Skip to main content

Advertisement

SpringerLink
Log in

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

  • Original Article
  • Published:
Marine Biotechnology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

CPPs:

Cell-penetrating peptides

Cy3:

Cyanine 3

DMSO:

Dimethyl sulfoxide

dsRNA:

Double-stranded RNA

EGFP:

Enhanced green fluorescent protein

FITC:

Fluorescein isothiocyanate

GFP:

Green fluorescent protein

HA2:

Hemagglutinin-2

6His:

Hexa-histidine

N/P:

Nitrogen/phosphate

MTT:

1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan

QDs:

Quantum dots

R9:

Nona-arginine

RFP:

Red fluorescent protein

siRNA:

Small interfering RNA

References

  • Bolhassani A, Safaiyan S, Rafati S (2011) Improvement of different vaccine delivery systems for cancer therapy. Mol Cancer 10:3

    Article  CAS  PubMed  Google Scholar 

  • Chang M, Chou JC, Lee HJ (2005a) Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells. Plant Cell Physiol 46:482–488

    Article  CAS  PubMed  Google Scholar 

  • Chang M, Hsu HY, Lee HJ (2005b) Dye-free protein molecular weight markers. Electrophoresis 26:3062–3068

    Article  CAS  PubMed  Google Scholar 

  • Chang M, Chou JC, Chen CP, Liu BR, Lee HJ (2007) Noncovalent protein transduction in plant cells by macropinocytosis. New Phytol 174:46–56

    Article  CAS  PubMed  Google Scholar 

  • Chen F, Gerion D (2004) Fluorescent CdSe/ZnS nanocrystal–peptide conjugates for long-term, nontoxic imaging and nuclear targeting in living cells. Nano Lett 4:1827–1832

    Article  CAS  Google Scholar 

  • 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 581:1891–1897

    Article  CAS  PubMed  Google Scholar 

  • 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 493:201–210

    Article  CAS  PubMed  Google Scholar 

  • Dahms HU, Hagiwara A, Lee JS (2011) Ecotoxicology, ecophysiology, and mechanistic studies with rotifers. Aquat Toxicol 101:1–12

    Article  CAS  PubMed  Google Scholar 

  • Dai YH, Liu BR, Chiang HJ, Lee HJ (2011) Gene transport and expression by arginine-rich cell-penetrating peptides in Paramecium. Gene 489:89–97

    Article  CAS  PubMed  Google Scholar 

  • 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 1798:2304–2314

    Article  CAS  PubMed  Google Scholar 

  • 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 cruzi–Rhodnius prolixus interaction using confocal microscopy. Parasitol Res 106:85–93

    Article  PubMed  Google Scholar 

  • Frankel AD, Pabo CO (1988) Cellular uptake of the Tat protein from human immunodeficiency virus. Cell 55:1189–1193

    Article  CAS  PubMed  Google Scholar 

  • Gama Sosa MA, De Gasperi R, Elder GA (2010) Animal transgenesis: an overview. Brain Struct Funct 214:91–109

    Article  CAS  PubMed  Google Scholar 

  • Green M, Loewenstein PM (1988) Autonomous functional domains of chemically synthesized human immunodeficiency virus Tat trans-activator protein. Cell 55:1179–1188

    Article  CAS  PubMed  Google Scholar 

  • Gump JM, Dowdy SF (2007) TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med 13:443–448

    Article  CAS  PubMed  Google Scholar 

  • Hannon GJ (2002) RNA interference. Nature 418:244–251

    Article  CAS  PubMed  Google Scholar 

  • Holbrook RD, Murphy KE, Morrow JB, Cole KD (2008) Trophic transfer of nanoparticles in a simplified invertebrate food web. Nat Nanotechnol 3:352–355

