Invading target cells: multifunctional polymer conjugates as therapeutic nucleic acid carriers

Abstract

Polymer-based conjugates are an interesting option and challenge for the design of nano-sized drug-delivery systems, as they require advanced conjugation chemistry and precise engineering. In the case of nucleic acid therapy, non-viral carriers face several biological barriers during the delivery process, namely 1) protection of the cargo from extracellular degradation, 2) avoidance of non-specific interactions with non-targeted tissues, 3) efficient entry into the target cells, 4) intracellular trafficking to the site of action and 5) cargo release. To take on these obstacles, multifunctional conjugates can act as “smart polymers” with microenvironment-sensing dynamics to facilitate the separate delivery steps. Synthesis of defined polymer architectures with precise functionalization enables structure-activity relationships to be investigated and the integration of key functions for efficient delivery. Thus bioresponsive polymer conjugates, which are equipped with molecular devices responding to the certain microenvironments within the delivery pathway (e.g. pH, redox potential, enzymes) can be assembled. This review focuses on the modular engineering and conjugation of multifunctional polymeric structures for the utilization as “tailor-made” nucleic acid carriers.

Key words： conjugate; DNA; gene transfer; polymer; RNA; targeting

Acknowledgment

We thank our secretary Mrs. Olga Brück (LMU Munich) for her skillful assistance in preparing the manuscript. Our work related to the topic of this review is supported by the DFG excellence cluster Nanosystems Initiative Munich (NIM), a grant by Roche Kulmbach, and the Munich Biotech Cluster m4 T12.

References

1 Famulok M, Ackermann D. RNA nanotechnology: inspired by DNA. Nature Nanotechnology, 2010, 5(9): 634–635

2 Guo P. The emerging field of RNA nanotechnology. Nature Nanotechnology, 2010, 5(12): 833–842

3 Shir A, Ogris M, Wagner E, Levitzki A. EGF receptor-targeted synthetic double-stranded RNA eliminates glioblastoma, breast cancer, and adenocarcinoma tumors in mice. PLoS Medicine, 2006, 3(1): e6

4 Schaffert D, Kiss M, Rödl W, Shir A, Levitzki A, Ogris M, Wagner E. Poly(I:C)-mediated tumor growth suppression in EGF-receptor overexpressing tumors using EGF-polyethylene glycol-linear polyethylenimine as carrier. Pharmaceutical Research, 2011, 28(4): 731–741

5 Shir A, Ogris M, Roedl W, Wagner E, Levitzki A. EGFR-homing dsRNA activates cancer-targeted immune response and eliminates disseminated EGFR-overexpressing tumors in mice. Clinical Cancer Research, 2011, 17(5): 1033–1043

6 Venkataraman S, Dirks R M, Ueda C T, Pierce N A. Selective cell death mediated by small conditional RNAs. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(39): 16777–16782

7 Ulrich H, Trujillo C A, Nery A A, Alves J M, Majumder P, Resende R R, Martins A H. DNA and RNA aptamers: from tools for basic research towards therapeutic applications. Combinatorial Chemistry & High Throughput Screening, 2006, 9(8): 619–632

8 Gutsaeva D R, Parkerson J B, Yerigenahally S D, Kurz J C, Schaub R G, Ikuta T, Head C A. Inhibition of cell adhesion by anti-P-selectin aptamer: a new potential therapeutic agent for sickle cell disease. Blood, 2011, 117(2): 727–735

9 Sassanfar M, Szostak J W. An RNA motif that binds ATP. Nature, 1993, 364(6437): 550–553

10 Ellington A D, Szostak J W. In vitro selection of RNA molecules that bind specific ligands. Nature, 1990, 346(6287): 818–822

11 Maasch C, Vater A, Buchner K, Purschke W G, Eulberg D, Vonhoff S, Klussmann S. Polyetheylenimine-polyplexes of Spiegelmer NOX-A50 directed against intracellular high mobility group protein A1 (HMGA1) reduce tumor growth in vivo. The Journal of Biological Chemistry, 2010, 285(51): 40012–40018

