Communication pubs.acs.org/Biomac
Polymer Nanocarrier System for Endosome Escape and Timed Release of siRNA with Complete Gene Silencing and Cell Death in Cancer Cells Wenyi Gu,†,‡ Zhongfan Jia,† Nghia P. Truong,† Indira Prasadam,‡ Yin Xiao,*,‡ and Michael J. Monteiro*,† †
Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove Campus, Brisbane, Queensland 4059, Australia
‡
S Supporting Information *
ABSTRACT: An influenza virus-inspired polymer mimic nanocarrier was used to deliver siRNA for specific and near complete gene knockdown of an osteoscarcom cell line (U2SO). The polymer was synthesized by single-electron transfer living radical polymerization (SET-LRP) at room temperature to avoid complexities of transfer to monomer or polymer. It was the only LRP method that allowed good block copolymer formation with a narrow molecular weight distribution. At nitrogen to phosphorus (N/P) ratios of equal to or greater than 20 (greater than a polymer concentration of 13.8 μg/mL) with polo-like kinase 1 (PLK1) siRNA gave specific and near complete (>98%) cell death. The polymer further degrades to a benign polymer that showed no toxicity even at polymer concentrations of 200 μg/mL (or N/P ratio of 300), suggesting that our polymer nanocarrier can be used as a very effective siRNA delivery system and in a multiple dose administration. This work demonstrates that with a well-designed delivery device, siRNA can specifically kill cells without the inclusion of an additional clinically used highly toxic cochemotherapeutic agent. Our work also showed that this excellent delivery is sensitive for the study of off-target knockdown of siRNA.
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rates.12 We further explore the effects of off-target knockdown using an siRNA not specific for PLK1 but specific for the E6 and E7 oncogenes (i.e., S10) expressed in cervical carcinomas and carcinoma-derived cell lines.13 Polymer nanocarriers usually incorporate pH buffering groups that act as “proton sponges” at low pH values or bind electrostatically to the negatively charged endosome membrane to facilitate endosome escape.14,15 Incorporation of aminebased buffering groups advantageously allows strong electrostatic binding to negatively charged siRNA but conversely through this strong binding inhibits siRNA release.16−18 Attempts to overcome the release problem has been through the incorporation of side chains molecules that can degrade and release siRNA upon an external or environmental stimulus, including temperature,19 pH,20 redox potential,21 light,22 electrical pulse,23 and enzymatic degradation.24,25 However, the use of remote triggers due to inaccessibility to the tumor and the variability of environmental triggers between cell lines and even within the same cell line has limited their use, requiring a more general and independent approach to siRNA release.
he ability for small interfering RNA (siRNA) to silence specific biological pathways by interfering with messenger RNA (mRNA) holds great promise in cancer and other disease treatments.1−3 Cationic polymers and liposomes represent the most widely tested nanocarriers for siRNA delivery.4,5 The greatest challenges for such delivery systems are the ability of the nanocarrier to first escape the endosome and then release siRNA into the cytosol where it can bind onto RNA-induced silencing complex (RISC) for silencing to take place.6 Escape from the endosome has been recently shown to be extremely inefficient (1−2%) for liposomes,7 which would presumably be similar for other cationic delivery vehicles. The cationic charge will also limit the release of free siRNA in the cytosol, and accumulation of these cationic nanocarriers will result in unwanted toxicity, especially when administered in multiple doses. Here, we describe a