Intranasal Drug Delivery into Mouse Nasal Mucosa and Brain Utilizing Arginine-Rich Cell-Penetrating Peptide-Mediated Protein Transduction

  • Toru MiwaEmail author
  • Kyoko Tachii
  • Fan-Yan Wei
  • Taku Kaitsuka
  • Kazuhito Tomizawa


The nasal pathway represents a non-invasive administration route of pharmaceutical agents with local, systemic, and central nervous system action. Intranasal application has a relatively short time to onset of effect and high bioavailability because it avoids hepatic first-pass metabolism. However, sustained delivery is important because drugs can be rapidly eliminated via mucociliary clearance. Protein transduction using arginine-rich cell-penetrating peptides (poly-arginine) shows high delivery efficiency, no cell specificity, and minimal cytotoxicity. We investigated the effect of poly-arginine on protein delivery into the nasal mucosa and brain of mice, and its ability to prolong contact between the delivered molecule and nasal mucosa by preventing mucociliary clearance. Enhanced green fluorescent protein (EGFP) fused to a nine-arginine peptide (EGFP-9R group) or EGFP (EGFP group) was administered once to the bilateral nasal cavities of mice. Histopathological evaluation was conducted for 3–120 h to evaluate side effects, and the number of sneezes was recorded before and after administration. EGFP was detected in cells lining the nasal cavity and their vicinity for 3–96 and 3–24 h in the EGFP-9R and EGFP groups, respectively. EGFP was detected in the brain at 3–96 h in the EGFP-9R group but not in the EGFP group. Nasal symptoms and histopathological assessment revealed no deterioration in either group. These results suggest that protein transduction using poly-arginine can deliver therapeutically relevant molecules for allergic rhinitis, and can be applied for olfactory disturbance and other central nervous system diseases. Further research is necessary to establish therapy protocols using this technique.


Cell-penetrating peptide Poly-arginine Intranasal drug delivery Nose-to-brain drug delivery Allergic rhinitis 



The authors thank members of the Molecular Physiology Department at Kumamoto University for their important contributions to these experiments. This research received no specific grants from funding agencies in the public, commercial, or not-for-profit sectors.

Author Contributions

Toru Miwa – Conceptualization; Investigation; Project administration; Software; Visualization; Writing – original draft; Writing – review & editing. Kyoko Tachii – Data curation; Formal analysis; Investigation. Fan-Yan Wei – Methodology; Resources. Taku Kaitsuka – Methodology; Resources; Validation. Kazuhito Tomizawa – Supervision; Conceptualization. All authors have approved the final version of this manuscript.

Compliance with Ethical Standards

Conflict of interest

Toru Miwa, Kyoko Tachii, Fan-Yan Wei, Taku Kaitsuka, and Kazuhito Tomizawa declare that they have no conflict of interest.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted (Committee on the Use and Care of Animals at Kumamoto University; Number: H28-053). This article does not contain any studies with human participants performed by any of the authors.


