Brevinin-2R-linked polyethylenimine as a promising hybrid nano-gene-delivery vector

Document Type : Original Article


1 Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

2 Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

3 Nuclear Medicine Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

4 Targeted Drug Delivery Research Center, Institute of Pharmaceutical Technology, Mashhad University of Medical Sciences, Mashhad, Iran


Objective(s): Polyethylenimine (PEI) is one of the most widely used polymers in gene delivery. The aim of this study was to modify PEI by replacing some of its primary amines with Brevinin 2R (BR-2R) peptide in order to increase the efficiency of gene delivery.
Materials and Methods: Polyethylenimine was modified by BR-2R peptide by two different approaches; A) conjugation methods including (І) using succinimidyl 3-(2-pyridyldithio) propionate (SPDP), (П) EDC/NHS protocol and (ПІ) EDC/NHS+6-bromohexanoic acid protocol, and B) physical interaction method. The modified polymers were characterized for their ability of plasmid condensation, number of primary amines, size and zeta potential. The transfection efficiency and cytotoxicity were evaluated on HEK293, L929, WEHI164 and Neuro2A cell lines by green fluorescent protein (GFP)-based plasmid (pGFP) reporter gene and viability assays, respectively. Apoptosis induction ability was also evaluated via PI/Annexin V assay.
Results: Polyplex had size and zeta potential between 200-270 nm and +21.5- +28.4 mV, respectively. All vectors were able to condense plasmid DNA in C/P=4 (carrier-plasmid ratio). Transfection results on the Neuro2A cell line showed that the vector containing the BR-2R peptide, which was synthesized using EDC-NHS protocol had the best transfection efficiency.
Conclusion: Our results showed that conjugation of Brevinin 2R as cell penetrating peptide to polyethyleneimine could enhance the transfection ability of the polymer.


