Skip to main content
SpringerLink
Log in

Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection

  • Research Paper
  • Published:
Journal of Nanoparticle Research Aims and scope Submit manuscript

Abstract

Aimed to prepare an enhanced gene delivery system with low cytotoxicity and high transfection efficiency, various cholesterol-conjugated derivates of low generation polyamidoamine (PAMAM) dendrimers were prepared. The conjugates were characterized by TNBS assay, FTIR, and 1H-NMR spectroscopy. Self-assembly of the dendrimer conjugates (G1-Chol, G2-Chol, and G3-Chol) was investigated by pyrene assay. Following formation of the complexes between enhanced green fluorescence protein plasmid and the dendrimer conjugates at various N (primary amine)/P (phosphate) mole ratios, plasmid condensation, biologic stability, cytotoxicity, and protein expression were investigated. The conjugates self-assembled into micellar dispersions with the critical micelle concentration values (<50 µg/ml) depending on the dendrimer generation and cholesterol/amine mole ratio. Cholesterol conjugation resulted in higher resistance of the condensed plasmid DNA in a competition assay with heparin sulfate. Also, the transfection efficiency was determined higher for the cholesterol conjugates than unmodified dendrimers in HepG2 cells, showing the highest for G2-Chol at 40 % degree of cholesterol modification (G2-Chol40 %) among various dendrimer generations. Interestingly, such conjugate showed a complete protection of plasmid against serum nucleases. Our results confirmed that the cholesterol conjugation to PAMAM dendrimers of low generations bearing little cytotoxicity improves their several physicochemical and biological characteristics required for an enhanced delivery of plasmid DNA into cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  • Alshamsan A, Haddadi A, Incani V, Samuel J, Lavasanifar A, Uludag H (2009) Formulation and delivery of siRNA by oleic acid and stearic acid modified polyethylenimine. Mol Pharm 6:121–133

    Article  Google Scholar 

  • Bielinska A, Kukowska-Latallo JF, Johnson J, Tomalia DA, Baker JR (1996) Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers. Nucleic Acids Res 24:2176–2182

    Article  Google Scholar 

  • Bielinska AU, Chen C, Johnson J, Baker JR (1999) DNA complexing with polyamidoamine dendrimers implications for transfection. Bioconjug Chem 10:843–850

    Article  Google Scholar 

  • Biswas S, Torchilin VP (2013) Dendrimers for siRNA Delivery. Pharmaceuticals 6:161–183

    Article  Google Scholar 

  • Biswas S, Deshpande PP, Navarro G, Dodwadkar NS, Torchilin VP (2013) Lipid modified triblock PAMAM-based nanocarriers for siRNA drug co-delivery. Biomaterials 34:1289–1301

    Article  Google Scholar 

  • Charest MC, Rhainds D, Falstrault L, Matzouranis T, Brissett L (1999) Selective uptake of cholesteryl ester from low density lipoprotein is involved in HepG2 cell cholesterol homeostasis. Eur J Biochem 263:402–409

    Article  Google Scholar 

  • Chen W, Turro NJ, Tomalia DA (2000) Using ethidium bromide to probe the interactions between DNA and dendrimers. Langmuir 16:15–19

    Article  Google Scholar 

  • Choi JS, Ko KS, Park JS, Kim YH, Kim SW, Lee M (2006) Dexamethasone conjugated poly(amidoamine) dendrimer as a gene carrier for efficient nuclear translocation. Int J Pharm 320:171–178

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Deniz A, Sade A, Severcan F, Keskin D, Tezcaner A, Banerjee S (2010) Celecoxib-loaded liposomes: effect of cholesterol on encapsulation and in vitro release characteristics. Biosci Rep 30:365–373

    Article  Google Scholar 

  • Dong X, Liu C (2010) Preparation and characterization of self-assembled nanoparticles of hyaluronic acid-deoxycholic acid conjugates. J Nanomater 12:1–9

    Article  Google Scholar 

  • Dung TH, Kim JS, Juliano RL, Yoo H (2008) Preparation and evaluation of cholesteryl PAMAM dendrimers as nano delivery agents for antisense oligonucleotides. Colloids Surf A 313–314:273–277

