**12. References**


showed a significant decrease in muscle and serum after the injection of the IL-1Ra protein demonstrating by direct evidence the efficacy of the administration technique to deliver efficient nanoparticles. Therefore, we have already shown it is possible to do gene therapy

This work was supported by the grants from National Natural Science Foundation of China (No. 30811120440 and 30911120261), The Ministry of Science and Technology of China (No. S2011GR0323), Shanghai International Collaboration Foundation (No. 08410701800) and Canadian Institutes of Health Research (CCI-92212, CCL-99636 and CCM 104888). Dr Fernandes and Dr Benderdour are research scholars of *Fonds de la Recherche en santé du* 

Alexander, B.L.; Ali, R.R.; Alton, E.W.; Bainbridge, J.W.; Braun, S.; Cheng, S.H.; Flotte, T.R.;

Aiba, S. (1993). Reactivity of partially N-acetylated chitosan in aqueous media,

Andersen, M.O.; Howard, K.A. & Kjems, J. (2009). RNAi using a chitosan/siRNA

Andersen, M.O.; Howard, K.A.; Paludan, S.R.; Besenbacher, F. & Kjems,J. (2008). Delivery of

Baker, M. (2010a). RNA interference: From tools to therapies. *Nature*, 464, 1225, ISSN 0028-

Baker, M. (2010b). RNA interference: Homing in on delivery. *Nature*, 464, 1225-1228. ISSN

Berger, J.; Reist, M.; Mayer, J.M.; Felt, O. & Gurny, R. (2004). Structure and interactions in

Berger, J.; Reist, M.; Mayer, J.M.; Felt, O.; Peppas, N.A. & Gurny, R. (2004). Structure and

biomedical applications. *Eur. J. Pharm. Biopharm.,* 57, 19-34, ISSN 0928-0987 Bertha, G.; Dautzenberg, H. & Peter, M.G. (1998). Physico-chemical characterization of

Bertha, G. & Dautzenberg, H. (2002). The degree of acetylation of chitosans and its effect on

applications. *Eur. J. Pharm. Biopharm.,* 57*,* 35-52, ISSN 0928-0987

Gaspar, H.B.; Grez, M.; Griesenbach, U.; Kaplit,t M.G.; Ott, M.G.; Seger, R.; Simons, M.; Thrasher, A.J.; Thrasher, A.Z. & Ylä-Herttuala, S. (2007). Progress and prospects: gene therapy clinical trials (part 1). *Gene Ther.,* 14, 1439-1447, ISSN 0969-

nanoparticle system: in vitro and in vivo applications. *Methods Mol. Biol.*, 555, 77-86,

siRNA from lyophilized polymeric surfaces. *Biomaterials,* 29, 506-512, ISSN 1043-

chitosan hydrogels formed by complexation or aggregation for biomedical

interactions in covalently and ionically crosslinked chitosan hydrogels for

chitosans varying in degree of acetylation. *Carbohydr. Polym*., 36, 205-216, ISSN

the chain conformation in aqueous solution. *Carbohydr. Polym.*, 47, 39-51, ISSN 1043-

with IL-1Ra to decrease arthritis and have a positive effect on inflammation.

*Québec* (FRSQ). Dr Tiera holds a post-PH D scholarship from UNESP - Brazil.

*Makromolekulare Chemie.,* 194(1), 65–75, ISSN 0025-116X

**11. Acknowledgments** 

**12. References** 

7128

1802

0836

0028-0836

0144-8617

1802

ISSN 1064-3745


Chitosan-DNA/siRNA Nanoparticles for Gene Therapy 473

Howard, K.A., Paludan, S.R., Behlke, M.A., Besenbacher, F., Deleuran, B. & Kjems, J. (2009).

Howard, K.A., Rahbek, U.L., Liu, X., Damgaard, C.K., Glud, S.Z., Andersen, M.O.,

Huang, M., Fong, C.W., Khor, E. & Lim, L.Y. (2005). Transfection efficiency of chitosan

Hung, T.H.; Chang, Y.M.; Sung, H.Y. & Chang, C.T. (2002). Purification and characterization

Hyde, S.C., Southern, K.W., Gileadi, U., Fitzjohn, E.M., Mofford, K.A., Waddell, B.E., Gooi,

Jackson, A.L. & Linsley, P.S. (2010). Recognizing and avoiding siRNA off-target effects for

Janes, K.A.; Calvo, P. & Alonso, M.J. (2001). Polysaccharide colloidal particles as delivery systems for macromolecules. *Adv. Drug Deliv. Rev.,* 47, 83–97, ISSN 0169-409X Jang, M.K.; Kong, B.G; Jeong, Y.I.; Lee, C.H. & Nah, J.W. (2004). Physicochemical

Jiang, H.L., Kim, Y.K., Arote, R., Nah, J.W., Cho, M.H., Choi, Y.J., Akaike, T. & Cho, C.S.

