Article pubs.acs.org/IC
Multimodal Upconversion Nanoplatform with a MitochondriaTargeted Property for Improved Photodynamic Therapy of Cancer Cells Xiaoman Zhang,†,‡,§ Fujin Ai,†,‡,§ Tianying Sun,⊥ Feng Wang,*,⊥,‡ and Guangyu Zhu*,†,‡ †
Department of Biology and Chemistry and ⊥Department of Physics and Materials Science, City University of Hong Kong, Kowloon Tong, Hong Kong SAR ‡ City University of Hong Kong Shenzhen Research Institute, Shenzhen 518057, P. R. China S Supporting Information *
ABSTRACT: Upconversion nanoparticles (UCNPs) with the capacity to emit highenergy visible or UV light under low-energy near-infrared excitation have been extensively explored for biomedical applications including imaging and photodynamic therapy (PDT) against cancer. Enhanced cellular uptake and controlled subcellular localization of a UCNP-based PDT system are desired to broaden the biomedical applications of the system and to increase its PDT effect. Herein, we build a multimodal nanoplatform with enhanced therapeutic efficiency based on 808 nm excited NaYbF4:Nd@NaGdF4:Yb/Er@NaGdF4 core−shell−shell nanoparticles that have a minimized overheating effect. The photosensitizer pyropheophorbide a (Ppa) is loaded onto the nanoparticles capped with biocompatible polymers, and the nanoplatform is functionalized with transcriptional activator peptides as targeting moieties. Significantly increased cellular uptake of the nanoparticles and dramatically elevated photocytotoxicity are achieved. Remarkably, colocalization of Ppa with mitochondria, a crucial subcellular organelle as a target of PDT, is proven and quantified. The subsequent damage to mitochondria caused by this colocalization is also confirmed to be significant. Our work provides a comprehensively improved UCNP-based nanoplatform that maintains great biocompatibility but shows higher photocytotoxicity under irradiation and superior imaging capabilities, which increases the biomedical values of UCNPs as both nanoprobes and carriers of photosensitizers toward mitochondria for PDT.
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years.10−13 The rare-earth element-doped UCNPs can absorb two or more low-energy photons and emit a high-energy photon, thus upconverting the deeper penetrable near-infrared (NIR) light to visible or even UV light.14,15 The visible emission from UCNPs excites the PS to generate 1O2 to kill cancer cells and is used as an imaging tool.16,17 UCNPs possess advantages in this process, such as a low autofluorescence background, a low photobleaching effect, and long lifetimes of sharp emission.18 The sharp and stable emission makes UCNPs ideal biosensors that can obtain higher sensitivity compared with quantum dots or other organic fluorescence probes.19 Effort is still required to develop the UCNP-based system as a more powerful and efficient tool for PDT. For example, a 980 nm laser, which has been extensively used to excite UCNPs, could cause great overheating issues because of the significant absorption of water at this wavelength.20−22 We have successfully solved the overheating problem by introducing novel kinds of core−shell−shell UCNPs that are excited by an 808 nm laser.21,23 Water has minimized absorption at this wavelength in the medical spectral window. Therefore, 808 nm irradiation causes much less damage to tissues around cancer
INTRODUCTION Photodynamic therapy (PDT) has been widely used as an effective approach to treating various types of cancer and has shown great advantages including minimized long-term side effects, precise targeting, and less cost.1−4 PDT kills cancer cells using singlet oxygen (1O2) and other reactive oxygen species (ROS), which are generated by photosensitizers (PSs) under certain wavelengths of excitation, usually in the visible region.2 The ROS have very short lifetimes; therefore, the action always occurs at the immediate site of the PS under light irradiation, making the subcellular localization of the PS in the cytoplasm crucial in PDT.5,6 The initiation of ROS-induced cell death is mostly believed to take place at the membranes of organelles in the cytoplasm, among which mitochondria have been found to be the primary target for the PS to produce apoptosis.3,7 Trials are still needed for improvements of the PS, such as to increase the subcellular localization selectivity of the PS and to shift the excitation wavelength for elevated tissue penetration depth.8 For the PSs of greatest interest up to now, the excitation wavelength is always in the visible region, which limits the preference of PDT within thin basal cancer cells due to the low penetration depth.3,9 In order to solve the penetration issue, upconversion nanoparticles (UCNPs) have been developed as a carrier and an energy transfer agent for the PS in recent © XXXX American Chemical Society
