View Article Online View Journal
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: J. Yu, S. Zhu, P. Feng, C. Qian, J. Huang, M. Sun and Q. Shen, Chem. Commun., 2015, DOI: 10.1039/C4CC09685A.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/chemcomm
Page 1 of 5
ChemComm
Journal Name
RSCPublishing View Article Online
DOI: 10.1039/C4CC09685A
COMMUNICATION
Received 00th January 2012, Accepted 00th January 2012
Ji-Cheng Yu, ‡a Sha Zhu, ‡ a Pei-Jian Feng, a Cheng-Gen Qian, a Jun Huang, a Min-Jie Sun b and Qun-Dong Shen*,a
DOI: 10.1039/x0xx00000x www.rsc.org/
A multifunctional nanocarrier for encapsulation and delivery of short interfering RNA (siRNA) has been realized using cationic fluorescent polymer core-shell nanoparticles. The nanocarrier has good biocompatibility and high transfection efficiency over the most popular transfection reagent, Lipofectamine 2000. Fluorescence resonance energy transfer within the nanocarrier provides a non-invasively and labelfree way to track the intracellular release of siRNA. RNA interference (RNAi), as a RNA-dependent gene silencing process, plays a significant role in life science. During the gene expression regulation process, RNA inhibits specific gene expression via causing the destruction of specific mRNA molecules. In recent decades, small interfering RNA (siRNA) has been exploited as potential means for the treatment of diseases such as cancers, Parkinson’s disease, and genetic disorders.[1-6] However, naked siRNA is vulnerable to ribonuclease (RNase) and difficult to cross through negatively charged cell membranes, leading to the low transfection efficiency.[7-8] Therefore, one of the most important challenges in siRNA therapy is to find an efficient non-viral gene delivery vehicle. To date, a large amount of materials including cationic lipids, polymers, dendrimers, and peptides have been developed.[9-15] Among them, cationic polymers have the abilities to condense anionic siRNA via electrostatic assembly into siRNA/polymer complexes, and to promote the complexes to release from endosomes via proton sponge. [16-18] Cationic pi-conjugated polymers have been developed as nonviral nucleic acid nanocarriers in recent years,[19-21] owing to their abundant positive charges, low cytotoxicity, and most importantly, intrinsic fluorescence, which enables real-time tracking the biodistribution of the delivery systems using non-invasive fluorescence imaging.[22] The behavior of delivery system and the function of nucleic acids are highly dependent on the environment of the living organisms or cells. Therefore, it is important to reveal how the nucleic acid molecules are released from the delivery vehicles. Generally, fluorophores can be chemically attached to the nucleic acids to assess the success of intracellular transfection. Förster resonance energy transfer (FRET) between the fluorescence-labeled nucleic acids and the fluorescent nanocarriers is a smart technique to understand in vitro or in vivo release kinetics of the nanoparticle-based delivery systems.[19, 23-26]
This journal is © The Royal Society of Chemistry 2012
The fluorescence off or on state depicts whether the nucleic acid molecules are trapped inside or released from the nanocarriers.
Scheme 1. Fluorescent nanoparticles as siRNA nanocarriers by sequentially electrostatic assembly of siRNA and ThPFN on BtPFN nanoparticles. In this study, we designed a simple multifunctional nanoparticle system, which can deliver siRNA free from fluorescence labeling to the cells and sense the delivery of the siRNA by fluorescence resonance energy transfer imaging approach. As illustrate in Scheme 1, the nanocarriers are comprised of three components: a cationic yellow-emissive conjugated polymer (BtPFN) nanoparticles as core, siRNA electrostatically attached to the BtPFN nanoparticles, and a sequentially electrostatically adsorbed cationic green-emissive conjugated polymer (ThPFN) as the shell. The cationic ThPFN shell is able to protect siRNA from RNase and to interact with the negatively charged cell membranes, which leads to high transfection efficiency of triplex nanoparticles. The key point is that, when negatively charged siRNA brings fluorescence acceptor (BtPFN) into the proximity of fluorescence donor (ThPFN), efficient energy transfer can take place within the electrostatic complexes. After the complexes are delivered into target cells, siRNA is gradually released from the complexes, where electrostatic repulsion between two cationic conjugated polymers leads to loss of the FRET effect. Such approach allows real-time visualization of the siRNA-release in a non-invasive and high-sensitive manner, which reduces the costs and avoid time-consuming and labor-intensive fluorescence labeling of siRNA.
J. Name., 2012, 00, 1‐3 | 1
ChemComm Accepted Manuscript
Published on 13 January 2015. Downloaded by New York University on 13/01/2015 12:12:44.
