Efficient Gene Delivery and Multimodal Imaging by Lanthanide-Based

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Efficient Gene Delivery and Multi-Modal Imaging by Lanthanide-Based Up-Conversion Nanoparticles Lin Wang, Jianhua Liu, Yunlu Dai, Qiang Yang, Yuanxin Zhang, Piaoping Yang, Ziyong Cheng, Hongzhou Lian, Chunxia Li, Zhiyao Hou, Ping'an Ma, and Jun Lin Langmuir, Just Accepted Manuscript • DOI: 10.1021/la503444g • Publication Date (Web): 07 Oct 2014 Downloaded from http://pubs.acs.org on October 10, 2014

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Efficient Gene Delivery and Multi-Modal Imaging by Lanthanide-Based UpConversion Nanoparticles Lin Wang,†,‡ Jianhua Liu,£ Yunlu Dai,†,± Qiang Yang,† Yuanxin Zhang,† Piaoping Yang,‡ Ziyong Cheng,† Hongzhou Lian,† Chunxia Li,† Zhiyao Hou,† Ping’an Ma,†,* and Jun Lin,†,*

†. State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, People’s Republic of China ‡ . Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education, Harbin Engineering University, Harbin, 150001, People’s Republic of China £. Department of Radiology, The Second Hospital of Jilin University, Changchun, 130022, People’s Republic of China ±. University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China

*

Corresponding Author:

*

Jun Lin: [email protected].

*

Ping’an Ma: [email protected].

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Abstract Nanoparticles have been explored as non-viral gene carriers for years because of the simplicity of surface modification and lack of immune response. Lanthanide-based up-conversion nanoparticles (UCNPs) are becoming attractive candidates for biomedical applications in virtue of their unique optical properties and multi-modality imaging ability. Here, we report a UCNPs-based structure with polyethylenimine coating for both efficient gene transfection and tri-modality imaging. Cytotoxicity tests demonstrated that the nanoparticles exhibited significantly decreased cytotoxicity compared to polyethylenimine polymer. Further, in vitro studies revealed that the gene carriers are able to transfer the enhanced green fluorescence protein (EGFP) plasmid DNA into Hela cells in higher transfection efficiency than PEI. Gene silencing was also examined by delivering bcl-2 siRNA into Hela cells, resulting in significant down-regulation of target bcl-2 mRNA. More importantly, we demonstrated the feasibility of up-conversion gene carriers to serve as effective contrast agents for MRI/CT/UCL tri-modality imaging both in vitro and in vivo. The facile fabrication process, great biocompatibility, enhanced gene transfection efficiency and great bioimaging ability can make it promising for application in gene therapy.

Keywords: up-conversion fluorescence, gene delivery, multi-modality imaging, nanoparticles, gene silence.


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INTRODUCTION Gene therapy to cure or prevent diseases has been persistently pursued as an alternative to drug treatment for decades. This approach requires a technology capable of delivering nucleic acids into a variety of cells, tissues and organs safely and effectively.1, 2 Physical methods, such as electroporation and gene gun, have been developed for the delivery of naked DNA,3, 4 while the expression level as well as the area after injection is limited owing to the degradation by nuclease. Using a vehicle to ferry the gene to target sites is an alternative method. Viral delivery systems, including retroviral vectors, lentiviral vectors and adenoviral vectors, have been proved to be effective both in vitro and in vivo.5 However, as these vectors exploit the transfection mechanism of viruses, they suffer from serious immunological problems despite these viruses have been disabled of pathogenic effects. In contrast, non-viral delivery systems have shown advantages in terms of ease of large-scale production, simplicity of further modification and lack of immune response.6-8 Cationic polymers such as polyethylenimine (PEI) and poly(L-lysine) (PLL) have been widely investigated as gene carriers in virtue of the ability to complex and condense DNA molecules resulting in the formation of polyplexes, while the high cytotoxicity impeded their clinical application.9,

10

Inorganic nanoparticles have been

employed as biomaterials in recent years. Many kinds of inorganic nanomaterials, such as gold,11, 12 silica,13, 14 calcium phosphate15, 16 and graphene,17, 18 have been explored as gene carriers. Although the transfection efficiency is moderate, inorganic nanoparticles are not subject to microbial attack and exhibit good storage stability. Therefore, the inorganic nanoparticles offer promising ways to prepare a suitable system for clinical use.