    Article  CAS  PubMed  Google Scholar 

  • 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 16:999–1006

    Article  CAS  PubMed  Google Scholar 

  • 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 30:1669–1678

    Article  CAS  PubMed  Google Scholar 

  • Jo J, Tabata Y (2008) Non-viral gene transfection technologies for genetic engineering of stem cells. Eur J Pharm Biopharm 68:90–104

    Article  CAS  PubMed  Google Scholar 

  • 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 33:1319–1326

    Article  CAS  Google Scholar 

  • 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 32:6264–6276

    CAS  PubMed  Google Scholar 

  • 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 30:2193–2202

    CAS  PubMed  Google Scholar 

  • 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 37:273–284

    Article  CAS  PubMed  Google Scholar 

  • 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 26:278–284

    Article  CAS  Google Scholar 

  • Liu BR, Chou JC, Lee HJ (2008) Cell membrane diversity in noncovalent protein transduction. J Membr Biol 222:1–15

    Article  CAS  PubMed  Google Scholar 

  • 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 10:6534–6543

    Article  CAS  PubMed  Google Scholar 

  • Liu BR, Huang YW, Chiang HJ, Lee HJ (2010b) Cell-penetrating peptide-functionalized quantum dots for intracellular delivery. J Nanosci Nanotechnol 10:7897–7905

    Article  CAS  PubMed  Google Scholar 

  • 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 32:3520–3537

    Article  CAS  PubMed  Google Scholar 

  • Liu BR, Lin MD, Chiang HJ, Lee HJ (2012) Arginine-rich cell-penetrating peptides deliver gene into living human cells. Gene 505:37–45

    Article  CAS  PubMed  Google Scholar 

  • 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 5:11–25

    CAS  Google Scholar 

  • 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 5:71–88

    Google Scholar 

  • 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 58:2288–2294

    Article  CAS  PubMed  Google Scholar 

  • Madani F, Lindberg S, Langel U, Futaki S, Graslund A (2011) Mechanisms of cellular uptake of cell-penetrating peptides. J Biophys 2011:414729

    PubMed  Google Scholar 

  • 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 1818:502–511

    Article  CAS  PubMed  Google Scholar 

  • May RC, Plasterk RH (2005) RNA interference spreading in C. elegans. Methods Enzymol 392:308–315

    Article  CAS  PubMed  Google Scholar 

  • 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 307:538–544

    Article  CAS  PubMed  Google Scholar 

  • Nakase I, Kobayashi S, Futaki S (2010) Endosome-disruptive peptides for improving cytosolic delivery of bioactive macromolecules. Biopolymers 94:763–770

    Article  CAS  PubMed  Google Scholar 

  • 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 180:105–116

    Article  CAS  PubMed  Google Scholar 

  • Pfannkuchen M, Brummer F (2009) Heterologous expression of DsRed2 in young sponges (Porifera). Int J Dev Biol 53:1113–1117

    Article  CAS  PubMed  Google Scholar 

  • Piedrahita JA, Olby N (2011) Perspectives on transgenic livestock in agriculture and biomedicine: an update. Reprod Fertil Dev 23:56–63

    Article  PubMed  Google Scholar 

  • 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 269:12918–12924

    CAS  PubMed  Google Scholar 

  • Schmidt N, Mishra A, Lai GH, Wong GC (2010) Arginine-rich cell-penetrating peptides. FEBS Lett 584:1806–1813

    Article  CAS  PubMed  Google Scholar 

  • 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 22:1567–1572

    Article  CAS  PubMed  Google Scholar 

  • Shearer TL, Snell TW (2007) Transfection of siRNA into Brachionus plicatilis (Rotifera). Hydrobiologia 593:141–150

    Article  CAS  Google Scholar 

  • Snell TW, Hicks DG (2011) Assessing toxicity of nanoparticles using Brachionus manjavacas (Rotifera). Environ Toxicol 26:146–152

    Article  CAS  PubMed  Google Scholar 

  • Snell TW, Shearer TL, Smith HA, Kubanek J, Gribble KE, Welch DBM (2009) Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera). BMC Biol 7:60

