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Dual-modal imaging and photodynamic therapy using upconversion nanoparticles for tumor cells† Chunna Yang,a Qiuling Liu,a Dacheng He,b Na Na,a Yunling Zhaoa and Jin Ouyang*a Here we synthesized silica-coated NaYF4:Yb,Tm@NaGdF4 nanoparticles with hypocrellin photosensitizers covalently incorporated inside the silica shells, combining dual modal imaging and photodynamic therapy (PDT) functions together. Under excitation at 980 nm, the tumor-targeting specificity of the asprepared nanomaterials efficiently enhanced as folic acid (FA) was conjugated. The internalization of UCNPs@SiO2@hypocrellin A–FA in HeLa cells and HEK-293 cells was observed by confocal microscopy and in vitro magnetic resonance imaging (MRI), which demonstrated that the as-prepared
Received 5th September 2014 Accepted 2nd October 2014
nanocomposites have the ability to target folate receptor (FR) (+) cells. Moreover, magnetic resonance (MR) measurements also demonstrated that the as-prepared nanocomposites could be used as a
DOI: 10.1039/c4an01642d
contrast agent for MRI. All these results showed the feasibility and potential of the as-prepared nanocomposites for simultaneous imaging and PDT application.
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1. Introduction Photodynamic therapy (PDT), as an effective therapeutic modality, utilizes photosensitizers (PSs) to generate cytotoxic reactive oxygen species (ROS) under an appropriate wavelength of light. These ROS kill cancer cells by damaging cellular compartment, including plasma membrane and lysosomes.1 However, most PSs with ring structure are hydrophobic and difficult to deliver.2 Strong self-aggregation in aqueous environment also signicantly reduces photodynamic efficiency as only monomers are appreciably photoactive.3 The classic method to improve activity of PSs is synthesizing their derivatives; however, the synthetic steps are complicated and not always possible. Moreover, several available PSs are excited by visible light, which limits tissue penetration, thereby the penetration depth is limited.4 To overcome these drawbacks of current PDT, upconversion nanoparticles with prominent advantages, such as low toxicity,5 good photostability,6 deep tissue penetration and little autouorescence of biological samples,7 have attracted various research interest and used as PSs carriers in recent years.8–10 Magnetic resonance imaging (MRI) is non-invasive with high spatial resolution and deep penetration depth; however, its sensitivity is lower than other imaging methods. Regrettably, the spatial resolution of uorescence imaging is limited, though it has good sensitivity and a
Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, 100875, Beijing, P. R. China. E-mail: [email protected]; Fax: +86-10-62799838
b
Key Laboratory for Cell Proliferation and Regulation Biology, Ministry of Education, Beijing Normal University, 100875, Beijing, P. R. China † Electronic supplementary 10.1039/c4an01642d
information
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available.
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short imaging time. It will offer a new possibility to compensate the shortcomings of single imaging approach to combine uorescence imaging and MRI. Nearly, Gd3+ doped dual-modal upconversion nanoprobes have attracted extensive interest and developed to be potential T1-weighted MRI contrast agents. Moreover for acquiring accurate high-resolution imaging, T1 contrast agent is more desirable than T2 agent as the intrinsic dark signal in T2-weighted MRI can mislead the clinical diagnosis.11 Unfortunately, the uorescent properties would observably decrease with magnetic Gd3+ ions co-doping inside an upconversion lanthanide nanocrystal, owing to the change of luminescent ions in the host environment. In addition, only a portion of the nanoparticle surface is occupied by Gd3+, thereby limiting the contact between Gd3+ ions and water protons. To solve these problems, NaYF4:Yb,Tm(Er)–NaGdF4 core–shell nanoparticles have been developed.12–15 NaYF4:Yb,Tm(Er) nanoparticles were used as the core, as they are the most efficient host material.16 Then, a NaGdF4 shell can be epitaxially grown on the core because of their similar crystal structure. This coating of shell not only enhances the intensity of luminescence through eliminating the surface defects, but also increases the number of Gd3+ on the surface; thus, improving their magnetic resonance (MR) relaxivity. It is also worth noting that Gd3+ iondoped nanocrystals might solve the problems of traditional gadolinium chelates used in clinical medicine, such as single modality, low sensitivity, lack of specic targeting and possible renal dysfunction.17,18 In addition to the carriers, the PDT effect is also inuenced by PSs loading efficiency. The previously reported physical adsorption of PSs possess relatively weak force and could result in the gradual detachment of PSs from nanoparticles.19,20 Covalent coupling of PSs with nanoparticles may be able to
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overcome these drawbacks. In our previous work, we reported the synthesis of silica-coated UCNPs with PSs that were covalently incorporated, which showed a better PDT performance.21,22 Another limitation of conventional PDT is the toxicity of PSs, such as Photofrin. Compared to many other PSs, hypocrellin A, a natural perylene quinone PS isolated from natural fungi sacs of Hypocrellin bambusae, are well-recognized as promising non-porphyrin photosensitizers for PDT applications, because of the high quantum yields of singlet oxygen and no toxicity in dark conditions.23,24 Recently, a few research about triple-function nanomaterials has been published.25,26 Moreover, the ability to target specic tissues at the same time is also important for biomedical imaging and PDT. In order to enhance tumor-targeting specicity, folic acid (FA) could be conjugated. Folate receptor (FR) is known to be over-expressed in various human cancerous cells, whereas the expression of FR on normal cells is low.27–29 For FR, FA is an ideal ligand with high affinity, stability, low cost and non-immunogenicity.30,31 In this study, hypocrellin A PSs-conjugated NaYF4: Yb,Tm@NaGdF4 (UCNPs@SiO2@hypocrellin A–FA) were synthesized for the T1-weighted MR/UCL imaging and PDT of cancer cells. A detailed scheme is illustrated in Scheme 1. The NaYF4:Yb,Tm@NaGdF4 (UCNPs) were successfully conjugated with hypocrellin A PSs by covalent binding during the silicacoating process. Compared with physical absorption, covalent conjugation eliminates the leakage of PSs and improves the PSs loading efficiency. The core–shell structure combines uorescent and magnetic properties compensating for the limitation of single imaging approach. Under the excitation from a 980 nm laser, UCNPs exhibit emission band at 470 nm, which overlaps with the absorption wavelength of hypocrellin A PSs, and then induce cancer cell apoptosis, which was investigated by ow cytometry, uorescence microscope imaging with Annexin VFITC/PI. MR studies both in aqueous and in vitro demonstrated that the as-prepared nanocomposites could be used as a contrast agent for MRI. To render the nanoparticles with targeting capability, FA was also conjugated. Confocal microscopy and MRI studies showed that UCNPs@SiO2@hypocrellin A–FA can specically target HeLa cells, which overexpress FAR. Therefore, we synthesized multifunctional nanomaterials with specicity, which can be the potential candidates for dual modal imaging and PDT applications.