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Another advancement in the design of delivery system is the combination of target imaging and therapeutic delivery. Molecular imaging methods such as optical imaging, magnetic resonance imaging (MRI), X-ray computed tomography (CT) and positron emission tomography (PET) are playing important roles in biotechnology research. The application of these combined systems allows for target imaging, early detection and treatment of diseases.19-21 In recent years, multi-functional platforms based on iron oxide nanoparticles (IONPs),22-24 and quantum dots (QDs)25, 26 have been developed. IONPs have a relatively long blood retention time and low inherent toxicity, while QDs have an edge over IONPs owing to the imaging ability and diverse surface modification methods. Besides the above mentioned candidates that hold promises, lanthanide-doped up-conversion nanoparticles (UCNPs) are attracting growing attention in this field because of their unique properties.27-31 First, UCNPs are capable of converting low-energy light to higher-energy photons via a two photons or multi-photons mechanism, which endows greater tissue penetration and low cytotoxicity.32-36 Second, Gd3+ with seven unpaired electrons shows high paramagnetic relaxivity and materials containing Gd3+ can act as T1-positive contrast agents,37, 38 , some MRI advances have been made by using materials such as NaGdF4.39, 40 Third, nanomaterials containing ytterbium (Yb), the commonly used sensitizer in UCNPs, have been explored as CT contrast agents. Products with higher CT contrast efficacy than the clinical used Iobitridol have been reported.41-43 Therefore, of interest to us in this work is the integration of efficient gene delivery and UCNP-based multi-modal imaging - a combination platform that may achieve optimized treatment efficacy in related diseases.


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Up to date, several studies on UCNPs-based gene carriers have been reported. Zhang’s group pioneered in the research of using UCNPs as gene vectors. Through amino-functionalized silica coating on UCNPs, they reported the efficient delivery and photo-controlled activation of small interiencing RNA (siRNA).44, 45 Xing’s group developed another strategy for targeted delivery of siRNA by introducing the photo-sensitive o-nitrobenzyl linker to UCNPs, which could split the amino group from UCNPs’ surface.46 He et al. synthesized Gd3+-doped UCNPs with multiple polymer layers for serum enhanced gene delivery and multimodal imaging.47 Though seems promising, further studies are still required to improve the transfection efficiency and in vivo imaging efficacy of the UCNPs-based gene carriers. Herein, based on our previous works on the fabrication of functionalized up-conversion drug delivery systems,48-50 we describe the UCNPs-based multi-functional gene carriers with cationic polymer coating via a facile ligand exchange method. The up-conversion luminescence property, loading capacity and cytotoxicity of the gene carriers were characterized in detail. In vitro transfection of enhanced green fluorescence protein (EGFP) plasmid and small siRNA by UCNP@PEI were fully examined and optimized in comparison with PEI polymer. Finally, the MRI, CT and up-conversion luminescence (UCL) tri-modal imaging properties of the platform were investigated both in vitro and in vivo. EXPERIMENTAL Chemicals. Gd2O3 (99.99%), Yb2O3 (99.99%) and Er2O3 (99.99%) were purchased from Science and Technology Parent Company of the Changchun Institute of Applied Chemistry. Oleic acid (OA), 1-Octadecene (ODE), Polyethylenimine (branched PEI polymer with average molecular weight of 25000) were purchased from Sigma Aldrich. The rare earth


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chlorides GdCl3, YbCl3 and ErCl3 were prepared by dissolving the corresponding rare earth oxides in hydrochloric acid at elevated temperature followed by evaporating the solvent under vacuum. siRNA that are sequence specific for human bcl-2 mRNA was synthesized by Sango Biotech, Shanghai, China. The sequence of the siRNA used as followed: sense strand, 5’-GUG AAG UCA ACA UGC CUGC-dTdT-3’; anti-sense strand, 5’-GCAG GCA UGU UGA CUU CAC-dTdT-3’. Ultrapure water (18.2 MΩ) was used throughout the experiments and all the chemicals and solvents were used without further purification. The plasmid DNA was purchased from Clontech (Palo Alto, CA). Cell Line and Animals. Hela cells (Human cervical cell line) and L929 cells (Human Fiberous Cell line) were chosen for cell tests. Both HeLa and L929 cells were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cell was first cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO), 100 units/mL penicillin and 100 units/mL streptomycin (Sigma). The culture medium was replaced once a day. Test mice were Kunming in a weight range of 20–25 g (6-8 weeks old) and purchased from a breeding base at Oncology Center, Changchun. All animal operations were conducted in accordance with the PRC national standards for laboratory animal quality and the Chinese guidelines for the care and use of laboratory animals. Characterization. The X-ray diffraction (XRD) measurements were performed on a D8 Focus diffractometer (Bruker) with Cu Kα radiation (λ = 0.15405 nm). Transmission electron microscopy (TEM) was conducted using a FEI Tecnai G2 S-Twin with a field emission gun operating at 200 kV. The UC emission spectra were taken on an F-7000 spectrophotometer


