Nerve Cells
NIR-Responsive Upconversion Nanoparticles Stimulate Neurite Outgrowth in PC12 Cells Yijia Guan, Meng Li, Kai Dong, Jinsong Ren, and Xiaogang Qu* Upconversion nanoparticles (UCNPs) which are capable of converting near-infrared (NIR) into higher energy light through sequential absorption of multiple photons or energy transfer have attracted tremendous interest.[1–3] Compared with the conventional down-conversion fluorescent materials that require higher energy ultra violet (UV) or visible light excitation, upconverting materials have many conceivable advantages including weak autofluorescence backgrounds, tunable emission fluorescence,[3] strong penetration abilities under near-infrared (NIR) radiation, resistance to photobleaching, and low toxicity, making them suitable for various biological applications.[4,5] Significant progress has been made towards the utilization of UCNPs in biosensors, nanocomposites, and the recent developed UCNP-based bioprobes for multi-modality imaging and drug delivery system.[6,7] Although it is widely used in biological area, there is still no report for employing UCNPs to manipulate and control behaviors and functions of biological systems especially nervous system. Enhancement of neuron development and outgrowth is of diagnostic importance in neuroscience since they are in regards to the treatment of injured neurons after accident or a degenerative disease, such as Alzheimer's disease (AD).[8] In past decades, nanoparticle platforms have been developed to aid in the diagnosis and therapy of such disease.[9–14] The effects of these nanomaterials on neuron cell outgrowth have received considerable attention, owing to their ability to interact with biological systems at fundamental, molecular levels with high specificity. Nanomaterials and nanotechnology have been reported to have the ability to affect the neurite outgrowth through changing the extracellular matrix (ECM),[15] whose morphology and composition can strongly affect the cell differentiation properties. With the ability to release inorganic ions into the target organ or its surroundings, several inorganic particles without modification can be used as promoter for cell differentiation.[16] Y. Guan, M. Li, K. Dong, Prof. J. Ren, Prof. X. Qu Laboratory of Chemical Biology Division of Biological Inorganic Chemistry State Key Laboratory of Rare Earth Resource Utilization Changchun Institute of Applied Chemistry University of the Chinese Academy of Sciences Chinese Academy of Sciences Changchun, Jilin 130022, China E-mail: [email protected] DOI: 10.1002/smll.201400612 small 2014, 10, No. 18, 3655–3661
Metal ions of therapeutic and diagnostic interest have been reported to enhance the neurite outgrowth.[17] Rare earth (RE) ions, especially lanthanide ions, which can modulate cell attachment and affect cell differentiation, have attracted intensive research interest in medical applications.[18] It has been reported that UCNPs have the potential to release lanthanum ions into the solution, particularly upon NIR irradiation. In addition, through the upconversion process, reactive oxygen species (ROS), which has the ability to enhance cell differentiation in low dose,[19] may also be probably generated. The unique properties of UCNPs inspire us to use them as promising promoters for neurite outgrowth. In this study, we investigated the effects of UCNPs on the neurite outgrowth during differentiation. PC12 cells were used as the neuronal differentiation model. It has been proposed that different charged coating materials play a critical role in cell attachment,[20] which further lead to different effects on cell differentiation. Different charges also modulate neurite outgrowth process by influencing calcium ions channel.[21] Herein, we systematically studied cell morphology and degree of differentiation upon the treatment of poly(ethylene imine) (PEI), polyethylene glycol (PEG) and poly(acrylic acid) (PAA)-decorated UCNPs (PEI-, PEG-, PAA-UCNPs). PEG-UCNPs, filled with hydroxyl-group in the surface of UCNPs, exhibit no charge. The PEI-UCNPs and PAA-UCNPs, which are coated with amino and carboxyl group, carry positive and negative charges, respectively. To the best of our knowledge, this is the first example of using UCNPs as neurite outgrowth promoters. Three different kinds of polymers capped NaYF4:Yb/ Er UCNPs (PEI-UCNPs, PEG-UCNPs and PAA-UCNPs) with an average particle diameter of 70 nm were first synthesized via hydrothermal method as previously reported[22] and were confirmed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) (Figure 1A,B). Coating the UCNPs with PEI, PEG and PAA instead of oleic acid can not only dramatically decrease the cytotoxicity of the materials to enhance their biocompatibility in biological application, but also make the UCNPs reveal different surface charges. Fourier-transform infrared spectroscopy (FTIR) results demonstrated that UCNPs were well covered by these three kinds of polymers (Figure S1, Supporting Information). The chemical compositions of these nanoparticles were further determined by X-ray diffraction (XRD) analysis (Figure 1C) and all diffraction peaks in the XRD pattern could be unambiguously assigned to the NaYF4 of cubic phase (JCPDS, Card No. 77–2042).[23] The absence of any
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Figure 1. A) SEM image, B) TEM image, C) wide-angle XRD pattern, D) upconversion fluorescence spectrum of the NaYF4:Yb/Er nanoparticles.
