Communication pubs.acs.org/bc
Artificial Control of Gene Silencing Activity Based on siRNA Conjugation with Polymeric Molecule Having Coil−Globule Transition Behavior Noor Faizah Che Harun, Hiroyasu Takemoto,* Takahiro Nomoto, Keishiro Tomoda, Makoto Matsui, and Nobuhiro Nishiyama* Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, R1-11, 4259, Nagatsuta, Midori-Ku, Yokohama, Kanagawa 226-8503, Japan S Supporting Information *
ABSTRACT: A new strategy for controlling gene silencing activity of siRNA in the cell was developed in the present study. siRNA was linearly conjugated with PNIPAAm, where coil−globule transition of the conjugated PNIPAAm allows thermoresponsive exposure of the vicinal siRNA molecule; a coil form of PNIPAAm (T < LCST) inhibits siRNA interaction with gene silencing-related proteins due to the steric hindrance effect, while a globule form of PNIPAAm (T > LCST) allows a ready access of siRNA to gene silencing pathway. As a result, at T > LCST, PNIPAAm-siRNA elicited effective association of siRNA with a gene silencing-related protein of Ago2, while siRNA recruitment into the gene silencing pathway was significantly suppressed at T < LCST. Ultimately, gene silencing efficacy of PNIPAAm-siRNA was close to unconjugated siRNA at T > LCST (∼80%), while it was dramatically decreased to ∼20% at T < LCST, suggesting that coil−globule transition of the conjugated polymer can control the bioactivity of the vicinal siRNA molecule.
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in the cytoplasm, leading to ineffective gene silencing activity.15 In a similar manner, modification of proteins or peptides with polymers has resulted in controlled bioactivity.16−18 The steric hindrance character of polymers enables molecular design for an artificial control of the siRNA recognition by the proteins in the cytoplasm, and thus, in the present study, a terminus of an siRNA molecule was conjugated with poly(N-isoproprylacrylamide) (PNIPAAm). PNIPAAm behaves as an extended coil form below the lower critical solution temperature (LCST) of 33 °C in an aqueous solution, whereas it shrinks into a globule form above the LCST.19,20 This coil−globule transition is associated with a considerable volume change and potentially leads to thermosensitive exposure of the vicinal siRNA molecule to gene silencing-related proteins in the cytoplasm, toward artificial induction of the gene silencing effect (Figure 1). This is the first report to demonstrate that the coil−globule transition of the conjugated polymer offers an artificial control of the vicinal siRNA recognition by proteins and associated gene silencing activity in the cell. First, PNIPAAm was prepared through reversible addition− fragmentation chain transfer (RAFT) polymerization,21,22 as described in the Supporting Information (Scheme S1, Figure S1). RAFT polymerization produces a polymer with a narrow molecular weight distribution, and provides functional groups
nduction of gene silencing in a controlled manner, e.g., specific time-point and targeted site, is an attractive approach to cure intractable diseases including cancer and genetic disorders without adverse effects.1−3 For gene silencing at the targeted site, tremendous efforts have been dedicated to creating carriers with/without target-specific ligands for small interfering RNA (siRNA) delivery; sophisticatedly designed polycations, lipids, and inorganic materials have been developed,4−6 and their siRNA carriers were often decorated with ligand molecules, such as tumor-targeted cyclic RGD peptide and folate, and liver-targeted galactose and cholesterol,7−10 in order to trigger gene silencing at the targeted site.7−11 Meanwhile, the development of the methodology that allows an artificial control of the therapeutic activity of siRNA in the cell remains a big challenge. Artificial induction of the therapeutic activity in the cell potentially provides another solution to realize targeted-site specific gene silencing toward the siRNAbased therapeutics without adverse effect. Herein, a new methodology for artificial control of gene silencing activity in the cell was successfully developed based on the construction of a new class of polymer-conjugated siRNA. For realization of the artificial control of the siRNA activity, in the present study, siRNA was linearly conjugated with the polymer that changes its hydrodynamic size in response to an external stimulus. In general, gene silencing is initiated with the siRNA recognition by gene silencing-related proteins in the cytoplasm.12−14 In this regard, siRNA conjugation with molecules possessing the steric hindrance effect, such as polymers, often undergoes weak recognition by the proteins © XXXX American Chemical Society
Received: June 20, 2016 Revised: August 5, 2016
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DOI: 10.1021/acs.bioconjchem.6b00322 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Communication
Bioconjugate Chemistry
Thermoresponsive behavior of the obtained PNIPAAmsiRNA was examined by light scattering analysis and fluorescence correlation spectroscopic (FCS) analysis. Upon heating solutions of unconjugated siRNA and polymerconjugated siRNAs from 20 to 40 °C, scattering light intensity started to increase in PNIPAAm-siRNA solution at 35 °C (5 μM siRNA, 10 mM HEPES pH 7.4), whereas PEG-siRNA and unconjugated siRNA solutions showed constant scattering light intensities over the heating process, suggesting that PNIPAAmsiRNA aggregated in a thermoresponsive manner because of the action of the PNIPAAm segment (Figure 2). Of note, the
Figure 2. Light scattering intensities of the solutions of siRNA series at indicated temperatures (5 μM siRNA, 10 mM HEPES pH 7.4). Results were shown as mean and standard deviation obtained from three measurements.