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(Hitachi) equipped with a 980 nm laser as the excitation source. Inductively Coupled Plasma-Mass Spectrometer (ICP-MS) was taken on an iCAP 6300 (Thermo scientific).

Synthesis of Rare Earth Oleate Complexes. A literature method for the synthesis of iron-oleate complex was used to prepare the rare earth oleate complexes.51 Briefly, 10 mmol of rare earth chloride RECl3 (RE = 80 %Gd + 17 %Yb + 3 %Er) and 30 mmol of sodium oleate were dissolved in a mixture solvent composed of 20 mL of ethanol, 15 mL of distilled water and 35 mL of hexane. The result solution was added into a 250 -mL round-bottomed flask with a reflux condenser, and heated to 70 °C for 4 h. Then the upper organic layer was separated and washed three times with distilled water. After washing, the remaining hexane was evaporated at 80 ℃ to yield rare earth oleate complexes. Synthesis of OA Stablized NaGdF4:Yb3+/Er3+. Oleic acid-capped NaGdF4:Yb3+/Er3+ were synthesized using a thermal decomposition methodology published previously with a slight modification.52 In a typical procedure, 1 mmol of RE(oleate)3 (RE = 80 %Gd+ 17 %Yb + 3 %Er), 12 mmol of NaF, 10 mL of OA, and 10 mL of ODE were added to a three-neck round-bottom reaction vessel and heated to 110 °C under vacuum with magnetic stirring for 30 min. The reaction was then flushed with N2 and kept at 320 °C for 90 min with vigorous stirring. When the reaction was completed, the transparent yellowish reaction mixture was allowed to cool to 60 °C. The UCNPs were precipitated with the addition of ethanol and isolated via centrifugation. Then the as prepared UCNPs were stored in 20 mL chloroform. Synthesis of PEI Coated NaGdF4:Yb3+/Er3+. 2.0g PEI was dissolved in 30 mL H2O. The above NaGdF4:Yb3+/Er3+ nanoparticles chloroform solution were added to the above


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solution and vigorously stired for 24 h. The nanoparticles were then separated by centrifugation and washed with deionized water.

Gel Retardation Assay. 0.5 µg of plasmid DNA was incubated with varying nanoparticles: pDNA weight ratios (N/P ratio) in room temperature in PBS. After 30 min incubation, the mixture with addition of loading buffer was loaded on a 1 % agarose gel. The electrophoresis was carried out at 80 V for 40 min in TAE buffer. Flow cytometry. 48h after Hela cells were transfected with UCNP@PEI and PEI nanoparticles, the medium was removed and washed 3 times with PBS. Single cell suspension was prepared by trypsinization, washing with PBS again for 3 times and filtration through 35 mm nylon mesh. Then the cells were detected by FACS-Calibure Flow Cytometry (BD Biosciences). The fluorescence scan was performed with 1×104 cells. Cytotoxicity of UCNP@PEI and PEI. In vitro cytotoxicity of NaGdF4:Yb3+/Er3+ nanoparticles were assessed against Hela cells and L929 cells. Hela cells were seeded in a 96-well plate at a density of 104 cells per well and cultured in 5 % CO2 at 37 ℃ for 24 h. Then certain amount of nanoparticles was added to the medium, and the cells were incubated in 5 % CO2 at 37 ℃

for another 48 h. At the end of the incubation, 20 µL of

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) solution (diluted in a culture media with a final concentration of 0.5 mg/mL) was added into each well and incubated for another 4 h. The supernatant in each well was removed. 150 µL of dimethyl sulfoxide (DMSO) was added to each well before the plate was examined using a microplate reader (Therom Multiskan MK3) at the wavelength of 490 nm. Tests against L929 cells were performed in the same method. 8 ACS Paragon Plus Environment

