Login or Register to make a submission.


Journal of Current Science and Technology

ISSN 2630-0656 (Online)

Cell-penetrating peptide nanocomplexes enhanced cellular uptake of dsRNA in Sf9 cell line

  • Narita Thungsatianpun, Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
  • Rapeepat Mavichak, Aquatic Animal Health Research Center, Charoen Pokphand Foods Public Company Limited, Samutsakorn 74000, Thailand
  • Nattanan T-Thienprasert, Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
  • Sasimanas Unajak, Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
  • Chomdao Sinthuvanich, Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand & Specialized center of Rubber and Polymer Materials in agriculture and industry (RPM), Faculty of, Corresponding author; E-mail: chomdao.si@ku.th


The use of double-stranded RNA (dsRNA) to knock down genes of interest has gained increased attention in arthropods for applications in insect pest management and vaccine development for aquatic animals.  However, its large size and highly anionic character impede the internalization of dsRNA into cells.  To improve cellular uptake, we utilized cell-penetrating peptides (CPPs) as a delivery vehicle to carry dsRNA across the cell membrane.  Here, nanocomplexes prepared from 600-bp dsRNA and CPPs, TAT or EB1, were characterized and their ability to carry dsRNA into cells was investigated.  The optimal positive to negative charge (P/N) ratio between CPPs and dsRNA was determined by electrophoresis mobility shift assay and fluorescence spectroscopy.  Hydrodynamic size and zeta potential of the complexes were assessed by the dynamic light scattering technique.  Morphology and size distribution of nanocomplexes were examined by transmission electron microscope.  Cellular uptake of CPP/dsRNA nanocomplexes was evaluated in Spodopteria frugiperda (Sf9) cell line.  Internalized dsRNA levels were assessed by semi-quantitative reverse transcription (RT)-PCR after 1, 6, and 48 h.  The results showed that at an appropriate charge ratio, cationic complexes can be formed with size in the range of nanometers.  Interestingly, regardless of CPPs used, the 600-bp dsRNA were internalized into the cell during the first hour of incubation.  However, levels of dsRNA delivered by TAT were diminished, comparing to EB1 after 48 h.  Overall, this work provides more insights into the factors involving nucleic acid delivery in arthropod cells.

Keywords: cell-penetrating peptide, cellular uptake, double-stranded RNA, nanocomplex, Spodopteria frugiperd

PDF (750.44 KB)

DOI: 10.14456/jcst.2021.30


Al Soraj, M., He, L., Peynshaert, K., Cousaert, J., Vercauteren, D., Braeckmans, K., De Smedt, S. C., Jones, A. T. (2012). siRNA and pharmacological inhibition of endocytic pathways to characterize the differential role of macropinocytosis and the actin cytoskeleton on cellular uptake of dextran and cationic cell penetrating peptides octaarginine (R8) and HIV-Tat. Journal of Controlled Release, 161(1), 132-141. DOI: https://doi.org/10.1016/j.jconrel.2012.03.015

Attasart, P., Kaewkhaw, R., Chimwai, C., Kongphom, U., Namramoon, O., & Panyim, S. (2009). Inhibition of white spot syndrome virus replication in Penaeus monodon by combined silencing of viral rr2 and shrimp PmRab7. Virus Research, 145(1), 127-133. DOI: https://doi.org/10.1016/j.virusres.2009.06.018

Brutscher, L. M., & Flenniken, M. L. (2015). RNAi and antiviral defense in the honey bee. Journal of Immunology Research, 2015, 941897. DOI: https://doi.org/10.1155/2015/941897

Cermenati, G., Terracciano, I., Castelli, I., Giordana, B., Rao, R., Pennacchio, F., & Casartelli, M. (2011). The CPP Tat enhances eGFP cell internalization and transepithelial transport by the larval midgut of Bombyx mori (Lepidoptera, Bombycidae). Journal of Insect Physiology, 57(12), 1689-1697. DOI: https://doi.org/10.1016/j.jinsphys.2011.09.004

Cerrato, C. P., Kivijärvi, T., Tozzi, R., Lehto, T., Gestin, M., & Langel, Ü. (2020). Intracellular delivery of therapeutic antisense oligonucleotides targeting mRNA coding mitochondrial proteins by cell-penetrating peptides. Journal of Materials Chemistry B, 8(47), 10825-10836. DOI: https://doi.org/10.1039/D0TB01106A