2. 2.1
of
the
design
of
This journal is © The Royal Society of Chemistry 2014
the
multifunctional
Chemicals
All the chemicals were of analytical grade and were used without further purication. LnCl3 (Ln ¼ Y, Yb, Gd and Tm) (99.9%), tetraethoxysilane (TEOS), (3-aminopropyl)triethoxysilane (APTES), (3-isocyanatopropyl)triethoxysilane (TESPIC), oleic acid (OA, 90%), octadecene (ODE), NH4F, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 3-[4,5dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT), N-hydroxysuccinimide (NHS), Igepal CO-520, dimethyl sulfoxide (DMSO) were purchased from Alfa Aesar. 9,10Anthracenediyl-bis(methylene)dimalonic acid (ABDA), Annexin V-FITC Apoptosis Detection Kit were purchased from SigmaAldrich. Hypocrellin A was purchased from Cenbeijia Bio-Tech Co. Ltd. PBS, Dulbecco's Modied Eagle Medium (DMEM), fetal bovine serum (FBS), pnicillin–streptomycin solution were obtained from Peking Union Medical College Hospital, NH3$H2O, NaOH were from Beijing Chemical Works. All the water used in the experiment was ultrapure water (Millipore, Bedford, MA). 2.2 Synthesis of NaYF4:Yb,Tm@NaGdF4 nanoparticles (UCNPs) The monodisperse NaYF4:Yb,Tm nanocrystals were prepared according to earlier published methods.12–15 In detail, YCl3 (0.80 mmol), YbCl3 (0.18 mmol), and TmCl3 (0.02 mmol) were mixed with 6 mL OA and 15 mL ODE and heated to 150 C under nitrogen protection to form a homogeneous solution. Aer the solution was cooled to room temperature (RT), 10 mL of methanol solution containing NaOH (2.5 mmol) and NH4F (4 mmol) was added dropwise. Subsequently, the solution was slowly heated for 30 min at 50 C to evaporate the methanol. Then, the mixture was heated to 300 C for 1 h under nitrogen atmosphere. A NaGdF4 shell was grown over the core using a seed-mediated method. 0.5 mmol GdCl3 was dissolved in 6 mL OA and 15 mL ODE at 150 C. 0.5 mmol NaYF4:Yb,Tm nanoparticles in 10 mL hexane were added to the solution and the hexane solution was evaporated. Next, a solution of NaOH (2.5 mmol) and NH4F (4 mmol) was added. Aer the evaporation of methanol, the solution was heated to 300 C for 1 h under nitrogen atmosphere before it was cooled to RT. The resultant nanoparticles were precipitated with ethanol and washed with ethanol/water (1 : 1 v/v) several times. 2.3
Scheme 1 Schematic nanocomposites.
Experimental section
Synthesis of UCNPs@SiO2@hypocrellin A–FA
Preparation of the hypocrellin A molecule precursor. 10 mg hypocrellin A in 20 mL ethanol and 14 mL TESPIC were mixed. The mixture was stirred overnight under nitrogen atmosphere at 80 C. Aer concentrating by rotary evaporator, the products were dissolved in 2 mL DMSO. Synthesis of UCNPs@SiO2@hypocrellin A. 30 mL of cyclohexane solution of 10 mg UCNPs and 1.6 g Igepal CO-520 was ultrasonicated for 20 min. Next, 230 mL NH3$H2O (25%) was added and the mixture was stirred for 1 h. Subsequently, 100 mL TEOS (98%), 15 mL APTES (98%) and the as-prepared Analyst, 2014, 139, 6414–6420 | 6415
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hypocrellin A molecule precursor were added and stirred for 48 h in the dark at RT. The nal nanomaterials were precipitated and washed several times with ethanol. FA conjugation. Covalent binding of FA to the UCNPs@SiO2@hypocrellin A was conducted using a modication of the standard EDC–NHS reaction. Carboxyl groups of FA (10 mg) were activated by an EDC (4.2 mg)/NHS (6.4 mg) solution for 30 min (FA/EDC/NHS molar ratio ¼ 1 : 1 : 2.5). Next, 10 mg of the as-prepared nanomaterials were added and stirred for 12 h in the dark. The resulting products were centrifuged and washed 3 times with PBS (pH ¼ 7.4) and dried for further use. 2.4
Singlet oxygen measurements
To detect the generation of singlet oxygen, UCNPs@SiO2@ hypocrellin A–FA (100 mg mL1) was dispersed in a solution of PBS containing 20 mM ABDA. The mixture was irradiated by a 980 nm laser for 0, 5, 10, 15 min. The uorescence emission of ABDA was measured by a spectrophotometer with an excitation of 380 nm. 2.5
Cell culture and MTT assay
HeLa cells were obtained from Peking Union Medical College Hospital, and cultured in DMEM supplemented with 10% FBS and 0.01% penicillin–streptomycin solution at 37 C in a humidied 5% CO2 atmosphere. For dark toxicity tests, HeLa cells (104 cells per well) were seeded in 96-well plates and incubated overnight with 5% CO2 at 37 C. Aer being washed with PBS, the cells were incubated with different concentration (0, 25, 50, 100, 200, 400 mg mL1) nanomaterials for 12 h at 37 C in the dark. Cell viability was determined by the standard MTT assay method. MTT (20 mL 5 mg mL1) was added to each well. Then, the culture medium was carefully removed and replaced with 200 mL DMSO. Aer 10 min of agitation on a shaker, absorbance was measured at 490 nm by a microplate reader. 2.6
Confocal luminescence imaging
HeLa cells were cultured and incubated with UCNPs@SiO2@ hypocrellin A–FA and UCNPs@SiO2@hypocrellin A for 2 h at concentration of 200 mg mL1, respectively. Cell imaging was performed by a Two-Photon Laser Scanning Confocal Microscope-510 Meta. The blue emissions and red emissions from UCNPs were collected at 476 nm and 650 nm. 2.7 Fluorescence imaging of cell apoptosis and ow cytometric analysis Aer culture with the UCNPs@SiO2@hypocrellin A–FA (250 mg mL1) for 4 h, the cells were exposed to NIR light for 15 min, and then stained with Annexin V-FITC/PI for 15 min. Aer washing three times with the binding buffer, the labeled cells were observed with an Olympus IX71 uorescence microscope. For ow cytometric analysis, aer incubating with UCNPs@SiO2@hypocrellin A–FA (250 mg mL1) for 4 h, the cells were exposed to 980 nm laser irradiation for 15 min. The laser power density was 0.3 W cm2 during irradiation. The cells
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were resuspended in binding buffer and stained with AnnexinVFITC/PI. Then, the samples were analyzed by a BD FACSVamtage SE ow cytometer. 2.8
Magnetic relaxivity of UCNPs@SiO2@hypocrellin A–FA
The relaxivity measurements were carried out on a 3.0 T clinical MRI instrument. A series of aqueous solutions with different concentrations of UCNPs@SiO2@hypocrellin A–FA nanoparticles were prepared and transferred into 1.5 mL Eppendorf tubes for longitudinal magnetic relaxivity measurements. For in vitro MRI, HeLa cells and HEK-293 cell were incubated with the nanocomposites for 4 h. Following the incubation, the cells were washed and centrifuged and the resulting cell pellets were covered with 1% agarose solution. 2.9
Characterization
Transmission electron microscopy (TEM) was performed with JEOL JEM-2010 high-resolution transmission microscope (HRTEM). Powder X-ray diffraction (PXRD) was recorded on a PANalytical X'Pert PRO MPD diffractometer. Dynamic light scattering (DLS) was performed on a Nano-ZS Zetzsozer ZEN3600 (Malvern Instruments Ltd., U.K.). UV/vis absorption spectra were obtained by TU-1901 UV-vis spectrophotometer (Beijing, China). Excitation and emission spectra were measured by SHIMADZU RF-5301PC spectrophotometer with a xenon lamp and an external 980 nm laser (Hi-tech Optoelectronics Co. Ltd. China). Fourier transform infrared (FTIR) spectra were obtained on a Nicolet 380. Biological analysis was obtained by Olympus IX71 uorescence microscope, BD FACSVamtage SE ow cytometry, Two-Photon Laser Scanning Confocal Microscope-510 Meta. The relaxivity measurements were carried out on a 3.0 T clinical MRI instrument (781278 Achieva 3.0 T TX).