other phase in the XRD indicated high purity of the UCNPs. Upon continuous excitation at 980 nm, the upconversion luminescence spectrums of the NaYF4:Yb/Er nanoparticles were recorded (Figure 1D). As expected, characteristic sharp emission peaks at around 524, 544, and 655 nm were observed as a result of the 2H11/2→4I15/2, 4S3/2→4I15/2 and 4F →4I 3+ respectively.[23,24] The results 9/2 15/2 transitions of Er demonstrated that the UCNPs could emit green light, which would be beneficial for imaging. To investigate the effect of UCNPs on mammalian neuron differentiation, PC12 cells were treated with a combination of nerve growth factor (NGF) and UCNPs. PC12 cell can sprout neuritis and differentiate into sympathetic ganglion-like cell in response to NGF, making it as a useful model system of neuronal differentiation. Morphological changes in PC12 cells affected by UCNPs were visualized by calcein-AM. Calcein-AM is a hydrophobic non-fluorescent probe that can permeate the plasma membrane and can be hydrolyzed to calcein, an extremely fluorescent and negatively charged molecule. As shown in Figure 2, the neurites started to elongate in the presence of NGF within 2 days when exposed to PEIUCNPs. Importantly, PC12 cells incubated with higher concentration of PEI-UCNPs were more elongated. PEG- and PAA-UCNPs also had the ability to enhance neurite outgrowth, although the differential effect was not as effective
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as that exposed to PEI decorated ones (Figure S2,S3, Supporting Information). The effect of PEI-UCNPs on neurite extension was sustained even up to 6 days (Figure S4, Supporting Information). Besides, the number of neurite-bearing cells and their length prominently increased with increase of the incubation time. When exposed to the highest dose of PEI- or PEG-UCNPs to the NGF-treated cells, neurite outgrowth was quite obvious compared with NGF-treated cells at day 6. However, when the treatment concentration of PAA-UCNPs was up to 40 µg/mL, neurite outgrowth decreased at day 6, mainly due to the cytotoxicity of PAAUCNPs (Figure S6, Supporting Information). Together with the SEM experiments (Figure S7, Supporting Information), compared with non-treated cells, our result indicated that UCNPs except PAA coated ones, promoted neurite outgrowth in a time- and dose-dependent manner. To quantitatively estimate the level of neurite outgrowth enhanced by UCNPs, the differentiated cells were divided into four groups (L0, L1, L2, and L3) according to the length of neurites which was described in detail in the experimental section. As shown in Figure 3, the percentage of the cells with longer neurite increased even within 2 days incubation in the presence of UCNPs. Taken PEI-UCNPs as an example, Figure 3A showed that co-incubated only with NGF, the percentage of L0 was 93.5%, while the percentage decreased to 77.9%,
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NIR-Responsive Upconversion Nanoparticles Stimulate Neurite Outgrowth in PC12 Cells
Figure 2. Images of PC12 after 2 days of treatment with NGF and 0 µg/mL, 10 µg/mL, 20 µg/mL, 40 µg/mL PEI-UCNPs. Each series can be classified to the bright light (left) and the green fluorescence from calcein (middle), and overlap of the bright and the green channels (right). The scale bar is 25 µm.
67.7% and 58.4% upon treatment with 10, 20, and 40 µg/ mL nanopaticles, respectively. On the other hand, the percentage of high differentiated cells (L2 and L3) increased from 0.27% to 12.7% with increase of PEI-UCNPs. Critically, PEG- and PAA-UCNPs performed similarly effects as PEI-UCNPs on PC12 cells differentiation with 2 days incubation (Figure 3B,C). That indicated that when incubated with UCNPs, the ratio of differentiated PC12 cells was significantly higher than the ones without UCNPs. It was worthy to note that the cells showed a slightly higher differentiated level for PEI-decorated UCNPs than PEG- and PAA-decorated ones (Figure 3D). It has been reported that PEI was an effective attachment factor in cell culture.[25] The effective attachment could enhance the UCNPs to exert the promoter effect on cell differentiation. Furthermore, the small 2014, 10, No. 18, 3655–3661
positive charges on the surface also had the ability to stimulate the neurite outgrowth through influencing calcium ion channel.[21] Next we studied NIR effect of the UCNPs on neurite outgrowth. Interestingly, with the stimulation of NIR, the number of neurite-bearing cells and their length were significantly increased when compared with the non-NIR-stimulated ones. Here we took PEI-UCNPs as an example since its effect on neurite outgrowth was more obvious. The differentiating status (the percentage of differentiated cells in the culture) was also monitored after 2 days of culture with different concentrations of UCNPs upon stimulated or non-stimulated with NIR. Figure 4A showed representative micrographs performed on all the groups after 2 days incubation with PEIUCNPs. Clearly, the lengths of neurites with NIR irradiation
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Figure 3. Distribution of the neurite length of PC12 cells 2 days after the inducement of A) PEI-UCNPs, B) PEG-UCNPs, and C) PAA-UCNPs with the increase of nanoparticles concentration. D) Distribution of the neurite length of PC12 cells 2 days after the inducement of various coated UCNPs. (L0 means the cells without neurites; L1 means the cells with neurites whose length is shorter than the size of the cell body; L2 means the cells with neurites whose length is between the original and twice the size of the cell body; L3 means the cells with neurites whose length is longer than twice the size of the cell body).