Jiang, H.L.; Kim, Y.K.; Lee, S.M.; Park, M.R.; Kim, E.M.; Jin, Y.M.; Arote, R.; Jeong, H.J.;

Jones, R.A., Cheung, C.Y., Black, F.E., Zia, J.K., Stayton, P.S., Hoffman, A.S. & Wilson, M.R.

Katas, H. & Alpar, H.O. (2006). Development and characterisation of chitosan nanoparticles

Katas, H., Chen, S., Osamuyimen, A.A., Cevher, E. & Oya, A.H. (2008). Effect of preparative

for siRNA delivery. *J. Control Release*, 115, 216-225, ISSN 0168-3659

patients with cystic fibrosis. *Gene Ther.,* 7, 1156-1165, ISSN 0969-7128 Ishii, T., Okahata, Y. & Sato, T. (2001). Mechanism of cell transfection with plasmid/chitosan

bromelain, *J. Agric. Food Chem*., 50, 4666-4673, ISSN 0021-8561

complexes. *Biochim. Biophys. Acta,* 1514, 51-64, ISSN 0006-3002

Resources. *J. Polym. Sci. Pol. Chem.*, 42, 3423–3432, ISSN 1099-0518.

gene delivery system. *Arch. Pharm. Res.,* 33, 551, ISSN 0253-6269

*Ther.,* 17, 162-168, ISSN 1525-0016

*Release*, 106, 391-406, ISSN 0168-3659

ISSN 1474-1776

273-280, ISSN 0168-3659

317, 580. ISSN 0036-8075

system. *Mol. Ther.,* 14, 476-484, ISSN 1525-0016

Chitosan/siRNA nanoparticle-mediated TNF-alpha knockdown in peritoneal macrophages for anti-inflammatory treatment in a murine arthritis model. *Mol.* 

Hovgaard, M.B., Schmitz, A., Nyengaard, J.R., Besenbacher, F. & Kjems, J. (2006). RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle

vectors: effect of polymer molecular weight and degree of deacetylation. *J. Control* 

of hydrolase with chitinase and chitosanase activity from commercial stem

H.C., Goddard, C.A., Hannavy, K., Smyth, S.E., Egan, J.J., Sorgi, F.L., Huang, L., Cuthbert, A.W., Evans, M.J., Colledge, W.H., Higgins, C.F., Webb, A.K. & Gill, D.R. (2000). Repeat administration of DNA/liposomes to the nasal epithelium of

target identification and therapeutic application. *Nat. Rev. Drug. Discov.,* 9, 57-67,

Characterization of α-Chitin, β-Chitin, and γ-Chitin Separated from Natural

(2007). Chitosan-graft-polyethylenimine as a gene carrier*. J. Control Release,* 117,

Song, S.C.; Cho, M.H. & Cho, C.S. (2010). Galactosylated chitosan-g-PEI/DNA complexes-loaded poly(organophosphazene) hydrogel as a hepatocyte targeting

(2003). Poly(2-alkylacrylic acid) polymers deliver molecules to the cytosol by pHsensitive disruption of endosomal vesicles. *Biochem. J.,* 372, 65-75, ISSN 0264-6021 Kaiser, J. (2007). Clinical research. Death prompts a review of gene therapy vector. *Science*,

variables on small interfering RNA loaded Poly(D,L-lactide-co-glycolide)-chitosan


Franco, P.; Senso, A.; Oliveros, L. & Minguillon, C. (2001). Covalently bonded

Gao, S.; Chen, J.; Xu, X.; Ding, Z.; Yang, Y.H.; Hua, Z. & Zhang, J. (2003). Galactosylated low

Gao, S.; Dagnaes-Hansen, F.; Nielsen, E.J.; Wengel, J.; Besenbacher, F.; Howard, K.A. &

Gao, X.; Kim, K.S. & Liu, D. (2007). Nonviral gene delivery: what we know and what is next.