12 Dua P, Kim S, Lee D K. Nucleic acid aptamers targeting cell-surface proteins. Methods (San Diego, Calif.), 2011

13 Keefe A D, Pai S, Ellington A. Aptamers as therapeutics. Nature Reviews. Drug Discovery, 2010, 9(7): 537–550

14 Li S D, Huang L. Non-viral is superior to viral gene delivery. Journal of Controlled Release: Official Journal of the Controlled Release Society, 2007, 123(3): 181–183

15 Tamura A, Nagasaki Y. Smart siRNA delivery systems based on polymeric nanoassemblies and nanoparticles. Nanomedicine (Lond), 2010, 5(7): 1089–1102

16 Mansouri S, Lavigne P, Corsi K, Benderdour M, Beaumont E, Fernandes J C. Chitosan-DNA nanoparticles as non-viral vectors in gene therapy: strategies to improve transfection efficacy. European Journal of Pharmaceutics and Biopharmaceutics: Official Journal of Arbeitsgemeinschaft für Pharmazeutische Verfahrenstechnik e.V, 2004, 57(1): 1–8

17 Behr J P. The proton sponge: A trick to enter cells the viruses did not exploit. Chimia, 1997, 51(1–2): 34–36

18 Kichler A, Leborgne C, Coeytaux E, Danos O. Polyethylenimine-mediated gene delivery: a mechanistic study. The Journal of Gene Medicine, 2001, 3(2): 135–144

19 von Harpe A, Petersen H, Li Y, Kissel T. Characterization of commercially available and synthesized polyethylenimines for gene delivery. Journal of Controlled Release : Official Journal of the Controlled Release Society, 2000, 69(2): 309–322

20 Werth S, Urban-Klein B, Dai L, Höbel S, Grzelinski M, Bakowsky U, Czubayko F, Aigner A. A low molecular weight fraction of polyethylenimine (PEI) displays increased transfection efficiency of DNA and siRNA in fresh or lyophilized complexes. Journal of Controlled Release: Official Journal of the Controlled Release Society, 2006, 112(2): 257–270

21 Cherng J Y, van de Wetering P, Talsma H, Crommelin D J A, Hennink W E. Effect of size and serum proteins on transfection efficiency of poly ((2-dimethylamino)ethyl methacrylate)-plasmid nanoparticles. Pharmaceutical Research, 1996, 13(7): 1038–1042

22 van de Wetering P, Cherng J Y, Talsma H, Hennink W E. Relation between transfection efficiency and cytotoxicity of poly(2-(dimethylamino)ethyl methacrylate)/plasmid complexes. Journal of Controlled Release, 1997, 49(1): 59–69

23 van de Wetering P, Cherng J Y, Talsma H, Crommelin D J, Hennink W E. 2-(Dimethylamino)ethyl methacrylate based (co)polymers as gene transfer agents. Journal of Controlled Release: Official Journal of the Controlled Release Society, 1998, 53(1–3): 145–153

24 Xu F J, Li H, Li J, Zhang Z, Kang E T, Neoh K G. Pentablock copolymers of poly(ethylene glycol), poly((2-dimethyl amino)ethyl methacrylate) and poly(2-hydroxyethyl methacrylate) from consecutive atom transfer radical polymerizations for non-viral gene delivery. Biomaterials, 2008, 29(20): 3023–3033

25 Krämer M, Stumbé J F, Grimm G, Kaufmann B, Krüger U, Weber M, Haag R. Dendritic polyamines: simple access to new materials with defined treelike structures for application in nonviral gene delivery. Chembiochem : a European Journal of Chemical Biology, 2004, 5(8): 1081–1087

26 Esfand R, Tomalia D A. Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discovery Today, 2001, 6(8): 427–436

27 Hartmann L. Polymers for control freaks: sequence-defined poly(amidoamine)s and their biomedical applications. Macromolecular Chemistry and Physics, 2011, 212(1): 8–13

28 Hartmann L, Krause E, Antonietti M, Börner H G. Solid-phase supported polymer synthesis of sequence-defined, multifunctional poly(amidoamines). Biomacromolecules, 2006, 7(4): 1239–1244

29 Hartmann L, Häfele S, Peschka-Süss R, Antonietti M, Börner H G. Tailor-made poly(amidoamine)s for controlled complexation and condensation of DNA. Chemistry, 2008, 14(7): 2025–2033