Christiaens, O., Niu, J., & Taning, C. N. T. (2020). RNAi in Insects: A Revolution in Fundamental Research and Pest Control Applications. Insects, 11(7). DOI: https://doi.org/10.3390/insects11070415

Clogston, J. D., & Patri, A. K. (2011). Zeta potential measurement. Methods Mol Biol, 697, 63-70. DOI: https://doi.org/10.1007/978-1-60327-198-1_6

Cooper, A. M., Song, H., Yu, Z., Biondi, M., Bai, J., Shi, X., Ren, Z., Weerasekara, S. M., Hua, D. H., Silver, K., Zhang, J., Zhu, K. Y. (2021). Comparison of strategies for enhancing RNA interference efficiency in Ostrinia nubilalis. Pest Management Science, 77(2), 635-645. DOI: https://doi.org/10.1002/ps.6114

Dhandapani, R. K., Gurusamy, D., Howell, J. L., & Palli, S. R. (2019). Development of CS-TPP-dsRNA nanoparticles to enhance RNAi efficiency in the yellow fever mosquito, Aedes aegypti. Scientific Reports, 9(1), 8775. DOI: https://doi.org/10.1038/s41598-019-45019-z

Edwards, C. H., Christie, C. R., Masotti, A., Celluzzi, A., Caporali, A., & Campbell, E. M. (2020). Dendrimer-coated carbon nanotubes deliver dsRNA and increase the efficacy of gene knockdown in the red flour beetle Tribolium castaneum. Scientific Reports, 10(1), 12422. DOI: https://doi.org/10.1038/s41598-020-69068-x

Eiríksdóttir, E., Mäger, I., Lehto, T., El Andaloussi, S., & Langel, U. (2010). Cellular internalization kinetics of (luciferin-)cell-penetrating peptide conjugates. Bioconjug Chem, 21(9), 1662-1672. DOI: https://doi.org/10.1021/bc100174y

El-Andaloussi, S., Järver, P., Johansson, H. J., & Langel, U. (2007). Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study. Biochem J, 407(2), 285-292. DOI: https://doi.org/10.1042/bj20070507

Feinberg, E. H., & Hunter, C. P. (2003). Transport of dsRNA into cells by the transmembrane protein SID-1. Science, 301(5639), 1545-1547. DOI: https://doi.org/10.1126/science.1087117

Fischer, D., Li, Y., Ahlemeyer, B., Krieglstein, J., & Kissel, T. (2003). In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials, 24(7), 1121-1131. DOI: https://doi.org/10.1016/S0142-9612(02)00445-3

Foroozandeh, P., & Aziz, A. A. (2018). Insight into Cellular Uptake and Intracellular Trafficking of Nanoparticles. Nanoscale research letters, 13(1), 339-339. DOI: https://doi.org/10.1186/s11671-018-2728-6

Frankel, A. D., & Pabo, C. O. (1988). Cellular uptake of the tat protein from human immunodeficiency virus. Cell, 55(6), 1189-1193. DOI: https://doi.org/10.1016/0092-8674(88)90263-2

Green, M., & Loewenstein, P. M. (1988). Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell, 55(6), 1179-1188. DOI: https://doi.org/10.1016/0092-8674(88)90262-0

Gurusamy, D., Mogilicherla, K., & Palli, S. R. (2020). Chitosan nanoparticles help double-stranded RNA escape from endosomes and improve RNA interference in the fall armyworm, Spodoptera frugiperda. Archives of Insect Biochemistry and Physiology, 104(4), e21677. DOI: https://doi.org/10.1002/arch.21677

Huang, Y.-W., Lee, H.-J., Tolliver, L. M., & Aronstam, R. S. (2015). Delivery of Nucleic Acids and Nanomaterials by Cell-Penetrating Peptides: Opportunities and Challenges. BioMed Research International, 2015, 834079. DOI: https://doi.org/10.1155/2015/834079

Joga, M. R., Zotti, M. J., Smagghe, G., & Christiaens, O. (2016). RNAi Efficiency, Systemic Properties, and Novel Delivery Methods for Pest Insect Control: What We Know So Far. Frontiers in Physiology, 7(553). DOI: https://doi.org/10.3389/fphys.2016.00553