  1. Altuntaş E, Yener G, Doğan R, Aksoy F, Şerif Aydın M, Karataş E (2018) Effects of a thermosensitive in situ gel containing mometasone furoate on a rat allergic rhinitis model. Am J Rhinol Allergy 32:132–138CrossRefGoogle Scholar
  2. Baig AM, Khan NA (2014) Novel chemotherapeutic strategies in the management of primary amoebic meningoencephalitis due to Naegleria fowleri. CNS Neurosci Ther 20:289–290CrossRefGoogle Scholar
  3. Bousquet J, Hellings PW, Agache I, Amat F, Annesi-Maesano I, Ansotegui IJ, Anto JM, Bachert C, Bateman ED, Bedbrook A, Bennoor K (2018) Allergic Rhinitis and its Impact on Asthma (ARIA) Phase 4 (2018): change management in allergic rhinitis and asthma multimorbidity using mobile technology. J Allergy Clin Immunol 143:864–879CrossRefGoogle Scholar
  4. Costantino HR, Illum L, Brandt G, Johnson PH, Quay SC (2007) Intranasal delivery: physicochemical and therapeutic aspects. Int J Pharm 337:1–24CrossRefGoogle Scholar
  5. Eguchi A, Meade BR, Chang YC, Fredrickson CT, Willert K, Puri N, Dowdy SF (2009) Efficient siRNA delivery into primary cells by a peptide transduction domain–dsRNA binding domain fusion protein. Nat Biotechnol 27:567–571CrossRefGoogle Scholar
  6. Erdő F, Bors LA, Farkas D, Bajza Á, Gizurarson S (2018) Evaluation of intranasal delivery route of drug administration for brain targeting. Brain Res Bull 143:155–170CrossRefGoogle Scholar
  7. Frankel AD, Pabo CO (1988) Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55:1189–1193CrossRefGoogle Scholar
  8. Fransén N (2008) Studies on a novel powder formulation for nasal drug delivery. Acta Universitatis Upsaliensis, UppsalaGoogle Scholar
  9. Futaki S (2002) Arginine-rich peptides: potential for intracellular delivery of macromolecules and the mystery of the translocation mechanisms. Int J Pharm 245:1–7CrossRefGoogle Scholar
  10. Futaki S (2005) Membrane-permeable arginine-rich peptides and the translocation mechanisms. Adv Drug Deliv Rev 57:547–558CrossRefGoogle Scholar
  11. Green M, Loewenstein PM (1988) Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell 55:1179–1188CrossRefGoogle Scholar
  12. Illum L (2004) Is nose-to-brain transport of drugs in man a reality? J Pharm Pharmacol 56:3–17CrossRefGoogle Scholar
  13. Järver P, Mäger I, Langel Ü (2010) In vivo biodistribution and efficacy of peptide mediated delivery. Trends Pharmacol Sci 31:528–535CrossRefGoogle Scholar
  14. Kamei N (2017) Nose-to-brain delivery of peptide drugs enhanced by coadministration of cell-penetrating peptides: therapeutic potential for dementia. Yakugaku Zasshi 137:1247–1253CrossRefGoogle Scholar
  15. Kandimalla KK, Donovan MD (2005) Transport of hydroxyzine and triprolidine across bovine olfactory mucosa: role of passive diffusion in the direct nose-to-brain uptake of small molecules. Int J Pharm 302:133–144CrossRefGoogle Scholar
  16. Kemp BE, Cheng HC, Walsh DA (1988) Peptide inhibitors of cAMP-dependent protein kinase. Methods Enzymol 159:173–183CrossRefGoogle Scholar
  17. Laakkonen P, Åkerman ME, Biliran H, Yang M, Ferrer F, Karpanen T, Hoffman RM, Ruoslahti E (2004) Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells. Proc Natl Acad Sci USA 101:9381–9386CrossRefGoogle Scholar
  18. Lindsay MA (2002) Peptide-mediated cell delivery: application in protein target validation. Curr Opin Pharmacol 2:587–594CrossRefGoogle Scholar
  19. Lungare S, Bowen J, Badhan R (2016) Development and evaluation of a novel intranasal spray for the delivery of amantadine. J Pharm Sci 105:1209–1220CrossRefGoogle Scholar
  20. Matsushita M, Tomizawa K, Moriwaki A, Li ST, Terada H, Matsui H (2001) A high-efficiency protein transduction system demonstrating the role of PKA in long-lasting long-term potentiation. J Neurosci 21:6000–6007CrossRefGoogle Scholar
  21. Miwa T, Minoda R, Kaitsuka T, Ise M, Tomizawa K, Yumoto E (2011) Protein transduction into the mouse otocyst using arginine-rich cell-penetrating peptides. NeuroReport 22:994–999CrossRefGoogle Scholar
  22. Monteillier A, Voisin A, Furrer P, Allémann E, Cuendet M (2018) Intranasal administration of resveratrol successfully prevents lung cancer in A/J mice. Sci Rep 8:14257CrossRefGoogle Scholar
  23. Nagahara H, Vocero-Akbani AM, Snyder EL, Ho A, Latham DG, Lissy NA, Becker-Hapak M, Ezhevsky SA, Dowdy SF (1998) Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat Med 4:1449–1452CrossRefGoogle Scholar
  24. Okubo K, Kurono Y, Ichimura K, Enomoto T, Okamoto Y, Kawauchi H, Suzaki H, Fujieda S, Masuyama K (2017) Japanese guidelines for allergic rhinitis 2017. Allergol Int 66:205–219CrossRefGoogle Scholar
  25. Pardeshi CV, Belgamwar VS (2013) Direct nose to brain drug delivery via integrated nerve pathways bypassing the blood–brain barrier: an excellent platform for brain targeting. Expert Opin Drug Deliv 10:957–972CrossRefGoogle Scholar
  26. Pardeshi CV, Rajput PV, Belgamwar VS, Tekade AR, Surana SJ (2013) Novel surface modified solid lipid nanoparticles as intranasal carriers for ropinirole hydrochloride: application of factorial design approach. Drug Deliv 20:47–56CrossRefGoogle Scholar
  27. Rothbard JB, Garlington S, Lin Q, Kirschberg T, Kreider E, McGrane PL, Wender PA, Khavari PA (2000) Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation. Nat Med 6(2000):1253–1257CrossRefGoogle Scholar
  28. Schmidt N, Mishra A, Lai GH, Wong GCL (2010) Arginine-rich cell-penetrating peptides. FEBS Lett 584:1806–1813CrossRefGoogle Scholar
  29. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF (1999) In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285:1569–1572CrossRefGoogle Scholar
  30. Sousa J, Alves G, Fortuna A, Falcão A (2017) Intranasal delivery of topically-acting levofloxacin to rats: a proof-of-concept pharmacokinetic study. Pharm Res 34:2260–2269CrossRefGoogle Scholar
  31. Takeda H, Kurioka T, Kaitsuka T, Tomizawa K, Matsunobu T, Hakim F, Mizutari K, Miwa T, Yamada T, Ise M, Shiotani A (2016) Protein transduction therapy into cochleae via the round window niche in guinea pigs. Mol Ther Methods Clin Dev 3:16055CrossRefGoogle Scholar
  32. Takenobu T, Tomizawa K, Matsushita M, Li ST, Moriwaki A, Lu YF, Matsui H (2002) Development of p53 protein transduction therapy using membrane-permeable peptides and the application to oral cancer cells. Mol Cancer Ther 1:1043–1049PubMedGoogle Scholar
  33. Touitou E, Illum L (2013) Nasal drug delivery. Drug Deliv Transl Res 3:1–3CrossRefGoogle Scholar
  34. Warnken ZN, Smyth HDC, Watts AB, Weitman S, Kuhn JG, Williams RO (2016) Formulation and device design to increase nose to brain drug delivery. J Drug Deliv Sci Technol 35:213–222CrossRefGoogle Scholar
  35. Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB (2000) The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc Natl Acad Sci USA 97:13003–13008CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Otolaryngology and Head and Neck Surgery, Graduate of School of MedicineKumamoto UniversityKumamotoJapan
  2. 2.Department of Otolaryngology-Head and Neck SurgeryJCHO Kumamoto General HospitalKumamotoJapan
  3. 3.Department of Molecular Physiology, Faculty of Life SciencesKumamoto UniversityKumamotoJapan

Personalised recommendations