Cite this: DOI: 10.1039/x0xx00000x
Cationic Fluorescent Polymer Core-Shell Nanoparticles for Encapsulation, Delivery, and Non-Invasively Tracking Intracellular Release of siRNA
ChemComm
Page 2 of 5
Journal Name
Figure 1. a) TEM image and b) hydrodynamic diameter distribution of ThPFN/siRNA/BtPFN NPs. (Scale bar: 200 nm)
2 | J. Name., 2012, 00, 1‐3
a) PL Intensity
1x10
6
9x10
5
8x10
5
View Article Online
7x10
5
DOI: 10.1039/C4CC09685A
6x10
5
5x10
5
4x10
5
3x10
5
2x10
5
1x10
5
BtPFN NPs and ThPFN ThPFN/siRNA/BtPFN
0
400
450
500
550
600
650
700
Wavelength (nm)
b)
3.2 3.0
pH=7.4
2.8 2.6 2.4 2.2 2.0
pH=5.7
1.8 1.6 1.4 1.2
0
1
2
3 4 Time (h)
5
6
Figure 2. a) Fluorescence emission spectra of a simple mixture of ThPFN and BtPFN NPs (solid line) and ThPFN/siRNA/BtPFN NPs (dash line) in aqueous solution. b) Time-lapse ratios of fluorescence intensities at 550 nm (BtPFN) and 454 nm (ThPFN) of the ThPFN/siRNA/BtPFN NPs under pH 5.7 and 7.4 environment. The excitation wavelength is 364 nm. To investigate the stabilities of ThPFN/siRNA/BtPFN NPs under physiological condition, fluorescence of the complexes was monitored in phosphate buffer at pH 5.7 or 7.4. We used I550/I454 to describe the change of FRET efficiency of the electrostatic complexes, where I550 and I454 are the fluorescence intensities at emission maxima of BtPFN and ThPFN respectively and the excitation wavelength is 364 nm. The FRET efficiency of the complexes under pH of 7.4 was almost constant, implying that siRNA is protected via association with the outermost shell of the polymer (Figure 2b). In acid environment (pH 5.7), the FRET efficiency reduced drastically, indicating disruption of the electrostatic complexes with the layer-by-layer structure and the consequent release of siRNA molecules. The FRET phenomenon existing within ThPFN/siRNA/BtPFN NPs also provides a useful optical tool for visualization of the distribution of siRNA-loaded NPs and the release of label-free siRNA from the NPs in live cells. Confocal laser scanning microscope (CLSM) images of HepG2 cells after 0- and 2-hour transfection are shown in Figure 3a-f. Bright-field images showed that the cells retain their normal morphology after being incubated with the electrostatic complexes. Efficient FRET between ThPFN and BtPFN resulted in the weak fluorescence of ThPFN and bright fluorescence of BtPFN. The clustered fluorescence revealed that most of the NPs were intact in the endosomes (pH 6.5-6.8 for early endosomes), and thus siRNA molecules did not release at this stage. After incubation for another 2 h, the fluorescence of ThPFN enhanced dramatically, whereas the fluorescence of BtPFN seemed to be considerably weakened. It suggests the electrostatic interactions between siRNA and cationic polymer are weakened and a large part of the NPs disassemble due to the protonation of the base groups of siRNA in acidic endosomes (pH 5.2-5.8 for late endosomes). Also, the protonation of the NPs results in the rupture of endosome by the proton sponge effect and escape of siRNA into cytoplasm, which leads to reduced FRET
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
BtPFN is a conjugated polymer with hydrophobic backbone and hydrophilic side chains, and therefore in aqueous solution exhibits a strong tendency to self-assemble into nanoparticles (NPs). The hydrodynamic diameters of BtPFN NPs were measured by dynamic light scattering. As shown in Figure S1 (details can be found in the ESI), the as-prepared NPs showed an average hydrodynamic diameter of 50.2 nm with a narrow size distribution. There was no aggregation of BtPFN NPs. It was confirmed by transmission electron microscopy imaging, where BtPFN NPs had an average diameter of about 50 nm with a spherical shape. The nanoparticles exhibited a zeta-potential value of +37 mV (Table S1). Due to their positive surface charges, BtPFN NPs electrostatically adsorbed anionic siRNA molecules, forming a siRNA/BtPFN shell/core structure. The average hydrodynamic diameter of the nanoparticles increased to 64.2 nm, and the zeta-potential value changed from +37 mV to -6 mV. siRNA/BtPFN NPs cannot enter cells efficiently because their negative charges lead to electrostatic repulsion from the anionic cell membranes. Moreover, siRNA molecules on the surface of the NPs are vulnerable to ribonuclease. Coating a cationic conjugated polymer (ThPFN) shell on siRNA/BtPFN NPs will significantly protect siRNA from attack by extracellular nucleases, and meanwhile allow facile cellular uptake via an endocytic pathway. The final three-layer NPs (ThPFN/siRNA/BtPFN) had an average hydrodynamic diameter of 82.8 nm and a zeta-potential value of +23 mV. The morphology of ThPFN/siRNA/BtPFN NPs was further investigated by transmission electron microscopy. Figure 1 shows that they had stratified structures, which confirms the layer-by-layer assembly process. To assess the protection of conjugated polymers to siRNA, we incubated the free siRNA or siRNA-loaded NPs with 10% fetal bovine serum (FBS) at 37oC for 4h. From the result shown in Figure S2, free siRNA was digested within 4 hours, whereas siRNA encapsulated in the NPs were obviously more stable in the presence of serum, which indicates the conjugated polymers are able to prevent siRNA from degradation by RNase in the serum. Polyfluorenes have high photostability and fluorescence quantum yields, and ThPFN and BtPFN emitted green and yellow light respectively upon excitation. The fluorescence emission spectra of ThPFN/siRNA/BtPFN NPs dispersed in aqueous solution, as well as a mixture of BtPFN NPs and ThPFN with the same polymer concentrations, were shown in Figure 2a. In contrast to that of the simple mixture, emission of ThPFN in the complexes was weakened, while the fluorescence intensity of BtPFN was invigorated dramatically. It is noteworthy that the emission spectrum of ThPFN overlaps well with the absorption spectrum of BtPFN (Figure S3), thus efficient energy transfer process through nonradiative dipoledipole coupling may occur if the donor (ThPFN) in excited state is in close proximity to the acceptor (BtPFN) in ground state. However, in the simple mixture, electrostatic repulsion is expected between these cationic polymers. When oppositely charged siRNA/BtPFN NPs and ThPFN are used, it brings the acceptor into proximity of the donor. As a result, fluorescence enhancement is more likely in the electrostatic complexes (ThPFN/siRNA/BtPFN NPs).
I550/I454
Published on 13 January 2015. Downloaded by New York University on 13/01/2015 12:12:44.