30 Schaffert D, Badgujar N, Wagner E. Novel Fmoc-polyamino acids for solid-phase synthesis of defined polyamidoamines. Organic Letters, 2011, 13(7): 1586–1589

31 Burke R S, Pun S H. Extracellular barriers to in vivo PEI and PEGylated PEI polyplex-mediated gene delivery to the liver. Bioconjugate Chemistry, 2008, 19(3): 693–704

32 Edinger D, Wagner E. Bioresponsive polymers for the delivery of therapeutic nucleic acids. Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology, 2011, 3(1): 33–46

33 Itaka K, Harada A, Yamasaki Y, Nakamura K, Kawaguchi H, Kataoka K. In situ single cell observation by fluorescence resonance energy transfer reveals fast intra-cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine. The Journal of Gene Medicine, 2004, 6(1): 76–84

34 Oupický D, Parker A L, Seymour L W. Laterally stabilized complexes of DNA with linear reducible polycations: strategy for triggered intracellular activation of DNA delivery vectors. Journal of the American Chemical Society, 2002, 124(1): 8–9

35 Neu M, Germershaus O, Mao S, Voigt K H, Behe M, Kissel T. Crosslinked nanocarriers based upon poly(ethylene imine) for systemic plasmid delivery: in vitro characterization and in vivo studies in mice. Journal of Controlled Release: Official Journal of the Controlled Release Society, 2007, 118(3): 370–380

36 Miyata K, Kakizawa Y, Nishiyama N, Harada A, Yamasaki Y, Koyama H, Kataoka K. Block catiomer polyplexes with regulated densities of charge and disulfide cross-linking directed to enhance gene expression. Journal of the American Chemical Society, 2004, 126(8): 2355–2361

37 Russ V, Fröhlich T, Li Y, Halama A, Ogris M, Wagner E. Improved in vivo gene transfer into tumor tissue by stabilization of pseudodendritic oligoethylenimine-based polyplexes. The journal of gene medicine, 2010, 12(2): 180–193

38 Hamidi M, Azadi A, Rafiei P. Pharmacokinetic consequences of pegylation. Drug Delivery, 2006, 13(6): 399–409

39 Itaka K, Yamauchi K, Harada A, Nakamura K, Kawaguchi H, Kataoka K. Polyion complex micelles from plasmid DNA and poly(ethylene glycol)-poly(L-lysine) block copolymer as serum-tolerable polyplex system: physicochemical properties of micelles relevant to gene transfection efficiency. Biomaterials, 2003, 24(24): 4495–4506

40 Venkataraman S, Ong W L, Ong Z Y, Joachim Loo S C, Ee P L, Yang Y Y. The role of PEG architecture and molecular weight in the gene transfection performance of PEGylated poly(dimethylaminoethyl methacrylate) based cationicβpolymers. Biomaterials, 2011, 32(9): 2369–2378

41 Lai T C, Bae Y, Yoshida T, Kataoka K, Kwon G S. pH-sensitive multi-PEGylated block copolymer as a bioresponsive pDNA delivery vector. Pharmaceutical Research, 2010, 27(11): 2260–2273

42 Tamura A, Oishi M, Nagasaki Y. Enhanced cytoplasmic delivery of siRNA using a stabilized polyion complex based on PEGylated nanogels with a cross-linked polyamine structure. Biomacromolecules, 2009, 10(7): 1818–1827

43 Meyer M, Dohmen C, Philipp A, Kiener D, Maiwald G, Scheu C, Ogris M, Wagner E. Synthesis and biological evaluation of a bioresponsive and endosomolytic siRNA-polymer conjugate. Molecular Pharmaceutics, 2009, 6(3): 752–762

44 Takae S, Miyata K, Oba M, Ishii T, Nishiyama N, Itaka K, Yamasaki Y, Koyama H, Kataoka K. PEG-detachable polyplex micelles based on disulfide-linked block catiomers as bioresponsive nonviral gene vectors. Journal of the American Chemical Society, 2008, 130(18): 6001–6009

