Preparation and transfection evaluation of modified multifunctional envelope-type nano device -DNA nanocomplexes based on low molecular weight protamine

Document Type : Research Paper


1 Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran

2 School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

3 Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

4 Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran



Objective(s): Gene therapy is a hopeful approach for treatment of a wide range of life threatening disease from infectious and inherited diseases to cancer. Multifunctional Envelope-type Nano Device (MEND) is a new carrier as non-viral genetic vector. Moreover, associating peptide structures with the nuclear localization signals (NLSs), which contains various functional groups enables them to condense DNA and specifically transfer genetic material to the nucleus.
Materials and Methods: In this study, two forms of low molecular weight protamine (LMWP) were used for preparation of MEND carrier. The MEND carriers were then targeted with GE11 ligand to obtain T-MEND structure. The size distribution of the resulting nanoparticles, as well as their transfection efficiency and cytotoxicity, were investigated on the A549 cell line.
Results: Results demonstrated that the size of polyplex carrier’s formulation by both peptides (VV45 and VV32) was below 200 nm and MEND formulations were between 200-300 nm. T-MEND formulations contained VV32 and VV45 peptides showed slightly higher transfection than similar MEND formulations. Also, MEND formulation showed increased transfection efficiency compared to similar PD complexes. The result of metabolic activity test showed that MEND lipopolyplex did not represent any remarkable cytotoxicity.
Conclusion: It can be concluded that multifunctional carriers designed based on LMWP are considered as the safe carrier for gene delivery. Presence of protamine and targeted ligand in the nanoparticulate structure did not increase the risk of cytotoxicity of carriers. So, MEND and T-MEND lipoplexes showed low cytotoxicity and acceptable transfection efficiency at the level of PEI 25 kDa.


