Alkyl cross-linked low molecular weight polypropyleneimine dendrimers as efficient gene delivery vectors

Document Type: Original Article

Authors

1 Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

2 Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

Abstract

Objective(s): First, 10-bromodecanoic acid was covalently attached to all available surface primary amines of PPI G2 and G3 to increase their lipophilicity. In the subsequent step, PPIs were conjugated to the alkylcarboxylate groups of alkylcarboxylate-PPI derivatives to increase the number of surface primary amines. Physicochemical properties of modified PPIs were determined. Transfection experiments (using both luciferase and green fluorescent protein (GFP)- expressing plasmids) and cytotoxicity assay were performed to evaluate the efficiency of the final derivatives.
Materials and Methods: First, 10-bromodecanoic acid was covalently attached to all available surface primary amines of PPI G2 and G3 to increase their lipophilicity. In the subsequent step, PPIs were conjugated to the alkylcarboxylate groups of alkylcarboxylate-PPI derivatives to increase the number of surface primary amines. Physicochemical properties of modified PPIs were determined. Transfection experiments (using both luciferase and green fluorescent protein (GFP)- expressing plasmids) and cytotoxicity assay were performed to evaluate the efficiency of the final derivatives.
Results: Fabricated vectors condensed DNA effectively so that polyplexes with appropriate size (below 155 nm) and positive surface charge were constructed. Cross-linked low molecular weight PPIs (G2 or G3) with decanoate linkage increased transfection efficiency significantly while maintaining the low cytotoxicity. PPI G2 derivative exhibited increased buffering capacity which is believed to be responsible for better proton sponge mechanism leading to higher transfection efficiency.
Conclusion: Our results indicated that oligomerization of low molecular weight PPI (PPI G2-alkyl-PPI G2 conjugate) could be an approach to increase the transfection efficiency and to lower the cytotoxicity of low molecular weight polycations.

Keywords


1. Mintzer MA, Simanek EE. Nonviral vectors for gene delivery. Chem Rev 2009; 109:259-302.

2. Parker AL, Newman C, Briggs S, Seymour L, Sheridan PJ. Nonviral gene delivery: techniques and implications for molecular medicine. Expert Rev Mol Med 2003; 5:1-15.

3. Dufes C, Uchegbu IF, Schatzlein AG. Dendrimers in gene delivery. Adv Drug Deliv Rev 2005; 57:2177-2202.

4. Parekh HS. The advance of dendrimers--a versatile targeting platform for gene/drug delivery. Curr Pharm Des 2007; 13:2837-2850.

5. Kunath K, von Harpe A, Fischer D, Petersen H, Bickel U, Voigt K, et al. Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine. J Control Release 2003; 89:113-125.

6. Shah N, Steptoe RJ, Parekh HS. Low-generation asymmetric dendrimers exhibit minimal toxicity and effectively complex DNA. J Pept Sci 2011; 17:470-478.

7. Park MR, Kim HW, Hwang CS, Han KO, Choi YJ, Song SC, et al. Highly efficient gene transfer with degradable poly(ester amine) based on poly(ethylene glycol) diacrylate and polyethylenimine in vitro and in vivo. J Gene Med 2008; 10:198-207.

8. Yu JH, Quan JS, Huang J, Nah JW, Cho CS. Degradable poly(amino ester) based on poly(ethylene glycol) dimethacrylate and polyethylenimine as a gene carrier: molecular weight of PEI affects transfection efficiency. J Mater Sci Mater Med 2009; 20:2501-2510.

9.   Bauhuber S, Liebl R, Tomasetti L, Rachel R, Goepferich A, Breunig M. A library of strictly linear poly(ethylene glycol)-poly(ethylene imine) diblock copolymers to perform structure-function relationship of non-viral gene carriers. J Control Release 2012; 162:446-455.

10. Zhang G, Liu J, Yang Q, Zhuo R, Jiang X. Disulfide-containing brushed polyethylenimine derivative synthesized by click chemistry for nonviral gene delivery. Bioconjug Chem 2012; 23:1290-1299.

11. Kim YH, Park JH, Lee M, Kim YH, Park TG, Kim SW. Polyethylenimine with acid-labile linkages as a biodegradable gene carrier. J Control Release 2005; 103:209-219.

12. Jiang D, Salem AK. Optimized dextran-polyethylenimine conjugates are efficient non-viral vectors with reduced cytotoxicity when used in serum containing environments. Int J Pharm 2012; 427:71-79.

13. Ahn CH, Chae SY, Bae YH, Kim SW. Biodegradable poly(ethylenimine) for plasmid DNA delivery. J Control Release 2002; 80:273-282.

14. Xu S, Chen M, Yao Y, Zhang Z, Jin T, Huang Y, et al. Novel poly(ethylene imine) biscarbamate conjugate as an efficient and nontoxic gene delivery system. J Control Release 2008; 130:64-68.

15. Wang YQ, Su J, Wu F, Lu P, Yuan LF, Yuan WE, et al. Biscarbamate cross-linked polyethylenimine derivative with low molecular weight, low cytotoxicity, and high efficiency for gene delivery. Int J Nanomedicine 2012; 7:693-704.

16. Liu H, Wang H, Yang W, Cheng Y. Disulfide cross-linked low generation dendrimers with high gene transfection efficacy, low cytotoxicity, and low cost. J Am Chem Soc 2012; 134:17680-17687.