45 Hatakeyama H, Akita H, Harashima H. A multifunctional envelope type nano device (MEND) for gene delivery to tumours based on the EPR effect: a strategy for overcoming the PEG dilemma. Advanced Drug Delivery Reviews, 2011, 63(3): 152–160

46 Dash P R, Read M L, Fisher K D, Howard K A, Wolfert M, Oupicky D, Subr V, Strohalm J, Ulbrich K, Seymour L W. Decreased binding to proteins and cells of polymeric gene delivery vectors surface modified with a multivalent hydrophilic polymer and retargeting through attachment of transferrin.The Journal of Biological Chemistry, 2000, 275(6): 3793–3802

47 Oupicky D, Ogris M, Howard K A, Dash P R, Ulbrich K, Seymour L W. Importance of lateral and steric stabilization of polyelectrolyte gene delivery vectors for extended systemic circulation. Molecular Therapy: the Journal of the American Society of Gene Therapy, 2002, 5(4): 463–472

48 Ito T, Yoshihara C, Hamada K, Koyama Y. DNA/polyethyleneimine/hyaluronic acid small complex particles and tumor suppression in mice. Biomaterials, 2010, 31(10): 2912–2918

49 Hornof M, de la Fuente M, Hallikainen M, Tammi R H, Urtti A. Low molecular weight hyaluronan shielding of DNA/PEI polyplexes facilitates CD44 receptor mediated uptake in human corneal epithelial cells. The Journal of Gene Medicine, 2008, 10(1): 70–80

50 Kircheis R, Wightman L, Schreiber A, Robitza B, Rössler V, Kursa M, Wagner E. Polyethylenimine/DNA complexes shielded by transferrin target gene expression to tumors after systemic application. Gene Therapy, 2001, 8(1): 28–40

51 Kircheis R, Schüller S, Brunner S, Ogris M, Heider K H, Zauner W, Wagner E. Polycation-based DNA complexes for tumor-targeted gene delivery in vivo. Journal of Gene Medicine, 1999, 1(2): 111–120

52 Li S D, Chono S, Huang L. Efficient oncogene silencing and metastasis inhibition via systemic delivery of siRNA. Molecular Therapy, 2008, 16(5): 942–946

53 Li S D, Huang L. Surface-modified LPD nanoparticles for tumor targeting. Annals of the New York Academy of Sciences, 2006, 1082(1): 1–8

54 Hong M S, Lim S J, Oh Y K, Kim C K. pH-sensitive, serum-stable and long-circulating liposomes as a new drug delivery system. Journal of Pharmacy and Pharmacology, 2002, 54(1): 51–58

55 Miller C R, Bondurant B, McLean S D, McGovern K A, O’Brien D F. Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes. Biochemistry, 1998, 37(37): 12875–12883

56 O’Riordan C R, Lachapelle A, Delgado C, Parkes V, Wadsworth S C, Smith A E, Francis G E. PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo. Human Gene Therapy, 1999, 10(8): 1349–1358

57 Croyle M A, Yu Q C, Wilson J M. Development of a rapid method for the PEGylation of adenoviruses with enhanced transduction and improved stability under harsh storage conditions. Human Gene Therapy, 2000, 11(12): 1713–1722

58 Wolfert M A, Schacht E H, Toncheva V, Ulbrich K, Nazarova O, Seymour L W. Characterization of vectors for gene therapy formed by self-assembly of DNA with synthetic block co-polymers. Human Gene Therapy, 1996, 7(17): 2123–2133

59 Erbacher P, Bettinger T, Belguise-Valladier P, Zou S, Coll J L, Behr J P, Remy J S. Transfection and physical properties of various saccharide, poly(ethylene glycol), and antibody-derivatized polyethylenimines (PEI). Journal of Gene Medicine, 1999, 1(3): 210–222

60 Kursa M, Walker G F, Roessler V, Ogris M, Roedl W, Kircheis R, Wagner E. Novel shielded transferrin-polyethylene glycol-polyethylenimine/DNA complexes for systemic tumor-targeted gene transfer. Bioconjugate Chemistry, 2003, 14(1): 222–231