Received: January 5, 2016
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DOI: 10.1021/acs.inorgchem.6b00020 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry Scheme 1. Functionalization of UCNPs with C18PMH−PEG−NH2, TAT Peptide, and PS (Ppa)
(dimethylamino)propyl]-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and 1,3-diphenylisobenzofuran (DPBF) were purchased from Sigma-Aldrich. The transcriptional activator (TAT) peptide (YGRKKRRQRRR) was from China Peptide Co. Ltd. MitoTracker green, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Life Technologies. JC-1 was purchased from Suzhou Amatek Scientific. All reagents were used as received. Synthesis of C18PMH−PEG−NH2. C18PMH−PEG−NH2 was synthesized according to a published protocol.28 Briefly, C18PMH (10 mg) was first activated with 10 mg of EDC and 10 mg of NHS in dichloromethane (DCM) at room temperature for 0.5 h, followed by the addition of 143 mg of mPEG−NH2, 50 mg of Boc−PEG−NH2, and 7 μL of triethylamine. The mixture was stirred vigorously for 24 h, and then the DCM solvent was evaporated at 30 °C. A 2 mL volume of trifluoroacetic acid (TFA) was added subsequently at room temperature and the solution was stirred for another 4 h to remove the Boc group. After evaporation of the TFA solvent, the residue was dissolved in water and dialyzed for 2 days at room temperature in a dialysis bag (MWCO = 10 kDa). The final product C18PMH−PEG− NH2 was stored at −20 °C after lyophilization. The structure and 1H NMR spectrum of C18PMH−PEG−NH2 is shown in Figure S1 (3.8− 3.5 ppm, CH2 of PEG; 1.3−1.1 ppm, CH2 of C18 chains; 0.88 ppm, CH3 of C18PMH). PEG Functionalization of UCNPs. A 500 μL volume of an UCNP stock solution in cyclohexane was added to 1 mL of ethanol followed by precipitation through centrifugation. The nanoparticles were subsequently washed twice with ethanol and redispersed in 1 mL of chloroform. C18PMH−PEG−NH2 (20 mg in 2 mL of chloroform) was added to the mixture and stirred for 2 h. After rotaevaporation, the residue was readily dissolved in Milli-Q water. Loading of PS Molecules. Ppa was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 0.5 mg/mL. From this point, all preparations and measurements of Ppa-related samples were kept in the dark as much as possible. Certain concentrations of PEGylated UCNPs in water were mixed with Ppa and stirred overnight at room temperature. The Ppa-loaded nanoparticles were spun down at 14500 rpm for 20 min to remove free Ppa. The nanoparticles were washed three times with water. The amount of Ppa
cells and has deeper penetration than 980 nm irradiation. For energy-transfer efficiency, enhancement can be achieved by choosing the proper PS, increasing the emission intensity of UCNPs in the region for PS absorption, and decreasing the distance between UCNPs and the PS.10,12,13,24−27 There are also other factors that limit the effectiveness of a UCNP-based PDT system, including the amount of nanoparticles taken by cells and colocalization with critical parts within cancer cells.10 To improve the above-mentioned limiting factors to increase the therapeutic outcome of the UCNP-based PDT system, herein we take advantage of the 808-nm-excited UCNP-based nanoplatform and create a new system with a clear imaging capability and high therapeutic efficiency. The core−shell−shell UCNPs were first capped with a poly(ethylene glycol) (PEG)functionalized polymer in a way that could ensure high morphological and functional stabilities in aqueous solutions.12 An arginine-rich cell-penetrating peptide, trans-activating transcriptional activator (TAT), was used as a targeting moiety to endow the nanoplatform with increased cellular uptake of nanoparticles and the capability to simultaneously target certain areas, e.g., mitochondria, within cancer cells. The high-energytransfer process and photocytotoxicity in the cells were confirmed. The detailed subcellular localization of UCNPs and Ppa was visualized by confocal microscopy, and the subsequent elevated effectiveness of photodamage to mitochondria was further monitored and proven, showing that our upconversion nanoplatform is not only a powerful PDT agent against cancers but also an advanced theranostic tool for diagnosis (Scheme 1).