Figure 1. Illustration of the chemical structure of the polymerconjugated siRNA (a), and its gene silencing ability based on coil− globule transition of the PNIPAAm segment (b), in the present study.
aggregation temperature of 35 °C is in good agreement with the original LCST of PNIPAAm (∼33 °C),19,20 indicating that the PNIPAAm segment undergoes LCST-related behavior even in the presence of the vicinal siRNA molecule. This LCSTrelated behavior of the conjugated PNIPAAm molecule potentially permits siRNA exposure, as a result of coil−globule transition and associated shrinkage, when the PNIPAAmsiRNA concentration is lower than the critical aggregation concentration, such as after entering the cell. Indeed, in FCS analysis for a diluted condition (100 nM siRNA, 10 mM HEPES pH 7.4), the hydrodynamic diameter of fluorescently labeled PNIPAAm-siRNA (TAMRA-labeled PNIPAAmsiRNA) decreased from 7.61 ± 0.23 nm at ambient temperature to 6.50 ± 0.21 nm at 37 °C (Table S1). In contrast, TAMRAlabeled unconjugated siRNA and PEG-siRNA maintained their hydrodynamic diameters regardless of a change in temperature. This result suggests the huge potential of the PNIPAAm segment that allows thermoresponsive siRNA exposure and associated siRNA recruitment into gene silencing pathway. Next, gene silencing activity for PNIPAAm-siRNA was investigated using Lipofectamine RNAiMAX for cultured human cervical cancer cells stably expressing luciferase (HeLa-Luc) at two different temperatures (30 and 37 °C). In order to confirm the sequence-specific gene silencing effect, the scrambled siRNA sequence (siScr) was utilized as well as the luciferase-targeting sequence (siLuc). At 37 °C, the treatment with PNIPAAm-siLuc suppressed ∼80% of luciferase expression at 10 nM siRNA, which is close to the suppression level after the unconjugated siLuc treatment (∼90%), while the PEG-siLuc treatment suppressed ∼20% of luciferase expression (Figure 3a). In sharp contrast, at 30 °C, the suppression level after the treatment with the 10 nM PNIPAAm-siLuc dramatically decreased to ∼20%, which is close to the suppression level after the treatment with PEG-siLuc (∼10%), while the unconjugated siLuc treatment maintained
at the polymer terminus for further modification, i.e., siRNA conjugation in the present study, according to the chemical structure of the selected RAFT reagent. The synthesized PNIPAAm with trithiocarbonate terminus and the other terminus of carboxyl moiety (PNIPAAm-CTA) had an average molecular weight of 39 000 g/mol with a polydispersity index (Mw/Mn) of 1.24, as confirmed by size exclusion chromatography (SEC) based on the standard curve of poly(ethylene glycol) (PEG). The trithiocarbonate terminus PNIPAAm-CTA was converted into an unreactive thioether group in order to circumvent unexpected side reactions in the following steps (PNIPAAm-TE, Scheme S1). The carboxyl terminus of PNIPAAm-TE was converted into the cyclooctyne moiety, for the subsequent copper-free click reaction with azideterminated siRNA (azide-siRNA) (DBCO-PNIPAAm-TE, Scheme S2, Figure S2). As a nonthermoresponsive control, PEG with a cyclooctyne terminus (PEG-DBCO, Mw ∼ 40 000 g/mol) was prepared in a similar synthetic procedure (Scheme S3, Figure S3). Then, an azide-siRNA (Mw ∼ 13 600 g/mol) was reacted with the DBCO-PNIPAAm-TE in 10 mM HEPES buffer (pH 7.4) (Scheme S4) to produce PNIPAAm-siRNA, according to the previous method,23 followed by a purification by ion exchange chromatography to completely remove the unreacted DBCO-PNIPAAm-TE and azide-siRNA (Figure S4). The PEGsiRNA was also synthesized in a similar manner (Scheme S5, Figure S5). The obtained PNIPAAm-siRNA and PEG-siRNA were further analyzed by agarose gel electrophoresis (Figure S6). The shorter migrations of the polymer-conjugated siRNAs, compared to unconjugated siRNA, without the presence of the unconjugated siRNA-related band, confirm the higher molecular weights as a result of polymer conjugation and the successful synthesis. B
DOI: 10.1021/acs.bioconjchem.6b00322 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Communication
Bioconjugate Chemistry