61 Kasper J C, Schaffert D, Ogris M, Wagner E, Friess W. Development of a lyophilized plasmid/LPEI polyplex formulation with long-term stability–A step closer from promising technology to application. Journal of Controlled Release, 2011, 151(3): 246–255

62 Kasper J C, Schaffert D, Ogris M, Wagner E, Friess W. The establishment of an up-scaled micro-mixer method allows the standardized and reproducible preparation of well-defined plasmid/LPEI polyplexes. European Journal of Pharmaceutics and Biopharmaceutics, 2011, 77(1): 182–185

63 Zhang X, Pan S R, Hu H M, Wu G F, Feng M, Zhang W, Luo X. Poly(ethylene glycol)-block-polyethylenimine copolymers as carriers for gene delivery: effects of PEG molecular weight and PEGylation degree. Journal of Biomedical Materials Research. Part A, 2008, 84A(3): 795–804

64 Walker G F, Fella C, Pelisek J, Fahrmeir J, Boeckle S, Ogris M, Wagner E. Toward synthetic viruses: endosomal pH-triggered deshielding of targeted polyplexes greatly enhances gene transfer in vitro and in vivo. Molecular Therapy, 2005, 11(3): 418–425

65 Fella C, Walker G F, Ogris M, Wagner E. Amine-reactive pyridylhydrazone-based PEG reagents for pH-reversible PEI polyplex shielding. European Journal of Pharmaceutical Sciences, 2008, 34(4–5): 309–320

66 Knorr V, Ogris M, Wagner E. An acid sensitive ketal-based polyethylene glycol-oligoethylenimine copolymer mediates improved transfection efficiency at reduced toxicity. Pharmaceutical Research, 2008, 25(12): 2937–2945

67 Knorr V, Allmendinger L, Walker G F, Paintner F F, Wagner E. An acetal-based PEGylation reagent for pH-sensitive shielding of DNA polyplexes. Bioconjugate Chemistry, 2007, 18(4): 1218–1225

68 Li W, Huang Z, MacKay J A, Grube S, Szoka F C Jr. Low-pH-sensitive poly(ethylene glycol) (PEG)-stabilized plasmid nanolipoparticles: effects of PEG chain length, lipid composition and assembly conditions on gene delivery. The Journal of Gene Medicine, 2005, 7(1): 67–79

69 Hatakeyama H, Akita H, Kogure K, Oishi M, Nagasaki Y, Kihira Y, Ueno M, Kobayashi H, Kikuchi H, Harashima H. Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid. Gene Therapy, 2007, 14(1): 68–77

70 Ikeda Y, Taira K. Ligand-targeted delivery of therapeutic siRNA. Pharmaceutical Research, 2006, 23(8): 1631–1640

71 Thurnher M, Wagner E, Clausen H, Mechtler K, Rusconi S, Dinter A, Birnstiel M L, Berger E G, Cotten M. Carbohydrate receptor-mediated gene transfer to human T leukaemic cells. Glycobiology, 1994, 4(4): 429–435

72 Spänkuch B, Steinhauser I, Wartlick H, Kurunci-Csacsko E, Strebhardt K I, Langer K. Downregulation of Plk1 expression by receptor-mediated uptake of antisense oligonucleotide-loaded nanoparticles. Neoplasia (New York, N.Y.), 2008, 10(3): 223–234

73 Saul J M, Annapragada A V, Bellamkonda R V. A dual-ligand approach for enhancing targeting selectivity of therapeutic nanocarriers. Journal of Controlled Release, 2006, 114(3): 277–287

74 Moffatt S, Papasakelariou C, Wiehle S, Cristiano R. Successful in vivo tumor targeting of prostate-specific membrane antigen with a highly efficient J591/PEI/DNA molecular conjugate. Gene Therapy, 2006, 13(9): 761–772

75 de Bruin K, Ruthardt N, von Gersdorff K, Bausinger R, Wagner E, Ogris M, Bräuchle C. Cellular dynamics of EGF receptor-targeted synthetic viruses. Molecular Therapy, 2007, 15(7): 1297–1305

