Nanoscale View Article Online

Published on 03 June 2014. Downloaded by Ondoku Mayis Universitesi on 02/11/2014 16:19:02.

PAPER

View Journal | View Issue

Cite this: Nanoscale, 2014, 6, 9198

Near-infrared light triggered photodynamic therapy in combination with gene therapy using upconversion nanoparticles for effective cancer cell killing† Xin Wang,a Kai Liu,b Guangbao Yang,b Liang Cheng,*b Lu He,a Yumeng Liu,a Yonggang Li,*a Liang Guo*a and Zhuang Liu*b Upconversion nanoparticles (UCNPs) have drawn much attention in cancer imaging and therapy in recent years. Herein, we for the first time report the use of UCNPs with carefully engineered surface chemistry for combined photodynamic therapy (PDT) and gene therapy of cancer. In our system, positively charged NaGdF4:Yb,Er UCNPs with multilayered polymer coatings are synthesized via a layer by layer strategy, and then loaded simultaneously with Chlorin e6 (Ce6), a photosensitizing molecule, and small interfering RNA (siRNA), which targets the Plk1 oncogene. On the one hand, under excitation by a near-infrared (NIR) light at 980 nm, which shows greatly improved tissue penetration compared with visible light, cytotoxic singlet oxygen can be generated via resonance energy transfer from UCNPs to photosensitizer Ce6, while the residual upconversion luminescence is utilized for imaging. On the other hand, the

Received 8th May 2014 Accepted 29th May 2014

silencing of Plk1 induced by siRNA delivered with UCNPs could induce significant cancer cell apoptosis. As the result of such combined photodynamic and gene therapy, a remarkably enhanced cancer cell

DOI: 10.1039/c4nr02495h

killing effect is realized. Our work thus highlights the promise of UCNPs for imaging guided combination

www.rsc.org/nanoscale

therapy of cancer.

1. Introduction Traditional therapy modalities such as surgery, chemotherapy, and radiotherapy used in current clinical cancer treatment show a number of disadvantages including toxic side effects, drug resistance, and high recurrence probability posttreatment.1–4 The development of new cancer therapy strategies that are highly specic to tumor cells for cancer treatment with high efficacy and low side effects is thus urgently needed. In the past few decades, researchers have proposed various types of alternative cancer treatment methods, among which gene therapy and photodynamic therapy (PDT) have been widely explored.5–10 Gene delivery, which usually involves transduction or knockdown of specic genes, has been playing a crucial role in molecular biomedical research.9 For example, small interfering RNA (siRNA) could be delivered into cells utilizing either viral vectors or non-viral carriers, causing degradation of targeted mRNA and subsequently leading to the silence of specic protein a

Department of Radiology, the First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China. E-mail: [email protected]; [email protected]

b Institute of Functional Nano & So Materials (FUNSOM) & Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou, Jiangsu, 215123, China. E-mail: [email protected]; [email protected]

† Electronic supplementary 10.1039/c4nr02495h

information

9198 | Nanoscale, 2014, 6, 9198–9205

(ESI)

available.

See

DOI:

expression. However, although viral vectors are very efficient in gene delivery into the host cells, their uses are limited by the safety concerns as well as the possible immune response caused to the host.9,11 Therefore, the development of safe and efficient non-viral based gene carriers has attracted tremendous interest in the area of gene therapy. Many non-viral based gene delivery vectors, such as cationic polymers,12,13 silica nanoparticles,14 iron oxide nanoparticles,15 and many other types,16–19 have been extensively explored in recent years. In addition to gene delivery, those nano-carriers in the mean time could possess imaging or other therapeutic functionalities, making them attractive platforms for various theranostic applications. On the other side, photodynamic therapy, another nonconventional cancer treatment approach, presents remarkably reduced side effects and improved selectivity compared to traditional chemotherapy and radiotherapy.20 Upon photon absorption, the photosensitizers could undergo energy transition to surrounding oxygen, and then generate singlet oxygen (SO) or other reactive oxygen species (ROS), which are cytotoxic to cancer cells. However, most of the commonly used photosensitizing molecules are excited by visible light, which has poor tissue penetration and thus restricts the efficacy of PDT in deep cancer treatment. Recently, upconversion nanoparticles (UCNPs), which are able to emit visible high-energy photons under excitation by near-infrared (NIR) light, have shown great

This journal is © The Royal Society of Chemistry 2014

View Article Online

Published on 03 June 2014. Downloaded by Ondoku Mayis Universitesi on 02/11/2014 16:19:02.