unique polymer that mimics the influenza virus escape mechanism from the endosome and releases the siRNA in a time-dependent manner through a selfcatalyzed degradation8 of the cationic to an anionic side groups made by single-electron transfer living radical polymerization (SET-LRP).9,10 This degradation process is independent of both the molecular weight of the polymer and pH of the environment, allowing a predictable release time of siRNA regardless of cellular environment.11 We target the knockdown of U-2OS cell line by inhibiting the polo-like kinase (PLK1) pathway as an in vitro model for osteosarcoma, a bone cancer disease prevalent in young children with very poor survival © 2013 American Chemical Society
Received: August 6, 2013 Revised: August 27, 2013 Published: August 30, 2013 3386
dx.doi.org/10.1021/bm401139e | Biomacromolecules 2013, 14, 3386−3389
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Scheme 1. Mechanism for Polymer Assembly, Binding with siRNA, Endosome Fusion and Escape, and Release through a SelfCatalyzed Degradation of PDMAEA
Poly(2-dimethylaminoethyl acrylate) (PDMAEA, pKa ∼ 7.1) is cationic at physiological pH and degrades into the negatively charged and nontoxic poly(acrylic acid) (PAA) (Scheme 1) in water through a self-catalyzed hydrolysis mechanism.8 It binds strongly to siRNA and can release it at a defined time, which is independent of the environmental conditions.11 The advantage of this polymer is its ability to release where external or environmental triggers are not accessible or can be variable, and that the polymer will be relatively nontoxic. Our recent work has demonstrated that a diblock copolymer (A-C3) consisting of a first block of PDMAEA blocked and a second block of P(N-(3-(1H-imidazol-1-yl)propyl)acrylamide (PImPAA) and poly(butyl acrylate) (PBA) worked effectively to deliver, release and thus knockdown biological pathways.26 The second block was designed to fuse with the endosome membrane in a similar way to how the influenza virus acts and allow escape from the endosome to the cytosol. Once in the cytosol, degradation of the PDMAEA to poly(acrylic acid) (PAA) results in ionic repulsion between the polymer nanocarrier and siRNA, providing a rapid mechanism for release after 17 h (see Scheme 1). For the U-2OS cell line, A-C3 showed specific knockdown of ∼80% of cells at an N/P ratio (i.e., nitrogen to phosphorus ratio) of 10 using a concentration of siRNA of 50 nM. Below this N/P ratio, there was little or no knockdown. Here, we wanted to determine whether greater knockdown could be achieved at higher N/P ratios, and determine the toxicity of the A-C3 polymer at these high N/P ratios before and after degradation using the U-2OS cell line. The toxicity of A-C3 was determined by a cell viability assay as described in Supporting Information. Briefly, the U-2OS cells were preseeded in 96-well plates at a density of 4000 cells/well in 100 μL of complete DMEM medium. The cells were treated with polymer, that is (i) freshly made with A-C3, and (ii) A-C3 degraded to a PAA block for a week in water (denoted as DegC3) at N/P ratios of 20, 50, 100, and 300, which corresponds to 13.8, 34.4, 68.8, and 206.5 μg/mL, respectively. The cell viability as shown in Figure 1 was not affected by A-C3 (fresh) until an N/P ratio of 300 (∼20%); while the degraded polymer (Deg-A-C3) showed no toxicity over the range of N/P ratios tested. These experiments were reproducibly repeated three times. The data suggest that at N/P ratios even 10 times greater than that used in our previous work,26 there was little or no
Figure 1. Effect of A-C3 polymer freshly prepared and A-C3 degraded after 1 week (Deg-C3) on the U-2OS cell viability with increasing N/P ratio from 20 to 300 (equivalent to polymer concentration from 13.8 to 206.5 μg/mL).