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In vitro EGFP Plasmid Transfection. HeLa cells were seeded in a 24-well plate at a density of 105 cells per well 24 h before transfection. EGFP plasmid were diluted in 250 µL serum-free DMEM medium, and appropriate amounts of UCNP@PEI or PEI polymer were diluted in 250 µL serum free DMEM medium. The two solutions were then mixed and incubated for 60 min at room temperature before being added into cells. All cells were washed 3 times with PBS before adding the transfection complexes. After 6 h incubation, the medium were removed. Cells were washed 3 times with PBS before the addition of 500 uL DMEM medium containing 10% fetal bovine serum. Different amount of incubation time was required for efficient EGFP expression based on the overall transfection time. Control experiment was carried out using naked plasmid DNA without any transfection agents. siRNA Delivery and Gene Silencing. Hela cells were seeded in a 6-well plate at a density of 106 per well before transfection. PBS, siRNA, Gene carriers (UCNP@PEI and PEI polymer) loaded bcl-2 siRNA or scramble sequence siRNA complexes were formed in 0.5 mL diethylpyrocarbonate (DEPC) treated water 30 min before transfection. After 6 h incubation of carriers/siRNA complex, cell medium was replaced with 2 mL fresh DMEM medium. The final siRNA concentration was 200 nM. For mRNA assay, all the transfected cells were washed three times with PBS. The total RNA was extracted using Trizo-Reagent according to the manufacturer’s instructions. The concentration and purity of the total RNA were determined spectrophotometrically at 260 nm and 280 nm. The total RNA (2 µg) was reverse-transcribed into first-strand cDNA in a 20 µL reaction volume using M-MLV Reverse Transcriptase (Promega). For the qRT-PCR analysis, the following pairs of bcl-2 mRNA were used to amplify cDNA: bcl-2—GGA TTG


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TGG CCT TCT TTG AG (sense), CCA AAC TGA GCA GAG TCT TC (anti-sense). Calculations were performed by the Sequence Detection System 1.2.3. (SDS 1.2.3.) computer software, provided by the manufacturer (Applied Biosystems). The reaction conditions included an initial step at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s (Melt) and 56 °C for 1 min (Anneal/Extend). In Vitro and In Vivo T1-Weighted MR Imaging. The MR imaging experiments were performed in a 1.5 T MR scanner (Shanghai Huantong). For in vitro MR Imaging, UCNP@PEI samples were dispersed in water at various Gd concentrations (by ICP-MS measurement). T1 measurements were performed using a nonlinear fit to changes in the mean signal intensity within each well as a function of repetition time. Finally, the r1 relaxivity values were determined through the curve fitting of 1/T1 relaxation time (s−1) versus the Gd concentration (mM).

For In vivo MR imaging experiments, the tumor-bearing mouse was anesthetized with 10 % chloral hydrate by intraperitoneal injection. The mouse was scanned before and after intratumoral injection of UCNP@PEI nanoparticles (50 µL, 10 mg/mL).

In Vivo X-Ray CT Imaging. The CT imaging experiments were performed at 120 kVp voltages on a Philips 256-slice CT scanner (Philips Medical System). The UCNP@PEI nanoparticles were dispersed in PBS. To perform in vivo CT imaging, the Kunming mice were first anesthetized with 10 % chloral hydrate. UCNP@PEI nanoparticles (50 µL, 20 mg/mL) were intratumorally injected into the tumor-bearing mouse in situ. The mouse was scanned before and after the injection. CT Imaging parameters were given as follows: thickness, 0.9 mm; pitch, 0.99; 120 kVp, 300 mA; field of view, 350 mm; gantry rotation time, 0.5 s; table speed, 10 ACS Paragon Plus Environment