COMMUNICATION
Page 3 of 5
ChemComm
Journal Name
COMMUNICATION
View Article Online
Figure 3. Confocal laser scanning microscopic images of HepG2 cells incubated with ThPFN/siRNA/BtPFN NPs after (a-c) 0h and (d-f) 2h transfection. The green channel was ThPFN (420-500 nm); the red channel was BtPFN (550-600 nm); and the last was bright field image. 3D sectional CLSM images of HepG2 cells: g) green channel (ThPFN), h) red channel (BtPFN), and i) merged. The light source was 405 nm. efficiency between ThPFN and BtPFN. 3D sectional CLSM images were taken to show the bio-distribution of ThPFN and BtPFN after 2-hour transfection. As shown in Figure 3g and h, the fluorescence signal of ThPFN distributed more evenly in the cytoplasm, whereas the signal of BtPFN was clustered in the cells. When the colocalization of two fluorescence signals were investigated, it seemed that only a small amount of ThPFN signal overlaps well with that of BtPFN (Figure 3i). It indicates that a large number of ThPFN molecules were dissociated from the ThPFN/siRNA/BtPFN NPs and escaped into cytoplasm from the endosomes, and stimulating the release of siRNA simultaneously. In order to further study the siRNA releasing from the nanoparticles in the cells, we utilized Cy5 groups to label the siRNA and observed the distribution of siRNA during transfection process. After 2-hour transfection, there were fluorescence signals of Cy5-siRNA in the whole cytoplasm (Figure S4). Of note, the fluorescence signals didn’t overlap with the signals of conjugated polymers completely, which means most of the NPs had disassembled and siRNAs had detached from the NPs. It is noteworthy that the conjugated polymers had much better photostability than the small organic molecule dye Cy5 after laser illumination for a short time (Figure S5). To evaluate the RNA interference efficiency using ThPFN/siRNA/BtPFN NPs, HepG2 (human hepatocellular carcinoma) cells stably transfected with luciferase were chosen. The transfer of exogenous nucleic acids into Hep G2 cell line is important for the study of human liver diseases, metabolism, tumor progression, and toxicity of xenobiotics. However, Hep G2 seems to be more resistant to lipid-mediated transfection than other cell lines. [27] ThPFN/siRNA/BtPFN NPs are able to successfully deliver siRNA into HepG2 cells for efficient transfection, and therefore reduce luciferase expression. As shown in Figure 4a, ThPFN/siRNA/BtPFN NPs can achieve approximately 70% gene silencing efficiency, which exceeds the efficiency of the most popular transfection reagents Lipofectamine 2000 (53%). Thus,
This journal is © The Royal Society of Chemistry 2012
Figure 4. a) Gene silencing efficiency of siRNA targeting luciferase. b) Cell viability of HepG2 cells after incubation with BtPFN, ThPFN, ThPFN/siRNA/BtPFN NPs, and Lipofectamine (Lipo) 2000/siRNA at 37oC for 5h. ThPFN/siRNA/BtPFN NPs might be a good choice for improving the transfection efficiency. ThPFN/negative control siRNA (N.C. siRNA)/BtPFN NPs were used to distinguish sequence-specific silencing from non-specific effects, and showed barely gene silencing. It is noteworthy that both free siRNA and siRNA/BtPFN NPs showed low gene silencing efficiency. It indicates that the ability of the cationic ThPFN shell to protect siRNA from RNase and cross negatively charged cell membranes is important for the high transfection efficiency. The biocompatibility of siRNA carriers is crucial for their potential application in clinical gene therapy. The standard methyl thiazolyl tetrazolium (MTT) assay was carried out to determine the relative cellular viabilities of HepG2 cells incubated with conjugated polymers (ThPFN or BtPFN), or ThPFN/siRNA/BtPFN NPs. Figure 4b shows no significant cytotoxicity of two polymers in transfection time (5 h). ThPFN/siRNA/BtPFN NPs had better biocompatibility than Lipofectamine 2000. ThPFN/siRNA/BtPFN NPs exhibit colloidal stability within 24 hours and efficient transfection of siRNA in 10% FBS. For further in vivo applications, cationic carriers with near infrared emission and surface modifications are necessary for fluorescence imaging and decreasing the uptake of the carriers by the macrophages. In conclusion, we constructed a simple nanocarrier to deliver siRNA by sequentially electrostatic adsorption of siRNA and ThPFN on BtPFN NPs. ThPFN/siRNA/BtPFN NPs have good biocompatibility and high transfection efficiency. Of note, the FRET between the ThPFN shell and BtPFN core provides an effective and label-free way to track the intracellular release of siRNA. The electrostatic complexes may have the potential for image-guided gene therapy in the future.
J. Name., 2012, 00, 1‐3 | 3
ChemComm Accepted Manuscript
Published on 13 January 2015. Downloaded by New York University on 13/01/2015 12:12:44.
DOI: 10.1039/C4CC09685A
ChemComm
Page 4 of 5
COMMUNICATION
Journal Name
Acknowledgements
8
S. Zhang, Y. Zhao, D. Zhi, S. Zhang, Bioorg. Chem. 2012, 40, 10.
This work was supported by National Natural Science Foundation of China (No. 21174060), Fundamental Research Funds for the Central Universities (No. 1116020509), Program for Changjiang Scholars and Innovative Research Team in University (IRT1252), and China Equipment and Education Resources System (CERS-1-45).
9
Y. M. Yang, F. Liu, X. G. Liu, B. G. Xing, Nanoscale 2013, 5, 231.
11 R. Kanasty, J. R. Dorkin, A. Vegas, D. Anderson, Nat. Mater. 2013,
Notes and references
12 H. Hatakeyama, H. Akita, E. Ito, Y. Hayashi, M. Oishi, Y. Nagasaki,
Therapy 2012, 20, 513. 12, 967. R. Danev, K. Nagayama, N. Kaji, H. Kikuchi, Y. Baba, H.