Paper

promise in the area of biomedicine for applications in both imaging and therapy.8,21–37 Owing to the absence of an autouorescence background in upconversion luminescence (UCL) imaging, UCNPs are excellent nano-probes for in vivo biomedical imaging and cell tracking with rather high sensitivities.30–35,38–41 With well engineered surface coatings, UCNPs could serve as nano-carriers for delivery of various biomolecules ranging from chemotherapy drugs to gene materials.10,38,42–44 It has also been found that UCNPs under NIR light irradiation could act as an energy donor to excite photosensitizers attached or adsorbed on their surface via resonance energy transfer, thus producing SO to kill cancer cells.8,21–30,38,45 Owing to the greatly reduced light scattering and absorption by biological tissue in the NIR window, the UCNP-based PDT triggered by NIR light could offer remarkably enhanced tissue penetration. Although the use of UCNPs for gene delivery and photodynamic therapy has been separately proposed in a number of recent studies,10,22,27,42,46 the integration of these two therapeutic approaches with UCNPs has never been achieved to the best of our knowledge. In the present study, a multifunctional

Nanoscale

upconversion nanoplatform which combines PDT with gene therapy is put forward to improve the cancer cell killing efficiency. NaGdF4:Yb,Er UCNPs are synthesized and then functionalized via layer by layer coating of counter-charged polymers. UCNPs prepared by this method have a hydrophobic oleic acid layer on top of their surface and can be loaded with lipophilic Chlorin Ce6 molecules by hydrophobic interaction. In the meantime, the positively charged nanoparticles could also be loaded with siRNA for gene delivery (Fig. 1a). Combining PDT and gene therapy both delivered by UCNPs, which at the same time could serve as imaging probes for real-time tracking, a signicant synergistic cancer cell killing effect is nally realized in our experiments, suggesting the great promise of UCNPs as a multifunctional theranostic nanoplatform.

2.

Materials & experiments

2.1. Materials Six-armed amine-terminated poly-(ethylene glycol) (PEG) with a molecular weight (MW) of 10 kDa was obtained from Sunbio

Fig. 1 Preparation and characterization of UCNPs. (a) Schematic illustration showing the functionalization of UCNPs, co-loading with Ce6 and siRNA, and then the combined PDT and gene therapy delivered by UCNPs. (b) A TEM image of as-made UCNPs. (c) Zeta potentials of UCNPs with various layers of polymer coatings measured in water.

This journal is © The Royal Society of Chemistry 2014

Nanoscale, 2014, 6, 9198–9205 | 9199

View Article Online

Published on 03 June 2014. Downloaded by Ondoku Mayis Universitesi on 02/11/2014 16:19:02.

Nanoscale

(South Korea). Branched poly(ethylenimine) (PEI) with a MW of 25 kDa, N-(3-dimethylaminopropyl-N0 -ethylcarbodiimide) hydrochloride (EDC), poly(acrylic acid) (PAA) with a MW of 1.8 kDa, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were purchased from Sigma-Aldrich (USA). Lipofectamine 2000 transfection kit, 40 ,6-diamidino-2-phenylindole (DAPI), and fetal bovine serum (FBS) were obtained from Invitrogen (USA). An Annexin V-FITC apoptosis detection kit was purchased from Biouniquer Technology Co, LTD (Nanjing, China). Dulbecco's modied Eagle's medium (DMEM) was purchased from Thermo Scientic (USA). Negative control siRNA with a scrambled sequence (siN.C., antisense strand, 50 ACGUGACACG UUCGGAGAAdTdT-30 ) and siRNA targeting Plk1 mRNA (siPlk1, antisense strand, 50 -AUAUUCGACUUU GGUUGCCdTdT-30 ) were synthesized by GenePharma Co, LTD (Suzhou, China). Antibodies were supplied by Abcam Co, LTD (USA). 2.2. Preparation of UCNP & UCNP–[email protected]PEI NaGdF4 (Gd : Yb : Er ¼ 78% : 20% : 2%) UCNPs were synthesized using a thermal decomposition method following a literature protocol.23 The as-made UCNPs with oleic acid coating were then precipitated by addition of ethanol, separated by centrifugation, washed with cyclohexane, and then washed twice with ethanol. The obtained nanoparticles could be dispersed well in organic solvents. UCNP–[email protected]PEI was obtained according to our previous work.42 Briey, the hydrophobic UCNPs were rst modied with an octylamine-graedPAA (OA–PAA) copolymer through hydrophobic interactions, yielding UCNP–PAA well dispersed in water. The obtained UCNP–PAA was further conjugated with PEG by mixing UCNPs (1 mg mL1) with six-armed PEG (1 mg mL1) under sonication for 5 min and then adding EDC (2 mg mL1) to induce amide formation. Aer being stirred at room temperature for 30 min, PEI (50 mg mL1) and EDC (2 mg mL1) were added to the mixture following another 5 min of sonication. Aer stirring at room temperature for 8 h, the mixture was puried by centrifugation (14 800 rpm, 5 min). The UCNP–[email protected]PEI (1 mg mL1) was further coated with PAA (10 mg mL1) and PEI (10 mg mL1) layer by layer to further improve the gene binding and delivery efficiency of UCNPs.42 The outermost layer of the nal UCNPs was composed of PEI molecules. Transmission electronic microscopy (TEM) images of UCNPs were taken by a Tecnai G2F20 transmission electronic microscope (FEI Co.). The concentrations of UCNPs were measured by inductively coupled plasma mass spectrometry (ICP-MS). Elemental analysis data were acquired by an elemental analyzer (EA1110 CHNO-S, Carlo Erba). Zeta potentials of nanoparticles were measured by a Nano-ZS90 nanoparticle analyzer (Malvern Instruments Ltd.). 2.3. Chlorin e6 molecules loading into UCNP–[email protected]PEI Chlorin e6 (Ce6) molecules were loaded onto UCNPs via hydrophobic interactions. Briey, 5 mg Ce6 was dissolved in 1 mL DMSO as the stock solution. Ce6 with the desired concentration (0.0125–0.25 mg mL1) was added into UCNP–