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158.9 mm s−1. Thin-section axial images were re-formed to coronal images by a computational technique referred to as multiplanar reconstruction. In Vivo Up-conversion Imaging. The UCNP@PEI nanoparticles were dispersed in PBS. To perform in vivo up-conversion imaging, the Kunming mouse was first anesthetized with 10 % chloral hydrate. UCNP@PEI nanoparticles (50 µL, 10 mg/mL) were intratumorally injected into the tumor-bearing mouse in situ. A 980 nm laser was used to demonstrate the luminescence before and after the injection. Results and Discussion Preparation and Characterization. Oleic acid stabilized NaGdF4:Yb/Er UCNPs were synthesized according to a thermal decomposition methodology with slight modifications.52 The NaGdF4 nanoparticles with PEI coating (UCNP@PEI) were then prepared through a facile ligand exchange method. PEI polymer was employed on the UCNPs due to its ability to effectively condense and complex DNA molecules as well as its considerable buffer capacity at the low pH environment of the endosomes, a mechanism generally known as proton sponge effect.53 The wide-angle XRD pattern of UCNPs is shown in Figure 1. All the diffraction peaks can be ascribed to the pure hexagonal phase structure known from NaGdF4 (JCPDS: 27-0699). The absence of any extra diffraction peaks indicates the high purity of the UCNPs. Representative TEM images of the resulting UCNPs and UCNP@PEI show the products with good monodispersity (Figure 2a). After being converted into hydrophilic, the UCNP@PEI nanoparticles are still well-dispersed. The PEI coating on UCNPs is so thin that it could hardly be observed, resulting in the unchanged size and morphology of UCNP@PEI. The HR-TEM image in Figure 2a exhibits lattice fringes of the (200) planes with interplanar spacing of 0.28


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nm. Most of the nanoparticles appear to be spheres with diameter between 15 nm and 25 nm (Figure 2c). The emission spectra of UCNPs and UCNPs@PEI under the 980 nm excitation are given in Figure 2b. The three emission bands centered in 522 nm, 541 nm and 655 nm can be assigned to 2H11/2 → 4I15/2, 4S3/2 → 4I15/2, 4F9/2 → 4I15/2 transition of Er3+, respectively. These characteristic peaks are similar to the previous study of the NaGdF4 crystals.54 The inset of Figure 2b exhibits the digital photographs of UCNPs (upper) and UCNP@PEI (lower) dispersed in cyclohexane and water with their corresponding UC luminescence under 980 nm excitation. The photographs demonstrate that the UCNP@PEI is hydrophilic and well-dispersed in water. Loading capacity of UCNP@PEI and PEI. Loading capacity is an important factor for gene carriers. It determines how many DNA molecules could be delivered and affects the transfection efficacy. To evaluate the gene loading capacity, a gel electrophoresis assay was performed at varying nanoparticles: pDNA weight ratios (N/P ratio) for the gene carriers (Figure 3a). For the UCNP@PEI gene carriers, DNA was completely retained at N/P ratios of 7 by carriers/pDNA complex. No retarded band was observed when the N/P ratio was greater than 7, indicating that all the pDNA was absorbed by UCNP@PEI in these conditions and 7 µg UCNP@PEI were needed for 1µg pDNA. However, in the case of PEI polymer, the N/P ratio to achieve the total retardation of pDNA is 0.7, which means to load 1 µg pDNA only 0.7 µg PEI was needed. The better loading capacity of PEI than UCNP@PEI could be attributed in following reasons. On the one hand, for PEI and UCNP@PEI in same mass, UCNP@PEI has


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less PEI molecules. Secondly, the water-dispersed UCNP@PEI may have spatial hindrance effect when PEI on the surface interact with DNA molecules. Zeta potential was then measured to examine the change of surface charges of UCNP@PEI and PEI polymer at different N/P ratios (Figure 3b, 3c). For both two carriers, the zeta potential was negative at their lowest N/P ratio and rose along with the increasing amount of carriers. The UCNP@PEI/pDNA complex showed a negative potential of -11.21 mV and -8.64 mV at the N/P ratios of 1 and 5, respectively. Then it came to 0.213 mV at the N/P ratios of 10 and finally arrived 15.20 mV at the N/P ratios of 50. For the PEI/pDNA complex, zeta potential was -8.5 mV at the N/P ratios of 0.1, then turned positive at the N/P ratios of 1 and surged to 31.7 mV when N/P ratios came to 5. For UCNP@PEI/pDNA and PEI@DNA complexes, the results of zeta potential are consistent with their performance at the gel electrophoresis assay. In vitro transfection of EGFP plasmid and siRNA. EGFP plasmid encodes the green fluorescence protein (GFP) and the expression of GFP induced by the carriers/pDNA complexes was examined. To determine the optimal tranfection efficiency of UCNP@PEI gene carriers, we investigated the transient transfection of EGFP plasmid at different transfection time and N/P ratios. Observation of EGFP plasmid transfection by UCNP@PEI and PEI at different transfection time was shown in Figure S1. PEI polymer showed a better transfection efficacy after 24 h and 36 h transfection while UCNP@PEI excelled after 60 h transfection, suggesting PEI may induce GFP expression in less time. For either UCNP@PEI or PEI, very few fluorescent cells can be observed after 72 h transfection. Both groups exhibited


