Marine Biotechnology

, Volume 15, Issue 5, pp 584–595

Delivery of Nucleic Acids, Proteins, and Nanoparticles by Arginine-Rich Cell-Penetrating Peptides in Rotifers

  • Betty Revon Liu
  • Ji-Sing Liou
  • Yung-Jen Chen
  • Yue-Wern Huang
  • Han-Jung Lee
Original Article


Cell-penetrating peptides (CPPs) are a group of short, membrane-permeable cationic peptides that represent a nonviral technology for delivering nanomaterials and macromolecules into live cells. In this study, two arginine-rich CPPs, HR9 and IR9, were found to be capable of entering rotifers. CPPs were able to efficiently deliver noncovalently associated with cargoes, including plasmid DNAs, red fluorescent proteins (RFPs), and semiconductor quantum dots, into rotifers. The functional reporter gene assay demonstrated that HR9-delivered plasmid DNAs containing the enhanced green fluorescent protein and RFP coding sequences could be actively expressed in rotifers. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan assay further confirmed that CPP-mediated cargo delivery was not toxic to rotifers. Thus, these two CPPs hold a great potential for the delivery of exogenous genes, proteins, and nanoparticles in rotifers.


Cell-penetrating peptides (CPPs) Green fluorescent protein (GFP) MTT assay Quantum dots (QDs) Red fluorescent protein (RFP) Transgenesis 



Cell-penetrating peptides


Cyanine 3


Dimethyl sulfoxide


Double-stranded RNA


Enhanced green fluorescent protein


Fluorescein isothiocyanate


Green fluorescent protein










Quantum dots




Red fluorescent protein


Small interfering RNA

Supplementary material

10126_2013_9509_MOESM1_ESM.docx (1.7 mb)
ESM 1(DOCX 1.72 mb)