Main Subjects

1. Nouri N, Talebi M, Palizban AA. Viral and nonviral delivery systems for gene delivery. Adv Biomed Res 2012; 1:1-11.
2. Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR, Anderson DG. Non-viral vectors for gene-based therapy. Nat Rev Genet 2014; 15:541-555.
3. Männistö M, Reinisalo M, Ruponen M, Honkakoski P, Tammi M, Urtti A. Polyplex-mediated gene transfer and cell cycle: effect of carrier on cellular uptake and intracellular kinetics, and significance of glycosaminoglycans. J Gene Med 2007; 9:479-487.
4. Zuhorn IS, Kalicharan R, Hoekstra D. Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis. J Biol Chem 2002; 277:18021-18028.
5. Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A 1995; 92:7297-7301.
6. Patnaik S, Gupta KC. Novel polyethylenimine-derived nanoparticles for in vivo gene delivery. Expert Opin Drug Deliv 2013; 10:215-228.
7. Akinc A, Thomas M, Klibanov AM, Langer R. Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis. J Gene Med 2005; 7:657-663.
8. Pack DW, Hoffman AS, Pun S, Stayton PS. Design and development of polymers for gene delivery. Nat Rev Drug Discov 2005; 4:581-593.
9. Parhamifar L, Larsen AK, Christy Hunter A. Andresen Thomas L, Moein Moghimi S. Polycation cytotoxicity: a delicate matter for nucleic acid therapy—focus on polyethylenimine. Soft Matter 2010; 61:4001-4009.
10. Taranejoo S, Liu J, Verma P, Hourigan K. A review of the developments of characteristics of PEI derivatives for gene delivery applications. J Appl Polym Sci 2015; 132:1-8.
11. Venkiteswaran S, Thomas T, Thomas TJ. Selectivity of polyethyleneimines on DNA nanoparticle preparation and gene transport. Chemistry Select 2016; 1:1144-1150.
12. Bronich T, Kabanov AV, Marky LA. A thermodynamic characterization of the interaction of a cationic copolymer with DNA J Phys Chem 2001; 105:6042-6050.
13. Rezvani Amin Z, Rahimizadeh M, Eshghi H, Dehshahri A, Ramezani M. The effect of cationic charge density change on transfection efficiency of polyethylenimine. Iran J Basic Med Sci 2013; 16:150-156.
14. Farkhani SM, Valizadeh A, Karami H, Mohammadi S, Sohrabi N, Badrzadeh F. Cell penetrating peptides: efficient vectors for delivery of nanoparticles, nanocarriers, therapeutic and diagnostic molecules. Peptides 2014; 57:78-94.
15. Koren E, Torchilin VP. Cell-penetrating peptides: breaking through to the other side. Trends Mol Med 2012; 18:385-393.
16. Elouahabi A, Ruysschaert JM. Formation and intracellular trafficking of lipoplexes and polyplexes. Mol Ther 2005; 11:336-347.
17. Zhang D, Wang J, Xu D. Cell-penetrating peptides as noninvasive transmembrane vectors for the development of novel multifunctional drug-delivery systems. J Controlled Release 2016; 229:130-139.
18. Conlon JM, Kolodziejek J, Nowotny N, Leprince J, Vaudry H, Coquet L, et al. Cytolytic peptides belonging to the brevinin-1 and brevinin-2 families isolated from the skin of the Japanese brown frog, Rana dybowskii. Toxicon 2007; 50:746-756.
19. Asoodeh A, Mashreghi M, Rezazade Bazaz M, Darroudi M, Kazemi Oskuee R. Antioxidant properties of brevinin-2R peptide conjugated with cerium oxide nanoparticle. Sjimu 2016; 23:142-151.
20. Ghavami S, Asoodeh A, Klonisch T, Halayko AJ, Kadkhoda K, Kroczak TJ, et al. Brevinin‐2R1 semi‐selectively kills cancer cells by a distinct mechanism, which involves the lysosomal‐mitochondrial death pathway. J Cell Mol Med 2008; 12:1005-1022.
21. Zohrab F, Askarian S, Jalili A, Kazemi Oskuee R. Biological properties, current applications and potential therapeautic applications of brevinin peptide superfamily. Int J Pept Res Ther 2018; 1:1-10.
22. Jacoby D, Fraefel C, Breakefield X. Hybrid vectors: a new generation of virus-based vectors designed to control the cellular fate of delivered genes. Gene Ther. 1997; 4:1281-1283
23. Huang S, Kamihira M. Development of hybrid viral vectors for gene therapy. Biotechnol Adv. 2013; 31:208-223.
24. Schmidt-Wolf GD, Schmidt-Wolf IG. Non-viral and hybrid vectors in human gene therapy: an update. Trends Mol Med 2003; 9:67-72.
25. Rajagopal P, Duraiswamy S, Sethuraman S, Giridhara Rao J, Krishnan UM. Polymer‐coated viral vectors: hybrid nanosystems for gene therapy. J Gene Med 2018; 20:e3011.
26. Meyer M, Philipp A, Kazemi Oskuee R, Schmidt C, Wagner E. Breathing life into polycations: functionalization with pH-responsive endosomolytic peptides and polyethylene glycol enables siRNA delivery. J Am Chem Soc 2008; 130:3272-3273.
27. Betancourt T, Byrne JD, Sunaryo N, Crowder SW, Kadapakkam M, Patel S, et al. Brannon-Peppas, PEGylation strategies for active targeting of PLA/PLGA nanoparticles. J Biomed Mater Res A 2009; 91:263-276.
28. Hashemi M, Parhiz B, Hatefi A, Ramezani M. Modified polyethyleneimine with histidine–lysine short peptides as gene carrier. Cancer Gene Ther 2011; 18:12.
29. Mah C, Byrne BJ, Flotte TR. Virus-based gene delivery systems. Clin Pharmacokinet 2002; 41:901-911.
30. Wu P, Chen H, Jin R, Weng T, Ho JK, You C, et al. Non-viral gene delivery systems for tissue repair and regeneration. J Transl Med 2018; 16:29.
31. Alsaggar M, Liu D. Physical methods for gene transfer. Adv Genet 2015; 89:1-24.
32. Lindgren M, Hällbrink M, Prochiantz A, Langel Ü. Cell-penetrating peptides. Trends Pharmacol Sci 2000; 21:99-103.
33. Lungwitz U, Breunig M, Blunk T, Göpferich A. Polyethylenimine-based non-viral gene delivery systems. Eur J Pharm Biopharm 2005; 60:247-266.
34. El-Sayed A, Futaki S, Harashima H. Delivery of macromolecules using arginine-rich cell-penetrating peptides: ways to overcome endosomal entrapment. The Aaps J 2009; 11:13-22.
35. Fischer D, Bieber T, Li Y, Elsässer HP, Kissel T. A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity. Pharm Res 1999; 16:1273-1279.
36. Florea BI, Meaney C. Junginger HE, Borchard G. Transfection efficiency and toxicity of polyethylenimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures. Aaps Pharmsci 2002; 4:1-11.
37. Gholami L, Sadeghnia HR, Darroudi M, Kazemi Oskuee R. Evaluation of genotoxicity and cytotoxicity induced by different molecular weights of polyethylenimine/DNA nanoparticles. Turk J Biol 2014; 38:380-387.
38. Moghimi SM, Symonds P, Murray JC, Hunter AC, Debska G, Szewczyk A. A two-stage poly (ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. Mol Ther 2005; 11:990-995.
39. Putnam D, Gentry CA, Pack DW, Langer R. Polymer-based gene delivery with low cytotoxicity by a unique balance of side-chain termini. Proc Natl Acad Sci U S A 2001; 98:1200-1205.
40. Wightman L, Kircheis R, Rössler V, Carotta S, Ruzicka R, Kursa M, Wagner E. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J Gene Med 2001; 3:362-372.
41. Dai Z, Gjetting T, Mattebjerg MA, Wu C, Andresen TL. Elucidating the interplay between DNA-condensing and free polycations in gene transfection through a mechanistic study of linear and branched PEI. Biomaterials 2011; 32:8626-8634.
42. Mao Z, Zhou X, Gao C. Influence of structure and properties of colloidal biomaterials on cellular uptake and cell functions. Biomater. Sci 2013; 1:896-911.
43. Gestin M, Dowaidar M, Langel Ü. Uptake mechanism of cell-penetrating peptides. Adv Exp Med Biol. 2017; 1030:255-264.