9200 | Nanoscale, 2014, 6, 9198–9205

Paper

[email protected]PEI (0.5 mg mL1) aqueous solution. The mixture was placed at room temperature with continuous stirring in the dark overnight. Free Ce6 was removed by centrifugation at 14 800 rpm for 10 minutes and washed 3 times with deionized water. The acquired UCNP–[email protected]PEI–Ce6 complex was resuspended in water by sonication and stored at 4  C in the dark. The UV-Vis-NIR absorbance spectra of UCNP–[email protected]PEI–Ce6 were measured by using a UV765 spectrometer (Shanghai Precision & Scientic Instrument Co. Ltd). The concentration of Ce6 loaded onto UCNPs was determined by its characteristic absorbance peak at 404 nm aer subtracting the absorbance from UCNP–[email protected]PEI at the same wavelength. The upconversion luminescence (UCL) spectra of UCNPs and UCNP– [email protected]PEI–Ce6 were measured by the FluoroMax 4 uorometer equipped with an external 980 nm laser (2 W) as the excitation source. To measure the release of Ce6 from the UCNP–[email protected]PEI– Ce6 nanocomplex, UCNP–[email protected]PEI–Ce6 was incubated in a phosphate buffer solution (PBS) (pH ¼ 7.4) or FBS for different periods of time (0, 1, 2, 4, 8, 12 and 24 h). The amount of released Ce6 in the supernatant was measured by UV-Vis-NIR absorbance spectroscopy aer centrifugation to remove nanoparticles. 2.4. Determination of singlet oxygen Singlet oxygen (SO) was determined according to the previously reported protocol.23,24 Soon aer, singlet oxygen sensor green (SOSG) which was extremely sensitive to singlet oxygen was used to detect SO generated during our experiments. Different samples were mixed with 2.5 mM SOSG, and then irradiated by a 980 nm laser (0.8 W cm2) for different periods of time. The generation of singlet oxygen by UCNP–[email protected]PEI–Ce6 would result in the recovered uorescence of SOSG at 525 nm. A free Ce6 solution was also irradiated by the 980 nm laser as a control. 2.5. Loading of siRNA onto UCNP–[email protected]PEI–Ce6 and agarose gel electrophoresis assay UCNP–[email protected]PEI–Ce6 was mixed with 1 mL siRNA in 20 mL deionized water at different nitrogen/phosphor (N/P) ratios (N/P ¼ 0, 5, 10, 15, 20). The mixtures were then incubated for 1 h at room temperature before they were analyzed by 1% agarose gel electrophoresis at 95 mV in a Tris-acetate-EDTA (TAE) buffer. 2.6. Cellular experiments HeLa cells initially obtained from American Type Culture Collection (ATCC) were cultured in Dulbecco's modied Eagle's medium (DMEM) containing 10% FBS and 1% penicillin/ streptomycin at 37  C in a humidied 5% CO2-containing atmosphere. For siRNA transfection experiment, HeLa cells were seeded in 35 mm culture dishes at a density of 1  105 cells per well. We diluted 200 pmol FAM-siRNA in 200 mL serum-free DMEM and different appropriate amounts of UCNP–[email protected]PEI–Ce6 in 200 mL serum-free DMEM. The two solutions were mixed together and incubated for 20 min at room temperature before

This journal is © The Royal Society of Chemistry 2014

View Article Online

Paper

Published on 03 June 2014. Downloaded by Ondoku Mayis Universitesi on 02/11/2014 16:19:02.