  1. Bolhassani A, Safaiyan S, Rafati S (2011) Improvement of different vaccine delivery systems for cancer therapy. Mol Cancer 10:3CrossRefPubMedGoogle Scholar
  2. Chang M, Chou JC, Lee HJ (2005a) Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells. Plant Cell Physiol 46:482–488CrossRefPubMedGoogle Scholar
  3. Chang M, Hsu HY, Lee HJ (2005b) Dye-free protein molecular weight markers. Electrophoresis 26:3062–3068CrossRefPubMedGoogle Scholar
  4. Chang M, Chou JC, Chen CP, Liu BR, Lee HJ (2007) Noncovalent protein transduction in plant cells by macropinocytosis. New Phytol 174:46–56CrossRefPubMedGoogle Scholar
  5. Chen F, Gerion D (2004) Fluorescent CdSe/ZnS nanocrystal–peptide conjugates for long-term, nontoxic imaging and nuclear targeting in living cells. Nano Lett 4:1827–1832CrossRefGoogle Scholar
  6. Chen CP, Chou JC, Liu BR, Chang M, Lee HJ (2007) Transfection and expression of plasmid DNA in plant cells by an arginine-rich intracellular delivery peptide without protoplast preparation. FEBS Lett 581:1891–1897CrossRefPubMedGoogle Scholar
  7. Chen YJ, Liu BR, Dai YH, Lee CY, Chan MH, Chen HH, Chiang HJ, Lee HJ (2012) A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides. Gene 493:201–210CrossRefPubMedGoogle Scholar
  8. Dahms HU, Hagiwara A, Lee JS (2011) Ecotoxicology, ecophysiology, and mechanistic studies with rotifers. Aquat Toxicol 101:1–12CrossRefPubMedGoogle Scholar
  9. Dai YH, Liu BR, Chiang HJ, Lee HJ (2011) Gene transport and expression by arginine-rich cell-penetrating peptides in Paramecium. Gene 489:89–97CrossRefPubMedGoogle Scholar
  10. Deshayes S, Konate K, Aldrian G, Crombez L, Heitz F, Divita G (2010) Structural polymorphism of non-covalent peptide-based delivery systems: highway to cellular uptake. Biochim Biophys Acta 1798:2304–2314CrossRefPubMedGoogle Scholar
  11. Feder D, Gomes SAO, de Thomaz AA, Almeida DB, Faustino WM, Fontes A, Stahl CV, Santos-Mallet JR, Cesar CL (2009) In vitro and in vivo documentation of quantum dots labeled Trypanosoma cruziRhodnius prolixus interaction using confocal microscopy. Parasitol Res 106:85–93CrossRefPubMedGoogle Scholar
  12. Frankel AD, Pabo CO (1988) Cellular uptake of the Tat protein from human immunodeficiency virus. Cell 55:1189–1193CrossRefPubMedGoogle Scholar
  13. Gama Sosa MA, De Gasperi R, Elder GA (2010) Animal transgenesis: an overview. Brain Struct Funct 214:91–109CrossRefPubMedGoogle Scholar
  14. Green M, Loewenstein PM (1988) Autonomous functional domains of chemically synthesized human immunodeficiency virus Tat trans-activator protein. Cell 55:1179–1188CrossRefPubMedGoogle Scholar
  15. Gump JM, Dowdy SF (2007) TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med 13:443–448CrossRefPubMedGoogle Scholar
  16. Hannon GJ (2002) RNA interference. Nature 418:244–251CrossRefPubMedGoogle Scholar
  17. Holbrook RD, Murphy KE, Morrow JB, Cole KD (2008) Trophic transfer of nanoparticles in a simplified invertebrate food web. Nat Nanotechnol 3:352–355CrossRefPubMedGoogle Scholar
  18. Hou YW, Chan MH, Hsu HR, Liu BR, Chen CP, Chen HH, Lee HJ (2007) Transdermal delivery of proteins mediated by non-covalently associated arginine-rich intracellular delivery peptides. Exp Dermatol 16:999–1006CrossRefPubMedGoogle Scholar
  19. Hu JW, Liu BR, Wu CY, Lu SW, Lee HJ (2009) Protein transport in human cells mediated by covalently and noncovalently conjugated arginine-rich intracellular delivery peptides. Peptides 30:1669–1678CrossRefPubMedGoogle Scholar
  20. Jo J, Tabata Y (2008) Non-viral gene transfection technologies for genetic engineering of stem cells. Eur J Pharm Biopharm 68:90–104CrossRefPubMedGoogle Scholar
  21. Kim GY, Moon JM, Han JH, Kim KH, Rhim H (2011) The sCMV IE enhancer/promoter system for high-level expression and efficient functional studies of target genes in mammalian cells and zebrafish. Biotechno Lett 33:1319–1326CrossRefGoogle Scholar