Kaplan, I. M., Wadia, J. S., & Dowdy, S. F. (2005). Cationic TAT peptide transduction domain enters cells by macropinocytosis. Journal of Controlled Release, 102(1), 247-253. DOI: https://doi.org/10.1016/j.jconrel.2004.10.018

Kasai, H., Inoue, K., Imamura, K., Yuvienco, C., Montclare, J. K., & Yamano, S. (2019). Efficient siRNA delivery and gene silencing using a lipopolypeptide hybrid vector mediated by a caveolae-mediated and temperature-dependent endocytic pathway. Journal of nanobiotechnology, 17(1), 11-11. DOI: https://doi.org/10.1186/s12951-019-0444-8

Kim, T. H., Kim, S. I., Akaike, T., & Cho, C. S. (2005). Synergistic effect of poly(ethylenimine) on the transfection efficiency of galactosylated chitosan/DNA complexes. J Control Release, 105(3), 354-366. DOI: https://doi.org/10.1016/j.jconrel.2005.03.024

Laisney, J., Gurusamy, D., Baddar, Z. E., Palli, S. R., & Unrine, J. M. (2020). RNAi in Spodoptera frugiperda Sf9 cells via nanomaterial mediated delivery of dsRNA: A comparison of poly-l-arginine polyplexes and poly-l-arginine-functionalized Au nanoparticles. ACS Applied Materials & Interfaces, 12(23), 25645-25657. DOI: https://doi.org/10.1021/acsami.0c06234

Lakshmanan, M., Kodama, Y., Yoshizumi, T., Sudesh, K., & Numata, K. (2013). Rapid and efficient gene delivery into plant cells using designed peptide carriers. Biomacromolecules, 14(1), 10-16. DOI: https://doi.org/10.1021/bm301275g

LeCher, J. C., Nowak, S. J., & McMurry, J. L. (2017). Breaking in and busting out: cell-penetrating peptides and the endosomal escape problem. Biomolecular concepts, 8(3-4), 131-141. DOI: https://doi.org/10.1515/bmc-2017-0023

Lin, Y.-H., Huang, J.-H., Liu, Y., Belles, X., & Lee, H.-J. (2017). Oral delivery of dsRNA lipoplexes to German cockroach protects dsRNA from degradation and induces RNAi response. Pest Management Science, 73(5), 960-966. DOI: https://doi.org/10.1002/ps.4407

Liu, B. R., Huang, Y.-W., Aronstam, R. S., & Lee, H.-J. (2016). Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells. PLOS ONE, 11(3), e0150439. DOI: https://doi.org/10.1371/journal.pone.0150439

Liu, C., Liu, X.-N., Wang, G.-L., Hei, Y., Meng, S., Yang, L.-F., Yuan, L., & Xie, Y. (2017). A dual-mediated liposomal drug delivery system targeting the brain: rational construction, integrity evaluation across the blood-brain barrier, and the transporting mechanism to glioma cells. International journal of nanomedicine, 12, 2407-2425. DOI: https://doi.org/10.2147/IJN.S131367

Liu, W.-J., Chang, Y.-S., Wang, C.-H., Kou, G.-H., & Lo, C.-F. (2005). Microarray and RT-PCR screening for white spot syndrome virus immediate-early genes in cycloheximide-treated shrimp. Virology, 334(2), 327-341. DOI: https://doi.org/10.1016/j.virol.2005.01.047

Lundberg, P., El-Andaloussi, S., Sütlü, T., Johansson, H., & Langel, Ü. (2007). Delivery of short interfering RNA using endosomolytic cell-penetrating peptides. The FASEB Journal, 21(11), 2664-2671. DOI: https://doi.org/10.1096/fj.06-6502com

McEwan, D. L., Weisman, A. S., & Hunter, C. P. (2012). Uptake of extracellular double-stranded RNA by SID-2. Molecular Cell, 47(5), 746-754. DOI: https://doi.org/10.1016/j.molcel.2012.07.014

Panariti, A., Miserocchi, G., & Rivolta, I. (2012). The effect of nanoparticle uptake on cellular behavior: disrupting or enabling functions? Nanotechnology, science and applications, 5, 87-100. DOI: https://doi.org/10.2147/NSA.S25515

Pei, D., & Buyanova, M. (2019). Overcoming endosomal entrapment in drug delivery. Bioconjug Chem, 30(2), 273-283. DOI: https://doi.org/10.1021/acs.bioconjchem.8b00778

