechst 33342 were obtained from Beyotime (Shanghai, China). A calcein AM/propidium iodide (PI) staining kit was provided by Shanghai Yu Bo Biotech Co., Ltd. Deionized (DI) water (18.2 MΩ cm), obtained from a water purification system (Synergy, Millipore, MA), was used in all preparation processes. Synthesis of PEGylated PDA NPs. PDA NPs were prepared according to the report with slight modification.36,46 Typically, DI water (9 mL), ethanol (4 mL), and ammonium hydroxide (0.5 mL) were initially mixed, whereafter dopamine hydrochloride (50 mg/mL, 1 mL) was feeded into the mixture. After mixing evenly, polymerization of dopamine monomers was performed in a room temperature water bath. After reaction for 24 h, the PDA NPs were collected and purified by centrifugation. Subsequently, the PDA NPs thus generated were modified with PEG chains to enhance their biocompatibility. In a typical procedure, PEG-NH2 was added into a PDA NP solution (10 mM Tris buffer, pH = 8.5) at a PEG/PDA weight ratio of 2:1. After 24 h of stirring, the generated PEGylated PDA (PP) NP products were separated via centrifugation and washed with DI water. Loading of PP NPs with IR820 and Iron Ions. A freshly prepared IR820 solution was mixed with PP NP aqueous solution at IR820/PP NP weight ratios of 0.02:1, 0.04:1, 0.06:1, and 0.08:1. This reaction was performed for 4 h in the dark. The formed NPs were collected and purified by centrifugation. Unloaded free IR820 was collected and analyzed using a UV−vis−NIR spectrophotometer. The loading content [weight of loaded IR820/(weight of loaded IR820 + weight of NPs) × 100%] and loading efficiency (weight of loaded IR820/weight of added IR820 × 100%) of IR820 on the NPs were calculated. The IR820-loaded PP NPs were further used to load iron ions. Typically, freshly prepared FeCl3 solution and NP solution were 9517
DOI: 10.1021/acs.langmuir.8b01769 Langmuir 2018, 34, 9516−9524
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Figure 1. (a) Schematic illustration of the synthesis of PEGylated PDA NPs. (b) 1H NMR spectra of PDA (1) and PEGylated PDA (2) NPs. (c) FESEM image and size distribution (inset) of the generated PEGylated PDA NPs.
Figure 2. (a) Schematic illustration of the synthesis of final PPIF NPs. (b) Loading content and loading efficiency of IR820 with a series of IR820/ PP feeding weight ratios. (c) UV−vis−NIR spectra of PP NPs with different loading amounts of IR820. (d) FESEM image and size distribution (inset) of the generated PPIF NPs. (e) Hydrodynamic size of the generated PPIF NPs.
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Figure 3. (a) PA and (b) MR images and the corresponding signals of PPIF NPs at different concentrations.
Figure 4. (a) Normalized absorbance of DPBF at 410 nm under laser irradiation (0.5 W/cm2) when mixed with IR820, PP NPs, and PPIF NPs. (b) Temperature curves of IR820, PP, and PPIF NP aqueous solutions. The concentration of PPIF was 200 ppm, whereas the concentrations of PP and IR820 were maintained constant with their contents in PPIF NPs. (c) Temperature curves of PPIF NPs at different concentrations. (d) Temperature curve of 200 ppm PPIF NP aqueous solution during five laser on−off cycles. An 808 nm laser (1.0 W/cm2) was used for irradiation procedures in (b−d). NP-free water was used as control. 33342 and IR820, respectively. The fluorescence emissions through two corresponding channels were obtained using confocal laser scanning microscopy (LSM 780, Carl Zeiss, Germany). Intracellular ROS Generation Detection. Briefly, HeLa cells (1.0 × 105) co-incubated with NPs at final concentrations of 0, 50, 100, and 150 ppm were added. After co-incubation for 6 h, the cells were incubated with the fluorescer 2,7-dichlorofluorescein diacetate (DCFH-DA; included in the purchased ROS assay kit) for 30 min to detect the levels of ROS in cells. Thereafter, free DCFH-DA was removed and the cells were irradiated with a laser (808 nm, 1.0 W/ cm2). Laser channels of excitation wavelength 519 and 773 nm were utilized to excite DCF and IR820, respectively. The fluorescence emissions through two corresponding channels and the cell images of the bright field were recorded by confocal laser scanning microscopy. In Vitro Combined Therapy of Cancer Cells. Briefly, 1.5 × 104 HeLa cells were co-incubated with PPIF NPs of different concentrations. After a 6 h incubation, the cells were treated with the same laser for 10 min. Their cell viabilities were evaluated using the aforementioned CCK-8 assay.