    Article  Google Scholar 

  • Escoffre J-M, Teissié J, Rols M-P (2010) Gene transfer: how can the biological barriers be overcome? J Membr Biol 236:61–74

    Article  Google Scholar 

  • Fant K, Esbjörner EK, Jenkins A, Grossel MC, Lincoln P, Nordén B (2010) Effects of PEGylation and acetylation of PAMAM dendrimers on DNA binding, cytotoxicity and in vitro transfection efficiency. Mol Pharm 7:1734–1746

    Article  Google Scholar 

  • Fitzsimmons REB, Uludağ H (2012) Specific effects of PEGylation on gene delivery efficacy of polyethylenimine: interplay between PEG substitution and N/P ratio. Acta Biomater 8:3941–3955

    Article  Google Scholar 

  • Freedman RB, Radda GK (1968) The reaction of 2,4,6-trinitrobenzenesulphonic acid with amino acids, peptides and proteins. Biochem J 108:383–391

    Article  Google Scholar 

  • Gao X, Huang L (1991) A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem Biophys Res Commun 179:280–285

    Article  Google Scholar 

  • Gao X, Kim K-S, Liu D (2007) Nonviral gene delivery: what we know and what is next. AAPS J 9:E92–E104

    Article  Google Scholar 

  • Geall AJ, Blagbrough IS (2000) 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 22:849–859

    Article  Google Scholar 

  • Gersdorff KV, Ogris M, Wagner E (2005) Cryoconserved shielded and EGF receptor targeted DNA polyplexes: cellular mechanisms. Eur J Pharm Biopharm 60:279–285

    Article  Google Scholar 

  • Guo S, Huang L (2011) Nanoparticles escaping RES and endosome: challenges for siRNA delivery for cancer therapy. J Nanomater 5:1–12

    Google Scholar 

  • Gusachenko Simonova O, Kravchuk Y, Konevets D, Silnikov V, Vlassov VV, Zenkova MA (2009) Transfection efficiency of 25-kDa PEI-cholesterol conjugates with different levels of modification. J Biomater Sci Polym Ed 20:1091–1110

    Article  Google Scholar 

  • Haensler J, Szoka FC Jr (1993) Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate Chem 4:372–379

    Article  Google Scholar 

  • Haririan I et al (2010) Cellular delivery of nanostructured poly(amido amine) dendrimers and establishment of a simple methodology upon ninhydrin reaction. Iran J Pharm Sci 6:71–82

    Google Scholar 

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

    Google Scholar 

  • Hirsch-Lernera D, Zhanga M, Eliyahua H, Ferrarib ME, Wheelerb CJ, Barenholza Y (2005) Effect of ‘‘helper lipid’’ on lipoplex electrostatics. Biochim Biophys Acta 1714(2):71–84

    Article  Google Scholar 

  • Hu Y et al (2003) Preparation and drug release behaviors of nimodipine-loaded poly(caprolactone)–poly(ethylene oxide)–polylactide amphiphilic copolymer nanoparticles. Biomater 24:2395–2404

    Article  Google Scholar 

  • Jafari M, Xu W, Pan R, Sweeting CM, Karunaratne DN, Chen P (2014) Serum stability and physicochemical characterization of a novel amphipathic peptide C6M1 for SiRNA delivery. PLoS One 9:e97797

    Article  Google Scholar 

  • Javitt NB (1990) Hep G2 cells as a resource for metabolic studies: lipoprotein, cholesterol, and bile acids. Faseb J 4:161–168

    Google Scholar 

  • Jevprasesphant R, Penny J, Jalal R, Attwood D, McKeown NB, D’Emanuele A (2003) The influence of surface modification on the cytotoxicity of PAMAM dendrimers. Int J Pharm 252:263–266

    Article  Google Scholar 

  • Ju J, Huan M-L, Wan N, Qiu H, Zhou S-Y, Zhang B-L (2015) Novel cholesterol-based cationic lipids as transfecting agents of DNA for efficient gene delivery. Int J Mol Sci 16:5666–5681

    Article  Google Scholar 

  • Junquera E, Aicart E (2014) Cationic lipids as transfecting agents of DNA in gene therapy. Curr Top Med Chem 14:649–663

    Article  Google Scholar 

  • Kalyanasundaram K, Thomas J (1977) Environmental effects on vibronic band intensities in pyrene monomer fluorescence and their application in studies of micellar systems. J Am Chem Soc 99:2039–2044