Gao, Y.; Xu, Z.H.; Chen, S.W.; Gu, W.W.; Chen, L.L. & Li, Y.P. (2008). Arginine-

Gary, D.J.; Puri, N. & Won, Y.Y. (2007). Polymer-based siRNA delivery: perspectives on the

Germershaus, O.; Mao, S.; Sitterberg, J.; Bakowsky, U. & Kissel, T. (2008). Gene delivery

Giannoudis, P.V.; Tzioupis, C.C. & Tsiridis, E. (2006). Gene therapy in orthopaedics. *Injury*,

Godbey, W.T.; Wu, K.K. & Mikos, A.G. (1999). Size matters: molecular weight affects the

Guibal, E. (2004). Interactions of metal ions with chitosan-based sorbents: a review. *Separ.* 

Guo, T.; Zhao, J.; Chang, J.; Ding, Z.; Hong, H.; Chen, J. & Zhang, J. (2006). Porous chitosan-

Hammond, S.M.; Bernstein, E.; Beach, D. & Hannon, G.J. (2000). An RNA-directed nuclease

Harris, J.M., Struk, E.C., Case, M.G., Paley, M.S., Yalpani, M., Van Alstin, J.M. & Brooks,

Hillaireau, H. & Couvreur, P. (2009). Nanocarriers' entry into the cell: relevance to drug

immunodeficiency. *N. Engl. J Med*, 348, 255-256, ISSN 0028-4793

Polym. Sci. Polym. Chem. Ed. *22*, 341–352, ISSN 0360-6376

delivery. *Cell Mol. Life Sci.,* 66, 2873-2896, ISSN 1420-682X

delivery. *J. Control Release*, 121, 64-73, ISSN 0168-3659

*Control Release*, 125, 145-154, ISSN 0168-3659

37 Suppl 1, S30-S40, ISSN 0020-1383

*Purific. Techn.,* 38*,* 43-74*,* ISSN 1383-5866

268-275, ISSN 1549-3296

296, ISSN 0028-0836

chromatography. *J. Chromatogr. A,* 906, 155-170, ISSN 0021-9673

255, 57-68, ISSN 0378-5173

*AAPS. J.*, 9, E92-104, ISSN 1550-7416

1525-0016

5173

polysaccharide derivatives as chiral stationary phases in high-performance liquid

molecular weight chitosan as DNA carrier for hepatocyte-targeting. *Int. J. Pharm.,*

Kjems, J. (2009). The effect of chemical modification and nanoparticle formulation on stability and biodistribution of siRNA in mice. *Mol. Ther.,* 17, 1225-1233, ISSN

chitosan/DNA self-assemble nanoparticles for gene delivery: In vitro characteristics and transfection efficiency*. Int. J. Pharm.*, 359, 241-246, ISSN 0378-

fundamental and phenomenological distinctions from polymer-based DNA

using chitosan, trimethyl chitosan or polyethylenglycol-graft-trimethyl chitosan block copolymers: establishment of structure-activity relationships in vitro*. J.* 

efficiency of poly(ethylenimine) as a gene delivery vehicle. *J. Biomed. Mater. Res.,* 45,

gelatin scaffold containing plasmid DNA encoding transforming growth factorbeta1 for chondrocytes proliferation. *Biomaterials*, 27, 1095-1103, ISSN 0142-9612 Hacein-Bey-Abina, S.; von, K.C.; Schmidt, M.; Le, D.F.; Wulffraat, N.; McIntyre, E.; Radford,

I.; Villeval, J.L.; Fraser, C.C.; Cavazzana-Calvo, M. & Fischer, A. (2003). A serious adverse event after successful gene therapy for X-linked severe combined

mediates post-transcriptional gene silencing in Drosophila cells. *Nature,* 404, 293-

D.E. (1984). Synthesis and Characterization of Poly( ethylene Glycol) Derivatives. J.


Chitosan-DNA/siRNA Nanoparticles for Gene Therapy 475

Lee, K.Y.; Ha, W.S. & Park, W.H. (1995) Blood compatibility and biodegradability of

Lee, K.Y., Kwon, I.C., Kim, Y.H., Jo, W.H. & Jeong, S.Y. (1998). Preparation of chitosan self-

Lee, D., Lockey, R. & Mohapatra, S. (2006). Folate receptor-mediated cancer cell specific gene

Lee, D., Zhang, W., Shirley, S.A., Kong, X., Hellermann, G.R., Lockey, R.F. & Mohapatra, S.S.