Published on 13 January 2015. Downloaded by New York University on 13/01/2015 12:12:44.
a
Harashima, Biomaterials 2011, 32, 4306. 13 M. L. Patil, M. Zhang, T. Minko, ACS Nano 2011, 5, 1877. 14 X. Liu, C. Liu, E. Laurini, P. Posocco, S. Pricl, F. Qu, P. Rocchi, L. Peng, Mol. Pharmaceutics 2012, 9, 470. 15 J. Hoyer, I. Neundorf, Acc. Chem. Res. 2012, 45, 1048. 16 C. J. Chen, J. C. Wang, E. Y. Zhao, L. Y. Gao, Q. Feng, X. Y. Liu, Z. X. Zhao, X. F. Ma, W. J. Hou, L. R. Zhang, W. L. Lu, Q. Zhang, Biomaterials 2013, 34, 5303.
1 N. A. Fonseca, A. C. Gregorio, A. Valerio-Fernandes, S. Simoes, J. N.
17 S. H. Hu, T. Y. Hsieh, C. S. Chiang, P. J. Chen, Y. Y. Chen, T. L. Chiu, S. Y. Chen, Adv. Healthcare Mater. 2014, 3, 273.
Moreira, Cancer Treat. Rev. 2014, 40, 626. 2
R. L. Bogorad, H. Yin, A. Zeigerer, H. Nonaka, V. M. Ruda, M.
18 S. K. Tripathi, V. P. Singh, K. C. Gupta, P. Kumar, J. Mater. Chem. B 2013, 1, 2515.
Zerial, D. G. Anderson, V. Koteliansky, Nat. Commun. 2014, 5, 3869. 3
6
20 X. Feng, F. Lv, L. Liu, Q. Yang, S. Wang, G. C. Bazan, Adv. Mater. 2012, 24, 5428.
A. Tan, J. Rajadas, A. M. Seifalian, Adv. Drug Deliver. Rev. 2013, 65, 357.
5
Biomacromolecules 2013, 14, 3643.
P. Resnier, T. Montier, V. Mathieu, J. P. Benoit, C. Passirani, Biomaterials 2013, 34, 6429.
4
19 R. Jiang, X. Lu, M. Yang, W. Deng, Q. Fan, W. Huang,
21 A. T. Silva, N. Alien, C. Ye, J. Verchot, J. H. Moon, BMC Plant Bio. 2010, 10, 291.
H. Meng, W. X. Mai, H. Zhang, M. Xue, T. Xia, S. Lin, X. Wang, Y. Zhao, Z. Ji, J. I. Zink, A. E. Nel, ACS Nano 2013, 7, 994.
22 D. Ding, J. Liu, G. Feng, K. Li, Y. Hu, B. Liu, Small 2013, 9, 3093.
J. Tabernero, G. I. Shapiro, P. M. LoRusso, A. Cervantes, G. K.
23 C. A. Alabi, K. T. Love, G. Sahay, T. Stutzman, W. T. Young, R. Langer, D. G. Anderson, ACS Nano 2012, 6, 6133.
Schwartz, G. J. Weiss, L. Paz-Ares, D. C. Cho, J. R. Infante, M. Alsina, M. M. Gounder, R. Falzone, J. Harrop, A. C. S. White, I.
24 Z. Cheng, Y. Dai, X. Kang, C. Li, S. Huang, H. Lian, Z. Hou, P. Ma, J. Lin, Biomaterials 2014, 35, 6359.
Toudjarska, D. Bumcrot, R. E. Meyers, G. Hinkle, N. Svrzikapa, R. M. Hutabarat, V. A. Clausen, J. Cehelsky, S. V. Nochur, C. Gamba-
25 X. Wang, F. He, L. Li, H. Wang, R. Yan, L. Li, ACS Appl. Mater. Interfaces 2013, 5, 5700.
Vitalo, A. K. Vaishnaw, D. W. Y. Sah, J. A. Gollob, H. A. Burris III, Cancer Discov. 2013, 3, 406. 7
26 J. Tang, B. Kong, H. Wu, M. Xu, Y. Wang, Y. Wang, D. Zhao, G. Zheng, Adv. Mater. 2013, 25, 6569.
A. de Fougerolles, H. P. Vornlocher, J. Maraganore, J. Lieberman, Nat. Rev. Drug Discov. 2007, 6, 443.
4 | J. Name., 2012, 00, 1‐3
27 S. Yamano, J. S. Dai, A. M. Moursi, Mol. Biotech. 2010, 46, 287.
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
Department of Polymer Science & Engineering and Key Laboratory of High Performance Polymer Materials & Technology of MOE, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing, 210093, China. E-mail: [email protected]; Fax: +86-25-8331 7761; Tel: +86-25-8331 7807 b Department of Pharmaceutics, China Pharmaceutical University, Nanjing, 210009, China. † Electronic Supplementary Information (ESI) available. See DOI: 10.1039/c000000x/ ‡ These authors contributed equally to this work.
View Article Online
10 R. L. Kanasty, K. A. Whitehead, A. J. Vegas, G. Anderson, Mol. DOI:D. 10.1039/C4CC09685A
Page 5 of 5
ChemComm
Journal Name
RSCPublishing View Article Online
DOI: 10.1039/C4CC09685A
COMMUNICATION
Published on 13 January 2015. Downloaded by New York University on 13/01/2015 12:12:44.
Ji-Cheng Yu, ‡a Sha Zhu, ‡ a Pei-Jian Feng, a Cheng-Gen Qian, a Jun Huang, a Min-Jie Sun b and Qun-Dong Shen*,a a
Department of Polymer Science & Engineering and Key Laboratory of High Performance Polymer Materials &
Technology of MOE, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing, 210093, China. E-mail: [email protected]; Fax: +86-25-8331 7761; Tel: +86-25-8331 7807 b
Department of Pharmaceutics, China Pharmaceutical University, Nanjing, 210009, China.
Table of contents:
Nanocarriers with core-shell structure for delivery and non-invasively tracking intracellular release of siRNA are developed.