76 Wolschek M F, Thallinger C, Kursa M, Rössler V, Allen M, Lichtenberger C, Kircheis R, Lucas T, Willheim M, Reinisch W, Gangl A, Wagner E, Jansen B. Specific systemic nonviral gene delivery to human hepatocellular carcinoma xenografts in SCID mice. Hepatology (Baltimore, Md.), 2002, 36(5): 1106–1114

77 Elfinger M, Pfeifer C, Uezguen S, Golas M M, Sander B, Maucksch C, Stark H, Aneja M K, Rudolph C. Self-assembly of ternary insulin-polyethylenimine (PEI)-DNA nanoparticles for enhanced gene delivery and expression in alveolar epithelial cells. Biomacromolecules, 2009, 10(10): 2912–2920

78 Furgeson D Y, Chan W S, Yockman J W, Kim S W. Modified linear polyethylenimine-cholesterol conjugates for DNA complexation. Bioconjugate Chemistry, 2003, 14(4): 840–847

79 Thomas M, Kularatne S A, Qi L, Kleindl P, Leamon C P, Hansen M J, Low P S. Ligand-targeted delivery of small interfering RNAs to malignant cells and tissues. Annals of the New York Academy of Sciences, 2009, 1175(1): 32–39

80 Xia W, Low P S. Folate-targeted therapies for cancer. Journal of Medicinal Chemistry, 2010, 53(19): 6811–6824

81 Wang S, Lee R J, Cauchon G, Gorenstein D G, Low P S.Delivery of antisense oligodeoxyribonucleotides against the human epidermal growth factor receptor into cultured KB cells with liposomes conjugated to folate via polyethylene glycol. Proc Natl Acad Sci USA, 1995, 92(0027–8424): 3318–3322

82 Kularatne S A, Low P S. Targeting of nanoparticles: folate receptor. Methods in Molecular Biology (Clifton, N.J.), 2010, 624: 249–265

83 Zhang K, Wang Q, Xie Y, Mor G, Sega E, Low P S, Huang Y. Receptor-mediated delivery of siRNAs by tethered nucleic acid base-paired interactions. RNA (New York), 2008, 14(3): 577–583

84 Elfinger M, Geiger J, Hasenpusch G, Uzgün S, Sieverling N, Aneja M K, Maucksch C, Rudolph C. Targeting of the beta(2)-adrenoceptor increases nonviral gene delivery to pulmonary epithelial cells in vitro and lungs in vivo. Journal of Controlled Release, 2009, 135(3): 234–241

85 Geiger J, Aneja M K, Hasenpusch G, Yüksekdag G, Kummerlöwe G, Luy B, Romer T, Rothbauer U, Rudolph C. Targeting of the prostacyclin specific IP1 receptor in lungs with molecular conjugates comprising prostaglandin I2 analogues. Biomaterials, 2010, 31(10): 2903–2911

86 Li S D, Chen Y C, Hackett M J, Huang L. Tumor-targeted delivery of siRNA by self-assembled nanoparticles. Molecular Therapy, 2008, 16(1): 163–169

87 Li S D, Huang L. Targeted delivery of antisense oligodeoxynucleotide and small interference RNA into lung cancer cells. Molecular Pharmaceutics, 2006, 3(5): 579–588

88 Oishi M, Kataoka K, Nagasaki Y. pH-responsive three-layered PEGylated polyplex micelle based on a lactosylated ABC triblock copolymer as a targetable and endosome-disruptive nonviral gene vector. Bioconjugate Chemistry, 2006, 17(3): 677–688

89 Oishi M, Nagatsugi F, Sasaki S, Nagasaki Y, Kataoka K. Smart polyion complex micelles for targeted intracellular delivery of PEGylated antisense oligonucleotides containing acid-labile linkages. ChemBioChem, 2005, 6(4): 718–725

90 Zauner W, Ogris M, Wagner E. Polylysine-based transfection systems utilizing receptor-mediated delivery. Advanced Drug Delivery Reviews, 1998, 30(1–3): 97–113

91 Oba M, Fukushima S, Kanayama N, Aoyagi K, Nishiyama N, Koyama H, Kataoka K. Cyclic RGD peptide-conjugated polyplex micelles as a targetable gene delivery system directed to cells possessing alphavbeta3 and alphavbeta5 integrins. Bioconjugate Chemistry, 2007, 18(5): 1415–1423