toxicity. Degrading the PDMAEA block to PAA (i.e., Deg-C3) resulted in no toxicity supporting the use of this polymer to be administered in multiple doses. The size of the nanoparticles before and after degradation remained relatively constant at approximately 20 nm (see Table S4 in reference26). Essentially, once A-C3 has degraded it no longer becomes toxic to cells, an important characteristic for any drug delivery device, and due to its small size can be cleared from the body and thus improve patient safety. The toxicity of A-C3 complexed with Universal and scrambled siRNAs (50 nM) to U-2OS cells was carried out using N/P ratios ranging from 20 to 300 (Figure 2). The Universal siRNA sequence has been found to have no effect on many cell lines and thus used as a control to determine the level of toxicity of the nanocarrier. Scrambled siRNA contains the same sequence as the siRNA to down regulate the PLK1 pathway (i.e., PLK1 siRNA) but with a scrambled nucleotide sequence, allowing determination of the effect of specific cell knockdown. The size of the polymer/siRNA complexes are approximated to be close to 180 nm.26 The Universal siRNA/ A-C3 complex showed a similar result to A-C3 alone, with cell viability at an N/P ratio of 300 close to 20% (Figure 2A). When the Universal siRNA was complex to Deg-C3 there was no affect of cell viability over the N/P ratios studied. In the case of scrambled siRNA complex with A-C3, there was no affect on 3387
dx.doi.org/10.1021/bm401139e | Biomacromolecules 2013, 14, 3386−3389
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Figure 2. Effect of A-C3 polymer freshly prepared and A-C3 degraded after 1 week (Deg-C3) complexed with either (A) Universal or (B) scrambled siRNA on the U-2OS cell viability with increasing N/P ratio from 20 to 300 (equivalent to polymer concentration from 13.8 to 206.5 μg/mL).
Figure 3. Effect of A-C3 polymer freshly prepared and A-C3 degraded after 1 week (Deg-C3) complexed with either (A) PLK1 siRNA or (B) S10 siRNA on the U-2OS cell viability with increasing N/P ratio from 20 to 300 (equivalent to polymer concentration from 6.9 to 206.5 μg/mL).
As shown above, the PKL1 siRNA showed specific knockdown when compared to the control Universal and scrambled siRNA’s. It has been proposed that siRNA is specific and there are no off-target knockdown using other nonspecific types of siRNA. We trialed an siRNA (S10) specific for the E6 and E7 oncogenes expressed in cervical carcinomas and carcinoma-derived cell lines.31 These overexpressed oncogenes should not be prevalent in the U-2OS cell line. However, complexing S10 siRNA with A-C3 at N/P ratios greater than 20 gave high levels of nonspecific knockdown (Figure 3B). This suggests that the S10 siRNA interferes with other biological pathways to induce cell death. The Deg-C3 and S10 siRNA also had no affect on the cell viability until an N/P ratio of 300, in which cell viability was 40%. This indicates that our delivery system is more sensitive at showing the side effects of siRNAs, which we postulate is due to its timely and complete release of siRNA in cytosol. The off-target effect of this siRNA was not demonstrated in other delivery systems32 or at low N/P ratio of 10. However, using A-C3 at higher N/P ratios we observed sever off-target effects, especially when compared to the universal and scrambled siRNA controls (Figure 3B). In summary, we demonstrate the use of a sophisticated polymer deliver nanocarrier with the ability to bind and release siRNA in a time-dependent manner, and which does not rely on an external release trigger (e.g., pH, enzymatic, light, or heat). The polymer also has the ability to change conformation at the endosome pH to allow for membrane fusion and escape. Here, we show that the in vitro knockdown of the U-2SO cell line (a model system for osetosarcoma) with PLK1 siRNA at N/P ratios greater than 20 (where an N/P ratio of 20 equals a polymer concentration of 13.8 μg/mL) gave near complete and