17. Asasutjarit R, Theerachayanan T, Kewsuwan P, Veeranodha S, Fuongfuchat A, Ritthidej GC. Development and evaluation of diclofenac sodium loaded-N-Trimethyl chitosan nanoparticles for ophthalmic use. Aaps Pharmscitech 2015; 16:1013-1024.

18. Hashemi M, Sahraie Fard H, Amel Farzad S, Parhiz H, Ramezani M. Gene transfer enhancement by alkylcarboxylation of poly (propylenimine). Nanomedicine J 2013; 1:55-62.

19. Snyder SL, Sobocinski PZ. An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines. Anal Biochem 1975; 64:284-288.

20. Geall AJ, Blagbrough IS. Rapid and sensitive ethidium bromide fluorescence quenching assay of polyamine conjugate-DNA interactions for the analysis of lipoplex formation in gene therapy. J pharm biomed anal 2000; 22:849-859.

21. Nanjwade BK, Bechra HM, Derkar GK, Manvi FV, Nanjwade VK. Dendrimers: emerging polymers for drug-delivery systems. Eur J Pharm Sci 2009; 38:185-196.

22. Tomalia DA, Baker H, Dewald J, Hall M, Kallos G, Martin S, et al. A new class of polymers: starburst-dendritic macromolecules. Polym J 1985; 17:117-132.

23. Stasko NA, Johnson CB, Schoenfisch MH, Johnson TA, Holmuhamedov EL. Cytotoxicity of polypropylenimine dendrimer conjugates on cultured endothelial cells. Biomacromolecules 2007; 8:3853-3859.

24. Cho C-S. Design and development of degradable Polyethylenimines for delivery of DNA and small interfering RNA: An updated review. ISRN Materials Science 2012; 2012.

25. Thomas M, Ge Q, Lu JJ, Chen J, Klibanov AM. Cross-linked small polyethylenimines: while still nontoxic, deliver DNA efficiently to mammalian cells in vitro and in vivo. Pharm Res  2005; 22:373-380.

26. Lee GJ, Ryu K, Kim K, Choi JY, Kim TI. Crosslinked polypropylenimine dendrimers with bioreducible linkages for gene delivery systems and their reductive degradation behaviors. Macromol Biosci 2015; 15:1595-1604.

27. Forrest ML, Meister GE, Koerber JT, Pack DW. Partial acetylation of polyethylenimine enhances in vitro gene delivery. Pharm Res 2004; 21:365-371.

28. Gabrielson NP, Pack DW. Acetylation of polyethylenimine enhances gene delivery via weakened polymer/DNA interactions. Biomacromolecules 2006; 7:2427-2435.

29. Zonghua Liu ZZ, Changren Zhou,Yanpeng Jiao. Hydrophobic modifications of cationic polymers for gene delivery. Prog Polym Sci 2010;35:1144-1162.

30. Dehshahri A, Oskuee RK, Shier WT, Hatefi A, Ramezani M. Gene transfer efficiency of high primary amine content, hydrophobic, alkyl-oligoamine derivatives of polyethylenimine. Biomaterials 2009; 30:4187-4194.

31. Ganjalikhani hakemi M, Hashemi M. siRNA delivery improvement by co-formulation of different modified polymers in erythroleukemic cell line K562. Iran J Basic Med Sci 2013;16:973-978.

32. Hashemi M, Parhiz H, Mokhtarzadeh A, Tabatabai SM, Farzad SA, Shirvan HR, et al. Preparation of effective and safe gene carriers by grafting alkyl chains to generation 5 polypropyleneimine. Aaps Pharmscitech 2015; 16:1002-1012.

33. 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; 377:159-169.

34. Dos Santos T, Varela J, Lynch I, Salvati A, Dawson KA. Effects of transport inhibitors on the cellular uptake of carboxylated polystyrene nanoparticles in different cell lines. PloS one 2011; 6:e24438.

35. Oh JM, Choi SJ, Lee GE, Kim JE, Choy JH. Inorganic metal hydroxide nanoparticles for targeted cellular uptake through clathrin-mediated endocytosis. Chem Asian J 2009; 4:67-73.

36. Xu Q, Wang CH, Pack DW. Polymeric carriers for gene delivery: chitosan and poly(amidoamine) dendrimers. Curr Pharm Des 2010; 16:2350-2368.

37. Midoux P, Pichon C, Yaouanc JJ, Jaffres PA. Chemical vectors for gene delivery: a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers. Br J Pharmacol 2009; 157:166-178.

38. Salmasi Z, Shier WT, Hashemi M, Mahdipour E, Parhiz H, Abnous K, et al. Heterocyclic amine-modified polyethylenimine as gene carriers for transfection of mammalian cells. Eur J Pharm Biopharm 2015; 96:76-88.

39. 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.

40. Sonawane ND, Szoka FC, Jr, Verkman AS. Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J Biol Chem 2003; 278:44826-44831.

41. Singh B, Maharjan S, Park TE, Jiang T, Kang SK, Choi YJ, et al. Tuning the buffering capacity of polyethylenimine with glycerol molecules for efficient gene delivery: staying in or out of the endosomes. Macromol Biosci 2015; 15:622-635.