Lee, M.Y.; Var, F.; Shin-ya, Y.; Kajiuchi, T. & Yang, J.W. (1999). Optimum conditions for the

Leong, K.W.; Mao, H.Q.; Truong-Le, V.L.; Roy, K.; Walsh, S.M. & August, J.T. (1998). DNA-

Li, S.D. & Huang, L. (2006). Gene therapy progress and prospects: non-viral gene therapy by

Lin, H.; Wang, H. Y.; Xue, C.H. M. Y, (2002). Preparation of chitosan oligomers by immobilized papain. *Enzyme Microbial Technology*, 31,588-592, ISSN 0141- 0229 Liu, D. & Knapp, J.E. (2001). Hydrodynamics-based gene delivery. *Curr. Opin. Mol. Ther.,* 3,

Liu, X., Howard, K.A., Dong, M., Andersen, M.O., Rahbek, U.L., Johnsen, M.G., Hansen,

Liu, W.G.; Zhang, J.R.; Cao, Z.Q.; Xu, F.Y. & Yao, K.D. (2004). A chitosan-arginine conjugate

Liu, W.G. & Yao, K.D. (2002). Chitosan and its derivatives—a promising non-viral vector for

Mansouri, S.; Cui, Y.; Winnik, F.; Shi, Q.; Benderdour, M.; Lavigne, P.; Beaumont, E. &

Mao*,* S.; Shuai, X.; Unger, F. Simona, M., Kissel, D., & Bi, T. (2004). The depolymerization of

Mingozzi, F. & High, K.A. (2007). Immune Responses to AAV in Clinical Trials. *Curr. Gene* 

Moghimi, S.M., Hunter, A.C. & Murray, J.C. (2005). Nanomedicine: current status and future

O.C., Besenbacher, F. & Kjems, J. (2007). The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencing. *Biomaterials,* 28, 1280-

as a novel anticoagulation biomaterial. *J. Mater. Sci. Mater. Med.,* 15, 1199–1203,

Fernandes, J.C. (2006). Characterization of Folate-Chitosan-DNA nanoparticles for

chitosan: effects on physicochemical and biological properties. *Int. J. Pharm*., 281,

gene delivery. *Pharm. Res.,* 24, 157-167, ISSN 0724-8741

at low temperature. *Proc. Biochem,* 34, 493–500, ISSN 1359-5113

systemic delivery. *Gene Ther.,* 13, 1313-1319, ISSN 0969-7128

gene transfection*. J. Control Release,* 83*,* 1-11, ISSN 0168-3659

gene therapy. *Biomaterials*, 27, 2060-2065, ISSN 0142-9612

prospects. *FASEB J.,* 19, 311-330, ISSN 0892-6638

1043-1802

2866, ISSN 1533-4880

183–193, ISSN 0168-3659

192-197, ISSN 1093-4715

1288, ISSN 1043-1802

45–54, ISSN 0378-5173

*Ther.,* 7, 316-324, ISSN 1566-5232

ISSN 0957-4530.

1802

molecular weight and degree of deacetylation. *Biomaterials,* 27, 4815-4824, ISSN

partially *N*-acylated chitosan derivatives*. Biomaterials,* 16, 1211-1216, ISSN 1043-

aggregates as a gene delivery system. *J. Control Release,* 51, 213-220, ISSN 0168-3659

delivery using folic acid-conjugated oligochitosans. *J. Nanosci. Nanotechnol.,* 6, 2860-

(2007). Thiolated chitosan/DNA nanocomplexes exhibit enhanced and sustained

precipitation of chitosan oligomers with DP 5–7 in concentrated hydrochloric acid

polycation nanospheres as non-viral gene delivery vehicles. *J. Control Release.,* 53,

submicron particles prepared by emulsification diffusion method. *J. Microencapsul.,* 25, 541-548, ISSN 0265-2048


Kato, Y.; Onishi, H. & Machida, Y. (2004). *N*-succinyl-chitosan as a drug carrier: waterinsoluble and water-soluble conjugates*. Biomaterials,* 25*,* 907-915*,* ISSN 0142-9612 Kean, T. & Thanou, M. (2010). Biodegradation, biodistribution and toxicity of chitosan. *Adv.* 

Khatri, K., Goyal, A.K., Gupta, P.N., Mishra, N. & Vyas, S.P. (2008). Plasmid DNA loaded