being added into cells with or without the serum, maintaining the nal volume at 2 mL. Here we used Lipofectamine 2000 as the positive transfection agent and siRNA with a scrambled sequence as the negative control. Aer 6 h of transfection, cells were washed twice with PBS (pH ¼ 7.4) and then imaged by a laser scanning confocal microscope (Leica SP5). Following the same method, we could also see the luminescence of UCNPs and uorescence of Ce6. The cell nuclei could be stained by 40 ,60 -diamidino-2-phenylindole (DAPI). 2.7. Photodynamic therapy For the PDT experiment, HeLa cells were seeded in a 96-well plate at a density of 1  104 cells per well for 24 h. Then different concentrations of UCNP–[email protected]PEI–Ce6 or UCNP– [email protected]PEI–Ce6–siRNA were added into each well and incubated for 6 h. Aer the removal of nanoparticles, cells were transferred into fresh media and then irradiated by the 980 nm laser at a power density of 0.8 W cm2 for 20 min, with a 1 min interval for every 1 min of light exposure to avoid heating. The laser power density was measured by a LPE-1C laser powermeter (Wuke Photoelectric Technique, Beijing, China). The cells were then incubated at 37  C for additional 48 h before the MTT assay to determine the cell viabilities. For gene therapy experiments, siRNA transfection was handled following the same protocol mentioned above. We transfered the cells into new fresh complete media and also incubated at 37  C for additional 48 h aer 6 h transfection without irradiation. Cells could be stained with the calcein-AM/propidium iodide (PI) to determine their viabilities. 2.8. RNA extraction and reverse transcription quantitative real-time PCR (RT-qPCR) All transfected cells were washed twice with PBS before their total RNA was extracted for isolation using TRIzol (Takara, Dalian, China) according to the manufacturer's protocol. The concentration and purity of RNA were determined using the NanoDrop (Thermo Fisher Scientic, Waltham, MA, USA). Then RNA was subsequently reversely transcribed to cDNA using an M-MLV First-Strand cDNA Synthesis Kit (Invitrogen, USA). Aerwards, real-time qRT-PCR analysis was carried out targeting Plk1 and b-actin using Platinum SYBR Green qPCR SuperMix-UDG kits (Invitrogen, USA) according to the manufacturer's instructions. Real-time PCR was performed on an ABI Prism 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). According to the DDCt method, the expression value of Plk1 was normalized to b-actin to calculate the relative amount of RNA present in each sample. Each sample was run in triplicate. The primer sequences used for Plk1 and b-actin were: 50 -AGCCTGAGGCCCGATACTACCTAC-30 (Plk1-forward), 50 -ATTAGGAGTCCCACACAGGGTCTTC-30 (Plk1-reverse), and 50 -GCACAGAGCCTCGCCTT-30 (b-actin-forward), 50 -GTTGTCGACGACGAGCG-30 (b-actin-reverse). 2.9. Western blotting analysis Cells were lysed using a BOSTER cell lysis buffer according to the manufacturer's protocol. The protein concentration of each

This journal is © The Royal Society of Chemistry 2014

Nanoscale

sample was determined using a Bradford BSA Protein Assay Kit. Then the cell lysate was mixed at a ratio of 5 : 1 with the loading buffer and heated at 95  C for 5 minutes followed by 5 minute incubation at room temperature. The samples were then loaded on 10% SDS-PAGE gel and subsequently electrotransferred to a polyvinylidence diuoride (PVDF) membrane, which was blocked for an hour at room temperature under a Western blocking buffer with 5% non-fat dry milk. Aer blocking, the membrane was incubated with a mouse anti-human Plk1 antibody at 1 : 1000 for 1 h at 4  C overnight followed by incubation with a goat anti-mouse IgG antibody at 1 : 2000 for 1 h at room temperature. The mouse anti-human b-actin antibody diluted at 1 : 1000 was used as a control for equal protein loading and transfer. Densitometric values of protein bands were quantied using Image Analysis soware on an Evolve-512 photometric system.

3.

Results and discussion

In our experiments, NaGdF4:Yb,Er (Gd : Yb : Er ¼ 78% : 20% : 2%) UCNPs were synthesized by a high temperature thermal decomposition method. The TEM image displayed in Fig. 1b shows that the synthesized UCNPs were monodispersed nanocrystals with a uniform average diameter of 30 nm. Following our previously published procedures,42 as-made UCNPs were rst modied with an OA–PAA copolymer to become water-soluble, conjugated with PEG to acquire stability against salts, and then subsequently coated with PEI, PAA, and another layer of PEI via electrostatic binding. The zeta potentials of UCNPs with different layers of polymer coatings are presented in Fig. 1c. The zeta potentials of UCNP–OA–PAA and UCNP–PEG were measured to be 21.4 and +4.5 mV respectively. UCNP–[email protected]PEI with positively charged PEI coating showed a further increased zeta potential to be +33.4 mV, which was decreased to 27.9 mV aer PAA coating, and then jumped back to +37.9 mV aer the second layer of PEI was coated. The nitrogen content of the nal nano-complex, which was predominately contributed by PEI, was determined by elemental analysis to be 4.2%. Due to the existence of Gd3+ in UCNPs, they can act as a T1 contrasting agent in magnetic resonance imaging37,47,48 (ESI Fig. S1†). To enable the use of UCNPs in PDT, we then loaded Ce6, a widely used photosensitizing agent, onto those nanoparticles. Various concentrations of Ce6 were added into a 0.5 mg mL1 UCNP–[email protected]PEI solution. Aer removal of the unbound Ce6, the UV–Vis spectra were recorded (Fig. 2a). We found that the amount of Ce6 loaded onto UCNPs showed a nearly linear increase with increasing Ce6 concentrations, and reached 10– 11% when the UCNP–Ce6 (w/w) ratio was 2 : 1 (Fig. 2b). Further increase of Ce6 loading, however, would result in decreased stability of those nanoparticles. The surface of UCNPs synthesized in the organic phase is coated with a layer of a capping agent, oleic acid in this case. Similar to our previous reports,29 the binding of Ce6 onto UCNPs happens mainly via hydrophobic interactions between Ce6 molecules and the hydrophobic oleic acid layer on the surface of UCNP cores beneath the PEG/PEI coating. Interestingly, the maximal Ce6 loading