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1‐3 | 5
ChemComm Accepted Manuscript
Cationic Fluorescent Polymer Core-Shell Nanoparticles for Encapsulation, Delivery, and Non-Invasively Tracking Intracellular Release of siRNA
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: J. Yu, S. Zhu, P. Feng, C. Qian, J. Huang, M. Sun and Q. Shen, Chem. Commun., 2015, DOI: 10.1039/C4CC09685A.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/chemcomm
Page 1 of 5
ChemComm
Journal Name
RSCPublishing View Article Online
DOI: 10.1039/C4CC09685A
COMMUNICATION
Received 00th January 2012, Accepted 00th January 2012
Ji-Cheng Yu, ‡a Sha Zhu, ‡ a Pei-Jian Feng, a Cheng-Gen Qian, a Jun Huang, a Min-Jie Sun b and Qun-Dong Shen*,a
DOI: 10.1039/x0xx00000x www.rsc.org/
A multifunctional nanocarrier for encapsulation and delivery of short interfering RNA (siRNA) has been realized using cationic fluorescent polymer core-shell nanoparticles. The nanocarrier has good biocompatibility and high transfection efficiency over the most popular transfection reagent, Lipofectamine 2000. Fluorescence resonance energy transfer within the nanocarrier provides a non-invasively and labelfree way to track the intracellular release of siRNA. RNA interference (RNAi), as a RNA-dependent gene silencing process, plays a significant role in life science. During the gene expression regulation process, RNA inhibits specific gene expression via causing the destruction of specific mRNA molecules. In recent decades, small interfering RNA (siRNA) has been exploited as potential means for the treatment of diseases such as cancers, Parkinson’s disease, and genetic disorders.[1-6] However, naked siRNA is vulnerable to ribonuclease (RNase) and difficult to cross through negatively charged cell membranes, leading to the low transfection efficiency.[7-8] Therefore, one of the most important challenges in siRNA therapy is to find an efficient non-viral gene delivery vehicle. To date, a large amount of materials including cationic lipids, polymers, dendrimers, and peptides have been developed.[9-15] Among them, cationic polymers have the abilities to condense anionic siRNA via electrostatic assembly into siRNA/polymer complexes, and to promote the complexes to release from endosomes via proton sponge. [16-18] Cationic pi-conjugated polymers have been developed as nonviral nucleic acid nanocarriers in recent years,[19-21] owing to their abundant positive charges, low cytotoxicity, and most importantly, intrinsic fluorescence, which enables real-time tracking the biodistribution of the delivery systems using non-invasive fluorescence imaging.[22] The behavior of delivery system and the function of nucleic acids are highly dependent on the environment of the living organisms or cells. Therefore, it is important to reveal how the nucleic acid molecules are released from the delivery vehicles. Generally, fluorophores can be chemically attached to the nucleic acids to assess the success of intracellular transfection. Förster resonance energy transfer (FRET) between the fluorescence-labeled nucleic acids and the fluorescent nanocarriers is a smart technique to understand in vitro or in vivo release kinetics of the nanoparticle-based delivery systems.[19, 23-26]
This journal is © The Royal Society of Chemistry 2012
The fluorescence off or on state depicts whether the nucleic acid molecules are trapped inside or released from the nanocarriers.
Scheme 1. Fluorescent nanoparticles as siRNA nanocarriers by sequentially electrostatic assembly of siRNA and ThPFN on BtPFN nanoparticles. In this study, we designed a simple multifunctional nanoparticle system, which can deliver siRNA free from fluorescence labeling to the cells and sense the delivery of the siRNA by fluorescence resonance energy transfer imaging approach. As illustrate in Scheme 1, the nanocarriers are comprised of three components: a cationic yellow-emissive conjugated polymer (BtPFN) nanoparticles as core, siRNA electrostatically attached to the BtPFN nanoparticles, and a sequentially electrostatically adsorbed cationic green-emissive conjugated polymer (ThPFN) as the shell. The cationic ThPFN shell is able to protect siRNA from RNase and to interact with the negatively charged cell membranes, which leads to high transfection efficiency of triplex nanoparticles. The key point is that, when negatively charged siRNA brings fluorescence acceptor (BtPFN) into the proximity of fluorescence donor (ThPFN), efficient energy transfer can take place within the electrostatic complexes. After the complexes are delivered into target cells, siRNA is gradually released from the complexes, where electrostatic repulsion between two cationic conjugated polymers leads to loss of the FRET effect. Such approach allows real-time visualization of the siRNA-release in a non-invasive and high-sensitive manner, which reduces the costs and avoid time-consuming and labor-intensive fluorescence labeling of siRNA.
J. Name., 2012, 00, 1‐3 | 1
ChemComm Accepted Manuscript
Published on 13 January 2015. Downloaded by New York University on 13/01/2015 12:12:44.