Kiang, T., Wen, J., Lim, H.W. & Leong, K.W. (2004). The effect of the degree of chitosan

Kim, T.H., Ihm, J.E., Choi, Y.J., Nah, J.W. & Cho, C.S. (2003). Efficient gene delivery by urocanic acid-modified chitosan. *J. Control Release,* 93, 389-402, ISSN 0168-3659 Kim, T.H., Kim, S.I., Akaike, T. & Cho, C.S. (2005). Synergistic effect of poly(ethylenimine)

Kim, T.H., Nah, J.W., Cho, M.H., Park, T.G. & Cho, C.S. (2006). Receptor-mediated gene

Kumar, M.N.V.R.; Muzzarelli, R.A.A.; Muzzarelli, C.; Sashiwa, H. & Domb, A.J. (2004).

Kumar, A.B.V. & Tharanathan, R.N. (2004). A comparative study on depolymerization of chitosan by proteolytic enzymes, *Carbohydr. Polym.* 58, 275-283, ISSN 0144-8617 Kumar, A.B.V.; Varadaraj, M. C.; Lalitha, R.G. & Tharanathan, R.N. (2004). Low molecular

Kurita. K. (2001). Controlled functionalization of polysaccharide chitin. *Prog. Polym. Sci.,* 26,

Kurita, K.; Ishii, S.; Tomita, K.; Nishimura, S.I. & Shlmoda, K. (1994) Reactivity

Kuroiwa, T.; Ichikawa, S.; Sato, S. & Mukataka, S. (2003). Improvement of the yield of

Lameiro, M.H., Malpique, R., Silva, A.C., Alves, P.M. & Melo, E. (2006). Encapsulation of

Lavertu, M., Methot, S., Tran-Khanh, N. & Buschmann, M.D. (2006). High efficiency gene

immobilized chitosanases. *Biotechn. Bioeng*., 84 , 121-127, ISSN 0006-3592. Kwon, S.; Park, J.H.; Chung, H.; Kwon, I.C. & Jeong, S.Y. (2003). Physicochemical

5β-Cholanic Acid. *Langmuir*, 19, 10188-10193, ISSN 0743-7463

nanoparticles. *J. Nanosci. Nanotechnol.,* 6, 2796-2803, ISSN 1533-4880

*Biophys. Acta-Gen. Sub*. 1670, 137-146, ISSN 0304-4165

*Polym. Sci. Pol. Chem.*, 32, 1027-1032, ISSN 1099-0518

*Biotechnol.,* 126, 152-162, ISSN 0168-1656

25, 541-548, ISSN 0265-2048

1043-1802

ISSN 0009-2665

1921-1971, ISSN 0340-255X

*Drug Deliv. Rev.,* 62, 3-11. ISSN 0169-409X

*Pharm.,* 354, 235-241, ISSN 0378-5173

*Release,* 105, 354-366, ISSN 0168-3659

submicron particles prepared by emulsification diffusion method. *J. Microencapsul.,*

chitosan nanoparticles for nasal mucosal immunization against hepatitis B. *Int. J.* 

deacetylation on the efficiency of gene transfection. *Biomaterials,* 25, 5293-5301, ISSN

on the transfection efficiency of galactosylated chitosan/DNA complexes. *J. Control* 

delivery into antigen presenting cells using mannosylated chitosan/DNA

Chitosan chemistry and pharmaceutical perspectives. *Chem. Rev.,* 104, 6017-6084*,*

weight chitosans: preparation with the aid of papain and characterization, *Biochim.* 

characteristics of squid beta-chitin as compared with those of shrimp chitin: High potentials of squid chitin as a starting material for facile chemical modifications. *J.* 

physiologically active oligosaccharides in continuous hydrolysis of chitosan using

Characteristics of Self-Assembled Nanoparticles Based on Glycol Chitosan Bearing

adenoviral vectors into chitosan-bile salt microparticles for mucosal vaccination. *J.* 

transfer using chitosan/DNA nanoparticles with specific combinations of

molecular weight and degree of deacetylation. *Biomaterials,* 27, 4815-4824, ISSN 1043-1802


Chitosan-DNA/siRNA Nanoparticles for Gene Therapy 477

Raper, S.E., Chirmule, N., Lee, F.S., Wivel, N.A., Bagg, A., Gao, G.P., Wilson, J.M. &

Richardson, S.C., Kolbe, H.V. & Duncan, R. (1999). Potential of low molecular mass chitosan