number of Ago2-associated asRNA with the elevated temperature for the unconjugated siRNA system can be explained by the less activity of the cell at lower temperature. Notably, the significantly higher multiplied increase (p < 0.05) for the PNIPAAm-siRNA system, compared to the unconjugated siRNA system, strongly suggests that the thermoresponsive behavior of the PNIPAAm segment, as well as the thermal effect on the cell activity, affected gene silencing efficacy; a coil form of the PNIPAAm segment in PNIPAAm-siRNA at 30 °C suppresses the siRNA access to RISC formation due to the steric hindrance, while a globular form of the PNIPAAm segment at 37 °C exposes the vicinal siRNA molecule for triggering the gene silencing effect, as suggested by the results in FCS analysis and gene silencing assay (Table S1, Figure 3a and b). In addition, the treatment with PEG-siRNA, where the conjugated PEG potentially hinders siRNA recruitment regardless of temperature, resulted in smaller numbers of Ago2-associated asRNA per cell compared to the treatments with unconjugated siRNA and PNIPAAm-siRNA, further supporting the suppressed gene silencing efficacy induced by a coil form of the vicinal polymer (Table S2). In summary, a polymer-conjugated siRNA was developed for an artificial control of gene silencing activity based on coil− globule transition of the conjugated polymer. The PNIPAAm segment in PNIPAAM-siRNA reduces its size with the elevated temperature and associated globule formation, leading to the decreased steric hindrance around the vicinal siRNA molecule. Ultimately, the coil−globule transition of the PNIPAAm segment controls the employment of the siRNA segment by Ago2 and subsequent gene silencing activity in the cytoplasm. Interestingly, molecular weight of the conjugated PNIPAAm also affected thermoresponsive change of gene silencing activity of the vicinal siRNA molecule; the conjugation of PNIPAAm with the molecular weights of 10 000 and 20 000 g/mol (termed PNIPAAm10k and PNIPAAm20k, respectively) resulted in ∼80% gene silencing at 37 °C regardless of the molecular weight of the conjugated PNIPAAm (10 nM siRNA, Figures S7a and S8a), while ∼50% and ∼40% gene silencing at 30 °C were observed for PNIPAAm10k system and PNIPAAm20k system, respectively (10 nM siRNA, Figures S7b and S8b). This result suggests that a larger hydrodynamic diameter of the conjugated polymer is suitable for inhibiting siRNA recruitment into the gene silencing pathway. Of note, the PEG10k-siRNA treatment induced ∼80% gene silencing, whereas the PEG20ksiRNA treatment resulted in ∼5% gene silencing (37 °C, 10 nM siRNA, Figures S7a and S8a), where the difference in hydrodynamic diameter of the two PEG-siRNA molecules is ∼1.1 nm (Table S2). This difference is close to the aforementioned thermoresponsive change in the hydrodynamic diameter of PNIPAAm-siRNA (∼1.1 nm, Table S1), suggesting that this small difference in the hydrodynamic diameter derived from the conjugated polymer plays a critical role in regulating siRNA bioactivity in the cell. In the present report, the response of the PNIPAAm segment is limited to the temperature change from 30 to 37 °C; however, the responsiveness of PNIPAAm to temperature can be tuned by introducing other acrylic monomers during polymerization.24 Furthermore, copolymerization of NIPAAm and acrylic monomers having ionic moiety gives pH-responsive coil−globule transition behavior,24,25 and the copolymer with enzymatic responsibility and disulfide linkage can be likewise prepared.26−28 Therefore, further investigation of our polymer-conjugated siRNA system will be challenged for the success in an artificial gene silencing in
Figure 3. Gene silencing efficacies of siRNA series (a,b) and relative numbers of Ago2-associated asRNA per cell (c) for HeLa-Luc cells. (a,b) HeLa-Luc cells were treated with siRNA series using Lipofectamine RNAiMAX at indicated siRNA concentrations and at 37 °C (a) or 30 °C (b). 48 h after the treatment, endogenous luciferase activities were measured by luminometer. Results were shown as mean and standard error of the mean obtained from six samples. (c) The number of the Ago2-associated asRNA per cell after the unconjugated siRNA (or PNIPAAm-siRNA) treatment at 37 °C was divided by the separately obtained value after the treatment with unconjugated siRNA (or PNIPAAm-siRNA) at 30 °C. The treatment condition was similar to the gene silencing assay. Results were shown as mean and standard error of the mean obtained from three samples. The p value was calculated according to the Student’s t test.