Cite this: DOI: 10.1039/x0xx00000x
Cationic Fluorescent Polymer Core-Shell Nanoparticles for Encapsulation, Delivery, and Non-Invasively Tracking Intracellular Release of siRNA
ChemComm
Page 2 of 5
Journal Name
Figure 1. a) TEM image and b) hydrodynamic diameter distribution of ThPFN/siRNA/BtPFN NPs. (Scale bar: 200 nm)
2 | J. Name., 2012, 00, 1‐3
a) PL Intensity
1x10
6
9x10
5
8x10
5
View Article Online
7x10
5
DOI: 10.1039/C4CC09685A
6x10
5
5x10
5
4x10
5
3x10
5
2x10
5
1x10
5
BtPFN NPs and ThPFN ThPFN/siRNA/BtPFN
0
400
450
500
550
600
650
700
Wavelength (nm)
b)
3.2 3.0
pH=7.4
2.8 2.6 2.4 2.2 2.0
pH=5.7
1.8 1.6 1.4 1.2
0
1
2
3 4 Time (h)
5
6
Figure 2. a) Fluorescence emission spectra of a simple mixture of ThPFN and BtPFN NPs (solid line) and ThPFN/siRNA/BtPFN NPs (dash line) in aqueous solution. b) Time-lapse ratios of fluorescence intensities at 550 nm (BtPFN) and 454 nm (ThPFN) of the ThPFN/siRNA/BtPFN NPs under pH 5.7 and 7.4 environment. The excitation wavelength is 364 nm. To investigate the stabilities of ThPFN/siRNA/BtPFN NPs under physiological condition, fluorescence of the complexes was monitored in phosphate buffer at pH 5.7 or 7.4. We used I550/I454 to describe the change of FRET efficiency of the electrostatic complexes, where I550 and I454 are the fluorescence intensities at emission maxima of BtPFN and ThPFN respectively and the excitation wavelength is 364 nm. The FRET efficiency of the complexes under pH of 7.4 was almost constant, implying that siRNA is protected via association with the outermost shell of the polymer (Figure 2b). In acid environment (pH 5.7), the FRET efficiency reduced drastically, indicating disruption of the electrostatic complexes with the layer-by-layer structure and the consequent release of siRNA molecules. The FRET phenomenon existing within ThPFN/siRNA/BtPFN NPs also provides a useful optical tool for visualization of the distribution of siRNA-loaded NPs and the release of label-free siRNA from the NPs in live cells. Confocal laser scanning microscope (CLSM) images of HepG2 cells after 0- and 2-hour transfection are shown in Figure 3a-f. Bright-field images showed that the cells retain their normal morphology after being incubated with the electrostatic complexes. Efficient FRET between ThPFN and BtPFN resulted in the weak fluorescence of ThPFN and bright fluorescence of BtPFN. The clustered fluorescence revealed that most of the NPs were intact in the endosomes (pH 6.5-6.8 for early endosomes), and thus siRNA molecules did not release at this stage. After incubation for another 2 h, the fluorescence of ThPFN enhanced dramatically, whereas the fluorescence of BtPFN seemed to be considerably weakened. It suggests the electrostatic interactions between siRNA and cationic polymer are weakened and a large part of the NPs disassemble due to the protonation of the base groups of siRNA in acidic endosomes (pH 5.2-5.8 for late endosomes). Also, the protonation of the NPs results in the rupture of endosome by the proton sponge effect and escape of siRNA into cytoplasm, which leads to reduced FRET
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
BtPFN is a conjugated polymer with hydrophobic backbone and hydrophilic side chains, and therefore in aqueous solution exhibits a strong tendency to self-assemble into nanoparticles (NPs). The hydrodynamic diameters of BtPFN NPs were measured by dynamic light scattering. As shown in Figure S1 (details can be found in the ESI), the as-prepared NPs showed an average hydrodynamic diameter of 50.2 nm with a narrow size distribution. There was no aggregation of BtPFN NPs. It was confirmed by transmission electron microscopy imaging, where BtPFN NPs had an average diameter of about 50 nm with a spherical shape. The nanoparticles exhibited a zeta-potential value of +37 mV (Table S1). Due to their positive surface charges, BtPFN NPs electrostatically adsorbed anionic siRNA molecules, forming a siRNA/BtPFN shell/core structure. The average hydrodynamic diameter of the nanoparticles increased to 64.2 nm, and the zeta-potential value changed from +37 mV to -6 mV. siRNA/BtPFN NPs cannot enter cells efficiently because their negative charges lead to electrostatic repulsion from the anionic cell membranes. Moreover, siRNA molecules on the surface of the NPs are vulnerable to ribonuclease. Coating a cationic conjugated polymer (ThPFN) shell on siRNA/BtPFN NPs will significantly protect siRNA from attack by extracellular nucleases, and meanwhile allow facile cellular uptake via an endocytic pathway. The final three-layer NPs (ThPFN/siRNA/BtPFN) had an average hydrodynamic diameter of 82.8 nm and a zeta-potential value of +23 mV. The morphology of ThPFN/siRNA/BtPFN NPs was further investigated by transmission electron microscopy. Figure 1 shows that they had stratified structures, which confirms the layer-by-layer assembly process. To assess the protection of conjugated polymers to siRNA, we incubated the free siRNA or siRNA-loaded NPs with 10% fetal bovine serum (FBS) at 37oC for 4h. From the result shown in Figure S2, free siRNA was digested within 4 hours, whereas siRNA encapsulated in the NPs were obviously more stable in the presence of serum, which indicates the conjugated polymers are able to prevent siRNA from degradation by RNase in the serum. Polyfluorenes have high photostability and fluorescence quantum yields, and ThPFN and BtPFN emitted green and yellow light respectively upon excitation. The fluorescence emission spectra of ThPFN/siRNA/BtPFN NPs dispersed in aqueous solution, as well as a mixture of BtPFN NPs and ThPFN with the same polymer concentrations, were shown in Figure 2a. In contrast to that of the simple mixture, emission of ThPFN in the complexes was weakened, while the fluorescence intensity of BtPFN was invigorated dramatically. It is noteworthy that the emission spectrum of ThPFN overlaps well with the absorption spectrum of BtPFN (Figure S3), thus efficient energy transfer process through nonradiative dipoledipole coupling may occur if the donor (ThPFN) in excited state is in close proximity to the acceptor (BtPFN) in ground state. However, in the simple mixture, electrostatic repulsion is expected between these cationic polymers. When oppositely charged siRNA/BtPFN NPs and ThPFN are used, it brings the acceptor into proximity of the donor. As a result, fluorescence enhancement is more likely in the electrostatic complexes (ThPFN/siRNA/BtPFN NPs).
I550/I454
Published on 13 January 2015. Downloaded by New York University on 13/01/2015 12:12:44.