treated with PBS were tested as control. After 24 and 48 h, the medium was removed and each well was washed using PBS. Thereafter, a standard CCK-8 assay was employed, following which the absorbance of the solution in each well was measured at 450 nm using a microplate reader (SPARK 10 M, Tecan). The mean and standard deviation of measurements for triplicate wells were reported for each sample. Cellular Uptake and Intracellular Localization. Flow cytometry analysis was used to investigate the cellular uptake behavior of the PPIF NPs. Briefly, 1.5 × 105 cells per well were co-incubated with NPs at concentrations of 50, 100, and 150 ppm. After coincubation for 3 or 6 h, the medium was removed and the wells were washed with PBS. The cells were then trypsinized, resuspended in PBS, and analyzed via flow cytometry (NovoCyte, ACEA). For each sample, 1 × 104 cell events were measured. For determination of intracellular localization, 1.0 × 105 cells per well were co-incubated with NPs at 150 ppm. After 6 h, the cell nuclei were stained using Hoechst 33342. Laser channels of excitation wavelength 455 and 773 nm were utilized to excite the Hoechst 9519
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Figure 5. Relative viability of HeLa and L929 cells co-incubated with PPIF NPs for 24 h (a) and 48 h (b) measured by CCK-8 assay.
chemical shift associated with the −CH2− protons of the PEG chain.51, Following PEGylation, the surface charge of the NPs decreased from −28.3 ± 1.3 to −13.1 ± 0.1 mV. The morphology of the PEGylated PDA NPs was determined by FESEM to be spherical or hemispherical in shape, with a mean diameter of 81.1 ± 7.8 nm, and the particles were found to have a relatively narrow size distribution (Figure 1c). Their hydrodynamic size was tested to be 161.1 ± 1.2 nm (Figure S1). The PEGylated PDA NPs thus generated were functionalized through loading with IR820 and Fe3+ (Figure 2a), which can be absorbed and chelated on the NP surface by means of π−π/electrostatic interactions and coordination reaction, respectively.42,45 By increasing the IR820/PP feeding weight ratio, the loading content of IR820 increased gradually to reach a plateau, indicating the utilization saturation of the PDA NP surface. However, the high loading content compromised their corresponding loading efficiency (Figure 2b). As a consequence of IR820 loading, a distinctive absorbance peak appeared at approximately 820 nm, which increased in height with an increase in IR820 feeding weight (Figure 2c). In overall consideration of the loading content and loading efficiency, a feed ratio of 0.06:1 was selected for further study. After loading IR820, iron ions were further loaded onto the NPs. The loading content of iron ions was determined to be 14.8 μg per mg of NPs. In addition to the reported desirable holding ability of PDA NPs for Fe3+,12,39 they could also stably absorb IR820 (Figure S2). The PPIF NPs eventually generated had a morphology and diameter similar to those of PP NPs (Figure 2d). Their hydrodynamic size was determined to be 211.7 ± 0.55 nm, with a relatively narrow polydispersity of 0.198 ± 0.005 (Figure 2e). After 6 months of storage at 4 °C, the formed PPIF displayed a UV−vis−NIR spectrum and hydrodynamic size similar to those of the as-prepared NPs (Figure S3), indicating their desirable stability. Their ζpotentials varied when dispersed in distinct solutions (Table S1), which may be caused by the absorption of inorganic salts onto the surface of the NPs. Evaluation of PA and MR Imaging Performance. Having generated the PPIF NPs, we went on to explore their potential applications for PA and MR imaging. Using a laser pulse of 706 nm, PPIF NPs at different concentrations yielded distinct PA signals (Figure 3a). Quantitative data analysis further indicated that these signals were enhanced with increasing concentration. In the concentration range of 0.1− 0.6 mg/mL, the corresponding PA signals increased linearly with NP concentration (Figure 3a, inset). It should be noted that PPIF NPs displayed higher PA signals than those of PP
The photoresponsive therapy effect of PPIF NPs on HeLa cells was further confirmed using the calcein AM/PI staining method. For this determination, HeLa cells were co-incubated with IR820, PP, and PPIF NPs of different concentrations. The concentrations of PP and IR820 were maintained constant with their contents in PPIF NPs. After 6 h, the cells were treated with the same laser for 10 min. After washing with PBS, the cells were stained with calcein AM/PI solution, whereafter the images were captured using an inverted microscope (IX73, Olympus).