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EXPERIMENTAL SECTION
Materials. Core−shell−shell upconversion nanoparticles (UCNPs; NaYbF4:Nd@NaGdF4:Yb/Er@NaGdF4) were synthesized following a previously established protocol.21 Pyropheophorbide a (Ppa) was obtained from Frontier Scientific. Methoxylpoly(ethylene glycol)amine (mPEG−NH2; 5K) was from Xiamen Sinopeg Co. Ltd. Boc− PEG−NH2 (5K) was purchased from Beijing Jenkem Technology Co. Ltd. Poly(maleic anhydride-alt-1-octadecene) (C18PMH), N-[3B
DOI: 10.1021/acs.inorgchem.6b00020 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry
Figure 1. (A) TEM images and (B) FT-IR spectra of OA−UCNPs, NH2−UCNPs, and TAT−UCNPs. Scale bar: 50 nm. (C) Photographs of NH2− UCNPs and TAT−UCNPs in aqueous solutions before and after centrifugation. in the supernatant solution was determined by UV−vis spectroscopy to calculate the loading capacity. 1 O2 Generated by a PS. 1O2 generation was determined by DPBF assay. DPBF was dissolved in DMSO at a low concentration. UCNPs loaded with different amounts of Ppa were dispersed in DMSO at a concentration of 1.0 mg/mL. A 50 μL volume of each Ppa−UCNP solution was added into 96-well microplate together with 50 μL of a DPBF solution per well to form a mixture, and the wells were irradiated by an 808 nm laser at a power density of 6 W/cm2. The absorption of DPBF at 418 nm was monitored every minute. Synthesis of TAT-Conjugated UCNPs. TAT peptide was covalently conjugated with UCNPs. TAT peptide (10 mg) was dissolved in 5 mL of a phosphate-buffered saline (PBS) buffer (pH 7.4), and 10 mg of EDC and 10 mg of NHS were added under stirring to activate the carboxylic groups in TAT peptides. After 15 min of activation, 5 mL of a PBS solution containing PEGylated UCNPs (2 mg/mL) was added followed by adjustment of the pH value to 8.0. The mixture was subsequently stirred for 24 h at room temperature. Excess amounts of EDC, NHS, and TAT peptide were removed by dialysis. Quantification of TAT peptide on UCNPs was carried out by measurement of the UV−vis absorbance of UCNPs before and after conjugation. The amount of TAT peptide was calculated to be 22 nmol/mg UCNPs. Characterizations of Modified UCNPs. The UV−vis spectrum was measured on a Shimazu UV-1700 UV−vis spectrophotometer. Fourier transform infrared (FT-IR) measurements were performed on an AVATAR-360 FT-IR spectrophotometer (Nicolet, USA). Transmission electron microscopy (TEM) images were acquired on a Philips CM-20 transmission electron microscope (Philips Technai 12). The ζ potential was measured by a dynamic light scattering (DLS) particle-size analyzer. Photoluminescence (PL) was measured by an F4600 spectrophotometer (Hitachi) with the excitation source adapted to fiber-coupled diode lasers. Cytotoxicity Assay. HeLa cells were seeded in 96-well culture plates at a density of 1500 cells/well and cultured in 5% CO2 at 37 °C for 48 h. The cells were then treated with various concentrations of
UCNPs for 2 h and irradiated with an 808 nm laser for 5 min/well (6 W/cm2). The standard MTT assay was carried out after another 48 h of incubation to determine the cell viabilities. Whole Cell Uptake. HeLa cells were seeded in 6 cm tissue culture dishes at a density of 300000 cells/dish and cultured in 5% CO2 at 37 °C for 48 h. The cells were then treated with 0.1 mg/mL concentrations of UCNPs. After 2 h of incubation, the cells were washed with PBS three times, harvested by trypsinization, and washed another three times. The cells were counted and centrifuged, followed by digestion and analysis of the gadolinium concentration using inductively coupled plasma-optical emission spectrometry (ICP-OES). Intracellular ROS Detection. HeLa cells were seeded in 35 mm confocal dishes (300000 cells/dish). After 24 h, the cells were treated with 100 μg/mL TAT−Ppa−UCNPs for 1 h, washed with PBS three times, and stained with 10 μM H2DCFDA in PBS for 40 min. The cells were washed with PBS. The cells treated with TAT−Ppa− UCNPs were irradiated by an 808 nm laser for 10 min (6 W/cm2). Cells without treatment and treated cells without irradiation were set as negative controls, and cells treated with 3 mM H2O2 for 15 min were used as positive controls. The confocal images were recorded with a confocal microscope (Leica SP5) with excitation at 488 nm and emission from 517 to 527 nm. Confocal Imaging of the Cells. HeLa cells were seeded onto glass slides and incubated under 5% CO2 at 37 °C. Then the cells were treated with UCNPs dispersed in a fresh medium at a concentration of 100 μg/mL for 4 h before imaging. MitoTracker green and JC-1 were also used to stain the mitochondria. In the case of MitoTracker green, the cells were washed at least three times with PBS to remove excess amounts of UCNPs and MitoTracker green. Imaging was performed on a Leica SP5 confocal microscope. Fluorescence of MitoTracker green (emission range 500−600 nm) could be detected under 488 nm excitation. Ppa (emission range 600−700 nm) and UCL emission of UCNPs (emission range 500−600 nm for the red band) was excited by an 808 nm laser. For JC-1 assay, the cells were irradiated first after treatment with UCNPs for 2 h and then stained by JC-1, washed, and taken for imaging on a Leica SPE confocal microscope. Fluorescence C
DOI: 10.1021/acs.inorgchem.6b00020 Inorg. Chem. XXXX, XXX, XXX−XXX
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Figure 2. (A) PL spectrum of OA−UCNPs under 808 nm laser irradiation (black) and UV−vis spectrum of Ppa (red). (B) Loading capacity (w/w) under different loading ratios. The ratio is the loading weight of Ppa to the weight of UCNPs. (C) 1O2 generated by NH2−Ppa−UCNPs with 808 nm irradiation. (D) 1O2 generated by NH2−Ppa−UCNPs without 808 nm irradiation. (E) PL spectra before and after loading with Ppa under 808 nm excitation.
images of OA−UCNPs, NH2−UCNPs, and TAT−UCNPs showed that the sizes of the as-synthesized nanoparticles were around 20−40 nm (Figure 1A). The processes of each functionalization step were validated by FT-IR spectroscopy (Figure 1B). For OA−UCNPs, two sharp peaks at 2852 and 2919 cm−1 are observed, which belong to the symmetric and asymmetric C−H stretching vibrations, respectively. After coating with C18PMH−PEG−NH2, the presence of oxygen atoms in PEG causes more intense symmetric stretching of C− H than asymmetric stretching;29 thus, one strong peak appears at 2875 cm−1. A CO stretching vibration at 1650 cm−1 and an N−H stretching vibration at 1562 cm−1 are also observed for NH2−UCNPs, indicating the successful coating of C18PMH− PEG−NH2 onto UCNPs. The CO stretching vibration at 1650 cm−1 and the N−H stretching vibration at 1562 cm−1 become stronger after covalent conjugation of TAT peptide because of the peptide linkages and the newly formed amide bonds (Figure 1B), confirming functionalization of UCNPs with TAT peptides. Both NH2−UCNPs and TAT−UCNPs are well dispersed in water, which is ideal for subsequent biological applications (Figure 1C). In addition, according to the DLS
of J-monomer (emission range 500−530 nm) and J-aggregate (emission range 550−600 nm) could be detected simultaneously under 488 nm excitation. Statistical Analysis. For all of the in vitro data in this work, calculations and statistical analysis were performed through one-way ANOVA in Microsoft Excel 2010.