    Article  Google Scholar 

  • Kamimura K, Suda T, Zhang G, Liu D (2011) Advances in gene delivery systems. Pharm Med 25:293–306

    Article  Google Scholar 

  • Kim TH, Yu GS, Choi H, Shim YJ, Lee M, Choi JS (2011) Preparation of dexamethasone-based cationic liposome and its application to gene delivery in vitro. J Nanosci Nanotech 11:1799–1802

    Article  Google Scholar 

  • Kleemann E et al (2005) Nano-carriers for DNA delivery to the lung based upon a TAT-derived peptide covalently coupled to PEG–PEI. J Control Release 109:299–316

    Article  Google Scholar 

  • Kukowska-Latallo JF, Bielinska AU, Johnson J, Spindler R, Tomalia DA, Baker JR (1996) Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers. PNAS 93:4897–4902

    Article  Google Scholar 

  • Labieniec M, Watala C (2009) PAMAM dendrimers—diverse biomedical applications. Facts and unresolved questions. Centeur Biol 4:434–451

    Google Scholar 

  • Lee ER et al (1996) Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung. Hum Gene Ther 7:1701–1717

    Article  Google Scholar 

  • Lee M, Rentz J, Han SO, Bull DA, Kim SW (2003) Water-soluble lipopolymer as an efficient carrier for gene delivery to myocardium. Gene Ther 10:585–593

    Article  Google Scholar 

  • Lepecq JB, Paoletti C (1967) A fluorescent complex between ethidium bromide and nucleic acids: physical—chemical characterization. J Mol Biol 27:87–106

    Article  Google Scholar 

  • Liu C-G, Desai KGH, Chen X-G, Park H-J (2005) Linolenic acid-modified chitosan for formation of self-assembled nanoparticles. J Agric Food Chem 53:437–441

    Article  Google Scholar 

  • Liu C et al (2009) Novel biodegradable lipid nano complex for siRNA delivery significantly improving the chemosensitivity of human colon cancer stem cells to paclitaxel. J Control Release 140:277–283

    Article  Google Scholar 

  • Liu Z, Zhang Z, Zhou C, Jiao Y (2010a) Hydrophobic modifications of cationic polymers for gene delivery. Prog Polym Sci 35:1144–1162

    Article  Google Scholar 

  • Liu XQ, Du JZ, Zhang CP, Zhao F, Yang XZ, Wang J (2010b) Brush-shaped polycation with poly(ethylenimine)-b-poly(ethylene glycol) side chains as highly efficient gene delivery vector. Int J Pharm Sci 392:118–126

    Article  Google Scholar 

  • Mahato RI, Kim SW (2005) Water soluble lipopolymers for gene delivery. Polym Gene Deliv, CRC Press, Boca Raton

  • Mahato RI, Rolland A, Tomlinson E (1997) Cationic lipid-based gene delivery systems: pharmaceutical perspectives. Pharm Res 14:853–859

    Article  Google Scholar 

  • Malik N et al (2000) Dendrimers: relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125I-labelled polyamidoamine dendrimers in vivo. J Control Release 65:133–148

    Article  Google Scholar 

  • Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

    Article  Google Scholar 

  • Mukherjee SP, Byrne HJ (2013) Polyamidoamine dendrimer nanoparticle cytotoxicity, oxidative stress, caspase activation and inflammatory response: experimental observation and numerical simulation. Nanomed 9:202–211

    Google Scholar 

  • Najafi H, Abolmaali S, Owrangi B, Ghasemi Y, Tamaddon A (2015) Serum resistant and enhanced transfection of plasmid DNA by PEG-stabilized polyplex nanoparticles of l-histidine substituted polyethyleneimine. Macromol Res 23:618–627

    Article  Google Scholar 

  • Nie Y, Ji L, Ding H, Xie L, Li L, He B, Wu Y, Gu Z (2012) Cholesterol derivatives based charged liposomes for doxorubicin delivery: preparation, in vitro and in vivo characterization. Theranostics 2:1092–1103

    Article  Google Scholar 

  • Park MR, Han KO, Han IK, Cho MHC, Nah WJ, Choi YJ, Cho CS (2009) Degradable polyethylenimine-alt-poly(ethylene glycol) copolymers as novel gene carriers. J Control Release 105:367–380