were longer than that without NIR irradiation. As predicted in the statistical results of the fluorescent images (Figure 4B,C), upon NIR irradiation, the percentage of cells in each differentiation level became significantly higher than those without NIR irradiation. With the irradiation of NIR, the percentage of high differentiated cells (L2 and L3) among the total differentiated cells was 49% at day 2 when treated with 20 µg/ mL of PEI-UCNPs, which was approximately 1.4-fold higher than that without NIR irradiation (Figure 4C). These results indicated that NIR irradiation could even increase UCNPs to enhance neurite outgrowth. It has been reported that RE ion exposure can enhance in vitro cell differentiation via extracellular signal-regulated kinase (ERK) signaling pathway.[26] The rare earth elements contained in these UCNPs may play a crucial role in facilitating PC12 differentiation. When exposed the NaYF4:Yb/ Er UCNPs to an aqueous phase, rare earth ions had the tendency to be released from the UCNPs due to the similar affinity of F and O to RE ions.[27] Furthermore, under NIR irradiation, more RE ions tended to be released from UCNPs because of the hot spot caused by photothermal effect. In order to estimate the ionic release from the UCNPs in an aqueous phase with or without NIR irradiation, inductively coupled plasma mass spectrometry (ICP-MS) was employed to measure the amount of Er3+ released from the UCNPs. A significant level of Er3+ was found in the sample of UCNPs compared to the control group (Table S1, Supporting Information). Noteworthy, under the same condition, when treated
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the sample with NIR irradiation, the released Er3+ increased 14.5 fold, leading to an enhanced effect of UCNPs on cell differentiation. On the other hand, NIR irradiation of nanoparticles always induces the production of reactive oxygen species (ROS)[28] (Figure S8, Supporting Information). Low concentrations of ROS are important for intracellular signaling mechanisms and regulating many cellular processes.[19] Oravecz have demonstrated that ROS had the ability to enhance nerve cells differentiation.[19] Therefore, the ROS produced by UCNPs under NIR may be another critical factor for enhancement of cell differentiation (Figure 5). The ability to induce cell differentiation is associated with cell viability. Therefore, it is important to check whether UNCPs with or without NIR irradiation can affect the growth of PC12 cells under our experimental conditions. As shown in Figure S9 (Supporting Information), UCNPs at concentrations ranging from 10 to 100 µg/mL treated with or without NIR irradiation showed no significant cytotoxicity on PC12 cells. As suitable candidates for neurite outgrowth, the nanoparticles should cross the blood-brain-barrier (BBB) and be uptaken by cells. To determine whether UCNPs can passively accumulate in the brain of living animals, we used ICP-MS to measure the amount of UCNPs in the brain after intraperitoneal injection for 12 h. A significant level of Er was found at the mouse brain that was treated with UCNPs compared to the control group. The efficiency of UCNPs accumulation in
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Figure 4. A) Images of calcein-labeled PC12 cells after 2 days of treatments (with and without UCNPs incubation; with and without NIR stimulation). The scale bar is 25 µm. B) Differentiation efficiency of PC12 cells as a function of the dose. C) The ratio of cells whose length was longer than the cell body to total differentiated cells ((L2+L3)/(L1+L2+L3)). (*p < 0.05, **p < 0.01, and ***p < 0.001.) Neurite-bearing cells were measured in three random images for each condition.
the brain was about 1.7%, indicating UCNPs possessed the ability to cross BBB, which was in agreement with previous report.[29] Similar to other nanoparticles, the UCNPs were taken into the brain by the endocytic pathway.[30] Critically, the uptake of UCNPs by PC12 cells was also investigated. The percentage of UCNPs internalized into the cell was 5.4%. These results further supported that UCNPs could act as promising therapeutic agents for neurite outgrowth. In summary, UCNPs can promote neurite outgrowth, PEI-decorated nanoparticles show better effect than PEGor PAA-decorated ones. The difference may be due to different surface charges of the UCNPs. NIR can even enhance the activity of UCNPs to promote the neurite outgrowth. Further studies indicate that the UCNPs can cross BBB with the potential as a promising therapeutic agent for neurite outgrowth. So far, there is no report of using UCNPs as neurite outgrowth promoters. Our results may accelerate small 2014, 10, No. 18, 3655–3661
biomedical applications of UCNPs as therapeutic agents for neurite outgrowth.
Experimental Section Preparation and Characterization of UCNPs: In a typical experiment, to a well-agitated transparent solution of ethylene glycol (EG) (25 mL) charged with NaCl (3.6 mmol), YCl3 (1.56 mmol), YbCl3 (0.4 mmol), ErCl3 (0.4 mmol), and PEI (0.15 g) was added a stoichiometric amount of NH4F in 18 mL of EG. The resulting mixture was agitated for another 15 min, then transferred to a 20 mL of Teflon-lined autoclave, and subsequently heated at 200 °C for 24 h. The obtained nanoparticles were collected by centrifugation, washed with ethanol and deionized water several times, and dried in an oven at 37 °C. The synthesized nanoparticles were observed using scan electron microscopy (SEM) and transmission electron microscopy (TEM).
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Figure 5. Schematic illustration of UCNPs enhance the differentiation of PC12 cells with the stimulation of NIR light.