3. 3.1
Results and discussion Characterization of UCNPs@SiO2@hypocrellin A–FA
NaYF4:Yb,Tm were prepared by an established protocol.12–15 TEM analysis showed that the NaYF4:Yb,Tm nanoparticles were monodispersed with an average size of ca. 21 nm (Fig. 1A). Fig. 1B shows the diameter of NaYF4:Yb,Tm@NaGdF4 increase to about 26 nm without a change in their structural and morphological uniformity. The diffraction peaks of NaYF4: Yb,Tm@NaGdF4 can be indexed to the pure hexagonal phase NaYF4 crystal (JCPDS 16-0334) in Fig. 1C. The formation of hypocrellin A molecule precursor was investigated by FTIR shown in Fig. S1.† In the FTIR spectrum of TESPIC (Fig. S1B†), the transmission band at 2270 cm1 was attributed to the –NCO stretching vibration of isocyanate group. However, in the FTIR spectrum of hypocrellin A molecule precursor (Fig. S1C†), the characteristic peak almost disappeared, which proved the successful synthesis of hypocrellin A molecule precursor. The as-prepared UCNPs were coated with a layer of silica to form UCNPs@SiO2@hypocrellin A nanoparticles, which were highly uniform in size, at about 38 nm in diameter (Fig. S2A†). The existence and identication of UCNPs@SiO2@hypocrellin A
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Emission spectra of UCNPs under 980 nm excitation and UV/vis absorption spectrum of hypocrellin A. Inset is the image of UCNPs under ambient light and 980 nm laser excitation.
Fig. 2
Fig. 1 TEM images of (A) NaYF4:Yb,Tm nanoparticles (B) NaYF4: Yb,Tm@NaGdF4 nanoparticles (UCNPs) and (C) XRD pattern of UCNPs. The JCPDS 16-0334 line pattern for pure hexagonal phase NaYF4 (red line) is shown for reference.
were also conrmed by FTIR spectroscopy (Fig. S2B†). Aer silica coating, the spectrum showed peaks around 1070, 953 and 459 cm1, which corresponded to the Si–O–Si asymmetric stretching vibration and Si–O deformation vibration. Fig. S3† shows that hydrodynamic diameter distribution of UCNPs@SiO2@hypocrellin A was centered at 38 nm, which is consistent with the results of TEM. These facts indicated that UCNPs@SiO2@hypocrellin A was successfully synthesized. In the next step, we investigated the existence of UCNPs @SiO2@hypocrellin A–FA using UV/vis spectroscopy (Fig. S4†). Aer modifying FA ligands, characteristic absorption peaks of FA at 280 nm and 360 nm appeared, indicating the immobilization of FA, which was further supported by FTIR spectral measurements. As shown in Fig. S5B,† a strong band at 1690 cm1 was attributed to the C]O stretching vibrations of carboxyl group. Nevertheless, aer functionalization with FA, characteristic peak of the amido bond at 1540 cm1 was observed. These results showed the successful modication of UCNPs@SiO2@hypocrellin A–FA. 3.2
Luminescence properties
As shown in Fig. 2, under the excitation of a 980 nm NIR laser, the UCNPs showed distinct Tm3+emission bands at 450, 476, 650 and 700 nm, which correspond to 1D2–3F4, 1G4–3H6, 1 G4–3F4, 3F2,3–3H6 transitions of the Tm3+ ions, respectively.32 Simultaneously, the hypocrellin A exhibited intense absorption bands at around 465 nm, which considerably overlapped with the blue emission of the UCNPs. This overlap ensures that hypocrellin A molecules are able to absorb the visible emission released by the nanoparticles aer being exposed to NIR irradiation at 980 nm, and consequently generate singlet oxygen to kill cancer cells.33,34 3.3
oxygen by the as-prepared nanomaterials aer irradiation by 980 nm laser. When excited at 380 nm, ABDA emits uorescence in the 400–500 nm region. When reacting with singlet oxygen, it can yield an endoperoxide, which is non-uorescent.35,36 As shown in Fig. 3, as the irradiation time increases, the absorbance of ABDA decreases, which accounted for the generation of singlet oxygen. Furthermore, the effect of pH on the generation of singlet oxygen was also investigated. The pH value of PBS were separately set at 5 (pH in endosome), 6.8 (pH around cancer cells) and 7.4 (pH of blood). The uorescence spectra of ABDA with UCNPs@SiO2@hypocrellin A–FA nanoparticles at different pH value were obtained. As shown in Fig. S6,† The degree of uorescence decrease has no obvious difference in all of the conditions, which indicated that pH has a little inuence on the generation of singlet oxygen in these physiological conditions. In addition, UCNPs@SiO2(hypocrellin A)–FA with hypocrellin A simply deposited were also prepared. As can be seen in Fig. S7,† under the same condition, UCNPs@SiO2@ hypocrellin A–FA with hypocrellin A that were covalently bound generated more singlet oxygen because of a higher loading efficiency. 3.4
MTT assay and cell uptake
Before further in vitro experiment, the cytotoxicity of the asprepared nanomaterials was checked. The cytotoxicity of UCNPs@SiO2@hypocrellin A–FA was evaluated on the HeLa cells and HEK-293 cells by a standard MTT assay. As shown in
Singlet oxygen measurements
9,10-Anthracenediyl-bis (methylene) dimalonic acid (ABDA) was used as the indicator to investigate the generation of singlet
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Fig. 3 Fluorescence spectra of ABDA with UCNPs@SiO2@hypocrellin A–FA nanoparticles exposed to 980 nm NIR laser for 0, 5, 10, 15 min.
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Fig. S8,† more than 90% of HeLa cells and HEK-293 cells survived with a concentration of the samples as high as 400 mg mL1. The result indicated that the as-prepared nanomaterials exhibited low toxicity. Photodynamic efficiency of the as-prepared nanomaterials was assessed by MTT assay. Aer the incubation with the different concentrations of UCNPs@SiO2@hypocrellin A–FA for 24 h, the cells were irradiated with NIR light for different time and incubated for an additional 24 h. As shown in Fig. 4, compared to the control group without NIR light irradiation, the cell viability in the NIR light irradiated groups dramatically decreased with an increase in the concentration of UCNPs@SiO2@hypocrellin A–FA. Little difference in the viability of incubated cells was observed before and aer irradiation. These results suggested that the cytotoxicity was mainly contributed to the PDT effect. To visualize the in vitro targeting ability of UCNPs@SiO2@ hypocrellin A–FA, we performed confocal imaging with FRpositive [FR (+)] HeLa cells incubated with UCNPs@ SiO2@hypocrellin A–FA, UCNPs@SiO2@hypocrellin A and FRnegative [FR ()] HEK-293 cells cultured with UCNPs@SiO2@ hypocrellin A–FA. As shown in Fig. 5, under excitation at 980 nm, blue light and red light of UCNPs could be observed. HEK293 cells incubated with UCNPs@SiO2@hypocrellin A–FA demonstrated lower luminescence when compared with that of HeLa cells. Moreover, compared to the nontargeted UCNPs@SiO2@hypocrellin A nanoparticles, HeLa cells incubated with UCNPs@SiO2@hypocrellin A–FA were more uorescent, indicating higher cellular uptake of the targeted particles. Serial layer scanning can be seen in Fig. S9,† UCNPs@SiO2@hypocrellin A–FA can enter the cytoplasm of tumor cells rather than merely staining the membrane surface. Taken together, these results showed that UCNPs@ SiO2@hypocrellin A–FA can specically target cancer cells overexpressing FAR by the receptor-mediated delivery.