    Article  PubMed  Google Scholar 

  • Snell TW, Shearer TL, Smith HA (2011) Exposure to dsRNA elicits RNA interference in Brachionus manjavacas (Rotifera). Mar Biotechnol 13:264–274

    Article  CAS  PubMed  Google Scholar 

  • 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 15:157–160

    Article  PubMed  Google Scholar 

  • Stylianou P, Skourides PA (2009) Imaging morphogenesis, in Xenopus with quantum dot nanocrystals. Mech Dev 126:828–841

    Article  CAS  PubMed  Google Scholar 

  • 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 9:54–64

    Article  CAS  PubMed  Google Scholar 

  • 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 229:866–875

    CAS  Google Scholar 

  • van den Berg A, Dowdy SF (2011) Protein transduction domain delivery of therapeutic macromolecules. Curr Opin Biotechnol 22:888–893

    Article  PubMed  Google Scholar 

  • Wadia JS, Dowdy SF (2002) Protein transduction technology. Curr Opin Biotechnol 13:52–56

    Article  CAS  PubMed  Google Scholar 

  • 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 346:758–767

    Article  CAS  PubMed  Google Scholar 

  • Wang YH, Hou YW, Lee HJ (2007) An intracellular delivery method for siRNA by an arginine-rich peptide. J Biochem Biophys Methods 70:579–586

    Article  CAS  PubMed  Google Scholar 

  • 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 2010:948543

    PubMed  Google Scholar 

  • Yu WW, Qu L, Guo W, Peng X (2003) Experimental determination of the extinction coefficient of CdTe, CdSe, and CdS nanocrystals. Chem Mater 15:2854–2860

    Article  CAS  Google Scholar 

  • Zhang Y, Wang TH (2012) Quantum dot enabled molecular sensing and diagnostics. Theranostics 2:631–654

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Roger Y. Tsien (University of California, San Diego, CA, USA) for provision of the mCherry plasmid, Goo-Young Kim and Hyangshuk Rhim (The Catholic University of Korea, Seoul, Korea) for the pCS2+ EGFP plasmid, Daniel Goldman (University of Michigan, Ann Arbor, MI, USA) for the pCS2+ DsRed plasmid, Chia-Wei Huang for the construction of the pCS2+ mCherry plasmid, Tze-Bin Chou (National Taiwan University, Taipei, Taiwan) and Core Instrument Center (National Health Research Institutes, Miaoli, Taiwan) for the Leica confocal systems, Institute of Cellular and Systems Medicine (National Health Research Institutes, Miaoli, Taiwan) for the Olympus microscope, and Robert S. Aronstam (Missouri University of Science and Technology, USA) for technical editing. This work was supported by Postdoctoral Fellowship NSC 101-2811-B-259-001 (to B.R.L.) and Grant Number NSC101-2311-B-259-003-MY3 (to H-J.L.) from the National Science Council of Taiwan.

Author information

Authors and Affiliations

  1. Department of Natural Resources and Environmental Studies, National Dong Hwa University, 1, Sec. 2, Da-Hsueh Road, Shoufeng, Hualien, 97401, Taiwan

    Betty Revon Liu, Ji-Sing Liou, Yung-Jen Chen & Han-Jung Lee

  2. Department of Biological Sciences, Missouri University of Science and Technology, Rolla, MO, 65409-1120, USA

    Yue-Wern Huang

Authors
  1. Betty Revon Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ji-Sing Liou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yung-Jen Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yue-Wern Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Han-Jung Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Han-Jung Lee.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 1.72 mb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, B.R., Liou, JS., Chen, YJ. et al. Delivery of Nucleic Acids, Proteins, and Nanoparticles by Arginine-Rich Cell-Penetrating Peptides in Rotifers. Mar Biotechnol 15, 584–595 (2013). https://doi.org/10.1007/s10126-013-9509-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10126-013-9509-0

Keywords

Search

Navigation