92 Taratula O, Garbuzenko O B, Kirkpatrick P, Pandya I, Savla R, Pozharov V P, He H, Minko T. Surface-engineered targeted PPI dendrimer for efficient intracellular and intratumoral siRNA delivery. Journal of Controlled Release, 2009, 140(3): 284–293

93 Han L, Huang R, Li J, Liu S, Huang S, Jiang C. Plasmid pORF-hTRAIL and doxorubicin co-delivery targeting to tumor using peptide-conjugated polyamidoamine dendrimer. Biomaterials, 2011, 32(4): 1242–1252

94 Klutz K, Schaffert D, Willhauck M J, Grünwald G K, Haase R, Wunderlich N, Zach C, Gildehaus F J, Senekowitsch-Schmidtke R, Göke B, Wagner E, Ogris M, Spitzweg C. Epidermal growth factor receptor-targeted (131)I-therapy of liver cancer following systemic delivery of the sodium iodide symporter gene. Molecular Therapy, 2011, 19(4): 676–685

95 Herbst R S. Review of epidermal growth factor receptor biology. International Journal of Radiation Oncology, Biology, Physics, 2004, 59(2, Suppl): S21–S26

96 Li Z, Zhao R, Wu X, Sun Y, Yao M, Li J, Xu Y, Gu J. Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics. The FASEB Journal, 2005, 19(14): 1978–1985

97 Leamon C P, Low P S. Folate-mediated targeting: from diagnostics to drug and gene delivery. Drug Discovery Today, 2001, 6(1): 44–51

98 Ross J F, Chaudhuri P K, Ratnam M. Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines. Physiologic and clinical implications. Cancer, 1994, 73(9): 2432–2443

99 Hwa Kim S, Hoon Jeong J, Chul Cho K, Wan Kim S, Gwan Park T. Target-specific gene silencing by siRNA plasmid DNA complexed with folate-modified poly(ethylenimine). Journal of Controlled Release, 2005, 104(1): 223–232

100 Bellocq N C, Pun S H, Jensen G S, Davis M E. Transferrin-containing, cyclodextrin polymer-based particles for tumor-targeted gene delivery. Bioconjugate Chemistry, 2003, 14(6): 1122–1132

101 Thorstensen K, Romslo I. The transferrin receptor: its diagnostic value and its potential as therapeutic target. Scandinavian Journal of Clinical and Laboratory Investigation. Supplementum, 1993, 53(s215): 113–120

102 Gatter K C, Brown G, Trowbridge I S, Woolston R E, Mason D Y. Transferrin receptors in human tissues: their distribution and possible clinical relevance. Journal of Clinical Pathology, 1983, 36(5): 539–545

103 Davis M E, Zuckerman J E, Choi C H, Seligson D, Tolcher A, Alabi C A, Yen Y, Heidel J D, Ribas A. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature, 2010, 464(7291): 1067–1070

104 Davis M E. The first targeted delivery of siRNA in humans via a self-assembling, cyclodextrin polymer-based nanoparticle: from concept to clinic. Molecular Pharmaceutics, 2009, 6(3): 659–668

105 Quan C Y, Chang C, Wei H, Chen C S, Xu X D, Cheng S X, Zhang X Z, Zhuo R X. Dual targeting of a thermosensitive nanogel conjugated with transferrin and RGD-containing peptide for effective cell uptake and drug release. Nanotechnology, 2009, 20(33): 335101

106 Kakimoto S, Moriyama T, Tanabe T, Shinkai S, Nagasaki T. Dual-ligand effect of transferrin and transforming growth factor alpha on polyethyleneimine-mediated gene delivery. Journal of Controlled Release, 2007, 120(3): 242–249

107 Wagner E, Cotten M, Foisner R, Birnstiel M L. Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells. Proceedings of the National Academy of Sciences of the United States of America, 1991, 88(10): 4255–4259

108 Han L, Huang R, Liu S, Huang S, Jiang C. Peptide-conjugated PAMAM for targeted doxorubicin delivery to transferrin receptor overexpressed tumors. Molecular Pharmaceutics, 2010, 7(6): 2156–2165