Romøren, K., Thu, B. J., Bols, N. C., & Evensen, Ø. (2004). Transfection efficiency and cytotoxicity of cationic liposomes in salmonid cell lines of hepatocyte and macrophage origin. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1663(1), 127-134. DOI: https://doi.org/10.1016/j.bbamem.2004.02.007

Sanitt, P., Apiratikul, N., Niyomtham, N., Yingyongnarongkul, B. E., Assavalapsakul, W., Panyim, S., & Udomkit, A. (2016). Cholesterol-based cationic liposome increases dsRNA protection of yellow head virus infection in Penaeus vannamei. Journal of Biotechnology, 228, 95-102. DOI: https://doi.org/10.1016/j.jbiotec.2016.04.049

Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nature methods, 9(7), 671-675. DOI: https://doi.org/10.1038/nmeth.2089

Shu, B., Zhang, J., Zeng, J., Cui, G., & Zhong, G. (2019). Stability of selected reference genes in Sf9 cells treated with extrinsic apoptotic agents. Scientific Reports, 9(1), 14147. DOI: https://doi.org/10.1038/s41598-019-50667-2

Shukla, J. N., Kalsi, M., Sethi, A., Narva, K. E., Fishilevich, E., Singh, S., Mogilicherla, K., & Palli, S. R. (2016). Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects. RNA biology, 13(7), 656-669. DOI: https://doi.org/10.1080/15476286.2016.1191728

Sifuentes-Romero, I., Milton, S. L., & García-Gasca, A. (2011). Post-transcriptional gene silencing by RNA interference in non-mammalian vertebrate systems: Where do we stand? Mutation Research/Reviews in Mutation Research, 728(3), 158-171. DOI: https://doi.org/10.1016/j.mrrev.2011.09.001

Theerawanitchpan, G., Saengkrit, N., Sajomsang, W., Gonil, P., Ruktanonchai, U., Saesoo, S., Flegel, T. W., & Saksmerprome, V. (2012). Chitosan and its quaternized derivative as effective long dsRNA carriers targeting shrimp virus in Spodoptera frugiperda 9 cells. Journal of Biotechnology, 160(3), 97-104. DOI: https://doi.org/10.1016/j.jbiotec.2012.04.011

Ufaz, S., Balter, A., Tzror, C., Einbender, S., Koshet, O., Shainsky-Roitman, J., Yaari, Z., & Schroeder, A. (2018). Anti-viral RNAi nanoparticles protect shrimp against white spot disease. Molecular Systems Design & Engineering, 3(1), 38-48. DOI: https://doi.org/10.1039/C7ME00092H

Uhl, P., Grundmann, C., Sauter, M., Storck, P., Tursch, A., Özbek, S., Leotta, K., Roth, R., Witzigmann, D., Kulkarni, J. A., Fidelj, V., Kleist, C., Cullis, P. R., Fricker, G., & Mier, W. (2020). Coating of PLA-nanoparticles with cyclic, arginine-rich cell penetrating peptides enables oral delivery of liraglutide. Nanomedicine: Nanotechnology, Biology and Medicine, 24, 102132. DOI: https://doi.org/10.1016/j.nano.2019.102132

Wei, Y., Niu, J., Huan, L., Huang, A., He, L., & Wang, G. (2015). Cell penetrating peptide can transport dsRNA into microalgae with thin cell walls. Algal Research, 8, 135-139. DOI: https://doi.org/10.1016/j.algal.2015.02.002

Xie, J., Bi, Y., Zhang, H., Dong, S., Teng, L., Lee, R. J., & Yang, Z. (2020). Cell-penetrating peptides in diagnosis and treatment of human diseases: From preclinical research to clinical application. Frontiers in pharmacology, 11, 697-697. DOI: https://doi.org/10.3389/fphar.2020.00697

Yamada, Y., Perez, S. M., Tabata, M., Abe, J., Yasuzaki, Y., & Harashima, H. (2015). Efficient and high-speed transduction of an antibody into living cells using a multifunctional nanocarrier system to control intracellular trafficking. Journal of Pharmaceutical Sciences, 104(9), 2845-2854. DOI: https://doi.org/10.1002/jps.24310

Approved By TCI (2020 - 2024)

Indexed in