Cell Culture: PC12 cells (rat pheochromocytoma, American Type Culture Collection) were cultured in DMEM (Gibco BRL) medium supplemented with 5% fetal bovine serum (FBS), 10% horse serum (HS) in a 5% CO2 humidified environment at 37 °C. PC12 cells were seeded (5000 cells/cm2) and incubated for 24 h in serum-reduced media (1% HS and 0.5% FBS) before treatment with UCNPs. UCNPs were added into the media at concentrations ranging from 10 to 40 µg/mL. Simultaneously, NGF (50 ng/mL) was added into the media to induce the differentiation of PC12 cells. Fresh NGF was added to the media every two days. Measurement of Neurite Outgrowth of PC12 Cells: Neurite outgrowth of PC12 cells was measured in a time- and dose-dependent manner, PC12 cells were treated with UCNPs and NGF. After induction of differentiation at 2, 4, and 6 days, the level of neurite outgrowth was quantified by counting the number of PC12 cells and measuring the neurite length in three random images from triplicate experiments. The level of differentiation was determined as L0, L1, L2 and L3 according to the length of the neurites. L0 was defined as cells with no neurites, L1 was defined as cells with neurites whose length was shorter than the size of the cell body, L2 was defined as cells with neurites whose length was between the original or twice the size of the cell body, and L3 was defined as cells with neurites whose length was longer than twice the size of the cell body. The differentiation efficiency was determined by dividing the number of neurite-bearing cells (L1, L2, plus L3) by the total number of cells. In NIR irradiation stimulation experiment, 4 hours After adding UCNPs, we irradiated the 24-well plates with 980 nm laser for 5 min (2.5 W/cm2). And after irradiation, fresh NGF was added to the media. Scanning Electron Microscopy (SEM) Study: In the SEM analysis, cells were seeded on a glass cover slip (10 mm in diameter). After treated with UCNPs, the differentiated PC12 cells were fixed with 2% glutaraldehyde and 2% paraformaldehyde in 0.01 m phosphate buffered saline (PBS) for 30 min at 4 °C. Cells were dehydrated in an ascending series of ethanol (50%, 70%, 80%, 90%, and 100%). Finally, cells were immersed in HMDS
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(hexamethyldizilasane) and dried, and sputter coated with Au. Changes in cell morphology were monitored using SEM. Cell Viability Assay: The MTT assay was used to examine the cytotoxicity of the UCNPs during the differentiation of PC12 cells. 2 × 103 cells were seeded onto the 96-well plates. After incubation with the UCNPs for 48 h at concentrations ranging from 10 to 100 µg/mL, the cells were treated with 10 µL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL, SigmaAldrich) was added to the media (0.5 mg/mL) for 4 h at 37 °C and then were lysed in DMSO for 10 min at room temperature in the dark. Absorbance values of formazan were determined at 490/630 nm with a Bio-Rad model-680 microplate reader. Ion Release and ROS Production Assay: To assess the ionic release of the UCNPs in an aqueous phase, the comparison of NaYF4:Yb/Er with and without NIR irradiation were employed to study the ionic release effect. The PEI-capped NPs were transferred into the aqueous phase. The irradiation sample had been irradiated with NIR for 30 minutes. Equal amounts of both samples of NPs experienced a 96 h dialysis in deionized water, and RE ion concentrations in the dialysate were analyzed by inductively coupled plasma mass spectrometry (ICP-MS). To evaluate the production of ROS through the upconversion process, the comparison of NaYF4:Yb/Er with and without NIR irradiation were employed to study the ROS production effect. The PEI-capped NPs were transferred into the aqueous phase. Both irradiated and nonirradiated samples of NPs were mixed with DCFH-DA (20 µM) in deionized water. The irradiation samples had been irradiated with NIR for 5 min and ROS concentrations were analyzed by fluorescence spectrum. Blood Brain Barrier Penetration and Cellular Uptake of UCNPs Assay: Wild-type Wistar rats were chosen as test animals, in a weight range of 200–250 g (8–12 weeks old) and random in sex. All animal studies were conducted in accordance with the principles and procedures outlined in “Regulations for the Administration of Affairs Concerning Laboratory Animals”, approved by the National Council of China on October 31, 1988, and “The National Regulation of China for Care and Use of Laboratory Animals”, promulgated by the National Science and Technology Commission of China, on November 14, 1988 as Decree No. 2. Protocols were approved by the Committee of Jilin University Institutional Animal Care and Use.[31] UCNPs in saline were intravenously administered to wild-type Wistar rats at 10 mg/kg body weight. Animals were killed 12 h after intraperitoneal injection of UCNPs. The brain was collected immediately, washed twice with normal saline solution, and dried under vacuum for 48 h at 80 °C. The dried samples were ground into powder, and the powders were acid-digested in 12 m aqua regia overnight. As a surrogate measure of drug levels, the brain homogenate was analysed[32] for level of rare earth (RE) ions by ICP-MS (Varian 720-ES). PC12 cells were grown in DMEM containing 10% horse serum (HS) and 5% fetal bovine serum (FBS). PC12 cells were seeded (5000 cells/cm2) and incubated for 24 h in serum-reduced media (1% HS and 0.5% FBS) before treatment with UCNPs. UCNPs were added into the media at 20 µg/ mL. Simultaneously, NGF (50 ng/mL) was added into the media to induce the differentiation of PC12 cells. After 4 h of adding UCNPs, PC12 cells were digested by trypsin and ultrasound to lyse cells. The samples washed with Ultrapure water for twice and experienced a 12 000 rpm centrifugation for 10 min. And the concentration of RE ions in the supernatant was analyzed by ICP-MS.
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Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This work was supported by 973 Project (2011CB936004, 2012CB720602), and NSFC (21210002, 91213302).