3.5
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Fig. 5 Confocal luminescence images of (A) FR (+) HeLa cells, (B) FR () HEK-293 cells incubated with UCNPs@SiO2@hypocrellin A–FA (200 mg mL1) and (C) FR (+) HeLa cells incubated with UCNPs@SiO2@hypocrellin A (200 mg mL1) for 1 h at 37 C.
FITC positive, PI positive), and necrotic cells (Annexin V-FITC negative, PI negative).37–39 HeLa cells were incubated with UCNPs@SiO2@hypocrellin A–FA (250 mg mL1) for 3 h at 37 C, and then exposed to NIR at 980 nm for 10 min. Aer Annexin V-FITC/PI staining, the cells were observed by an Olympus IX71 uorescence microscope. As shown in Fig. 6A–C, no obvious cell apoptosis was observed in control groups. However, apoptosis could be seen in Fig. 6D in the experimental group indicating that both UCNPs@SiO2@ hypocrellin A–FA and irradiation were indispensable to induce the apoptosis of the cells. Simultaneously, the results also indicated that the mechanism of cell death induced by UCNPs@SiO2@hypocrellin A–FA was apoptosis but not necrosis.
Fluorescence imaging and ow cytometry
To conrm that UCNPs@SiO2@hypocrellin A–FA may induce apoptotic death in tumor cells, the cells were stained with Annexin V-FITC/PI to distinguish early apoptotic cells (Annexin V-FITC positive, PI negative), late apoptotic cells (Annexin V-
Fig. 4 Viability of HeLa cells incubated with UCNPs@SiO2@ hypocrellin A–FA at different concentrations (0, 100, 200, 400 mg mL1) after irradiation with 980 nm of different time (0, 10 and 20 min).
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Fig. 6 UCNPs@SiO2@hypocrellin A–FA induced cell apoptosis as determined by fluorescence imaging using Annexin V-FITC/PI staining on HeLa cells. (A) HeLa cells without UCNPs@SiO2@hypocrellin A–FA treatment and NIR irradiation. (B) HeLa cells incubated without UCNPs@SiO2@hypocrellin A–FA and exposed to NIR laser for 15 min. (C) HeLa cells treated with UCNPs@SiO2@hypocrellin A–FA (250 mg mL1), without NIR irradiation. (D) HeLa cells incubated with UCNPs@SiO2@hypocrellin A–FA (250 mg mL1) and exposed to NIR laser for 15 min.
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conrmed that the as-prepared nanocomposites can be used as a targeted T1 MRI contrast agent to cancer cells that overexpress FR.
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4. Conclusions
Fig. 7 Flow cytometry of apoptosis of HeLa cells stained with Annexin V-FITC/PI induced by UCNPs@SiO2@hypocrellin A–FA.
We have developed a multifunctional nanosystem, which could serve as a platform for simultaneous bioimaging and PDT. NaYF4:Yb,Tm@NaGdF4 were encapsulated inside the silica shell in which hypocrellin A was covalently attached. The nanoparticles are able to enter cancer cells and induce cell death aer being exposed to the NIR light. Tailoring FA at the end improves the targeting efficacy in cancer cells, which was investigated by confocal microscopy and in vitro MRI. Moreover, owing to the presence of Gd3+ ions, the composites could also act as a targeted MR contrast agent, which was further conrmed by MR research both in aqueous and inside cells. Our study promises potential applications in clinical treatment and detection.
Acknowledgements Fig. 8 (A) Relaxation rate r1 for the different Gd3+ concentrations.
Inset: t1-weighted magnetic resonance images, (B) MR imaging of HeLa (a) and HEK-293 cells (b) after incubation with various concentrations of the as-prepared nanocomposites.
Apoptosis of HeLa cells induced by the as-prepared nanomaterials were also quantied with ow cytometry. Aer incubation with UCNPs@SiO2@hypocrellin A–FA (250 mg mL1) for 4 h, the HeLa cells were exposed to NIR irradiation for 15 min, and then stained with Annexin V-FITC/PI. As can be seen from Fig. 7, the percentage of early apoptosis and late apoptosis was 31.01%, further conrming apoptosis process occurring in cells. By comparison, apoptosis of HeLa cells induced by UCNPs@SiO2(hypocrellin A)–FA with hypocrellin A simply deposited were also determined by ow cytometry. As shown in Fig. S10,† UCNPs@SiO2(hypocrellin A)–FA showed lower percentage of apoptosis, which was consistent with the results of measurement of singlet oxygen production.
3.6
Magnetic relaxivity of UCNPs@SiO2@hypocrellin A–FA
The MRI capability of UCNPs@SiO2@hypocrellin A–FA was measured in water. As shown in Fig. 8A, with increasing concentration of Gd3+ ions, MR signal intensity enhanced gradually. The r1 relaxivity value was calculated to be 2.88 through the curve tting analysis with a coefficient of determination (R) of 0.998. MRI was also used to further conrm the specicity of UCNPs@SiO2@hypocrellin A–FA. HeLa cells and HEK-293 cells were treated with UCNPs@SiO2@hypocrellin A– FA with different Gd concentrations. As can be seen in Fig. 8B, MR signal intensity of HeLa cells treated with the as-prepared nanocomposites was higher than that of HEK-293 cells, which was because of FR mediated endocytosis. These results
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The authors gratefully acknowledge the support from the National Nature Science Foundation of China (21175014, 21475011, 21422503), the Fundamental Research Funds for the Central Universities, National Grant of Basic Research Program of China (No. 2011CB915504).