    Article  Google Scholar 

  • Perez AP, Romero EL, Morilla MJ (2009) Ethylendiamine core PAMAM dendrimers/siRNA complexes as in vitro silencing agents. Int J Pharm 380:189–200

    Article  Google Scholar 

  • Pozzi D, Marchini C, Cardarelli F, Amenitsch H, Garulli C, Bifone A, Caracciolo G (2012) Transfection efficiency boost of cholesterol-containing lipoplexes. Biochim Biophys Acta 1818:2335–2343

    Article  Google Scholar 

  • Raffy S, Teissie J (1999) Control of lipid membrane stability by cholesterol content. Biophys J 76:2072–2080

    Article  Google Scholar 

  • Remant Bahadur KC, Landry B, Aliabadi HM, Lavasanifar A, Uludağ H (2011) Lipid substitution on low molecular weight (0.6–2.0 kDa) polyethylenimine leads to a higher zeta potential of plasmid DNA and enhances transgene expression. Acta Biomater 7:2209–2217

    Article  Google Scholar 

  • Rezvani Amin Z, Rahimizadeh M, Eshghi H, Dehshahri A, Ramezani M (2013) The effect of cationic charge density change on transfection efficiency of polyethylenimine. Iran J Basic Med Sci 16:150–156

    Google Scholar 

  • Sadekar S, Ghandehari H (2012) Transepithelial transport and toxicity of PAMAM dendrimers: implications for oral drug delivery. Adv Drug Deliv Rev 64:571–588

    Article  Google Scholar 

  • Santos JL, Pandita D, Rodrigues J, Pêgo AP, Granja PL, Balian G, Tomás H (2010) Receptor-mediated gene delivery using PAMAM dendrimers conjugated with peptides recognized by mesenchymal stem cells. Mol Pharm 7:763–774

    Article  Google Scholar 

  • Sara Movassaghian HRM, Shirazi Farshad H, Torchilin Vladimir P (2011) Dendrosome-dendriplex inside liposomes: as a gene delivery system. J Drug Tar 19:925–932

    Article  Google Scholar 

  • Sarkar K, Kundu PP (2012) Preparation of low molecular weight N-maleated chitosan-graft-PAMAM copolymer for enhanced DNA complexation. Int J Biol Macromol 51:859–867

    Article  Google Scholar 

  • Shah DS, Sakthivel T, Toth I, Florence AT, Wilderspin AF (2000) DNA transfection and transfected cell viability using amphipathic asymmetric dendrimers. Int J Pharm 208:41–48

    Article  Google Scholar 

  • Shigematsu M (1996) Concentration dependence of hydrophobicity of monosaccharides estimated by fluorescence of pyrene. Carbohydr Res 292:165–172

    Article  Google Scholar 

  • Shim MS, Kwon YJ (2010) Efficient and targeted delivery of siRNA in vivo. FEBS J 277:4814–4827

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Han S-O, Mahato RI, Kim SW (2001) Water-soluble lipopolymer for gene delivery. Bioconjugate Chem 12:337–345

    Article  Google Scholar 

  • Stephen MS, Avery LM, Huan H, Kerstin KL, Gregory GM, Danilo LH, Ross P, Barbara PA, Ann BK, Friedhelm S (2012) Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes. Am J Physiol Gastrointest Liver Physiol 302:G824–G839

    Article  Google Scholar 

  • Sternberg B, Hong K, Zheng W, Papahadjopoulos D (1998) Ultrastructural characterization of cationic liposome–DNA complexes showing enhanced stability in serum and high transfection activity in vivo. Biochim Biophys Acta 1375(1–2):23–35

    Article  Google Scholar 

  • Swami A, Aggarwal A, Pathak A, Patnaik S, Kumar P, Singh Y, Gupta KC (2007) Imidazolyl-PEI modified nanoparticles for enhanced gene delivery. Int J Pharm 335:180–192

    Article  Google Scholar 

  • Tan Y-L, Liu C-G (2009) Self-aggregated nanoparticles from linoleic acid modified carboxymethyl chitosan: synthesis, characterization and application in vitro. Colloids Surf B 69:178–182