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Received: March 7, 2014 Revised: April 14, 2014 Published online: May 20, 2014
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NIR-Responsive Upconversion Nanoparticles Stimulate Neurite Outgrowth in PC12 Cells Yijia Guan, Meng Li, Kai Dong, Jinsong Ren, and Xiaogang Qu* Upconversion nanoparticles (UCNPs) which are capable of converting near-infrared (NIR) into higher energy light through sequential absorption of multiple photons or energy transfer have attracted tremendous interest.[1–3] Compared with the conventional down-conversion fluorescent materials that require higher energy ultra violet (UV) or visible light excitation, upconverting materials have many conceivable advantages including weak autofluorescence backgrounds, tunable emission fluorescence,[3] strong penetration abilities under near-infrared (NIR) radiation, resistance to photobleaching, and low toxicity, making them suitable for various biological applications.[4,5] Significant progress has been made towards the utilization of UCNPs in biosensors, nanocomposites, and the recent developed UCNP-based bioprobes for multi-modality imaging and drug delivery system.[6,7] Although it is widely used in biological area, there is still no report for employing UCNPs to manipulate and control behaviors and functions of biological systems especially nervous system. Enhancement of neuron development and outgrowth is of diagnostic importance in neuroscience since they are in regards to the treatment of injured neurons after accident or a degenerative disease, such as Alzheimer's disease (AD).[8] In past decades, nanoparticle platforms have been developed to aid in the diagnosis and therapy of such disease.[9–14] The effects of these nanomaterials on neuron cell outgrowth have received considerable attention, owing to their ability to interact with biological systems at fundamental, molecular levels with high specificity. Nanomaterials and nanotechnology have been reported to have the ability to affect the neurite outgrowth through changing the extracellular matrix (ECM),[15] whose morphology and composition can strongly affect the cell differentiation properties. With the ability to release inorganic ions into the target organ or its surroundings, several inorganic particles without modification can be used as promoter for cell differentiation.[16] Y. Guan, M. Li, K. Dong, Prof. J. Ren, Prof. X. Qu Laboratory of Chemical Biology Division of Biological Inorganic Chemistry State Key Laboratory of Rare Earth Resource Utilization Changchun Institute of Applied Chemistry University of the Chinese Academy of Sciences Chinese Academy of Sciences Changchun, Jilin 130022, China E-mail: [email protected] DOI: 10.1002/smll.201400612 small 2014, 10, No. 18, 3655–3661
Metal ions of therapeutic and diagnostic interest have been reported to enhance the neurite outgrowth.[17] Rare earth (RE) ions, especially lanthanide ions, which can modulate cell attachment and affect cell differentiation, have attracted intensive research interest in medical applications.[18] It has been reported that UCNPs have the potential to release lanthanum ions into the solution, particularly upon NIR irradiation. In addition, through the upconversion process, reactive oxygen species (ROS), which has the ability to enhance cell differentiation in low dose,[19] may also be probably generated. The unique properties of UCNPs inspire us to use them as promising promoters for neurite outgrowth. In this study, we investigated the effects of UCNPs on the neurite outgrowth during differentiation. PC12 cells were used as the neuronal differentiation model. It has been proposed that different charged coating materials play a critical role in cell attachment,[20] which further lead to different effects on cell differentiation. Different charges also modulate neurite outgrowth process by influencing calcium ions channel.[21] Herein, we systematically studied cell morphology and degree of differentiation upon the treatment of poly(ethylene imine) (PEI), polyethylene glycol (PEG) and poly(acrylic acid) (PAA)-decorated UCNPs (PEI-, PEG-, PAA-UCNPs). PEG-UCNPs, filled with hydroxyl-group in the surface of UCNPs, exhibit no charge. The PEI-UCNPs and PAA-UCNPs, which are coated with amino and carboxyl group, carry positive and negative charges, respectively. To the best of our knowledge, this is the first example of using UCNPs as neurite outgrowth promoters. Three different kinds of polymers capped NaYF4:Yb/ Er UCNPs (PEI-UCNPs, PEG-UCNPs and PAA-UCNPs) with an average particle diameter of 70 nm were first synthesized via hydrothermal method as previously reported[22] and were confirmed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) (Figure 1A,B). Coating the UCNPs with PEI, PEG and PAA instead of oleic acid can not only dramatically decrease the cytotoxicity of the materials to enhance their biocompatibility in biological application, but also make the UCNPs reveal different surface charges. Fourier-transform infrared spectroscopy (FTIR) results demonstrated that UCNPs were well covered by these three kinds of polymers (Figure S1, Supporting Information). The chemical compositions of these nanoparticles were further determined by X-ray diffraction (XRD) analysis (Figure 1C) and all diffraction peaks in the XRD pattern could be unambiguously assigned to the NaYF4 of cubic phase (JCPDS, Card No. 77–2042).[23] The absence of any
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Figure 1. A) SEM image, B) TEM image, C) wide-angle XRD pattern, D) upconversion fluorescence spectrum of the NaYF4:Yb/Er nanoparticles.