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14 F. Chen, W. B. Bu, S. J. Zhang, J. N. Liu, W. P. Fan, L. P. Zhou, W. J. Peng and J. L. Shi, Adv. Funct. Mater., 2013, 23, 298–307. 15 Y. I. Park, H. M. Kim, J. H. Kim, K. C. Moon, B. Yoo, K. T. Lee, N. Lee, Y. Choi, W. Park and D. Ling, Adv. Mater., 2012, 24, 5755–5761. 16 Z. Q. Li, Y. Zhang and S. Jiang, Adv. Mater., 2008, 20, 4765– 4769. 17 P. Hermann, J. Kotek, V. Kub´ıˇ cek and I. Lukeˇs, Dalton Trans., 2008, 3027–3047. 18 K. N. Raymond and V. C. Pierre, Bioconjugate Chem., 2005, 16, 3–8. 19 J. N. Shan, S. J. Budijono, G. H. Hu, N. Yao, Y. B. Kang, Y. G. Ju and R. K. Prud'homme, Adv. Funct. Mater., 2011, 21, 2488–2495. 20 C. Wang, H. Q. Tao, L. Cheng and Z. Liu, Biomaterials, 2011, 32, 6145–6154. 21 F. F. Wang, X. J. Yang, L. Ma, B. R. Huang, N. Na, E. Yingchun, D. C. He and J. Ouyang, J. Mater. Chem., 2012, 22, 24597–24604. 22 X. J. Yang, Q. Q. Xiao, C. X. Niu, N. Jin, J. Ouyang, X. Y. Xiao and D. C. He, J. Mater. Chem. B, 2013, 1, 2757–2763. 23 Z. Diwu, Photochem. Photobiol., 1995, 61, 529–539. 24 S. M. Ali, S. K. Chee, G. Y. Yuen and M. Olivo, J. Photochem. Photobiol., B, 2001, 65, 59–73. 25 Y. I. Park, H. M. Kim, J. H. Kim, K. C. Moon, B. Yoo, K. T. Lee, N. Lee, Y. Choi, W. Park, D. Ling, K. Na, W. K. Moon, S. H. Choi, H. S. Park, S. Y. Yoon, Y. D. Suh, S. H. Lee and T. Hyeon, Adv. Mater., 2012, 24, 5755–5761. 26 X. F. Qiao, J. C. Zhou, J. W. Xiao, Y. F. Wang, L. D. Sun and C. H. Yan, Nanoscale, 2012, 4, 4611–4623.
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Dual-modal imaging and photodynamic therapy using upconversion nanoparticles for tumor cells† Chunna Yang,a Qiuling Liu,a Dacheng He,b Na Na,a Yunling Zhaoa and Jin Ouyang*a Here we synthesized silica-coated NaYF4:Yb,Tm@NaGdF4 nanoparticles with hypocrellin photosensitizers covalently incorporated inside the silica shells, combining dual modal imaging and photodynamic therapy (PDT) functions together. Under excitation at 980 nm, the tumor-targeting specificity of the asprepared nanomaterials efficiently enhanced as folic acid (FA) was conjugated. The internalization of UCNPs@SiO2@hypocrellin A–FA in HeLa cells and HEK-293 cells was observed by confocal microscopy and in vitro magnetic resonance imaging (MRI), which demonstrated that the as-prepared
Received 5th September 2014 Accepted 2nd October 2014
nanocomposites have the ability to target folate receptor (FR) (+) cells. Moreover, magnetic resonance (MR) measurements also demonstrated that the as-prepared nanocomposites could be used as a
DOI: 10.1039/c4an01642d
contrast agent for MRI. All these results showed the feasibility and potential of the as-prepared nanocomposites for simultaneous imaging and PDT application.
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1. Introduction Photodynamic therapy (PDT), as an effective therapeutic modality, utilizes photosensitizers (PSs) to generate cytotoxic reactive oxygen species (ROS) under an appropriate wavelength of light. These ROS kill cancer cells by damaging cellular compartment, including plasma membrane and lysosomes.1 However, most PSs with ring structure are hydrophobic and difficult to deliver.2 Strong self-aggregation in aqueous environment also signicantly reduces photodynamic efficiency as only monomers are appreciably photoactive.3 The classic method to improve activity of PSs is synthesizing their derivatives; however, the synthetic steps are complicated and not always possible. Moreover, several available PSs are excited by visible light, which limits tissue penetration, thereby the penetration depth is limited.4 To overcome these drawbacks of current PDT, upconversion nanoparticles with prominent advantages, such as low toxicity,5 good photostability,6 deep tissue penetration and little autouorescence of biological samples,7 have attracted various research interest and used as PSs carriers in recent years.8–10 Magnetic resonance imaging (MRI) is non-invasive with high spatial resolution and deep penetration depth; however, its sensitivity is lower than other imaging methods. Regrettably, the spatial resolution of uorescence imaging is limited, though it has good sensitivity and a
Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, 100875, Beijing, P. R. China. E-mail: [email protected]; Fax: +86-10-62799838
b
Key Laboratory for Cell Proliferation and Regulation Biology, Ministry of Education, Beijing Normal University, 100875, Beijing, P. R. China † Electronic supplementary 10.1039/c4an01642d
information
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(ESI)
available.
See
DOI:
short imaging time. It will offer a new possibility to compensate the shortcomings of single imaging approach to combine uorescence imaging and MRI. Nearly, Gd3+ doped dual-modal upconversion nanoprobes have attracted extensive interest and developed to be potential T1-weighted MRI contrast agents. Moreover for acquiring accurate high-resolution imaging, T1 contrast agent is more desirable than T2 agent as the intrinsic dark signal in T2-weighted MRI can mislead the clinical diagnosis.11 Unfortunately, the uorescent properties would observably decrease with magnetic Gd3+ ions co-doping inside an upconversion lanthanide nanocrystal, owing to the change of luminescent ions in the host environment. In addition, only a portion of the nanoparticle surface is occupied by Gd3+, thereby limiting the contact between Gd3+ ions and water protons. To solve these problems, NaYF4:Yb,Tm(Er)–NaGdF4 core–shell nanoparticles have been developed.12–15 NaYF4:Yb,Tm(Er) nanoparticles were used as the core, as they are the most efficient host material.16 Then, a NaGdF4 shell can be epitaxially grown on the core because of their similar crystal structure. This coating of shell not only enhances the intensity of luminescence through eliminating the surface defects, but also increases the number of Gd3+ on the surface; thus, improving their magnetic resonance (MR) relaxivity. It is also worth noting that Gd3+ iondoped nanocrystals might solve the problems of traditional gadolinium chelates used in clinical medicine, such as single modality, low sensitivity, lack of specic targeting and possible renal dysfunction.17,18 In addition to the carriers, the PDT effect is also inuenced by PSs loading efficiency. The previously reported physical adsorption of PSs possess relatively weak force and could result in the gradual detachment of PSs from nanoparticles.19,20 Covalent coupling of PSs with nanoparticles may be able to
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overcome these drawbacks. In our previous work, we reported the synthesis of silica-coated UCNPs with PSs that were covalently incorporated, which showed a better PDT performance.21,22 Another limitation of conventional PDT is the toxicity of PSs, such as Photofrin. Compared to many other PSs, hypocrellin A, a natural perylene quinone PS isolated from natural fungi sacs of Hypocrellin bambusae, are well-recognized as promising non-porphyrin photosensitizers for PDT applications, because of the high quantum yields of singlet oxygen and no toxicity in dark conditions.23,24 Recently, a few research about triple-function nanomaterials has been published.25,26 Moreover, the ability to target specic tissues at the same time is also important for biomedical imaging and PDT. In order to enhance tumor-targeting specicity, folic acid (FA) could be conjugated. Folate receptor (FR) is known to be over-expressed in various human cancerous cells, whereas the expression of FR on normal cells is low.27–29 For FR, FA is an ideal ligand with high affinity, stability, low cost and non-immunogenicity.30,31 In this study, hypocrellin A PSs-conjugated NaYF4: Yb,Tm@NaGdF4 (UCNPs@SiO2@hypocrellin A–FA) were synthesized for the T1-weighted MR/UCL imaging and PDT of cancer cells. A detailed scheme is illustrated in Scheme 1. The NaYF4:Yb,Tm@NaGdF4 (UCNPs) were successfully conjugated with hypocrellin A PSs by covalent binding during the silicacoating process. Compared with physical absorption, covalent conjugation eliminates the leakage of PSs and improves the PSs loading efficiency. The core–shell structure combines uorescent and magnetic properties compensating for the limitation of single imaging approach. Under the excitation from a 980 nm laser, UCNPs exhibit emission band at 470 nm, which overlaps with the absorption wavelength of hypocrellin A PSs, and then induce cancer cell apoptosis, which was investigated by ow cytometry, uorescence microscope imaging with Annexin VFITC/PI. MR studies both in aqueous and in vitro demonstrated that the as-prepared nanocomposites could be used as a contrast agent for MRI. To render the nanoparticles with targeting capability, FA was also conjugated. Confocal microscopy and MRI studies showed that UCNPs@SiO2@hypocrellin A–FA can specically target HeLa cells, which overexpress FAR. Therefore, we synthesized multifunctional nanomaterials with specicity, which can be the potential candidates for dual modal imaging and PDT applications.