Nanoscale, 2014, 6, 9198–9205 | 9201

View Article Online

Published on 03 June 2014. Downloaded by Ondoku Mayis Universitesi on 02/11/2014 16:19:02.

Nanoscale

Paper

Characterization of Ce6 loaded nano-complexes. (a) UV-Vis absorbance spectra of UCNP–[email protected] (0.5 mg mL1) loaded with different concentrations of Ce6. (b) Quantification of Ce6 loadings at different feeding Ce6 concentrations. The Ce6 loading capacity increased with the rise of Ce6 concentrations. (c) UCL emission spectra of various UCNP samples after different layers of polymer coatings and the subsequent Ce6 loading under 980 nm excitation recorded at the same UCNP concentration. The inset shows photographs of the UCNP– [email protected]PEI sample under ambient light (left) or exposed to the 980 nm laser (right). (d) The generation of singlet oxygen by measuring the fluorescence intensity changes of SOSG at 525 nm as a functional of 980 nm light radiation time (0.8 W cm2). Fig. 2

capacity in the current formulation showed a slight increase from 8% for the previous formulation to 11%, likely owing to the extra electrostatic interactions between Ce6 molecules and the cationic polymer PEI in the present case. In fact the zeta potential of UCNP–[email protected]PEI nanoparticles decreased slightly from 37.9 mV to 30.4 mV aer Ce6 loading. Notably, Ce6 loaded on those nanoparticles appeared to be rather stable with only a small percentage of release (4–6%) aer 24 h incubation in PBS or FBS (ESI Fig. S2†), enabling us to use the obtained UCNP–[email protected]–Ce6 complex for various further applications. The UCL emission spectra of various samples were then measured under 980 nm excitation at the same UCNP concentration (Fig. 2c). While different layers of the polymer coating showed no signicant effect on the UCL intensity of UCNPs, an obvious decrease of UCL intensity of UCNPs aer Ce6 loading was observed, particularly for its emission peak at 660 nm, which overlapped with the absorbance peak of Ce6 at the same wavelength. Such a phenomenon is likely a result of resonance energy transfer from UCNPs to Ce6, which then may produce SO for PDT application. In fact, we did observe effective generation of SO by UCNP–[email protected]PEI–Ce6 under 980 nm laser irradiation (0.8 W cm2) using the SOSG probe (Fig. 2d). In contrast,

9202 | Nanoscale, 2014, 6, 9198–9205

free Ce6 under excitation by such NIR light was not able to produce SO. Therefore, our UCNP–[email protected]PEI–Ce6 could be a useful PDT agent under 980 nm NIR light exposure. It is known that Polo-like kinase 1 (Plk1) plays a crucial role in DNA replication, and is overexpressed in many types of cancer cells. Silencing of Plk1, on the other hand, would trigger cell apoptosis. We thus would like to use our UCNP–[email protected]PEI– Ce6 as a multifunctional vector for the delivery of Plk1 siRNA, aiming at gene therapy to kill cancer cells. An agarose gel electrophoresis experiment was thus conducted aer mixing UCNP– [email protected]PEI–Ce6 with siRNA at different N/P ratios (ESI Fig. S3†). At N/P ratios over 10, the majority of siRNA was effectively loaded onto nanoparticles. In our previous work, it was found that different from other conventional transfection agents, the plasmid DNA transfection efficiency of UCNP– [email protected]PEI would be enhanced, rather than be decreased, by the presence of serum. In our current study, we also checked the serum effects on the siRNA transfection by using uorescently labeled siRNA. Consistent with our previous results,42 the siRNA delivery ability of UCNP–[email protected]PEI–Ce6 was also increased by the serum in the transfection medium (ESI Fig. S4†). Next, we used a confocal uorescence microscope to study the cellular uptake of a UCNP–[email protected]PEI–Ce6–siRNA

This journal is © The Royal Society of Chemistry 2014

View Article Online

Published on 03 June 2014. Downloaded by Ondoku Mayis Universitesi on 02/11/2014 16:19:02.