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great transfction efficacy after 48 h. We further evaluate EGFP transfection at different N/P ratio after 48 h qualititively and quantitively. Figure 4a shows the direct observation of EGFP transfected Hela cells at 48 h. The control group for both UCNP@PEI and PEI carriers did not show any fluorescence as naked DNA alone could not cross the cell membrane. The expression of GFP increased along with the increasing N/P ratios at first, and then decreased after a certain N/P ratio point. For UCNP@PEI, the number of fluorescent cells increased from N/P ratios of 5 and maximized at N/P ratios of 20. Then it decreased at N/P ratios of 50. The expression of GFP induced by PEI@DNA was barely observed at N/P ratios of 0.1. It then increased to maximum at N/P ratios of 2.5, and fell at N/P ratios of 5. The observation with fluorescence microscopy of the EGFP transfection is a qualitative method. It demonstrated that the UCNP@PEI could transfer the EGFP plasmid into Hela cells successfully without the exact effectiveness of gene transfection quantitatively. To measure the transfection efficiency of UCNP@PEI and PEI precisely, flow cytometry was employed (Figure 4b). UCNP@PEI showed very low efficiency of 7.4 % at N/P ratios of 5 and increased to 23 % when N/P ratios came to 10. The gene carriers exhibited a maximam efficiency of 29 % at N/P ratios of 20, and droped to 20 % when N/P ratios increased to 50. The PEI polymer exhibited a similar tendency between N/P ratios of 0.1 and 5. It showed very low efficiency at N/P ratios of 0.1 and achieved a maximum of 29 % at N/P ratios of 2.5. Then it fell to 22 % at N/P ratios of 5. The efficiency results are in accordance with the observation of GFP fluorescence through microscopy showed in Figure 6a, and suggest that


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the UCNP@PEI gene carriers had a slightly higher transfection efficiency than PEI in EGFP plasmid transfection in Hela cells. RNA interference (RNAi) is a powerful technique to modify the genome function and regulate gene expression. To examine the gene silencing effect of UCNP@PEI delivered siRNA, we analyzed bcl-2 mRNA expression level by quantitative real time PCR (qRT-PCR) (Figure 4c). No down-regulation of bcl-2 mRNA level was observed in cells transfected by siRNA alone, indicating that the naked siRNA could barely cross the cell membrane and induce gene silencing. It was uncovered that the UCNP@PEI and PEI polymer could remarkably enhance the gene silencing efficacy resulting in 42 % and 55 % of gene knockdown, respectively. As negative controls, Hela cells were also transfected by UCNP@PEI and PEI complex with scramble sequence (UCNP-NC, PEI-NC), showing slight decrease of bcl-2 mRNA expression level, indicating the specific gene regulation effect by bcl-2 siRNA. It is shown that the gene knockdown efficacy of UCNP@PEI is not as good as PEI polymer, which is in disagreement with the transfection efficiency results of GFP transfection results. Despite the transfer of EGFP plasmid into cell nucleus is indispensable for the transfection, it is sufficient to deliver siRNA into cytoplasm for gene silencing. Therefore, the optimal condition for the two applications is likely to be different and work related to the optimized delivery of siRNA by UCNPs is being carried out in our group. It is worth noting that neither UCNP@PEI nor PEI showed the maximum transfection efficiency at their maxmium loaded N/P ratios. The maxmium loaded nanoparticles indicated the zeta potential of 0 mV or greater. However, this state of balance could easily be broken in the delivery process as the cellular environment changes quickly and rapidly. It


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is believed that after carriers/DNA complexes cross cell membranes, they enter and then escape from endosomes, which are full of emzymes that could degrade nucleic acids.55, 56 Although the nanocarriers could keep the loaded DNA from degradation, the low pH in endosome actuated the carriers/DNA complexes to disassemble and free DNA were then subjected to degradation. As the weight of DNA was fixed, fewer DNA were transfected and the transfection efficency dropped off. Cellular Cytotoxicity Assay. The cytotoxicity is a key factor for the materials in biomedical application. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to test the relative cell viability of Hela cells (Figure 5a) and L929 cells (Figure