other phase in the XRD indicated high purity of the UCNPs. Upon continuous excitation at 980 nm, the upconversion luminescence spectrums of the NaYF4:Yb/Er nanoparticles were recorded (Figure 1D). As expected, characteristic sharp emission peaks at around 524, 544, and 655 nm were observed as a result of the 2H11/2→4I15/2, 4S3/2→4I15/2 and 4F →4I 3+ respectively.[23,24] The results 9/2 15/2 transitions of Er demonstrated that the UCNPs could emit green light, which would be beneficial for imaging. To investigate the effect of UCNPs on mammalian neuron differentiation, PC12 cells were treated with a combination of nerve growth factor (NGF) and UCNPs. PC12 cell can sprout neuritis and differentiate into sympathetic ganglion-like cell in response to NGF, making it as a useful model system of neuronal differentiation. Morphological changes in PC12 cells affected by UCNPs were visualized by calcein-AM. Calcein-AM is a hydrophobic non-fluorescent probe that can permeate the plasma membrane and can be hydrolyzed to calcein, an extremely fluorescent and negatively charged molecule. As shown in Figure 2, the neurites started to elongate in the presence of NGF within 2 days when exposed to PEIUCNPs. Importantly, PC12 cells incubated with higher concentration of PEI-UCNPs were more elongated. PEG- and PAA-UCNPs also had the ability to enhance neurite outgrowth, although the differential effect was not as effective
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as that exposed to PEI decorated ones (Figure S2,S3, Supporting Information). The effect of PEI-UCNPs on neurite extension was sustained even up to 6 days (Figure S4, Supporting Information). Besides, the number of neurite-bearing cells and their length prominently increased with increase of the incubation time. When exposed to the highest dose of PEI- or PEG-UCNPs to the NGF-treated cells, neurite outgrowth was quite obvious compared with NGF-treated cells at day 6. However, when the treatment concentration of PAA-UCNPs was up to 40 µg/mL, neurite outgrowth decreased at day 6, mainly due to the cytotoxicity of PAAUCNPs (Figure S6, Supporting Information). Together with the SEM experiments (Figure S7, Supporting Information), compared with non-treated cells, our result indicated that UCNPs except PAA coated ones, promoted neurite outgrowth in a time- and dose-dependent manner. To quantitatively estimate the level of neurite outgrowth enhanced by UCNPs, the differentiated cells were divided into four groups (L0, L1, L2, and L3) according to the length of neurites which was described in detail in the experimental section. As shown in Figure 3, the percentage of the cells with longer neurite increased even within 2 days incubation in the presence of UCNPs. Taken PEI-UCNPs as an example, Figure 3A showed that co-incubated only with NGF, the percentage of L0 was 93.5%, while the percentage decreased to 77.9%,
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Figure 2. Images of PC12 after 2 days of treatment with NGF and 0 µg/mL, 10 µg/mL, 20 µg/mL, 40 µg/mL PEI-UCNPs. Each series can be classified to the bright light (left) and the green fluorescence from calcein (middle), and overlap of the bright and the green channels (right). The scale bar is 25 µm.
67.7% and 58.4% upon treatment with 10, 20, and 40 µg/ mL nanopaticles, respectively. On the other hand, the percentage of high differentiated cells (L2 and L3) increased from 0.27% to 12.7% with increase of PEI-UCNPs. Critically, PEG- and PAA-UCNPs performed similarly effects as PEI-UCNPs on PC12 cells differentiation with 2 days incubation (Figure 3B,C). That indicated that when incubated with UCNPs, the ratio of differentiated PC12 cells was significantly higher than the ones without UCNPs. It was worthy to note that the cells showed a slightly higher differentiated level for PEI-decorated UCNPs than PEG- and PAA-decorated ones (Figure 3D). It has been reported that PEI was an effective attachment factor in cell culture.[25] The effective attachment could enhance the UCNPs to exert the promoter effect on cell differentiation. Furthermore, the small 2014, 10, No. 18, 3655–3661
positive charges on the surface also had the ability to stimulate the neurite outgrowth through influencing calcium ion channel.[21] Next we studied NIR effect of the UCNPs on neurite outgrowth. Interestingly, with the stimulation of NIR, the number of neurite-bearing cells and their length were significantly increased when compared with the non-NIR-stimulated ones. Here we took PEI-UCNPs as an example since its effect on neurite outgrowth was more obvious. The differentiating status (the percentage of differentiated cells in the culture) was also monitored after 2 days of culture with different concentrations of UCNPs upon stimulated or non-stimulated with NIR. Figure 4A showed representative micrographs performed on all the groups after 2 days incubation with PEIUCNPs. Clearly, the lengths of neurites with NIR irradiation
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Figure 3. Distribution of the neurite length of PC12 cells 2 days after the inducement of A) PEI-UCNPs, B) PEG-UCNPs, and C) PAA-UCNPs with the increase of nanoparticles concentration. D) Distribution of the neurite length of PC12 cells 2 days after the inducement of various coated UCNPs. (L0 means the cells without neurites; L1 means the cells with neurites whose length is shorter than the size of the cell body; L2 means the cells with neurites whose length is between the original and twice the size of the cell body; L3 means the cells with neurites whose length is longer than twice the size of the cell body).