2. 2.1
of
the
design
of
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the
multifunctional
Chemicals
All the chemicals were of analytical grade and were used without further purication. LnCl3 (Ln ¼ Y, Yb, Gd and Tm) (99.9%), tetraethoxysilane (TEOS), (3-aminopropyl)triethoxysilane (APTES), (3-isocyanatopropyl)triethoxysilane (TESPIC), oleic acid (OA, 90%), octadecene (ODE), NH4F, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 3-[4,5dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT), N-hydroxysuccinimide (NHS), Igepal CO-520, dimethyl sulfoxide (DMSO) were purchased from Alfa Aesar. 9,10Anthracenediyl-bis(methylene)dimalonic acid (ABDA), Annexin V-FITC Apoptosis Detection Kit were purchased from SigmaAldrich. Hypocrellin A was purchased from Cenbeijia Bio-Tech Co. Ltd. PBS, Dulbecco's Modied Eagle Medium (DMEM), fetal bovine serum (FBS), pnicillin–streptomycin solution were obtained from Peking Union Medical College Hospital, NH3$H2O, NaOH were from Beijing Chemical Works. All the water used in the experiment was ultrapure water (Millipore, Bedford, MA). 2.2 Synthesis of NaYF4:Yb,Tm@NaGdF4 nanoparticles (UCNPs) The monodisperse NaYF4:Yb,Tm nanocrystals were prepared according to earlier published methods.12–15 In detail, YCl3 (0.80 mmol), YbCl3 (0.18 mmol), and TmCl3 (0.02 mmol) were mixed with 6 mL OA and 15 mL ODE and heated to 150 C under nitrogen protection to form a homogeneous solution. Aer the solution was cooled to room temperature (RT), 10 mL of methanol solution containing NaOH (2.5 mmol) and NH4F (4 mmol) was added dropwise. Subsequently, the solution was slowly heated for 30 min at 50 C to evaporate the methanol. Then, the mixture was heated to 300 C for 1 h under nitrogen atmosphere. A NaGdF4 shell was grown over the core using a seed-mediated method. 0.5 mmol GdCl3 was dissolved in 6 mL OA and 15 mL ODE at 150 C. 0.5 mmol NaYF4:Yb,Tm nanoparticles in 10 mL hexane were added to the solution and the hexane solution was evaporated. Next, a solution of NaOH (2.5 mmol) and NH4F (4 mmol) was added. Aer the evaporation of methanol, the solution was heated to 300 C for 1 h under nitrogen atmosphere before it was cooled to RT. The resultant nanoparticles were precipitated with ethanol and washed with ethanol/water (1 : 1 v/v) several times. 2.3
Scheme 1 Schematic nanocomposites.
Experimental section
Synthesis of UCNPs@SiO2@hypocrellin A–FA
Preparation of the hypocrellin A molecule precursor. 10 mg hypocrellin A in 20 mL ethanol and 14 mL TESPIC were mixed. The mixture was stirred overnight under nitrogen atmosphere at 80 C. Aer concentrating by rotary evaporator, the products were dissolved in 2 mL DMSO. Synthesis of UCNPs@SiO2@hypocrellin A. 30 mL of cyclohexane solution of 10 mg UCNPs and 1.6 g Igepal CO-520 was ultrasonicated for 20 min. Next, 230 mL NH3$H2O (25%) was added and the mixture was stirred for 1 h. Subsequently, 100 mL TEOS (98%), 15 mL APTES (98%) and the as-prepared Analyst, 2014, 139, 6414–6420 | 6415
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hypocrellin A molecule precursor were added and stirred for 48 h in the dark at RT. The nal nanomaterials were precipitated and washed several times with ethanol. FA conjugation. Covalent binding of FA to the UCNPs@SiO2@hypocrellin A was conducted using a modication of the standard EDC–NHS reaction. Carboxyl groups of FA (10 mg) were activated by an EDC (4.2 mg)/NHS (6.4 mg) solution for 30 min (FA/EDC/NHS molar ratio ¼ 1 : 1 : 2.5). Next, 10 mg of the as-prepared nanomaterials were added and stirred for 12 h in the dark. The resulting products were centrifuged and washed 3 times with PBS (pH ¼ 7.4) and dried for further use. 2.4
Singlet oxygen measurements
To detect the generation of singlet oxygen, UCNPs@SiO2@ hypocrellin A–FA (100 mg mL1) was dispersed in a solution of PBS containing 20 mM ABDA. The mixture was irradiated by a 980 nm laser for 0, 5, 10, 15 min. The uorescence emission of ABDA was measured by a spectrophotometer with an excitation of 380 nm. 2.5
Cell culture and MTT assay
HeLa cells were obtained from Peking Union Medical College Hospital, and cultured in DMEM supplemented with 10% FBS and 0.01% penicillin–streptomycin solution at 37 C in a humidied 5% CO2 atmosphere. For dark toxicity tests, HeLa cells (104 cells per well) were seeded in 96-well plates and incubated overnight with 5% CO2 at 37 C. Aer being washed with PBS, the cells were incubated with different concentration (0, 25, 50, 100, 200, 400 mg mL1) nanomaterials for 12 h at 37 C in the dark. Cell viability was determined by the standard MTT assay method. MTT (20 mL 5 mg mL1) was added to each well. Then, the culture medium was carefully removed and replaced with 200 mL DMSO. Aer 10 min of agitation on a shaker, absorbance was measured at 490 nm by a microplate reader. 2.6
Confocal luminescence imaging
HeLa cells were cultured and incubated with UCNPs@SiO2@ hypocrellin A–FA and UCNPs@SiO2@hypocrellin A for 2 h at concentration of 200 mg mL1, respectively. Cell imaging was performed by a Two-Photon Laser Scanning Confocal Microscope-510 Meta. The blue emissions and red emissions from UCNPs were collected at 476 nm and 650 nm. 2.7 Fluorescence imaging of cell apoptosis and ow cytometric analysis Aer culture with the UCNPs@SiO2@hypocrellin A–FA (250 mg mL1) for 4 h, the cells were exposed to NIR light for 15 min, and then stained with Annexin V-FITC/PI for 15 min. Aer washing three times with the binding buffer, the labeled cells were observed with an Olympus IX71 uorescence microscope. For ow cytometric analysis, aer incubating with UCNPs@SiO2@hypocrellin A–FA (250 mg mL1) for 4 h, the cells were exposed to 980 nm laser irradiation for 15 min. The laser power density was 0.3 W cm2 during irradiation. The cells
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were resuspended in binding buffer and stained with AnnexinVFITC/PI. Then, the samples were analyzed by a BD FACSVamtage SE ow cytometer. 2.8
Magnetic relaxivity of UCNPs@SiO2@hypocrellin A–FA
The relaxivity measurements were carried out on a 3.0 T clinical MRI instrument. A series of aqueous solutions with different concentrations of UCNPs@SiO2@hypocrellin A–FA nanoparticles were prepared and transferred into 1.5 mL Eppendorf tubes for longitudinal magnetic relaxivity measurements. For in vitro MRI, HeLa cells and HEK-293 cell were incubated with the nanocomposites for 4 h. Following the incubation, the cells were washed and centrifuged and the resulting cell pellets were covered with 1% agarose solution. 2.9
Characterization
Transmission electron microscopy (TEM) was performed with JEOL JEM-2010 high-resolution transmission microscope (HRTEM). Powder X-ray diffraction (PXRD) was recorded on a PANalytical X'Pert PRO MPD diffractometer. Dynamic light scattering (DLS) was performed on a Nano-ZS Zetzsozer ZEN3600 (Malvern Instruments Ltd., U.K.). UV/vis absorption spectra were obtained by TU-1901 UV-vis spectrophotometer (Beijing, China). Excitation and emission spectra were measured by SHIMADZU RF-5301PC spectrophotometer with a xenon lamp and an external 980 nm laser (Hi-tech Optoelectronics Co. Ltd. China). Fourier transform infrared (FTIR) spectra were obtained on a Nicolet 380. Biological analysis was obtained by Olympus IX71 uorescence microscope, BD FACSVamtage SE ow cytometry, Two-Photon Laser Scanning Confocal Microscope-510 Meta. The relaxivity measurements were carried out on a 3.0 T clinical MRI instrument (781278 Achieva 3.0 T TX).