5b)

incubated

with

UCNP@PEI,

PEI,

pDNA

loaded

UCNP@PEI

(UCNP@PEI@DNA) and pDNA loaded PEI (PEI@DNA). The UCNP@PEI@DNA refers to what entered and enabled the transfection process, and UCNP@PEI refers to what it remained after DNA was released. Both of them were investigated in the cytotoxicity assays to make sure the cell viability is not affected by the material. Experiments for PEI and PEI@DNA were setup for the same reason. The N/P ratios of UCNP@PEI@DNA and PEI@DNA were fixed as 20 and 2.5, respectively, as they were tested as the most efficient N/P ratios on the previous experiments. The MTT results in both cell lines showed similar patterns and no significant difference was observed between the DNA loaded gene carriers and none loaded gene carriers. The UCNP@PEI and UCNP@PEI@DNA remained a high cell viability of more than 80 % up to the concentration of 400 µg/mL, indicating good biocompatibility. However, cells incubated with PEI polymer and PEI@DNA behaved differently. The relative cell viability for both


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PEI and PEI@DNA decreased significantly with the increasing amount of PEI and PEI@DNA. The cell viability in both two cell lines was lower than 80 % at the concentration of 25 µg/mL and less than half cells were still alive at the concentration of 400 µg/mL. In general, the cytotoxicity induced by cationic polymers, such as PEI, increases along with their charge density.57, 58 PEI can induce disruption of either the cell membrane or the mitochondrial membrane, leading to necrotic cell death or apoptosis.10, 59 The less amount of PEI on the surface of UCNP@PEI therefore can be accounted for the better cytotoxicity results than pure PEI. Researchers also found that PEI exhibits less cytotoxicity when they were incorperated in certain core-shell structures due to a lower density of the PEI’s cationic residuals that interact with cells.60, 61 The decreased cytotoxicity of UCNP@PEI and UCNP@PEI@DNA can also be attributed to a similar mechanism. MRI/CT/UCL Tri-modal Imaging of UCNP@PEI. The UCNP@PEI gene carriers could be used as T1 contrast agent for MR imaging because of the signal enhancement ability of Gd3+ ions. The performance of the UCNP@PEI as contrast agent both in vitro and in vivo was examined on a 1.5 T MRI scanner, showed in Figure 7. The T1-weighted images in the range of 0-4.8 mM of Gd3+ were shown in Figure 6a. It can be seen that the positive enhancement effect of UCNP@PEI was significant with increased concentration of Gd3+ ions. The experimentally determined longitudinal relaxivity (r1) of water protons was plotted against the molar concentration of Gd3+ (Figure 6b). The molar relaxivity r1 extracted from the linear regression fits of the experimental data is 1.6709 mM-1s-1. For in vivo MR imaging experiments, a tumor-bearing kunming mouse was used as a model. The solution of UCNP@PEI was


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intratumorally injected to the mouse. As is shown in Figure 7c, d, the tumor site shows higher MRI signal intensity after injection of UCNP@PEI, indicating the feasibility of UCNP@PEI as potential contrast agent for T1 MR Imaging. X-ray computed tomography (CT) has become an important tool in biomedical imaging due to its high-contrast resolution. To evaluate the feasibility of UCNP@PEI as contrast agent for CT imaging, in vivo CT experiments were performed by intratumorally injecting the solution of UCNP@PEI to the tumor site (Figure 7). The CT value was 37 Hounsfield units (HU) before injection and significantly increased to 2299 HU after injection (Figure 8a). The enhancement of signal can also be observed in the corresponding 3D rendering of CT images before and after injection (Figure 7b, 7c). The up-conversion luminescence (UCL) has proven to be a promising method for bioimaging. Here, we evaluate the UCL imaging properties both in vitro and in vivo. Hela cells were incubated with UCNP@PEI for 0.5 h, 1 h, 3 h and 6 h at 37 ℃, an inverted fluorescence microscope was used to take the UCL images with an external 980 nm excitation source (Figure 8). The green luminescence stemmed from the UCNP@PEI gradually moves from the location near cell membrane to the internal part of the cells from 0.5 h to 6 h, indicating the internalization process of the UCNP@PEI. It can also be observed that the green luminescence derived from UCNP@PEI stayed at the nucleus area after 6 h incubation, which would be helpful for the loading gene to enter the nucleus for transfection. The results demonstrate that the UCNP@PEI gene carriers could serve as luminescence probes for cell imaging and monitoring the intracellular pathway. In addition, we used tumor-bearing kunming mouse to