were longer than that without NIR irradiation. As predicted in the statistical results of the fluorescent images (Figure 4B,C), upon NIR irradiation, the percentage of cells in each differentiation level became significantly higher than those without NIR irradiation. With the irradiation of NIR, the percentage of high differentiated cells (L2 and L3) among the total differentiated cells was 49% at day 2 when treated with 20 µg/ mL of PEI-UCNPs, which was approximately 1.4-fold higher than that without NIR irradiation (Figure 4C). These results indicated that NIR irradiation could even increase UCNPs to enhance neurite outgrowth. It has been reported that RE ion exposure can enhance in vitro cell differentiation via extracellular signal-regulated kinase (ERK) signaling pathway.[26] The rare earth elements contained in these UCNPs may play a crucial role in facilitating PC12 differentiation. When exposed the NaYF4:Yb/ Er UCNPs to an aqueous phase, rare earth ions had the tendency to be released from the UCNPs due to the similar affinity of F and O to RE ions.[27] Furthermore, under NIR irradiation, more RE ions tended to be released from UCNPs because of the hot spot caused by photothermal effect. In order to estimate the ionic release from the UCNPs in an aqueous phase with or without NIR irradiation, inductively coupled plasma mass spectrometry (ICP-MS) was employed to measure the amount of Er3+ released from the UCNPs. A significant level of Er3+ was found in the sample of UCNPs compared to the control group (Table S1, Supporting Information). Noteworthy, under the same condition, when treated
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the sample with NIR irradiation, the released Er3+ increased 14.5 fold, leading to an enhanced effect of UCNPs on cell differentiation. On the other hand, NIR irradiation of nanoparticles always induces the production of reactive oxygen species (ROS)[28] (Figure S8, Supporting Information). Low concentrations of ROS are important for intracellular signaling mechanisms and regulating many cellular processes.[19] Oravecz have demonstrated that ROS had the ability to enhance nerve cells differentiation.[19] Therefore, the ROS produced by UCNPs under NIR may be another critical factor for enhancement of cell differentiation (Figure 5). The ability to induce cell differentiation is associated with cell viability. Therefore, it is important to check whether UNCPs with or without NIR irradiation can affect the growth of PC12 cells under our experimental conditions. As shown in Figure S9 (Supporting Information), UCNPs at concentrations ranging from 10 to 100 µg/mL treated with or without NIR irradiation showed no significant cytotoxicity on PC12 cells. As suitable candidates for neurite outgrowth, the nanoparticles should cross the blood-brain-barrier (BBB) and be uptaken by cells. To determine whether UCNPs can passively accumulate in the brain of living animals, we used ICP-MS to measure the amount of UCNPs in the brain after intraperitoneal injection for 12 h. A significant level of Er was found at the mouse brain that was treated with UCNPs compared to the control group. The efficiency of UCNPs accumulation in
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Figure 4. A) Images of calcein-labeled PC12 cells after 2 days of treatments (with and without UCNPs incubation; with and without NIR stimulation). The scale bar is 25 µm. B) Differentiation efficiency of PC12 cells as a function of the dose. C) The ratio of cells whose length was longer than the cell body to total differentiated cells ((L2+L3)/(L1+L2+L3)). (*p < 0.05, **p < 0.01, and ***p < 0.001.) Neurite-bearing cells were measured in three random images for each condition.
the brain was about 1.7%, indicating UCNPs possessed the ability to cross BBB, which was in agreement with previous report.[29] Similar to other nanoparticles, the UCNPs were taken into the brain by the endocytic pathway.[30] Critically, the uptake of UCNPs by PC12 cells was also investigated. The percentage of UCNPs internalized into the cell was 5.4%. These results further supported that UCNPs could act as promising therapeutic agents for neurite outgrowth. In summary, UCNPs can promote neurite outgrowth, PEI-decorated nanoparticles show better effect than PEGor PAA-decorated ones. The difference may be due to different surface charges of the UCNPs. NIR can even enhance the activity of UCNPs to promote the neurite outgrowth. Further studies indicate that the UCNPs can cross BBB with the potential as a promising therapeutic agent for neurite outgrowth. So far, there is no report of using UCNPs as neurite outgrowth promoters. Our results may accelerate small 2014, 10, No. 18, 3655–3661
biomedical applications of UCNPs as therapeutic agents for neurite outgrowth.
Experimental Section Preparation and Characterization of UCNPs: In a typical experiment, to a well-agitated transparent solution of ethylene glycol (EG) (25 mL) charged with NaCl (3.6 mmol), YCl3 (1.56 mmol), YbCl3 (0.4 mmol), ErCl3 (0.4 mmol), and PEI (0.15 g) was added a stoichiometric amount of NH4F in 18 mL of EG. The resulting mixture was agitated for another 15 min, then transferred to a 20 mL of Teflon-lined autoclave, and subsequently heated at 200 °C for 24 h. The obtained nanoparticles were collected by centrifugation, washed with ethanol and deionized water several times, and dried in an oven at 37 °C. The synthesized nanoparticles were observed using scan electron microscopy (SEM) and transmission electron microscopy (TEM).
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Figure 5. Schematic illustration of UCNPs enhance the differentiation of PC12 cells with the stimulation of NIR light.