Paper

complex (Fig. 3a). The UCL signals from UCNPs, together with uorescence signals from both Ce6 and uorescently labeled siRNA were simultaneously detected. Clear co-localization of signals from three different channels was observed, suggesting that both Ce6 and siRNA were successfully shuttled into cells by UCNPs. Aerwards, Western blotting was carried out to determine the expression of Plk1 aer UCNP-induced siRNA transfection with or without FBS (Fig. 3b). The Lipofectamine-mediated transfection of Plk1 siRNA (siPlk1) (Fig. 3b, lane 3) led to an

Nanoscale

obvious down-regulation of Plk1, while transfection of siRNA with a scramble sequence (siN.C.) showed no appreciable effect on Plk1 expression (Fig. 3b, lane 2). For cells treated with UCNP–[email protected]PEI–Ce6–siPlk1, obviously decreased Plk1 expression was observed with the increase of the N/P ratio. Quantication of Western blotting data is shown in Fig. 3c. Consistent with our previous study using [email protected]PEI for DNA plasmid delivery,42 it was found that the presence of FBS could also enhance the siRNA-induced down-regulation of Plk1 protein expression, especially at the N/P ratio of 15.

Cell uptake and in vitro siRNA transfection. (a) Confocal microscopy images of HeLa cells after incubation with UCNP–[email protected]–Ce6– FAM-siRNA for 4 h. The UCL emission from UCNPs (green colored), FAM-siRNA fluorescence (blue colored) and Ce6 fluorescence (red colored) showed obvious co-localization inside cells. (b) Western blotting results to determine Plk1 expression of HeLa cells after various treatments indicated. b-Actin was also detected as the internal control. (c) Quantitative determination of Plk1 expression for different samples based on Western blotting data from (b). (d) The expression levels of Plk1 mRNA determined by qRT-PCR. Plk1 mRNA levels were expressed as a relative index normalized against b-actin. Error bars were based on triplicated samples. Fig. 3

This journal is © The Royal Society of Chemistry 2014

Nanoscale, 2014, 6, 9198–9205 | 9203

View Article Online

Published on 03 June 2014. Downloaded by Ondoku Mayis Universitesi on 02/11/2014 16:19:02.

Nanoscale

Paper

Fig. 4 In vitro NIR-induced PDT and combination cancer therapy. (a) Relative cell viability data of HeLa cells after being treated with UCNP– [email protected]–Ce6 without or with the 980 nm NIR light exposure. (b) In vitro cytotoxicity effect induced by PDT, gene therapy, or combination therapy. The combined photodynamic & gene therapy offered a significantly higher cancer cell killing effect compared to mono-therapy by either gene therapy or PDT alone. (c) Fluorescence micrographs showing the calcein-AM (green, for living cells) and PI (red, for dead cells) double stained HeLa cells. Scale bar: 100 mm. Error bars in (a and b) are based on triplicated samples.

To further verify Plk1 down-regulation and the specicity of the knockdown, the Plk1 mRNA expression levels were analyzed 48 h aer transfection by RT-qPCR (Fig. 3d). Consistent with Western blotting results, the mRNA levels of UCNP– [email protected]PEI–siPlk1 treated cells showed signicant downregulation, and the RNAi effects appeared to be more effective when transfection was conducted in the presence of FBS. Both Western blotting and qPCR results indicated that our UCNPbased siRNA transfection could offer a rather high efficiency, which was comparable to that achieved using the commercial transfection agent, Lipofectamine 2000, under its recommended optimal conditions (serum-free). Before UCNP–[email protected]PEI–Ce6–siPlk1 was used for combined PDT/gene therapy, we rst checked whether the production of singlet oxygen during PDT would affect siRNA loaded on UCNPs. UCNP–[email protected]PEI–Ce6–siPlk1 treated HeLa under the optimized transfection conditions were treated with or without 980 nm laser irradiation (0.8 W cm2) for 20 min. The Plk1 protein levels in those samples were measured by Western blotting (ESI Fig. S5†). It was found that PDT, which although could produce cytotoxic singlet oxygen, would not effect the siRNA-induced gene silencing, making the combination of PDT with gene therapy a reasonable approach. At last, cell viability assays were conducted for HeLa cells aer various treatments including PDT, gene therapy and combined PDT/gene therapy (Fig. 4a and b). For the control experiment where the cells were incubated with UCNP– [email protected]PEI–Ce6 in the absence of laser irradiation, no

9204 | Nanoscale, 2014, 6, 9198–9205

appreciable dark toxicity of our nanocomplex was observed. Upon 980 nm laser irradiation (0.8 W cm2) for 20 min, the cell viabilities gradually decreased with the rise of UCNP– [email protected]PEI–Ce6 concentrations, indicating the effectiveness of NIR-induced PDT mediated by UCNPs (Fig. 4a). Owing to the silencing of Plk1 expression, a concentration-dependent reduction of cell viabilities was also observed for cells treated with UCNP–[email protected]PEI–siPlk1 for gene therapy (Fig. 4b). Importantly, the combination of PDT and gene therapy for cells treated with UCNP–[email protected]PEI–Ce6–siPlk1 and then exposed to 980 nm light showed much lower remaining cell viabilities compared to single PDT or gene therapy alone (Fig. 4b). Microscopy images of calcein-AM & PI double stained (living & dead cells) HeLa cells aer different treatments further conrmed that remarkably enhanced cancer cell killing was realized by the combined photodynamic + gene therapy delivered with UCNPs (Fig. 4c).