3. 3.1
Results and discussion Characterization of UCNPs@SiO2@hypocrellin A–FA
NaYF4:Yb,Tm were prepared by an established protocol.12–15 TEM analysis showed that the NaYF4:Yb,Tm nanoparticles were monodispersed with an average size of ca. 21 nm (Fig. 1A). Fig. 1B shows the diameter of NaYF4:Yb,Tm@NaGdF4 increase to about 26 nm without a change in their structural and morphological uniformity. The diffraction peaks of NaYF4: Yb,Tm@NaGdF4 can be indexed to the pure hexagonal phase NaYF4 crystal (JCPDS 16-0334) in Fig. 1C. The formation of hypocrellin A molecule precursor was investigated by FTIR shown in Fig. S1.† In the FTIR spectrum of TESPIC (Fig. S1B†), the transmission band at 2270 cm1 was attributed to the –NCO stretching vibration of isocyanate group. However, in the FTIR spectrum of hypocrellin A molecule precursor (Fig. S1C†), the characteristic peak almost disappeared, which proved the successful synthesis of hypocrellin A molecule precursor. The as-prepared UCNPs were coated with a layer of silica to form UCNPs@SiO2@hypocrellin A nanoparticles, which were highly uniform in size, at about 38 nm in diameter (Fig. S2A†). The existence and identication of UCNPs@SiO2@hypocrellin A
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Emission spectra of UCNPs under 980 nm excitation and UV/vis absorption spectrum of hypocrellin A. Inset is the image of UCNPs under ambient light and 980 nm laser excitation.
Fig. 2
Fig. 1 TEM images of (A) NaYF4:Yb,Tm nanoparticles (B) NaYF4: Yb,Tm@NaGdF4 nanoparticles (UCNPs) and (C) XRD pattern of UCNPs. The JCPDS 16-0334 line pattern for pure hexagonal phase NaYF4 (red line) is shown for reference.
were also conrmed by FTIR spectroscopy (Fig. S2B†). Aer silica coating, the spectrum showed peaks around 1070, 953 and 459 cm1, which corresponded to the Si–O–Si asymmetric stretching vibration and Si–O deformation vibration. Fig. S3† shows that hydrodynamic diameter distribution of UCNPs@SiO2@hypocrellin A was centered at 38 nm, which is consistent with the results of TEM. These facts indicated that UCNPs@SiO2@hypocrellin A was successfully synthesized. In the next step, we investigated the existence of UCNPs @SiO2@hypocrellin A–FA using UV/vis spectroscopy (Fig. S4†). Aer modifying FA ligands, characteristic absorption peaks of FA at 280 nm and 360 nm appeared, indicating the immobilization of FA, which was further supported by FTIR spectral measurements. As shown in Fig. S5B,† a strong band at 1690 cm1 was attributed to the C]O stretching vibrations of carboxyl group. Nevertheless, aer functionalization with FA, characteristic peak of the amido bond at 1540 cm1 was observed. These results showed the successful modication of UCNPs@SiO2@hypocrellin A–FA. 3.2
Luminescence properties
As shown in Fig. 2, under the excitation of a 980 nm NIR laser, the UCNPs showed distinct Tm3+emission bands at 450, 476, 650 and 700 nm, which correspond to 1D2–3F4, 1G4–3H6, 1 G4–3F4, 3F2,3–3H6 transitions of the Tm3+ ions, respectively.32 Simultaneously, the hypocrellin A exhibited intense absorption bands at around 465 nm, which considerably overlapped with the blue emission of the UCNPs. This overlap ensures that hypocrellin A molecules are able to absorb the visible emission released by the nanoparticles aer being exposed to NIR irradiation at 980 nm, and consequently generate singlet oxygen to kill cancer cells.33,34 3.3
oxygen by the as-prepared nanomaterials aer irradiation by 980 nm laser. When excited at 380 nm, ABDA emits uorescence in the 400–500 nm region. When reacting with singlet oxygen, it can yield an endoperoxide, which is non-uorescent.35,36 As shown in Fig. 3, as the irradiation time increases, the absorbance of ABDA decreases, which accounted for the generation of singlet oxygen. Furthermore, the effect of pH on the generation of singlet oxygen was also investigated. The pH value of PBS were separately set at 5 (pH in endosome), 6.8 (pH around cancer cells) and 7.4 (pH of blood). The uorescence spectra of ABDA with UCNPs@SiO2@hypocrellin A–FA nanoparticles at different pH value were obtained. As shown in Fig. S6,† The degree of uorescence decrease has no obvious difference in all of the conditions, which indicated that pH has a little inuence on the generation of singlet oxygen in these physiological conditions. In addition, UCNPs@SiO2(hypocrellin A)–FA with hypocrellin A simply deposited were also prepared. As can be seen in Fig. S7,† under the same condition, UCNPs@SiO2@ hypocrellin A–FA with hypocrellin A that were covalently bound generated more singlet oxygen because of a higher loading efficiency. 3.4
MTT assay and cell uptake
Before further in vitro experiment, the cytotoxicity of the asprepared nanomaterials was checked. The cytotoxicity of UCNPs@SiO2@hypocrellin A–FA was evaluated on the HeLa cells and HEK-293 cells by a standard MTT assay. As shown in
Singlet oxygen measurements
9,10-Anthracenediyl-bis (methylene) dimalonic acid (ABDA) was used as the indicator to investigate the generation of singlet
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Fig. 3 Fluorescence spectra of ABDA with UCNPs@SiO2@hypocrellin A–FA nanoparticles exposed to 980 nm NIR laser for 0, 5, 10, 15 min.