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evaluate the in vivo imaging ability by intratumorally injection (50 µL, 10 mg/mL), the results show that the UCNP@PEI gene carriers have good tissue penetration ability (Figure S2). Conclusion In summary, we developed a up-conversion nanoparticle/polymer composite system capable of efficiently delivering nucleic acids into tumor cells, and have combined it with multi-modal imaging properties as a trackable probe in vitro and in vivo. The UCNP@PEI gene carriers can be fabricated in a facile method and decreased cytotoxicity compared with PEI polymer. The performance of UCNP@PEI enabled EGFP plasmid transfection was intensively investigated and optimized, exhibiting slightly higher transfection efficiency than PEI. Down-regulation of bcl-2 mRNA expression induced by UCNP@PEI delivered siRNA was also demonstrated by quantitative real-time PCR. In addition, the UCNP@PEI gene carriers showed great MRI/CT/UCL tri-modal imaging capacities in vitro and in vivo, suggesting their potential application for target imaging and cancer early detection. This work underlined the feasibility of UCNP@PEI nanoparticles for combined efficient gene delivery and bioimaging, and will advance the fabrication and application of non-viral gene carriers. Conflict of Interest The authors declare no competing financial interest. Acknowledgement This project is financially supported by the National Natural Science Foundation of China (NSFC 21101149, 51372241, 51332008, 21221061) and the National Basic Research Program of China (2014CB643803).


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Supporting information available: Fluorescence images of EGFP expression in Hela cells using UCNP@PEI and PEI with different transfection time (Figure S1). In vivo up-conversion luminescence of UCNP@PEI under 980 nm excitation in kunming mouse: before injection and after injection in situ. (Figure S2); This material is available free of charge via the Internet at http://pubs.acs.org.

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Captions of Figures Figure 1. Wide-angle XRD pattern of as-prepared NaGdF4:Yb/Er and standard data for βNaGdF4 (JCPDS No. 27-0699). Figure 2. (a) TEM images of as-prepared UCNPs, UCNP@PEI and HR-TEM images of UCNPs, (b) 980nm laser excited up-conversion emission spectra and digital photographs of UCNPs and UCNP@PEI. (c) The particle size distribution histogram of UCNP@PEI. Figure 3. (a) Agarose gel electrophoresis of UCNP@PEI and PEI loaded with pDNA at different weight ratios (N/P). Zeta potential of (b) UCNP@PEI and (c) PEI loaded with pDNA at different weight ratios. Figure 4. In vitro transfection of EGFP plasmid and bcl-2 siRNA with UCNP@PEI and PEI after 48h at different weight ratios. (a) Fluorescence images of EGFP plasmid expression of UCNP@PEI and PEI with increasing weight ratios. (b) Quantification of EGFP plasmid transfection efficiency by flow cytometry of UCNP@PEI and PEI at different weight ratios. (c) Expression level of bcl-2 mRNA in Hela cells determined by qRT-PCR. Scale bar, 100 µm. All samples in flow cytometry were run in triplicate. Figure 5. In vitro cell viability of (a) Hela and (b) L929 after 48h incubation with UCNP, pDNA loaded UCNP (UCNP@DNA) and pDNA loaded PEI (PEI@DNA). All samples were run in triplicate. Figure 6. (a) T1-weighted MR images of UCNP@PEI dispersed in water at different Gd concentration. (b) A plot of T1 relaxation rate R1 (1/T1) against Gd concentration of UCNP@PEI. In vivo T1-weighted MR images of a tumor-bearing kunming mouse: (c) before injection and (d) after injection in situ. Figure 7. CT imagings of a tumor-bearing kunming mouse: (a) before and post injection in situ, and the corresponding 3D rendering of the CT images (b,c). Figure 8. Inverted fluorescence microscope images of Hela cells incubated with UCNP@PEI for 0.5h (a-d), 1h (e-h), 3h (i-l) and 6h (m-p). Each column can be classified to the DAPI (left), bright-field (middle left), up-conversion luminescence images (middle right) in dark field and overlay of all above (right), respectively. Scale bar, 20 µm


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