1.    Mak KY, Rajapaksha IG, Angus PW, Herath CB. The Adeno-associated Virus - A Safe and Promising Vehicle for Liverspecific Gene Therapy of Inherited and Non-inherited Disorders. Curr Gene Ther. 2017;17(1):4-16.
2.    Samson A, Bentham MJ, Scott K, Nuovo G, Bloy A, Appleton E, et al. Oncolytic reovirus as a combined antiviral and anti-tumour agent for the treatment of liver cancer. Gut. 2018;67(3):562-573.
3.    Kirschner J, Cathomen T. Gene Therapy for Monogenic Inherited Disorders: Opportunities and Challenges. Dtsch Arztebl Int. 2020;117(51-52): 878–885.
4.    Zhan W, Muhuri M, Tai PWL, Gao G. Vectored Immunotherapeutics for Infectious Diseases: Can rAAVs Be The Game Changers for Fighting Transmissible Pathogens? Front Immunol. 2021;12:673699.
5.    Montaño-Samaniego M, Bravo-Estupiñan DM, Méndez-Guerrero O, Alarcón-Hernández E, Ibáñez-Hernández M. Strategies for Targeting Gene Therapy in Cancer Cells With Tumor-Specific Promoters. Front Oncol. 2020;10:605380.
6.    Ramamoorth M, Narvekar A. Non viral vectors in gene therapy- an overview. J Clin Diagn Res. 2015;9(1):GE01-GE6.
7.    Foldvari M, Chen DW, Nafissi N, Calderon D, Narsineni L, Rafiee A. Non-viral gene therapy: Gains and challenges of non-invasive administration methods. J Control Release. 2016;240:165-190.
8.    O’Reilly M, Jambou R, Rosenthal E, Montgomery M, Hassani M, Gargiulo L, et al. The national institutes of health oversight of human gene transfer research: Enhancing science and safety. Adv Exp Med Biol. 2015:871:31-47.
9.    Gantenbein B, Tang S, Guerrero J, Higuita-Castro N, Salazar-Puerta AI, Croft AS, et al. Non-viral gene delivery methods for bone and joints. Front Bioeng Biotechnol. 2020;8: 598466.
10.    Deng W, Chen W, Clement S, Guller A, Zhao Z, Engel A, et al. Controlled gene and drug release from a liposomal delivery platform triggered by X-ray radiation. Nature Communications. 2018;9(1):2713.
11.    Nakamura T, Akita H, Yamada Y, Hatakeyama H, Harashima H. A Multifunctional envelope-type nanodevice for use in nanomedicine: Concept and applications. Acc Chem Res. 2012;45(7):1113-1121.
12.    Kamiya H, Akita H, Harashima H. Pharmacokinetic and pharmacodynamic considerations in gene therapy. Drug Discov Today. 2003;8(21):990-996.
13.    Kogure K, Moriguchi R, Sasaki K, Ueno M, Futaki S, Harashima H. Development of a non-viral multifunctional envelope-type nano device by a novel lipid film hydration method. J Control Release. 2004;98(2):317-323.
14.    Hatakeyama H, Akita H, Harashima H. The Polyethyleneglycol Dilemma: Advantage and Disadvantage of PEGylation of Liposomes for Systemic Genes and Nucleic Acids Delivery to Tumors. Biol Pharm Bull. 2013;36(6):892-899.
15.    Kogure K, Akita H, Yamada Y, Harashima H. Multifunctional envelope-type nano device (MEND) as a non-viral gene delivery system. Adv Drug Deliv Rev.. 2008;60(4):559-71.
16.    Puras G, Martínez-Navarrete G, Mashal M, Zárate J, Agirre M, Ojeda E, et al. Protamine/DNA/Niosome ternary nonviral vectors for gene delivery to the retina: The role of protamine. Mol Pharm. 2015;12(10):3658-3671.
17.    Motta S, Brocca P, Favero ED, Rondelli V, Cantù L, Amici A, et al. Nanoscale structure of protamine/DNA complexes for gene delivery. App Phys Lett. 2013;102(5):053703.
18.    Kuzmich A, Rakitina O, Didych D, Potapov V, Zinovyeva M, Alekseenko I, et al. Novel histone-based DNA carrier targeting cancer-associated fibroblasts. Polymers. 2020;12(8): 1695.
19.    He H, Ye J, Liu E, Liang Q, Liu Q, Yang VC. Low molecular weight protamine (LMWP): a nontoxic protamine substitute and an effective cell-penetrating peptide. J Control Release. 2014;193:63-73.
20.    Xia H, Gao X, Gu G, Liu Z, Zeng N, Hu Q, et al. Low molecular weight protamine-functionalized nanoparticles for drug delivery to the brain after intranasal administration. Biomaterials. 2011;32(36):9888-9898.
21.    Park YJ, Liang JF, Ko KS, Kim SW, Yang VC. Low molecular weight protamine as an efficient and nontoxic gene carrier: In vitro study. J Gene Med. 2003;5(8):700-711.
22.    Jafari M, Soltani M, Naahidi S, Karunaratne DN, Chen P. Nonviral approach for targeted nucleic acid delivery. Curr Med Chem. 2012;19(2):197-208.
23.    Zarei H, Malaekeh-Nikouei B, Ramezani M, Soltani F. Multifunctional peptides based on low molecular weight protamine (LMWP) in the structure of polyplexes and lipopolyplexes: Design, preparation and gene delivery characterization. Journal of Drug Delivery Science and Technology. 2021;62:102422.
24.    Li Z, Zhao R, Wu X, Sun Y, Yao M, Li J, et al. Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics. FASEB J. 2005;19(14):1978-1985.
25.    Cheng L, Huang FZ, Cheng LF, Zhu YQ, Hu Q, Li L, et al. GE11-modified liposomes for non-small cell lung cancer targeting: Preparation, ex vitro and in vivo evaluation. Int J Nanomedicine. 2014;9:921-935.
26.    Niesner U, Halin C, Lozzi L, Günthert M, Neri P, Wunderli-Allenspach H, et al. Quantitation of the tumor-targeting properties of antibody fragments conjugated to cell-permeating HIV-1 TAT peptides. Bioconjug Chem. 2002;13(4):729-736.
27.    Dos Santos T, Varela J, Lynch I, Salvati A, Dawson KAJPo. Effects of transport inhibitors on the cellular uptake of carboxylated polystyrene nanoparticles in different cell lines. PLoS One. 2011;6(9):e24438.
28.    Song Y, Shi Y, Zhang L, Hu H, Zhang C, Yin M, et al. Oral delivery system for low molecular weight protamine-dextran-poly(lactic-co-glycolic acid) carrying exenatide to overcome the mucus barrier and improve intestinal targeting efficiency. Nanomedicine. 2019;14(8):989-1009.
29.    Welser K, Campbell F, Kudsiova L, Mohammadi A, Dawson N, Hart SL, et al. Gene delivery using ternary lipopolyplexes incorporating branched cationic peptides: The role of peptide sequence and branching. Mol Pharm. 2013;10(1):127-141.
30.    Rejman J, Oberle V, Zuhorn IS, Hoekstra D. Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J. 2004;377(Pt 1):159-169.
31.    Khalil IA, Kogure K, Akita H, Harashima H. Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacological reviews. Pharmacol Rev. 2006;58(1):32-45.
32.    Tang Q, Cao B, Wu H, Cheng G. Cholesterol-peptide hybrids to form liposome-like vesicles for gene delivery. PLoS One. 2013;8(1):e54460.
33.    Nakamura K, Yamashita K, Itoh Y, Yoshino K, Nozawa S, Kasukawa H. Comparative studies of polyethylene glycol-modified liposomes prepared using different PEG-modification methods. Biochim Biophys Acta. 2012;1818(11):2801-2807.
34.    Sabourian P, Yazdani G, Ashraf SS, Frounchi M, Mashayekhan S, Kiani S, et al. Effect of physico-chemical properties of nanoparticles on their intracellular uptake. Int J Mol Sci. 2020;21(21):8019.