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RESULTS AND DISCUSSION
NaYbF 4 :Nd@NaGdF 4 :Yb/Er@NaGdF 4 core−shell−shell UCNPs were synthesized according to our previously published protocol.21 The nanoparticles utilize excitation at 808 nm to generate luminescence emission at around 550 and 660 nm, which can be used for imaging and therapy, respectively. Oleic acid (OA)-capped UCNPs were transferred to an aqueous solution by coating C18PMH−PEG−NH2 to obtain NH2− PEG−C18PMH−UCNPs (NH2−UCNPs).12 In this case, C18PMH−PEG−NH2 was wrapped around the nanoparticles, and the amino groups in the hydrophilic region allowed for subsequent functionalization. TAT peptide was then covalently loaded onto the surface of the nanoparticles to obtain TAT− PEG−C18PMH−UCNPs (TAT−UCNPs; Scheme 1). TEM D
DOI: 10.1021/acs.inorgchem.6b00020 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry results, the ζ potentials of NH2−UCNPs and TAT−UCNPs are 13.5 ± 0.7 and 28.1 ± 1.8 mV, respectively. The positive charge of NH2−UCNPs confirms the presence of amino groups, and the increase in the ζ potential after TAT functionalization further reveals the successful loading of TAT peptide because it contains a number of arginines that are positively charged under neutral or acidic conditions.30 During the steps of functionalization with TAT peptide, we also set a control group simply by mixing TAT peptide and NH2− UCNPs together without adding any coupling agent. The ζ potential of the nanoparticles obtained in this way is 13.5 ± 0.9 mV, which is identical with that of NH2−UCNPs, further confirming that, in the TAT−UCNPs, TAT peptide is covalently conjugated. The coating of C18PMH−PEG−NH2 on the surface of the nanoparticles also allows for the further loading of hydrophobic molecules. As a PS of high hydrophobicity,3,12 Ppa was stabilized on the C18 units of the polymer coated on UCNPs. Ppa is a type of second-generation PS, which has high quantum yield of 1O2 and low dark toxicity.31 In addition, mitochondria are reported to be a sensitive target for Ppamediated photodamage, making Ppa more desirable for our system.32 However, Ppa can easily aggregate, leading to a marked decrease in the efficiency of PDT.33 For our system, we first measured the PL spectrum of OA−UCNPs and the UV− vis spectrum of Ppa, both of which have a strong peak observed at 660 nm (Figure 2A). This high degree of overlap at 660 nm indicates the possible efficient energy transfer from UCNPs to Ppa. The loading capacity was then studied, and the result shows that the loading capacity of Ppa reaches saturation when a ratio of 0.10 (mg of Ppa to mg of UCNPs) is used, and the actual loading amount is 0.06 (mg of Ppa to mg of UCNPs; Figure 2B). 1 O2 generation by 808 nm excitation was subsequently confirmed. DPBF assay was utilized to measure 1O2 generation. DPBF is a chemical probe commonly used to determine 1O2 generation by monitoring the decrease in the absorption of DPBF at 418 nm due to the photobleaching effect.27 UCNPs loaded with different ratios of Ppa (0, 0.025, 0.05, 0.1, and 0.2) were tested. A decrease of absorption at 418 nm indicates the generation of 1O2. The result shows that the UCNP with a loading ratio of 0.05, which is not the highest ratio, is able to generate the highest amount of 1O2 (Figure 2C). This could be explained by the effect that the quantum yield of 1O2 would significantly decrease with increasing concentration and aggregation of the PS.34 In the control group without 808 nm irradiation, there are minimum photobleaching effects for UCNPs with different loading ratios (Figure 2D). Thus, UCNPs with a loading ratio of 0.05 are used for the following experiments. This proved 1O2 generation evidences the efficient energy transfer from UCNPs to Ppa, which is further confirmed by quenching of the red emission band at 660 nm emitted from UCNPs after the loading of Ppa (Figure 2E). The stability of Ppa in NH2−Ppa−UCNPs and TAT−Ppa− UCNPs was tested. A good stability of Ppa in the nanoplatform is required for downstream biological applications. The Ppaloaded UCNPs were suspended in a PBS buffer (pH 7.4) at a concentration of 1.0 mg/mL in a dialysis cassette, and the release of Ppa from UCNPs was monitored by UV−vis spectroscopy. Ppa itself was included as a control. The results show that free Ppa is able to easily release from the dialysis cassette. After 1 h, only 33.4% of free Ppa remains. In stark contrast, no detectable Ppa releases from both NH2−Ppa−