4. Conclusion In conclusion, a new multifunctional UCNP-based gene vector is successfully developed for combined PDT and gene therapy of cancer. In this system, UCNPs with the unique upconversion photoluminescence could serve as an imaging probe, as well as an energy donor useful in triggering PDT under NIR light, which offers greatly improved tissue penetration as demonstrated in previous studies.29 With well engineered surface coatings, these nanoparticles appear to be an effective siRNA delivery vector

This journal is © The Royal Society of Chemistry 2014

View Article Online

Paper

Published on 03 June 2014. Downloaded by Ondoku Mayis Universitesi on 02/11/2014 16:19:02.

that works well in the presence of serum. Utilizing such multifunctional nano-complex with both Ce6 and siRNA loading, we are able to combine PDT, which is triggered by NIR light, with gene therapy, which is a result of Plk1 silencing, to achieve effective cancer cell killing. Therefore, our study demonstrates that UCNPs with a well designed and engineered surface could serve as a multifunctional nano-platform that offers novel opportunities in biomedical imaging and therapy.

Acknowledgements This work was partially supported by the National Basic Research Programs of China (973 Program) (2012CB932600, 2011CB911002), the National Natural Science Foundation of China (81171394, 51302180, 81171392, 51222203), a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and the Natural Science Foundation of Jiangsu Province (BK2011307). Z Liu acknowledges the Natural Science Fund for Distinguished Young Scholars of Jiangsu Province.

References 1 J. H. Maduro, E. Pras, P. H. B. Willemse and E. G. E. de Vries, Cancer Treat. Rev., 2003, 29, 471–488. 2 J. W. Mier, F. R. Aronson, R. P. Numerof, G. Vachino and M. B. Atkins, Pathol. Immunopathol. Res., 1988, 7, 459–476. 3 J. W. Mier and M. B. Atkins, Curr. Opin. Oncol., 1993, 5, 1067– 1072. 4 A. E. Omoti and C. E. Omoti, Pharm Pract, 2006, 4, 55–59. 5 D. E. J. G. J. Dolmans, D. Fukumura and R. K. Jain, Nat. Rev. Cancer, 2003, 3, 380–387. 6 L. Feng, X. Yang, X. Shi, X. Tan, R. Peng, J. Wang and Z. Liu, Small, 2013, 9, 1989–1997. 7 R. Ghosh, L. C. Singh, J. M. Shohet and P. H. Gunaratne, Biomaterials, 2013, 34, 807–816. 8 Z. Li, C. Wang, L. Cheng, H. Gong, S. Yin, Q. Gong, Y. Li and Z. Liu, Biomaterials, 2013, 34, 9160–9170. 9 Z. Zhang, L. Song, J. Dong, D. Guo, X. Du, B. Cao, Y. Zhang, N. Gu and X. Mao, J. Nanopart. Res., 2013, 15, 1–11. 10 Y. Yang, F. Liu, X. Liu and B. Xing, Nanoscale, 2013, 5, 231–238. 11 N. Bessis, F. J. GarciaCozar and M. C. Boissier, Gene Ther., 2004, 11, S10–S17. 12 X. Gao and L. Huang, Gene Ther., 1995, 2, 710–722. 13 S. K. Samal, M. Dash, S. Van Vlierberghe, D. L. Kaplan, E. Chiellini, C. Van Blitterswijk, L. Moroni and P. Dubruel, Chem. Soc. Rev., 2012, 41, 7147–7194. 14 K. K. Singh, Technol. Cancer Res. Treat., 2005, 4, 583–584. 15 S. K. Sahoo and V. Labhasetwar, Drug Discovery Today, 2003, 8, 1112–1120. 16 K. Bates and K. Kostarelos, Adv. Drug Delivery Rev., 2013, 65, 2023–2033. 17 A. Bianco, Expert Opin. Drug Delivery, 2004, 1, 57–65. 18 L. Feng, S. Zhang and Z. Liu, Nanoscale, 2011, 3, 1252–1257. 19 A. Bianco, K. Kostarelos, C. D. Partidos and M. Prato, Chem. Commun., 2005, 571–577.