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RESULTS AND DISCUSSION Synthesis and Characterization of the PPIF NPs. The preparation process for PEGylated PDA NPs is depicted in
Figure 6. Flow cytometry analysis of HeLa cells after co-incubation with PPIF NPs for 3 and 6 h.
Figure 1a. Dopamine monomers self-polymerized in an alkaline medium to form the PDA NPs. The NP surface was then modified by the addition of PEG chains (containing −NH2 groups) in Tris buffer (pH 8.5) via the Michael addition and/ or Schiff base reaction.50 Successful PEG chain modification was confirmed by 1H NMR spectra analysis (Figure 1b). Compared with PDA NPs, a new peak appeared at approximately 3.5 ppm, which could be attributed to the 9520
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Figure 7. Intracellular detection of ROS generated by PPIF NPs in response to an 808 nm laser (1.0 W/cm2) for 10 min. Scale bar: 20 μm.
Figure 8. Relative viability (a) and fluorescence images (b) of HeLa cells co-incubated with IR820, PP, or PPIF NPs, respectively, for 6 h and then irradiated using an 808 nm laser (1.0 W/cm2) for 10 min. The concentrations of PPIF were indicated in the horizontal axis of (a) and 150 ppm in (b), whereas the concentrations of PP and IR820 were maintained constant with their contents in PPIF NPs. The green and red fluorescence in cells stained with calcein AM and PI indicate live and dead cells, respectively. Scale bar: 100 μm.
increase in NP concentration, with the quantitatively measured signals showing a positive linear relationship with NP concentration (Figure 3b). Detection of Reactive Oxygen Species. The ROS generation under laser irradiation was examined using DPBF as a probe. A relative low power density (0.5 W/cm2) was
NPs at the same concentration (Figure S4), indicating that the loaded IR820 can improve the NIR-responsive properties of the PDA NPs. This is due to the obvious absorbance of IR820 around 706 nm, which could also provide PA signals (Figure S5). Furthermore, the T1-weighted MR images obtained using the generated PPIF NPs became increasingly brighter with an 9521
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generated ROS indicate the potential utility of PPIF NPs in PDT of cancer cells. In Vitro Combined Cancer Therapy. The photothermal/ photodynamic therapeutic properties of PPIF NPs were investigated using the CCK-8 method (Figure 8a). IR820 and PP NPs were used as control. When incubated with IR820 for 6 h and treated with laser irradiation, the viability of cells showed a decrease of 13.7%, which could be attributed to the PDT effect of IR820. After 6 h co-incubation with PP NPs, cell viability following laser irradiation decreased to 51.0%, which was caused by the PTT effect of PP NPs. Furthermore, in response to treatment with PPIF NPs and laser, the relative cell viabilities decreased markedly with an increase of the NP concentration. Notably, after 10 min of laser irradiation, the cells co-incubated with 150 ppm PPIF NPs showed the cell viability less than 10%, indicating a significant photoresponsive ablation effect on HeLa cells. Calcein AM/PI staining method was further performed, in which the green and red fluorescence indicate live and dead cells, respectively. After a 6 h coincubation with PPIF and a 10 min laser irradiation, the green fluorescence of cells in the laser-irradiated area was substantially weaker than that of blank cells and cells treated with PP NPs (Figure 8b). This is consistent with the cell viability data obtained using the CCK-8 assay.