Romero, N.B., Braun, S., Benveniste, O., Leturcq, F., Hogrel, J.Y., Morris, G.E., Barois, A.,

muscular dystrophy. *Hum. Gene Ther.,* 15, 1065-1076. ISSN 1043-0342 Sakurai, H., Kawabata, K., Sakurai, F., Nakagawa, S. & Mizuguchi, H. (2008). Innate

Saito, H.; Wu, Sato, T.; Nagasaki, T.; Sakairi, N. & Shinkai, S. (2004). 6-Amino-6-

Saito, H.; Wu, X.; Harris, J.M. & Hoffman, A.S. (1997). Graft copolymers of poly(ethylene glycol) (PEG) and chitosan. *Macromol. Rapid Commun.,* 18, 547-550, ISSN 1022-1336 Sato, T., Ishii, T. & Okahata,Y. (2001). In vitro gene delivery mediated by chitosan. effect of

Satoh, T.; Kakimoto, S.; Kano, H.; Nakatani, M.; Shinkai, S. & Nagasaki, T. (2007). In vitro

Schaffer, D.V., Fidelman, N.A., Dan, N. & Lauffenburger, D.A. (2000). Vector unpacking as a

Schatz, C.; Viton, C.; Delair, T.; Pichot, C. & Domard, A. (2003). Typical physicochemical

Seiler, M.P., Cerullo, V. & Lee, B. (2007). Immune response to helper dependent adenoviral

Shimojohay, M.; Fukushimab, K. & Kurita. K. (1998). Low-molecular-weight chitosans

Signini, R.; Desbrieres, J. & Campana Filho, S.P. (2000). On the stiffness of chitosan

DNA carrier. *Carbohydr. Res.*, 342, 1427–1433, ISSN 0008-6215

activity. *Carbohydr. Polym*.*,* 35, 223-231, ISSN 0144-8617

complex and protect DNA. *Int. J. Pharm.,* 178, 231-243, ISSN 0378-5173 Roberts, G.A.F. (1992). *Chitin Chemistry*, Mc Millan Press Ltd, London, ISBN 0333524179 Rojanarata, T., Opanasopit, P., Techaarpornkul, S., Ngawhirunpat, T. & Ruktanonchai, U.

*Mol. Genet. Metab,* 80, 148-158, ISSN 1096-7192

*Pharm. Res.,* 25, 2807-2814. ISSN 0724-8741

*Chem. Lett.*, 33: 340-341, ISSN 0366-7022

*Biomaterials,* 22, 2075-2080, ISSN 0142-9612

67, 598-606, ISSN 0006 3592

0378-5173

7797

ISSN 1566-5232

1043-1802

Batshaw, M.L. (2003). Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer.

as a DNA delivery system: biocompatibility, body distribution and ability to

(2008). Chitosan-thiamine pyrophosphate as a novel carrier for siRNA delivery.

Eymard, B., Payan, C., Ortega, V., Boch, A.L., Lejean, L., Thioudellet, C., Mourot, B., Escot, C., Choquel, A., Recan, D., Kaplan, J.C., Dickson, G., Klatzmann, D., Molinier-Frenckel, V., Guillet, J.G., Squiban, P., Herson, S. & Fardeau, M. (2004). Phase I study of dystrophin plasmid-based gene therapy in Duchenne/Becker

immune response induced by gene delivery vectors. *Int. J Pharm.,* 354, 9-15, ISSN

deoxychitosan (2004) Preparation and application as plasmid vector in COS-1 cells.

pH, serum, and molecular mass of chitosan on the transfection efficiency.

gene delivery to HepG2 cells using galactosylated 6-amino-6-deoxychitosan as a

potential barrier for receptor-mediated polyplex gene delivery. *Biotechnol. Bioeng.,*

behaviors of chitosan in aqueous solution. *Biomacromolecules,* 4, 641-648, ISSN 1525-

mediated liver gene therapy: challenges and prospects. *Curr. Gene Ther.,* 7, 297-305,

derived from P-chitin: preparation, molecular characteristics and aggregation

hydrochloride in acid-free aqueous solutions. *Carbohydr. Polym*. 43, 351–357. ISSN


Mook, O.R., Baas, F., de Wissel, M.B. & Fluiter, K. (2007). Evaluation of locked nucleic acid-

Morris, V.B. & Sharma C.P. (2010). Folate mediated in vitro targeting of depolymerised