  22. Lee CY, Li JF, Liou JS, Charng YC, Huang YW, Lee HJ (2011) A gene delivery system for human cells mediated by both a cell-penetrating peptide and a piggyBac transposase. Biomaterials 32:6264–6276PubMedGoogle Scholar
  23. Li JF, Huang Y, Chen RL, Lee HJ (2010) Induction of apoptosis by gene transfer of human TRAIL mediated by arginine-rich intracellular delivery peptides. Anticancer Res 30:2193–2202PubMedGoogle Scholar
  24. Liou JS, Liu BR, Martin AL, Huang YW, Chiang HJ, Lee HJ (2012) Protein transduction in human cells is enhanced by cell-penetrating peptides fused with an endosomolytic HA2 sequence. Peptides 37:273–284CrossRefPubMedGoogle Scholar
  25. Liu K, Lee HJ, Leong SS, Liu CL, Chou JC (2007) A bacterial indole-3-acetyl-l-aspartic acid hydrolase inhibits mung bean (Vigna radiata L.) seed germination through arginine-rich intracellular delivery. J Plant Growth Regul 26:278–284CrossRefGoogle Scholar
  26. Liu BR, Chou JC, Lee HJ (2008) Cell membrane diversity in noncovalent protein transduction. J Membr Biol 222:1–15CrossRefPubMedGoogle Scholar
  27. Liu BR, Li JF, Lu SW, Lee HJ, Huang YW, Shannon KB, Aronstam RS (2010a) Cellular internalization of quantum dots noncovalently conjugated with arginine-rich cell-penetrating peptides. J Nanosci Nanotechnol 10:6534–6543CrossRefPubMedGoogle Scholar
  28. Liu BR, Huang YW, Chiang HJ, Lee HJ (2010b) Cell-penetrating peptide-functionalized quantum dots for intracellular delivery. J Nanosci Nanotechnol 10:7897–7905CrossRefPubMedGoogle Scholar
  29. Liu BR, Huang YW, Winiarz JG, Chiang HJ, Lee HJ (2011) Intracellular delivery of quantum dots mediated by a histidine- and arginine-rich HR9 cell-penetrating peptide through the direct membrane translocation mechanism. Biomaterials 32:3520–3537CrossRefPubMedGoogle Scholar
  30. Liu BR, Lin MD, Chiang HJ, Lee HJ (2012) Arginine-rich cell-penetrating peptides deliver gene into living human cells. Gene 505:37–45CrossRefPubMedGoogle Scholar
  31. Liu BR, Huang YW, Chiang HJ, Lee HJ (2013a) Primary effectors in the mechanisms of transmembrane delivery of arginine-rich cell-penetrating peptides. Adv Stud Biol 5:11–25Google Scholar
  32. Liu MJ, Chou JC, Lee HJ (2013b) A gene delivery method mediated by three arginine-rich cell-penetrating peptides in plant cells. Adv Stud Biol 5:71–88Google Scholar
  33. Lu SW, Hu JW, Liu BR, Lee CY, Li JF, Chou JC, Lee HJ (2010) Arginine-rich intracellular delivery peptides synchronously deliver covalently and noncovalently linked proteins into plant cells. J Agric Food Chem 58:2288–2294CrossRefPubMedGoogle Scholar
  34. Madani F, Lindberg S, Langel U, Futaki S, Graslund A (2011) Mechanisms of cellular uptake of cell-penetrating peptides. J Biophys 2011:414729PubMedGoogle Scholar
  35. Mager I, Langel K, Lehto T, Eiriksdottir E, Langel U (2012) The role of endocytosis on the uptake kinetics of luciferin-conjugated cell-penetrating peptides. Biochim Biophys Acta 1818:502–511CrossRefPubMedGoogle Scholar
  36. May RC, Plasterk RH (2005) RNA interference spreading in C. elegans. Methods Enzymol 392:308–315CrossRefPubMedGoogle Scholar
  37. Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307:538–544CrossRefPubMedGoogle Scholar
  38. Nakase I, Kobayashi S, Futaki S (2010) Endosome-disruptive peptides for improving cytosolic delivery of bioactive macromolecules. Biopolymers 94:763–770CrossRefPubMedGoogle Scholar
  39. Oo AKS, Kaneko G, Hirayama M, Kinoshita S, Watabe S (2010) Identification of genes differentially expressed by calorie restriction in the rotifer (Brachionus plicatilis). J Comp Physiol B 180:105–116CrossRefPubMedGoogle Scholar
  40. Pfannkuchen M, Brummer F (2009) Heterologous expression of DsRed2 in young sponges (Porifera). Int J Dev Biol 53:1113–1117CrossRefPubMedGoogle Scholar