selected in this study to avoid the damage of DPBF by heat, during which condition all samples showed a temperature evaluation less than 10 °C (Figure S6). The absorbance of DPBF was measured and analyzed according to the irradiation time (Figures 4a and S7). It can be seen that the relative absorbance of DPBF in the PPIF and IR820 groups decreased with an increase in irradiation time, indicating the effective generation of ROS.47 They showed similar absorbance decrease at the same IR820 concentration, indicating ROS was mainly generated by IR820. The similar curves of PP NPs and water indicated no generation of ROS. Evaluation of Photothermal Performance. The heatgenerating property of the formed NPs was evaluated under irradiation of an 808 nm laser. At 200 ppm, PPIF showed a considerably higher photothermal performance, which was due to the combination of two kinds of photothermal agents PP and IR820 (Figure 4b). The photothermal performance of PPIF NPs was enhanced by increasing the NP concentration (Figure 4c). Furthermore, we assessed the photothermal reproducibility of PPIF NPs by irradiating them with five laser on (10 min)−off (20 min) cycles (Figure 4d). The evaluated temperature remained almost the same throughout the five cycles, indicating that there was no photobleaching. After the five cycles of irradiation, the PPIF NPs had a morphology and size distribution comparable to those of the prepared NPs. Their hydrodynamic size was measured as 221.3 ± 1.5 nm, with a polydispersity of 0.161 ± 0.023. Their optical absorbance was also similar to that of the as-prepared NPs (Figure S8). These data indicate that the generated PPIF NPs had a more desirable photothermal stability compared with conventional small molecular organic photothermal agents. In Vitro Cytotoxicity Assay. An evaluation of in vitro cytocompatibility is essential for assessing the biomedical applications of nanomaterials. In the present study, we selected HeLa cells and L929 cells to evaluate the cytotoxicity of the generated PPIF NPs using a well-established CCK-8 method. After co-incubation with PPIF NPs, cell viabilities were measured to be similar to those of PBS-treated cells (Figure 5). Given that we observed no obvious decrease in cell viability, it can be assumed that the generated PPIF NPs own desirable cytocompatibility. Cellular Uptake and Intracellular Localization. Using HeLa cells as a model system, the cellular uptake behavior of PPIF NPs was analyzed using flow cytometry (Figure 6). After a 3h co-incubation, the percentage of fluorescence-positive cells was found to have increased with the NP concentrations. The cells co-incubated with 150 ppm PPIF NPs displayed a fluorescence-positive percentage of 97.67%, indicating the effective uptake of PPIF NPs by the HeLa cells. Furthermore, by increasing the incubation time to 6 h, we demonstrated that their cellular uptake behavior was incubation time-dependent. Using confocal laser scanning microscopy, we subsequently assessed the intracellular localization of the internalized PPIF NPs (Figure S9). The imaging results revealed that all internalized PPIF NPs were surrounding the cell nuclei, thereby implying a cytoplasmic localization. Detection of Intracellular ROS Generation. DCFH-DA was employed as a probe to assess the intracellular ROS generation. As a standard fluorescent indicator, DCFH-DA can be converted to 2,7-dichlorofluorescin (DCF) when mixed with ROS. According to Figure 7, a considerably more intense green fluorescence appeared with an increase in NP concentration, indicating the generation of ROS. These
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CONCLUSIONS In summary, a type of multifunctional nanoplatform was developed using PEGylated PDA NPs loaded with IR820 and iron ions for cancer theranostic applications. The generated PPIF NPs were uniformly spherical in shape. The absorbed IR820 not only improved the NIR-responsive property of the NPs to enhance PA imaging and PTT efficiency but also induced ROS generation to endow the NPs with photodynamic therapeutic properties. The photostability of PPIF NPs was demonstrated to be desirable. The generated PPIF NPs displayed considerable contrast ability for T1-weighted MR imaging, and CCK-8 assays showed that these NPs exhibited desirable cytocompatibility within the studied concentration range. The cellular uptake behavior of these particles was shown to be incubation time- and concentrationdependent. Confocal images revealed that PPIF NPs were effectively internalized into HeLa cells. Following internalization, the intracellular generation of ROS was clearly detected, whereas in response to laser irradiation, PPIF NPs were found to show effective photoresponsive therapeutic properties. We accordingly believe that the theranostic PDA NPs developed in this study are promising candidates for further preclinical studies.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.8b01769. Characterization data of the formed NPs, NIRresponsive data, photothermal stability data, as well as intracellular localization images (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (Y.C.). *E-mail:
[email protected] (H.L.). 9522
DOI: 10.1021/acs.langmuir.8b01769 Langmuir 2018, 34, 9516−9524
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Yuejun Kang: 0000-0002-1021-0349 Hui Liu: 0000-0002-7648-0915 Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was funded by the National Natural Science Foundation of China (51703184 and 31671037), the Chongqing Research Program of Basic Research and Frontier Technology (cstc2017jcyjAX0066), the Fundamental Research Funds for the Central Universities from Southwest University (XDJK2018B007), a startup grant from Southwest University (SWU116027), China Postdoctoral Science Foundation funded projects (2015T80963 and 2016M590869), and Chongqing Postdoctoral Science Foundation funded project (Xm2015089).
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