COMMUNICATION
Page 3 of 5
ChemComm
Journal Name
COMMUNICATION
View Article Online
Figure 3. Confocal laser scanning microscopic images of HepG2 cells incubated with ThPFN/siRNA/BtPFN NPs after (a-c) 0h and (d-f) 2h transfection. The green channel was ThPFN (420-500 nm); the red channel was BtPFN (550-600 nm); and the last was bright field image. 3D sectional CLSM images of HepG2 cells: g) green channel (ThPFN), h) red channel (BtPFN), and i) merged. The light source was 405 nm. efficiency between ThPFN and BtPFN. 3D sectional CLSM images were taken to show the bio-distribution of ThPFN and BtPFN after 2-hour transfection. As shown in Figure 3g and h, the fluorescence signal of ThPFN distributed more evenly in the cytoplasm, whereas the signal of BtPFN was clustered in the cells. When the colocalization of two fluorescence signals were investigated, it seemed that only a small amount of ThPFN signal overlaps well with that of BtPFN (Figure 3i). It indicates that a large number of ThPFN molecules were dissociated from the ThPFN/siRNA/BtPFN NPs and escaped into cytoplasm from the endosomes, and stimulating the release of siRNA simultaneously. In order to further study the siRNA releasing from the nanoparticles in the cells, we utilized Cy5 groups to label the siRNA and observed the distribution of siRNA during transfection process. After 2-hour transfection, there were fluorescence signals of Cy5-siRNA in the whole cytoplasm (Figure S4). Of note, the fluorescence signals didn’t overlap with the signals of conjugated polymers completely, which means most of the NPs had disassembled and siRNAs had detached from the NPs. It is noteworthy that the conjugated polymers had much better photostability than the small organic molecule dye Cy5 after laser illumination for a short time (Figure S5). To evaluate the RNA interference efficiency using ThPFN/siRNA/BtPFN NPs, HepG2 (human hepatocellular carcinoma) cells stably transfected with luciferase were chosen. The transfer of exogenous nucleic acids into Hep G2 cell line is important for the study of human liver diseases, metabolism, tumor progression, and toxicity of xenobiotics. However, Hep G2 seems to be more resistant to lipid-mediated transfection than other cell lines. [27] ThPFN/siRNA/BtPFN NPs are able to successfully deliver siRNA into HepG2 cells for efficient transfection, and therefore reduce luciferase expression. As shown in Figure 4a, ThPFN/siRNA/BtPFN NPs can achieve approximately 70% gene silencing efficiency, which exceeds the efficiency of the most popular transfection reagents Lipofectamine 2000 (53%). Thus,
This journal is © The Royal Society of Chemistry 2012
Figure 4. a) Gene silencing efficiency of siRNA targeting luciferase. b) Cell viability of HepG2 cells after incubation with BtPFN, ThPFN, ThPFN/siRNA/BtPFN NPs, and Lipofectamine (Lipo) 2000/siRNA at 37oC for 5h. ThPFN/siRNA/BtPFN NPs might be a good choice for improving the transfection efficiency. ThPFN/negative control siRNA (N.C. siRNA)/BtPFN NPs were used to distinguish sequence-specific silencing from non-specific effects, and showed barely gene silencing. It is noteworthy that both free siRNA and siRNA/BtPFN NPs showed low gene silencing efficiency. It indicates that the ability of the cationic ThPFN shell to protect siRNA from RNase and cross negatively charged cell membranes is important for the high transfection efficiency. The biocompatibility of siRNA carriers is crucial for their potential application in clinical gene therapy. The standard methyl thiazolyl tetrazolium (MTT) assay was carried out to determine the relative cellular viabilities of HepG2 cells incubated with conjugated polymers (ThPFN or BtPFN), or ThPFN/siRNA/BtPFN NPs. Figure 4b shows no significant cytotoxicity of two polymers in transfection time (5 h). ThPFN/siRNA/BtPFN NPs had better biocompatibility than Lipofectamine 2000. ThPFN/siRNA/BtPFN NPs exhibit colloidal stability within 24 hours and efficient transfection of siRNA in 10% FBS. For further in vivo applications, cationic carriers with near infrared emission and surface modifications are necessary for fluorescence imaging and decreasing the uptake of the carriers by the macrophages. In conclusion, we constructed a simple nanocarrier to deliver siRNA by sequentially electrostatic adsorption of siRNA and ThPFN on BtPFN NPs. ThPFN/siRNA/BtPFN NPs have good biocompatibility and high transfection efficiency. Of note, the FRET between the ThPFN shell and BtPFN core provides an effective and label-free way to track the intracellular release of siRNA. The electrostatic complexes may have the potential for image-guided gene therapy in the future.
J. Name., 2012, 00, 1‐3 | 3
ChemComm Accepted Manuscript
Published on 13 January 2015. Downloaded by New York University on 13/01/2015 12:12:44.
DOI: 10.1039/C4CC09685A
ChemComm
Page 4 of 5
COMMUNICATION
Journal Name
Acknowledgements
8
S. Zhang, Y. Zhao, D. Zhi, S. Zhang, Bioorg. Chem. 2012, 40, 10.
This work was supported by National Natural Science Foundation of China (No. 21174060), Fundamental Research Funds for the Central Universities (No. 1116020509), Program for Changjiang Scholars and Innovative Research Team in University (IRT1252), and China Equipment and Education Resources System (CERS-1-45).
9
Y. M. Yang, F. Liu, X. G. Liu, B. G. Xing, Nanoscale 2013, 5, 231.
11 R. Kanasty, J. R. Dorkin, A. Vegas, D. Anderson, Nat. Mater. 2013,
Notes and references
12 H. Hatakeyama, H. Akita, E. Ito, Y. Hayashi, M. Oishi, Y. Nagasaki,
Therapy 2012, 20, 513. 12, 967. R. Danev, K. Nagayama, N. Kaji, H. Kikuchi, Y. Baba, H.