    Article  Google Scholar 

  • Takahashi T, Kono K, Itoh T, Emi N, Takagishi T (2003) Synthesis of novel cationic lipids having polyamidoamine dendrons and their transfection activity. Bioconje Chem 14:764–773

    Article  Google Scholar 

  • Tamaddon A, Hosseini-Shirazi F, Moghimi H (2007) Preparation of oligodeoxynucleotide encapsulated cationic liposomes and release study with models of cellular membranes. DARU J Pharm Sci 15:61–70

    Google Scholar 

  • Tang MX, Redemann CT, Szoka FC (1996) In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjugate Chem 7:703–714

    Article  Google Scholar 

  • Tavakoli S, Tamaddon AM, Golkar N, Samani SM (2014) Microencapsulation of (deoxythymidine) 20-DOTAP complexes in stealth liposomes optimized by Taguchi design. J Liposome Res 25:67–77

    Article  Google Scholar 

  • Templeton N, Lasic D, Frederik P, Strey H, Roberts D, Pavlakis G (1997) Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat Biotechnol 15:647–652

    Article  Google Scholar 

  • Vigneron J-P et al (1996) Guanidinium-cholesterol cationic lipids: efficient vectors for the transfection of eukaryotic cells. PNAS 93:9682–9686

    Article  Google Scholar 

  • Wang DA, Narang AS, Kotb M, Gaber AO, Miller DD, Kim SW, Mahato RI (2002) Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery. Biomacromol 3:1197–1207

    Article  Google Scholar 

  • Wang J, Lu Z, Wientjes MG, Au JLS (2010) Delivery of siRNA Therapeutics: barriers and Carriers. AAPS J 12:492–503

    Article  Google Scholar 

  • Yang Z, Sahay G, Sriadibhatla S, Kabanov AV (2008) Amphiphilic block copolymers enhance cellular uptake and nuclear entry of polyplex-delivered DNA. Bioconje Chem 19:1987–1994

    Article  Google Scholar 

  • Yang Y-L, Chang W-T, Shih Y-W (2011) Gene therapy using RNAi. INTECH open Access Publisher

  • Yoo H, Juliano R (2000) Enhanced delivery of antisense oligonucleotides with fluorophore-conjugated PAMAM dendrimers. Nucleic Acids Res 28:4225–4231

    Article  Google Scholar 

  • Yoo H, Sazani P, Juliano RL (1999) PAMAM dendrimers as delivery agents for antisense oligonucleotides. Pharm Res 16:1799–1804

    Article  Google Scholar 

  • Zhou J, Wu J, Hafdi N, Behr JP, Erbacher P, Peng L (2006) PAMAM dendrimers for efficient siRNA delivery and potent gene silencing. Chem Commun 22:2362–2364

    Article  Google Scholar 

  • Zhu L, Mahato RI (2010) Lipid and polymeric carrier-mediated nucleic acid delivery. Exp Opin Drug Deliv 7:1209–1226

    Article  Google Scholar 

  • Zuhorn I, Visser W, Bakowsky U, Engberts J, Hoekstra D (2002) Interference of serum with lipoplex–cell interaction: modulation of intracellular processing. Biochim Biophys Acta 1560:25–36

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported financially by the grant from Shiraz University of Medical Sciences as a part of Ms. Nasim Golkar Ph.D. thesis. The facility support of “Center for Nanotechnology in Drug Delivery” is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Department of Pharmaceutics, School of Pharmacy, Shiraz University of Medical Sciences, 71345, Shiraz, Iran

    Nasim Golkar, Soliman Mohammadi Samani & Ali Mohammad Tamaddon

  2. Center for Nanotechnology in Drug Delivery, Shiraz University of Medical Sciences, 71345, Shiraz, Iran

    Soliman Mohammadi Samani & Ali Mohammad Tamaddon

Authors
  1. Nasim Golkar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Soliman Mohammadi Samani
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ali Mohammad Tamaddon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ali Mohammad Tamaddon.

Ethics declarations

Conflicts of interest

The authors declare that they have no conflict of interest

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 649 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Golkar, N., Samani, S.M. & Tamaddon, A.M. Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection. J Nanopart Res 18, 107 (2016). https://doi.org/10.1007/s11051-016-3413-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11051-016-3413-2

Keywords

Search

Navigation