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Fig. S8,† more than 90% of HeLa cells and HEK-293 cells survived with a concentration of the samples as high as 400 mg mL1. The result indicated that the as-prepared nanomaterials exhibited low toxicity. Photodynamic efficiency of the as-prepared nanomaterials was assessed by MTT assay. Aer the incubation with the different concentrations of UCNPs@SiO2@hypocrellin A–FA for 24 h, the cells were irradiated with NIR light for different time and incubated for an additional 24 h. As shown in Fig. 4, compared to the control group without NIR light irradiation, the cell viability in the NIR light irradiated groups dramatically decreased with an increase in the concentration of UCNPs@SiO2@hypocrellin A–FA. Little difference in the viability of incubated cells was observed before and aer irradiation. These results suggested that the cytotoxicity was mainly contributed to the PDT effect. To visualize the in vitro targeting ability of UCNPs@SiO2@ hypocrellin A–FA, we performed confocal imaging with FRpositive [FR (+)] HeLa cells incubated with UCNPs@ SiO2@hypocrellin A–FA, UCNPs@SiO2@hypocrellin A and FRnegative [FR ()] HEK-293 cells cultured with UCNPs@SiO2@ hypocrellin A–FA. As shown in Fig. 5, under excitation at 980 nm, blue light and red light of UCNPs could be observed. HEK293 cells incubated with UCNPs@SiO2@hypocrellin A–FA demonstrated lower luminescence when compared with that of HeLa cells. Moreover, compared to the nontargeted UCNPs@SiO2@hypocrellin A nanoparticles, HeLa cells incubated with UCNPs@SiO2@hypocrellin A–FA were more uorescent, indicating higher cellular uptake of the targeted particles. Serial layer scanning can be seen in Fig. S9,† UCNPs@SiO2@hypocrellin A–FA can enter the cytoplasm of tumor cells rather than merely staining the membrane surface. Taken together, these results showed that UCNPs@ SiO2@hypocrellin A–FA can specically target cancer cells overexpressing FAR by the receptor-mediated delivery.
3.5
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Fig. 5 Confocal luminescence images of (A) FR (+) HeLa cells, (B) FR () HEK-293 cells incubated with UCNPs@SiO2@hypocrellin A–FA (200 mg mL1) and (C) FR (+) HeLa cells incubated with UCNPs@SiO2@hypocrellin A (200 mg mL1) for 1 h at 37 C.
FITC positive, PI positive), and necrotic cells (Annexin V-FITC negative, PI negative).37–39 HeLa cells were incubated with UCNPs@SiO2@hypocrellin A–FA (250 mg mL1) for 3 h at 37 C, and then exposed to NIR at 980 nm for 10 min. Aer Annexin V-FITC/PI staining, the cells were observed by an Olympus IX71 uorescence microscope. As shown in Fig. 6A–C, no obvious cell apoptosis was observed in control groups. However, apoptosis could be seen in Fig. 6D in the experimental group indicating that both UCNPs@SiO2@ hypocrellin A–FA and irradiation were indispensable to induce the apoptosis of the cells. Simultaneously, the results also indicated that the mechanism of cell death induced by UCNPs@SiO2@hypocrellin A–FA was apoptosis but not necrosis.
Fluorescence imaging and ow cytometry
To conrm that UCNPs@SiO2@hypocrellin A–FA may induce apoptotic death in tumor cells, the cells were stained with Annexin V-FITC/PI to distinguish early apoptotic cells (Annexin V-FITC positive, PI negative), late apoptotic cells (Annexin V-
Fig. 4 Viability of HeLa cells incubated with UCNPs@SiO2@ hypocrellin A–FA at different concentrations (0, 100, 200, 400 mg mL1) after irradiation with 980 nm of different time (0, 10 and 20 min).
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Fig. 6 UCNPs@SiO2@hypocrellin A–FA induced cell apoptosis as determined by fluorescence imaging using Annexin V-FITC/PI staining on HeLa cells. (A) HeLa cells without UCNPs@SiO2@hypocrellin A–FA treatment and NIR irradiation. (B) HeLa cells incubated without UCNPs@SiO2@hypocrellin A–FA and exposed to NIR laser for 15 min. (C) HeLa cells treated with UCNPs@SiO2@hypocrellin A–FA (250 mg mL1), without NIR irradiation. (D) HeLa cells incubated with UCNPs@SiO2@hypocrellin A–FA (250 mg mL1) and exposed to NIR laser for 15 min.
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conrmed that the as-prepared nanocomposites can be used as a targeted T1 MRI contrast agent to cancer cells that overexpress FR.
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4. Conclusions
Fig. 7 Flow cytometry of apoptosis of HeLa cells stained with Annexin V-FITC/PI induced by UCNPs@SiO2@hypocrellin A–FA.
We have developed a multifunctional nanosystem, which could serve as a platform for simultaneous bioimaging and PDT. NaYF4:Yb,Tm@NaGdF4 were encapsulated inside the silica shell in which hypocrellin A was covalently attached. The nanoparticles are able to enter cancer cells and induce cell death aer being exposed to the NIR light. Tailoring FA at the end improves the targeting efficacy in cancer cells, which was investigated by confocal microscopy and in vitro MRI. Moreover, owing to the presence of Gd3+ ions, the composites could also act as a targeted MR contrast agent, which was further conrmed by MR research both in aqueous and inside cells. Our study promises potential applications in clinical treatment and detection.
Acknowledgements Fig. 8 (A) Relaxation rate r1 for the different Gd3+ concentrations.
Inset: t1-weighted magnetic resonance images, (B) MR imaging of HeLa (a) and HEK-293 cells (b) after incubation with various concentrations of the as-prepared nanocomposites.
Apoptosis of HeLa cells induced by the as-prepared nanomaterials were also quantied with ow cytometry. Aer incubation with UCNPs@SiO2@hypocrellin A–FA (250 mg mL1) for 4 h, the HeLa cells were exposed to NIR irradiation for 15 min, and then stained with Annexin V-FITC/PI. As can be seen from Fig. 7, the percentage of early apoptosis and late apoptosis was 31.01%, further conrming apoptosis process occurring in cells. By comparison, apoptosis of HeLa cells induced by UCNPs@SiO2(hypocrellin A)–FA with hypocrellin A simply deposited were also determined by ow cytometry. As shown in Fig. S10,† UCNPs@SiO2(hypocrellin A)–FA showed lower percentage of apoptosis, which was consistent with the results of measurement of singlet oxygen production.
3.6
Magnetic relaxivity of UCNPs@SiO2@hypocrellin A–FA
The MRI capability of UCNPs@SiO2@hypocrellin A–FA was measured in water. As shown in Fig. 8A, with increasing concentration of Gd3+ ions, MR signal intensity enhanced gradually. The r1 relaxivity value was calculated to be 2.88 through the curve tting analysis with a coefficient of determination (R) of 0.998. MRI was also used to further conrm the specicity of UCNPs@SiO2@hypocrellin A–FA. HeLa cells and HEK-293 cells were treated with UCNPs@SiO2@hypocrellin A– FA with different Gd concentrations. As can be seen in Fig. 8B, MR signal intensity of HeLa cells treated with the as-prepared nanocomposites was higher than that of HEK-293 cells, which was because of FR mediated endocytosis. These results
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The authors gratefully acknowledge the support from the National Nature Science Foundation of China (21175014, 21475011, 21422503), the Fundamental Research Funds for the Central Universities, National Grant of Basic Research Program of China (No. 2011CB915504).
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