Published on 13 January 2015. Downloaded by New York University on 13/01/2015 12:12:44.
a
Harashima, Biomaterials 2011, 32, 4306. 13 M. L. Patil, M. Zhang, T. Minko, ACS Nano 2011, 5, 1877. 14 X. Liu, C. Liu, E. Laurini, P. Posocco, S. Pricl, F. Qu, P. Rocchi, L. Peng, Mol. Pharmaceutics 2012, 9, 470. 15 J. Hoyer, I. Neundorf, Acc. Chem. Res. 2012, 45, 1048. 16 C. J. Chen, J. C. Wang, E. Y. Zhao, L. Y. Gao, Q. Feng, X. Y. Liu, Z. X. Zhao, X. F. Ma, W. J. Hou, L. R. Zhang, W. L. Lu, Q. Zhang, Biomaterials 2013, 34, 5303.
1 N. A. Fonseca, A. C. Gregorio, A. Valerio-Fernandes, S. Simoes, J. N.
17 S. H. Hu, T. Y. Hsieh, C. S. Chiang, P. J. Chen, Y. Y. Chen, T. L. Chiu, S. Y. Chen, Adv. Healthcare Mater. 2014, 3, 273.
Moreira, Cancer Treat. Rev. 2014, 40, 626. 2
R. L. Bogorad, H. Yin, A. Zeigerer, H. Nonaka, V. M. Ruda, M.
18 S. K. Tripathi, V. P. Singh, K. C. Gupta, P. Kumar, J. Mater. Chem. B 2013, 1, 2515.
Zerial, D. G. Anderson, V. Koteliansky, Nat. Commun. 2014, 5, 3869. 3
6
20 X. Feng, F. Lv, L. Liu, Q. Yang, S. Wang, G. C. Bazan, Adv. Mater. 2012, 24, 5428.
A. Tan, J. Rajadas, A. M. Seifalian, Adv. Drug Deliver. Rev. 2013, 65, 357.
5
Biomacromolecules 2013, 14, 3643.
P. Resnier, T. Montier, V. Mathieu, J. P. Benoit, C. Passirani, Biomaterials 2013, 34, 6429.
4
19 R. Jiang, X. Lu, M. Yang, W. Deng, Q. Fan, W. Huang,
21 A. T. Silva, N. Alien, C. Ye, J. Verchot, J. H. Moon, BMC Plant Bio. 2010, 10, 291.
H. Meng, W. X. Mai, H. Zhang, M. Xue, T. Xia, S. Lin, X. Wang, Y. Zhao, Z. Ji, J. I. Zink, A. E. Nel, ACS Nano 2013, 7, 994.
22 D. Ding, J. Liu, G. Feng, K. Li, Y. Hu, B. Liu, Small 2013, 9, 3093.
J. Tabernero, G. I. Shapiro, P. M. LoRusso, A. Cervantes, G. K.
23 C. A. Alabi, K. T. Love, G. Sahay, T. Stutzman, W. T. Young, R. Langer, D. G. Anderson, ACS Nano 2012, 6, 6133.
Schwartz, G. J. Weiss, L. Paz-Ares, D. C. Cho, J. R. Infante, M. Alsina, M. M. Gounder, R. Falzone, J. Harrop, A. C. S. White, I.
24 Z. Cheng, Y. Dai, X. Kang, C. Li, S. Huang, H. Lian, Z. Hou, P. Ma, J. Lin, Biomaterials 2014, 35, 6359.
Toudjarska, D. Bumcrot, R. E. Meyers, G. Hinkle, N. Svrzikapa, R. M. Hutabarat, V. A. Clausen, J. Cehelsky, S. V. Nochur, C. Gamba-
25 X. Wang, F. He, L. Li, H. Wang, R. Yan, L. Li, ACS Appl. Mater. Interfaces 2013, 5, 5700.
Vitalo, A. K. Vaishnaw, D. W. Y. Sah, J. A. Gollob, H. A. Burris III, Cancer Discov. 2013, 3, 406. 7
26 J. Tang, B. Kong, H. Wu, M. Xu, Y. Wang, Y. Wang, D. Zhao, G. Zheng, Adv. Mater. 2013, 25, 6569.
A. de Fougerolles, H. P. Vornlocher, J. Maraganore, J. Lieberman, Nat. Rev. Drug Discov. 2007, 6, 443.
4 | J. Name., 2012, 00, 1‐3
27 S. Yamano, J. S. Dai, A. M. Moursi, Mol. Biotech. 2010, 46, 287.
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
Department of Polymer Science & Engineering and Key Laboratory of High Performance Polymer Materials & Technology of MOE, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing, 210093, China. E-mail: [email protected]; Fax: +86-25-8331 7761; Tel: +86-25-8331 7807 b Department of Pharmaceutics, China Pharmaceutical University, Nanjing, 210009, China. † Electronic Supplementary Information (ESI) available. See DOI: 10.1039/c000000x/ ‡ These authors contributed equally to this work.
View Article Online
10 R. L. Kanasty, K. A. Whitehead, A. J. Vegas, G. Anderson, Mol. DOI:D. 10.1039/C4CC09685A
Page 5 of 5
ChemComm
Journal Name
RSCPublishing View Article Online
DOI: 10.1039/C4CC09685A
COMMUNICATION
Published on 13 January 2015. Downloaded by New York University on 13/01/2015 12:12:44.
Ji-Cheng Yu, ‡a Sha Zhu, ‡ a Pei-Jian Feng, a Cheng-Gen Qian, a Jun Huang, a Min-Jie Sun b and Qun-Dong Shen*,a a
Department of Polymer Science & Engineering and Key Laboratory of High Performance Polymer Materials &
Technology of MOE, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing, 210093, China. E-mail: [email protected]; Fax: +86-25-8331 7761; Tel: +86-25-8331 7807 b
Department of Pharmaceutics, China Pharmaceutical University, Nanjing, 210009, China.
Table of contents:
Nanocarriers with core-shell structure for delivery and non-invasively tracking intracellular release of siRNA are developed.
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1‐3 | 5
ChemComm Accepted Manuscript
Cationic Fluorescent Polymer Core-Shell Nanoparticles for Encapsulation, Delivery, and Non-Invasively Tracking Intracellular Release of siRNA