UCNPs and TAT−Ppa−UCNPs even after 4 h, indicating great stability of Ppa in UCNPs in an aqueous solution (Figure S2). This high stability could be attributed to the high hydrophobicity of Ppa, which leads to the strong binding energy within the hydrophobic region of the nanoparticles,28 and the close proximity between Ppa and UCNPs ensures efficient energy transfer. The advantages of conjugating TAT peptide in the UCNPbased nanoplatform were examined. TAT peptide has long been utilized to transduce proteins and nanomaterials across cell membranes or even into intracellular organelles.35 Theories vary in the study of this transduction mechanism. The most advanced one suggested that transduction of TAT is energydependent on the cross plasma membrane, but a receptor- and energy-independent process is required when crossing into mitochondria.36 Despite all of the controversial hypotheses, one commonly believed point is that the highly positive charge of the peptide could provide an electrostatic driving force toward the negative potential of both the cellular and mitochondrial membranes, which makes it appealing to be utilized to increase uptake and to target subcellular organelles.37−39 TAT peptide is able to increase cellular uptake and direct small nanoparticles into the nucleus,40,41 but its potential to approach specific organelles within the cytoplasm and raise a subsequent PDT effect on the UCNP-based nanoplatform has not been reported previously. We first measured the cellular uptake of NH2−Ppa− UCNPs and TAT−Ppa−UCNPs. Human cervical cancer cells (HeLa) were incubated with the same concentration of NH2− Ppa−UCNPs and TAT−Ppa−UCNPs, and the uptake of the nanoparticle was measured by quantifying the concentration of gadolinium in the whole cell using ICP-OES. For NH2−Ppa− UCNPs, the cellular level is 0.17 ± 0.05 μg of Gd/106 cells. In contrast, for TAT−Ppa−UCNPs, the level significantly increases to 0.45 ± 0.02 μg of Gd/106 cells (Figure 3),
Figure 3. Whole cell uptake of NH2−Ppa−UCNPs and TAT−Ppa− UCNPs after 2 h in HeLa cells: **, p < 0.01.
which is 2.6-fold the level from the UCNPs that do not contain TAT peptide, indicating significantly increased cellular uptake of UCNPs due to functionalization with TAT peptide. The effectiveness caused by the increased uptake from TAT peptide was subsequently evidenced by photocytotoxicity assays. HeLa cells were treated with functionalized NH2− Ppa−UCNPs or TAT−Ppa−UCNPs for 2 h and then irradiated by an 808 nm continuous-wave diode laser at a power density of 6 W/cm2 for 5 min. At this power density, the laser itself has negligible effects on the cell viability.23 For NH2−Ppa−UCNPs, the cells remain confluence without E
DOI: 10.1021/acs.inorgchem.6b00020 Inorg. Chem. XXXX, XXX, XXX−XXX
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Figure 4. Cell viability of the HeLa cells treated with different concentrations of (A) NH2−Ppa−UCNPs and (B) TAT−Ppa−UCNPs with and without 808 nm laser irradiation at 6 W/cm2: *, p < 0.05; **, p < 0.01.
Figure 5. Upconversion luminescence imaging at the green (500−600 nm) and red (600−700 nm) channels of the HeLa cells incubated with 0.1 mg/mL TAT−Ppa−UCNPs for 4 h at 37 °C. An 808 nm multiphoton pulse laser was used as the excitation source. Scale bar: 25 μm.
Ppa−UCNPs, and the cells were imaged by a laser-scanning confocal fluorescence microscope equipped with a pulsed multiphoton NIR laser. Under 808 nm irradiation, UCNPs display bright upconversion luminescence in the green channel (500−600 nm, Figure 5). We also observed strong emission in the red channel (600−700 nm, Figure 5) for TAT−Ppa− UCNPs, which is unlikely from UCNPs because their emission at 660 nm is quenched by the loaded Ppa (Figure 2E). We then treated the cells with Ppa only and visualized fluorescence under the same condition. Strong emission was observed in the red channel (600−700 nm) but not the green channel (500− 600 nm) because of the usage of the multiphoton laser and the high quantum yield of Ppa (Figure S3).42 Therefore, the strong emission from Ppa-loaded UCNPs is majorly from Ppa when a multiphoton laser is applied. We next studied colocalization of UCNPs and Ppa within the cells. Pearson’s colocalization coefficiency (PCC), given as a number of the percentage overlapped by two images, which ranges between 0 and 1, with 0 being uncorrelated and 1 being perfectly correlated,43 was calculated by the software ImageJ. For TAT−Ppa−UCNPs, PCC of UCNPs and Ppa is 0.74 ± 0.15. This result shows that the majority of Ppa colocalizes with UCNPs (Figure 5), indicating that most of Ppa still remains in UCNPs after their entrance into cells, further proves the stability of the system. This colocalization also ensures the excellent PDT effect for TAT−Ppa−UCNPs. We also find that TAT−Ppa−UCNPs localize in the cytoplasmic region but not the nucleus. Therefore, although functionalization of UCNPs with TAT peptide significantly increases the cellular uptake of the nanoparticles, TAT peptide is not able to direct the particles