cell viability up to an N/P ratio of 100. Interestingly, the cell viability for the fresh A-C3 at an N/P ratio of 300 was ∼75%, suggesting that this scramble siRNA could provide some protection against cell death (Figure 2B). The reason for this is not known and is part of future work. As shown above, Deg-C3 complexed with scrambled siRNA also showed no affect on cell viability, further supporting the inert behavior of our polymer. PKL1 plays an important role in maintaining the tumorgenic phenotype of osteosarcoma cells,12 and knockdown using siRNA specific to PLK1 should induce selective growth arrest and cell death of the U-2OS cell line as an in vitro model. We evaluated the knockdown of U-2OS cells using our polymerloaded siRNA complex using the same cell viability assay as above. In this work, the N/P ratio was increased from 10 to 300 (Figure 3A). It was found that at an N/P ratio of 10, cell viability was close to 20%, a similar and reproducible result found from previous work with this polymer.26 Most research using many different types of nanocarriers for siRNA delivery usually results in cell viability of only 40−60% even at high N/P ratios,27,28 percentages that would have limited therapeutic value in many applications. This has led researchers to propose a dual delivery system that includes both siRNA delivery and chemotherapeutic to achieve 100% cell death.29,30 When we increased the N/P ratio to 20 and greater, cell death was nearly complete (>98%). This result is the highest found in the literature for siRNA in vitro knockdown and highlights that an effective delivery system can produce specific, effective, and complete cell death. The addition of Deg-C3 and PLK1 siRNA had no affect on the cell viability until an N/P ratio of 300 (60%). We postulate that this level of knockdown was due to some nonspecific transfection of the siRNA into the cells. 3388
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(19) Kurisawa, M.; Yokoyama, M.; Okano, T. J. Controlled Release 2000, 69, 127−137. (20) Mehrotra, S.; Lee, I.; Chan, C. Acta Biomaterialia 2009, 5, 1474−1488. (21) McKenzie, D. L.; Smiley, E.; Kwok, K. Y.; Rice, K. G. Bioconjug. Chem. 2000, 11, 901−9. (22) Handwerger, R. G.; Diamond, S. L. Bioconjug. Chem. 2007, 18, 717−723. (23) Dieguez, L.; Darwish, N.; Graf, N.; Voros, J.; Zambelli, T. Soft Matter 2009, 5, 2415−2421. (24) Ren, K. F.; Ji, J.; Shen, J. C. Bioconjug. Chem. 2006, 17, 77−83. (25) Samarajeewa, S.; Ibricevic, A.; Gunsten, S. P.; Shrestha, R.; Elsabahy, M.; Brody, S. L.; Wooley, K. L. Biomacromolecules 2013, 14, 1018−1027. (26) Truong, N. P.; Gu, W.; Prasadam, I.; Jia, Z.; Crawford, R.; Xiao, Y.; Monteiro, M. J. Nat. Commun. 2013, 4, 1902 DOI: 10.1038/ ncomms2905. (27) Smith, D. D.; Holley, A. C.; McCormick, C. L. Polym. Chem. 2011, 2 (7), 1428−1441. (28) Cui, L. N.; Cohen, J. L.; Chu, C. K.; Wich, P. R.; Kierstead, P. H.; Frechet, J. M. J. J. Am. Chem. Soc. 2012, 134, 15840−15848. (29) Elsabahy, M.; Shrestha, R.; Clark, C.; Taylor, S.; Leonard, J.; Wooley, K. L. Nano Lett. 2013, 13, 2172−2181. (30) Wiradharma, N.; Tong, Y. W.; Yang, Y. Y. Biomaterials 2009, 30, 3100−3109. (31) Putral, L. N.; Bywater, M. J.; Gu, W. Y.; Saunders, N. A.; Gabrielli, B. G.; Leggatt, G. R.; McMillan, N. A. J. Mol. Pharmacol. 2005, 68, 1311−1319. (32) Hartono, S. B.; Gu, W. Y.; Kleitz, F.; Liu, J.; He, L. Z.; Middelberg, A. P. J.; Yu, C. Z.; Lu, G. Q.; Qiao, S. Z. ACS Nano 2012, 6, 2104−2117.
specific knockdown with greater than 98% cell death. This work demonstrates that with a well-designed delivery device, siRNA can specifically kill cells without the requirement of a dual delivery system that includes both siRNA and a chemotherapeutic. Our work also showed that due to this polymer’s excellent delivery and release of siRNA to the cytoplasm, the sensitive off-target knockdown from other siRNAs (e.g., S10) can be determined.
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ASSOCIATED CONTENT
S Supporting Information *
Experimental procedures and data. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Funding Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was funded by an Australian Research Council (ARC) Discovery grant. REFERENCES
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