strong gene silencing activity (∼80%) with a slightly decreased efficacy compared to the treatment at 37 °C, possibly due to the reduced activity of the cell at lower temperature (Figure 3b). Note that the treatment with siRNA series did not induce cellular death (Figure S9a and b) as well as significant luciferase suppression by siScr, confirming the negligible cytotoxicity with a sequence-specific gene silencing. Considering the fact that the cellular uptake efficacies for the PNIPAAm-siRNA and PEGsiRNA systems were similar for both temperatures (Figure S10a and b), the huge drop in the gene silencing efficacy at 30 °C for the PNIPAAm-siRNA system would be attributed to the events inside the cell. To further elucidate the underlying mechanism in the thermoresponsive gene silencing for PNIPAAm-siRNA system, Argonaute 2 protein (Ago2)-associated asRNA was counted after collecting Ago2 from the siRNA-treated cells by the immunoprecipitation method. Ago2 is a catalytic component inside RNA-induced silencing complex (RISC), that plays a prime role in gene silencing activity, asRNA in siRNA is employed by Ago2 and packaged into RISC, leading to cleavage of its complementary sequence in mRNA in the cytoplasm.13,14 The siRNA series were prepared with TAMRA-labeled asRNA for calculating the number of Ago2-associated asRNA per cell, and were applied for the cultured HeLa-Luc cells in a similar manner to the gene silencing assay. The number of Ago2associated asRNA per cell after the treatment with PNIPAAMsiRNA at 37 °C was 2.78 times higher compared to the treatment at 30 °C, whereas the treatment with unconjugated siRNA at 37 °C led to 1.84 times increase in Ago2-associated asRNA per cell (Figure 3c, Table S3). The increase in the C
DOI: 10.1021/acs.bioconjchem.6b00322 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Communication
Bioconjugate Chemistry
(14) Wang, H. W., Noland, C., Siridechadilok, B., Taylor, D. W., Ma, E., Felderer, K., Doudna, J. A., and Nogales, E. (2009) Nat. Struct. Mol. Biol. 16, 1148. (15) Takemoto, H., Miyata, K., Hattori, S., Ishii, T., Suma, T., Uchida, S., Nishiyama, N., and Kataoka, K. (2013) Angew. Chem., Int. Ed. 52, 6218. (16) Stayton, P. S., Shimoboji, T., Long, C., Chilkoti, A., Ghen, G., Harris, J. M., and Hoffman, A. S. (1995) Nature 378, 472. (17) Molawi, K., and Studer, A. (2007) Chem. Commun., 5173. (18) De, P., Li, M., Gondi, S. R., and Sumerlin, B. S. (2008) J. Am. Chem. Soc. 130, 11288. (19) Fujishige, S., Kubota, K., and Ando, I. (1989) J. Phys. Chem. 93, 3311. (20) Schild, H. G. (1992) Prog. Polym. Sci. 17, 163. (21) Li, Y., Akiba, I., Harrisson, S., and Wooley, K. L. (2008) Adv. Funct. Mater. 18, 551. (22) Moad, C., Rizzardo, E., and Thang, S. H. (2009) Aust. J. Chem. 62, 1402. (23) Takemoto, H., Miyata, K., Ishii, T., Hattori, S., Osawa, S., Nishiyama, N., and Kataoka, K. (2012) Bioconjugate Chem. 23, 1503. (24) Jochum, F. D., and Theato, P. (2013) Chem. Soc. Rev. 42, 7468. (25) Hiruta, Y., Funatsu, T., Matsuura, M., Wang, J., Ayano, E., and Kanazawa, H. (2015) Sens. Actuators, B 207, 724. (26) Katayama, Y., Sonoda, T., and Maeda, M. (2001) Macromolecules 34, 8569. (27) Klaikherd, A., Nagamani, C., and Thayumanavan, S. (2009) J. Am. Chem. Soc. 131, 4830. (28) Bajgiran, K. R., Chan, N., Zhang, Q., Noh, S. M., Lee, H. I., and Oh, J. K. (2013) Chem. Commun. 49, 807.
response to site-specific biological stimuli (e.g., pH, redox potential, and enzyme). The combination with the developed delivery carriers, such as liposome and micelle,2,4,5 is also one of the future tactics, in order to realize the therapeutics highly specific for the targeted site.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00322. Experimental procedures, supporting schemes, supporting tables, and supporting figures (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Division of Materials Analysis Suzukake-dai, Technical Department, Tokyo Institute of Technology, for 1H NMR analysis. This work was supported by Basic Science and Platform Technology Program for Innovative Biological Medicine (Project No. 15am0301008h0002) from Japan Agency for Medical Research and Development (AMED), the Project for Cancer Research And Therapeutic Evolution (PCREATE) (Project No. 16 cm0106202h0001) from AMED, and a JSPS KAKENHI Grant-in-Aid for Young Scientists (B) (15K16331) to H. T., and by JSPS KAKENHI Grant Numbers 15H04635 and 16K15104 to N. N.
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DOI: 10.1021/acs.bioconjchem.6b00322 Bioconjugate Chem. XXXX, XXX, XXX−XXX
