In vitro cellular uptake and neuroprotective efficacy of poly-arginine-18 (R18) and poly-ornithine-18 (O18) peptides: critical role of arginine guanidinium head groups for neuroprotection

  • Gabriella MacDougallEmail author
  • Ryan S. Anderton
  • Eden Ouliel
  • Junjie Gao
  • Sharon L. Redmond
  • Neville W. Knuckey
  • Bruno P. Meloni


We have previously demonstrated that Cationic Arginine-Rich Peptides (CARPs) and in particular poly-arginine-18 (R18; 18-mer of arginine) exhibit potent neuroprotective properties in both in vitro and in vivo neuronal injury models. Based on the current literature, there is a consensus that arginine residues by virtue of their positive charge and guanidinium head group is the critical element for imparting CARP neuroprotective properties and their ability to traverse cell membranes. This study examined the importance of guanidinium head groups in R18 for peptide cellular uptake, localization, and neuroprotection. This was achieved by using poly-ornithine-18 (O18; 18-mer of ornithine) as a control, which is structurally identical to R18, but possesses amino head groups rather than guanidino head groups. Epifluorescence and confocal fluorescence microscopy was used to examine the cellular uptake and localization of the FITC-conjugated R18 and O18 in primary rat cortical neurons and SH-SY5Y human neuroblastoma cell cultures. An in vitro cortical neuronal glutamic acid excitotoxicity model was used to compare the effectiveness of R18 and O18 to inhibit cell death and intracellular calcium influx, as well as caspase and calpain activation. Fluorescence imaging studies revealed cellular uptake of both FITC-R18 and FITC-O18 in neuronal and SH-SY5Y cells; however, intracellular localization of the peptides differed in neurons. Following glutamic acid excitotoxicity, only R18 was neuroprotective, prevented caspases and calpain activation, and was more effective at reducing neuronal intracellular calcium influx. Overall, this study demonstrated that for long chain cationic poly-arginine peptides, the guanidinium head groups provided by arginine residues are an essential requirement for neuroprotection but are not required for entry into neurons.


Neuroprotection Poly-arginine Poly-ornithine Excitotoxicity R18 Guanidinium 



Cell-penetrating peptide


Cationic arginine-rich peptide


Hypoxic-ischaemic encephalopathy


Matrix metalloproteinases


‘Trans-activator of transcription’ HIV-1 protein


Traumatic brain injury


Poly-arginine-18 peptide


Poly-ornithine-18 peptide



The authors would like to acknowledge the Pierce-Armstrong Foundation and the Ian Potter Foundation for funding.


This work was supported in part by University Postgraduate Award (UPA) from the University of Notre Dame, Australia.

Compliance with ethical standards

Conflict of interest

B.P. Meloni and N.W. Knuckey are named inventors of several patent applications (Provisional Patents: 2013904197; 30/10/2013 and 2014902319; 17/6/2014 and PCT/AU2014/050326; 30/10/2104) regarding the use of arginine-rich peptides as neuroprotective agents. The other authors declare they have no conflict of interest.

Supplementary material

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Supplementary material 1 (PPTX 52 kb)
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Supplementary material 2 (PPTX 48480 kb)
11010_2019_3646_MOESM3_ESM.pptx (23.7 mb)
Supplementary material 3 (PPTX 24285 kb)


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Authors and Affiliations

  1. 1.Department of NeurosurgerySir Charles Gairdner Hospital, QEII Medical CentreNedlandsAustralia
  2. 2.Perron Institute for Neurological and Translational ScienceNedlandsAustralia
  3. 3.School of Heath Sciences, The University Notre DameFremantleAustralia
  4. 4.Centre for Neuromuscular and Neurological Disorders, The University of Western AustraliaNedlandsAustralia
  5. 5.Centre for Orthopaedic Research, Faculty of Health and Medical Sciences, The University of Western AustraliaNedlandsAustralia
  6. 6.Department of Orthopaedic SurgeryShanghai Jiao Tong University Affiliated Sixth People’s HospitalShanghaiChina
  7. 7.Ear Science Institute AustraliaSubiacoAustralia
  8. 8.Ear Sciences Centre, School of Surgery, The University of Western AustraliaNedlandsAustralia

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