109 Xia H, Anderson B, Mao Q, Davidson B L. Recombinant human adenovirus: targeting to the human transferrin receptor improves gene transfer to brain microcapillary endothelium. Journal of Virology, 2000, 74(23): 11359–11366

110 Li D, Tang G P, Li J Z, Kong Y, Huang H L, Min L J, Zhou J, Shen F P, Wang Q Q, Yu H. Dual-targeting non-viral vector based on polyethylenimine improves gene transfer efficiency. Journal of Biomaterials Science. Polymer Edition, 2007, 18(5): 545–560

111 Suh W, Han S O, Yu L, Kim S W. An angiogenic, endothelial-cell-targeted polymeric gene carrier. Molecular Therapy, 2002, 6(5): 664–672

112 Bai M, Campisi L, Freimuth P. Vitronectin receptor antibodies inhibit infection of HeLa and A549 cells by adenovirus type 12 but not by adenovirus type 2. Journal of Virology, 1994, 68(9): 5925–5932

113 Nie Y, Schaffert D, Rödl W, Ogris M, Wagner E, Günther M. Dual-targeted polyplexes: One step towards a synthetic virus for cancer gene therapy. Journal of Controlled Release, 2011, DOI: 10.1016/j.jconrel.2011.02.028

114 Wagner E. Effects of membrane-active agents in gene delivery. Journal of Controlled Release, 1998, 53(1–3): 155–158

115 Curiel D T, Agarwal S, Wagner E, Cotten M. Adenovirus enhancement of transferrin-polylysine-mediated gene delivery. Proceedings of the National Academy of Sciences of the United States of America, 1991, 88(19): 8850–8854

116 Meyer M, Philipp A, Oskuee R, Schmidt C, Wagner E. Breathing life into polycations: functionalization with pH-responsive endosomolytic peptides and polyethylene glycol enables siRNA delivery. Journal of the American Chemical Society, 2008, 130(11): 3272–3273

117 Meyer M, Zintchenko A, Ogris M, Wagner E. A dimethylmaleic acid-melittin-polylysine conjugate with reduced toxicity, pH-triggered endosomolytic activity and enhanced gene transfer potential. The Journal of Gene Medicine, 2007, 9(9): 797–805

118 Wagner E, Plank C, Zatloukal K, Cotten M, Birnstiel M L. Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle. Proceedings of the National Academy of Sciences of the United States of America, 1992, 89(17): 7934–7938

119 Plank C, Zatloukal K, Cotten M, Mechtler K, Wagner E. Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand. Bioconjugate Chemistry, 1992, 3(6): 533–539

120 Roy R, Jerry D J, Thayumanavan S. Virus-inspired approach to nonviral gene delivery vehicles. Biomacromolecules, 2009, 10(8): 2189–2193

121 Wang H, Liu K, Chen K J, Lu Y, Wang S, Lin W Y, Guo F, Kamei K, Chen Y C, Ohashi M, Wang M, Garcia M A, Zhao X Z, Shen C K, Tseng H R. A rapid pathway toward a superb gene delivery system: programming structural and functional diversity into a supramolecular nanoparticle library. ACS Nano, 2010, 4(10): 6235–6243

122 Kleemann E, Neu M, Jekel N, Fink L, Schmehl T, Gessler T, Seeger W, Kissel T. Nano-carriers for DNA delivery to the lung based upon a TAT-derived peptide covalently coupled to PEG-PEI. Journal of Controlled Release, 2005, 109(1–3): 299–316

123 Pun S H, Bellocq N C, Liu A, Jensen G, Machemer T, Quijano E, Schluep T, Wen S, Engler H, Heidel J, Davis M E. Cyclodextrin-modified polyethylenimine polymers for gene delivery. Bioconjugate Chemistry, 2004, 15(4): 831–840

124 Sletten E M, Bertozzi C R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angewandte Chemie (International ed. in English), 2009, 48(38): 6974–6998

125 Navath R S, Menjoge A R, Wang B, Romero R, Kannan S, Kannan R M. Amino acid-functionalized dendrimers with heterobifunctional chemoselective peripheral groups for drug delivery applications. Biomacromolecules, 2010, 11(6): 1544–1563