This journal is © The Royal Society of Chemistry 2014

Nanoscale

20 B. Tian, C. Wang, S. Zhang, L. Feng and Z. Liu, ACS Nano, 2011, 5, 7000–7009. 21 D. J. Burgess, Nat. Rev. Cancer, 2012, 12, 737–737. 22 D. K. Chatterjee and Z. Yong, Nanomedicine, 2008, 3, 73–82. 23 Q. Chen, C. Wang, L. Cheng, W. He, Z. Cheng and Z. Liu, Biomaterials, 2014, 35, 2915–2923. 24 H. Gong, L. Cheng, J. Xiang, H. Xu, L. Feng, X. Shi and Z. Liu, Adv. Funct. Mater., 2013, 23, 6059–6067. 25 N. M. Idris, M. K. Gnanasammandhan, J. Zhang, P. C. Ho, R. Mahendran and Y. Zhang, Nat. Med., 2012, 18, 1580–1585. 26 S. Jiang and Y. Zhang, Langmuir, 2010, 26, 6689–6694. 27 K. Liu, X. Liu, Q. Zeng, Y. Zhang, L. Tu, T. Liu, X. Kong, Y. Wang, F. Cao, S. A. G. Lambrechts, M. C. G. Aalders and H. Zhang, ACS Nano, 2012, 6, 4054–4062. 28 J. R. Starkey, A. K. Rebane, M. A. Drobizhev, F. Meng, A. Gong, A. Elliott, K. McInnerney and C. W. Spangler, Clin. Cancer Res., 2008, 14, 6564–6573. 29 C. Wang, H. Tao, L. Cheng and Z. Liu, Biomaterials, 2011, 32, 6145–6154. 30 B. G. Yust, D. K. Sardar, L. C. Mimun, A. K. Gangadharan and A. T. Tsin, Proc. SPIE, 2013, 8594, 85940D-9. 31 D. K. Chatterjee, A. J. Rufaihah and Y. Zhang, Biomaterials, 2008, 29, 937–943. 32 N. M. Idris, Z. Li, L. Ye, E. K. Wei Sim, R. Mahendran, P. C.-L. Ho and Y. Zhang, Biomaterials, 2009, 30, 5104–5113. 33 Q. Liu, Y. Sun, T. Yang, W. Feng, C. Li and F. Li, J. Am. Chem. Soc., 2011, 133, 17122–17125. 34 C. Wang, L. Cheng, H. Xu and Z. Liu, Biomaterials, 2012, 33, 4872–4881. 35 L. Xiong, Z. Chen, Q. Tian, T. Cao, C. Xu and F. Li, Anal. Chem., 2009, 81, 8687–8694. 36 J. Liu, W. Bu, L. Pan and J. Shi, Angew. Chem., Int. Ed., 2013, 52, 4375–4379. 37 D. Yang, Y. Dai, J. Liu, Y. Zhou, Y. Chen, C. Li, P. a. Ma and J. Lin, Biomaterials, 2014, 35, 2011–2023. 38 L. Cheng, C. Wang and Z. Liu, Nanoscale, 2013, 5, 23–37. 39 L. Cheng, C. Wang, X. Ma, Q. Wang, Y. Cheng, H. Wang, Y. Li and Z. Liu, Adv. Funct. Mater., 2013, 23, 272–280. 40 L. Cheng, K. Yang, S. Zhang, M. Shao, S. Lee and Z. Liu, Nano Res., 2010, 3, 722–732. 41 T. Yang, Q. Liu, J. Li, S. Pu, P. Yang and F. Li, RSC Adv., 2014, 4, 15613–15619. 42 L. He, L. Feng, L. Cheng, Y. Liu, Z. Li, R. Peng, Y. Li, L. Guo and Z. Liu, ACS Appl. Mater. Interfaces, 2013, 5, 10381–10388. 43 J. Shen, L. Zhao and G. Han, Adv. Drug Delivery Rev., 2013, 65, 744–755. 44 G. Tian, W. Ren, L. Yan, S. Jian, Z. Gu, L. Zhou, S. Jin, W. Yin, S. Li and Y. Zhao, Small, 2013, 9, 1929–1938. 45 S. Cui, D. Yin, Y. Chen, Y. Di, H. Chen, Y. Ma, S. Achilefu and Y. Gu, ACS Nano, 2012, 7, 676–688. 46 M. K. G. Jayakumar, N. M. Idris and Y. Zhang, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 8483–8488. 47 J. Zhou, Y. Sun, X. Du, L. Xiong, H. Hu and F. Li, Biomaterials, 2010, 31, 3287–3295. 48 D. Ni, J. Zhang, W. Bu, H. Xing, F. Han, Q. Xiao, Z. Yao, F. Chen, Q. He, J. Liu, S. Zhang, W. Fan, L. Zhou, W. Peng and J. Shi, ACS Nano, 2014, 8, 1231–1242.

Nanoscale, 2014, 6, 9198–9205 | 9205

Near-infrared light triggered photodynamic therapy in combination with gene therapy using upconversion nanoparticles for effective cancer cell killing.

Upconversion nanoparticles (UCNPs) have drawn much attention in cancer imaging and therapy in recent years. Herein, we for the first time report the u...
3MB Sizes 0 Downloads 5 Views