irradiation even under treatment with high concentrations of the nanoparticles, indicating the great biocompatibility of this nanoplatform. Upon irradiation, NH2−Ppa−UCNPs are able to kill cancer cells, especially at higher concentrations. For example, the cell viabilities upon treatment with 400 μg/mL NH2−Ppa−UCNPs without and with NIR irradiation are 84.5 ± 6.3% and 50.3 ± 5.2%, respectively, showing that NH2− Ppa−UCNPs are able to execute the PDT function (Figure 4A). Remarkably, TAT−Ppa−UCNPs show a different photocytotoxicity profile than NH2−Ppa−UCNPs. Without irradiation, the cells remain confluent upon treatment with low concentrations of NH2−Ppa−UCNPs, and the cell viabilities are lower upon high-concentration treatment. This effect is probably due to the higher uptake of TAT−Ppa−UCNPs. With irradiation, however, TAT−Ppa−UCNPs are able to kill HeLa cells much more effectively than NH2−Ppa−UCNPs. For example, upon treatment with TAT−Ppa−UCNPs at a low concentration of 62.5 μg/mL, the cell viabilities without and with irradiation are 89.2 ± 11.8% and 33.7 ± 2.9%, respectively, and this dramatic cell-killing effect is also observed at all of the concentrations that we used (Figure 4B). Therefore, functionalization of UCNPs with TAT peptide increases the photocytotoxicity of this nanoplatform, which could be ascribed to the increased cellular uptake by TAT peptides as described above. In a traditional PDT process, cellular distribution of the PS is not easy to control.6 We next studied the subcellular localization of Ppa and UCNPs, aimed at determining the distribution of Ppa in cells through delivery by the nanocarriers. HeLa cells were treated with NH2−Ppa−UCNPs and TAT− F
DOI: 10.1021/acs.inorgchem.6b00020 Inorg. Chem. XXXX, XXX, XXX−XXX
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Figure 6. Imaging at the green (500−600 nm) and red (650−700 nm) channels of the HeLa cells under 488 nm excitation. The cells were incubated with 0.1 mg/mL nanoparticles for 4 h at 37 °C and then stained with 50 nM MitoTracker green for 20 min. The line-scan profile of a selected cell is also given below. Scale bar: 50 μm for the upper row and 10 μm for the lower row.
Figure 7. Confocal imaging of HeLa cells under 488 nm excitation. The cells were incubated with 0.1 mg/mL TAT−Ppa−UCNPs for 2 h at 37 °C , irradiated by an 808 nm laser, and then stained by 10 μg/mL JC-1 for 15 min. J-monomer and J-aggregate were detected in the green (500−530 nm) and red (550−600 nm) channels, respectively. Scale bar: 25 μm. The ratio of the emission intensity from J-monomer to J-aggregate is shown in the column graph on the right.
cells treated with TAT−Ppa−UCNPs without irradiation, the treated and irradiated cells show increased flourescent intensities, indicating that our nanoplatform is able to generate ROS in the cells upon NIR irradiation (Figure S4). Because of the short half-life of ROS, it is crucial for the PS to localize precisely in cells to cause effective photodamage, and the mitochondria are proposed to be the most effective sites for PDT.3 We thus studied colocalization of Ppa with mitochondria. HeLa cells were treated with TAT−Ppa−UCNPs, stained with MitoTracker green,47 and the emissions were monitored upon 488 nm irradiation. The emission in the green channel (500−600 nm; Figure S5) is from MitoTracker green, and the one in the red channel (650−700 nm; Figure S6) is from Ppa. We found that most of Ppa colocalizes with mitochondria in HeLa cells, and the PCC is 0.79 ± 0.11. The line-scan profile of
into the nucleus within 4 h; instead, TAT−Ppa−UCNPs can effectively enter the cytoplasm. This effect is consistent with some other reports showing that TAT-functionalized nanoparticles can only enter into the nucleus after more than 4 h of treatment.44 In addition, the size of our UCNPs (∼40 nm) is slightly bigger than the normal size of a particle (