  41. Piedrahita JA, Olby N (2011) Perspectives on transgenic livestock in agriculture and biomedicine: an update. Reprod Fertil Dev 23:56–63CrossRefPubMedGoogle Scholar
  42. Plank C, Oberhauser B, Mechtler K, Koch C, Wagner E (1994) The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems. J Biol Chem 269:12918–12924PubMedGoogle Scholar
  43. Schmidt N, Mishra A, Lai GH, Wong GC (2010) Arginine-rich cell-penetrating peptides. FEBS Lett 584:1806–1813CrossRefPubMedGoogle Scholar
  44. Shaner NC, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572CrossRefPubMedGoogle Scholar
  45. Shearer TL, Snell TW (2007) Transfection of siRNA into Brachionus plicatilis (Rotifera). Hydrobiologia 593:141–150CrossRefGoogle Scholar
  46. Snell TW, Hicks DG (2011) Assessing toxicity of nanoparticles using Brachionus manjavacas (Rotifera). Environ Toxicol 26:146–152CrossRefPubMedGoogle Scholar
  47. Snell TW, Shearer TL, Smith HA, Kubanek J, Gribble KE, Welch DBM (2009) Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera). BMC Biol 7:60CrossRefPubMedGoogle Scholar
  48. Snell TW, Shearer TL, Smith HA (2011) Exposure to dsRNA elicits RNA interference in Brachionus manjavacas (Rotifera). Mar Biotechnol 13:264–274CrossRefPubMedGoogle Scholar
  49. Son SW, Kim JH, Kim SH, Kim H, Chung AY, Choo JB, Oh CH, Park HC (2009) Intravital imaging in zebrafish using quantum dots. Skin Res Technol 15:157–160CrossRefPubMedGoogle Scholar
  50. Stylianou P, Skourides PA (2009) Imaging morphogenesis, in Xenopus with quantum dot nanocrystals. Mech Dev 126:828–841CrossRefPubMedGoogle Scholar
  51. Suhr ST, Ramachandran R, Fuller CL, Veldman MB, Byrd CA, Goldman D (2009) Highly-restricted, cell-specific expression of the simian CMV-IE promoter in transgenic zebrafish with age and after heat shock. Gene Expr Patterns 9:54–64CrossRefPubMedGoogle Scholar
  52. Swanson KS, Mazur MJ, Vashisht K, Rund LA, Beever JE, Counter CM, Schook LB (2004) Genomics and clinical medicine: rationale for creating and effectively evaluating animal models. Exp Biol Med 229:866–875Google Scholar
  53. van den Berg A, Dowdy SF (2011) Protein transduction domain delivery of therapeutic macromolecules. Curr Opin Biotechnol 22:888–893CrossRefPubMedGoogle Scholar
  54. Wadia JS, Dowdy SF (2002) Protein transduction technology. Curr Opin Biotechnol 13:52–56CrossRefPubMedGoogle Scholar
  55. Wang YH, Chen CP, Chan MH, Chang M, Hou YH, Chen HH, Hsu HR, Liu K, Lee HJ (2006) Arginine-rich intracellular delivery peptides noncovalently transport protein into living cells. Biochem Biophys Res Commun 346:758–767CrossRefPubMedGoogle Scholar
  56. Wang YH, Hou YW, Lee HJ (2007) An intracellular delivery method for siRNA by an arginine-rich peptide. J Biochem Biophys Methods 70:579–586CrossRefPubMedGoogle Scholar
  57. Xu Y, Liu BR, Lee HJ, Shannon KS, Winiarz JG, Wang TC, Chiang HJ, Huang YW (2010) Nona-arginine facilitates delivery of quantum dots into cells via multiple pathways. J Biomed Biotechnol 2010:948543PubMedGoogle Scholar
  58. Yu WW, Qu L, Guo W, Peng X (2003) Experimental determination of the extinction coefficient of CdTe, CdSe, and CdS nanocrystals. Chem Mater 15:2854–2860CrossRefGoogle Scholar
  59. Zhang Y, Wang TH (2012) Quantum dot enabled molecular sensing and diagnostics. Theranostics 2:631–654CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Betty Revon Liu
    • 1
  • Ji-Sing Liou
    • 1
  • Yung-Jen Chen
    • 1
  • Yue-Wern Huang
    • 2
  • Han-Jung Lee
    • 1
  1. 1.Department of Natural Resources and Environmental StudiesNational Dong Hwa UniversityShoufengTaiwan
  2. 2.Department of Biological SciencesMissouri University of Science and TechnologyRollaUSA

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