Cell Culture: PC12 cells (rat pheochromocytoma, American Type Culture Collection) were cultured in DMEM (Gibco BRL) medium supplemented with 5% fetal bovine serum (FBS), 10% horse serum (HS) in a 5% CO2 humidified environment at 37 °C. PC12 cells were seeded (5000 cells/cm2) and incubated for 24 h in serum-reduced media (1% HS and 0.5% FBS) before treatment with UCNPs. UCNPs were added into the media at concentrations ranging from 10 to 40 µg/mL. Simultaneously, NGF (50 ng/mL) was added into the media to induce the differentiation of PC12 cells. Fresh NGF was added to the media every two days. Measurement of Neurite Outgrowth of PC12 Cells: Neurite outgrowth of PC12 cells was measured in a time- and dose-dependent manner, PC12 cells were treated with UCNPs and NGF. After induction of differentiation at 2, 4, and 6 days, the level of neurite outgrowth was quantified by counting the number of PC12 cells and measuring the neurite length in three random images from triplicate experiments. The level of differentiation was determined as L0, L1, L2 and L3 according to the length of the neurites. L0 was defined as cells with no neurites, L1 was defined as cells with neurites whose length was shorter than the size of the cell body, L2 was defined as cells with neurites whose length was between the original or twice the size of the cell body, and L3 was defined as cells with neurites whose length was longer than twice the size of the cell body. The differentiation efficiency was determined by dividing the number of neurite-bearing cells (L1, L2, plus L3) by the total number of cells. In NIR irradiation stimulation experiment, 4 hours After adding UCNPs, we irradiated the 24-well plates with 980 nm laser for 5 min (2.5 W/cm2). And after irradiation, fresh NGF was added to the media. Scanning Electron Microscopy (SEM) Study: In the SEM analysis, cells were seeded on a glass cover slip (10 mm in diameter). After treated with UCNPs, the differentiated PC12 cells were fixed with 2% glutaraldehyde and 2% paraformaldehyde in 0.01 m phosphate buffered saline (PBS) for 30 min at 4 °C. Cells were dehydrated in an ascending series of ethanol (50%, 70%, 80%, 90%, and 100%). Finally, cells were immersed in HMDS
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(hexamethyldizilasane) and dried, and sputter coated with Au. Changes in cell morphology were monitored using SEM. Cell Viability Assay: The MTT assay was used to examine the cytotoxicity of the UCNPs during the differentiation of PC12 cells. 2 × 103 cells were seeded onto the 96-well plates. After incubation with the UCNPs for 48 h at concentrations ranging from 10 to 100 µg/mL, the cells were treated with 10 µL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL, SigmaAldrich) was added to the media (0.5 mg/mL) for 4 h at 37 °C and then were lysed in DMSO for 10 min at room temperature in the dark. Absorbance values of formazan were determined at 490/630 nm with a Bio-Rad model-680 microplate reader. Ion Release and ROS Production Assay: To assess the ionic release of the UCNPs in an aqueous phase, the comparison of NaYF4:Yb/Er with and without NIR irradiation were employed to study the ionic release effect. The PEI-capped NPs were transferred into the aqueous phase. The irradiation sample had been irradiated with NIR for 30 minutes. Equal amounts of both samples of NPs experienced a 96 h dialysis in deionized water, and RE ion concentrations in the dialysate were analyzed by inductively coupled plasma mass spectrometry (ICP-MS). To evaluate the production of ROS through the upconversion process, the comparison of NaYF4:Yb/Er with and without NIR irradiation were employed to study the ROS production effect. The PEI-capped NPs were transferred into the aqueous phase. Both irradiated and nonirradiated samples of NPs were mixed with DCFH-DA (20 µM) in deionized water. The irradiation samples had been irradiated with NIR for 5 min and ROS concentrations were analyzed by fluorescence spectrum. Blood Brain Barrier Penetration and Cellular Uptake of UCNPs Assay: Wild-type Wistar rats were chosen as test animals, in a weight range of 200–250 g (8–12 weeks old) and random in sex. All animal studies were conducted in accordance with the principles and procedures outlined in “Regulations for the Administration of Affairs Concerning Laboratory Animals”, approved by the National Council of China on October 31, 1988, and “The National Regulation of China for Care and Use of Laboratory Animals”, promulgated by the National Science and Technology Commission of China, on November 14, 1988 as Decree No. 2. Protocols were approved by the Committee of Jilin University Institutional Animal Care and Use.[31] UCNPs in saline were intravenously administered to wild-type Wistar rats at 10 mg/kg body weight. Animals were killed 12 h after intraperitoneal injection of UCNPs. The brain was collected immediately, washed twice with normal saline solution, and dried under vacuum for 48 h at 80 °C. The dried samples were ground into powder, and the powders were acid-digested in 12 m aqua regia overnight. As a surrogate measure of drug levels, the brain homogenate was analysed[32] for level of rare earth (RE) ions by ICP-MS (Varian 720-ES). PC12 cells were grown in DMEM containing 10% horse serum (HS) and 5% fetal bovine serum (FBS). PC12 cells were seeded (5000 cells/cm2) and incubated for 24 h in serum-reduced media (1% HS and 0.5% FBS) before treatment with UCNPs. UCNPs were added into the media at 20 µg/ mL. Simultaneously, NGF (50 ng/mL) was added into the media to induce the differentiation of PC12 cells. After 4 h of adding UCNPs, PC12 cells were digested by trypsin and ultrasound to lyse cells. The samples washed with Ultrapure water for twice and experienced a 12 000 rpm centrifugation for 10 min. And the concentration of RE ions in the supernatant was analyzed by ICP-MS.
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Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This work was supported by 973 Project (2011CB936004, 2012CB720602), and NSFC (21210002, 91213302).
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Received: March 7, 2014 Revised: April 14, 2014 Published online: May 20, 2014
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