Muzzarelli, R.A.A.; Terbojevich, M.; Muzzarelli, C. & Francescangeli, O. (2002). Chitosans

Nakashima-Matsushita, N., Homma, T., Yu, S., Matsuda, T., Sunahara, N., Nakamura, T.,

No, H.K. & Meyers, S.P. (1989). Crawfish chitosan as a coagulant in recovery of organic

Okamoto, H., Shiraki, K., Yasuda, R., Danjo, K. & Watanabe, Y. (2010). Chitosan-interferon-

Ouchi, T.; Nishizawa, H. & Ohya, Y. (1998). Aggregation phenomenon of PEG-grafted chitosan in aqueous solution. *Polymer*, 39, 5171-5175, ISSN 0032-3861 Pa, J.H. & Yu T.L. (2001). Light scattering study of chitosan in acetic acid aqueous solutions.

Park, I.K.; Ihm, J.E.; Park, Y.H.; Choi Y.J.; Kim, S.I.; Kim W.J.; Akaike, T. & Cho, C.S. (2003).

graft-PVP/DNA complex. *J. Control Release*, 86: 349-359, ISSN 0168-3659. Park, I.K., Park, Y.H., Shin, B.A., Choi, E.S., Kim, Y.R., Akaike, T. & Cho, C.S. (2000).

Pelletier, J.P., Caron, J.P., Evans, C., Robbins, P.D., Georgescu, H.I., Jovanovic, D.,

Ping, Y.A.; Liu, C.D.; Tang, G.P.; Li, J.S.; Li, J.; Yang, W.T. & Xu, F.J. (2010). Functionalization

Rabea, E.I.; Badawy, M.E.T.; Stevens, C.V.; Smagghe, G. & Steurbaut, W. (2003). Chitosan as

Ramesh, R., Saeki, T., Templeton, N.S., Ji, L., Stephens, L.C., Ito, I., Wilson, D.R., Wu, Z.,

using an improved liposome vector. *Mol. Ther.,* 3, 337-350, ISSN 1525-0016

*Control Release.* 2010 Dec 23. [Epub ahead of print, ISSN 0168-3659

*Macromol. Chem. Phys.,* 202, 985-991, ISSN 1022-1352

*Control Release,* 69, 97-108, ISSN 0168-3659

Arthritis Rheum., 40, 1012-9, ISSN 1529-0131

*Mater.*, 20, 3106–3116, ISSN 1616-3028

1465, ISSN 1525-7797

ISSN 1535-7163

ISSN 0021-9797

1616, ISSN 1529-0131

0021-8561

*Polym.,* 50, 69-78, ISSN 0144-8617

modified small interfering RNA in vitro and in vivo. *Mol. Cancer Ther.,* 6, 833-843,

trimethylated chitosan having arginine functionality. *J. Coll. Interf. Sci.,* 348, 360-368,

depolymerized with the aid of papain and stabilized as glycosylamines. *Carbohydr.* 

Tsukano, M., Ratnam, M. & Matsuyama, T. (1999). Selective expression of folate receptor beta and its possible role in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis. *Arthritis Rheum.,* 42, 1609-

compounds from seafood processing streams. *J. Agric. Food Chem.* 37*,* 580-583, ISSN

beta gene complex powder for inhalation treatment of lung metastasis in mice. *J.* 

Galactosylated chitosan (GC)-graft-poly(vinyl pyrrolidone) (PVP) as hepatocytetargeting DNA carrier Preparation and physicochemical characterization of GC-

Galactosylated chitosan-graft-dextran as hepatocyte-targeting DNA carrier. *J.* 

Fernandes, J.C. & Martel-Pelletier, J. (1997). In vivo suppression of early experimental osteoarthritis by interleukin-1 receptor antagonist using gene therapy.

of chitosan via atom transfer radical polymerization for gene delivery. *Adv. Funct.* 

Antimicrobial Agent: applications and mode of action. *Biomacromolecules,* 4, 1457-

Branch, C.D., Minna, J.D. & Roth, J.A. (2001). Successful treatment of primary and disseminated human lung cancers by systemic delivery of tumor suppressor genes


Chitosan-DNA/siRNA Nanoparticles for Gene Therapy 479

Tsaih, M.L. & Chen, R.H. (2003). Effect of degree of deacetylation of chitosan on the kinetics

Turk, M.J., Breur, G.J., Widmer, W.R., Paulos, C.M., Xu, L.C., Grote, L.A. & Low, P.S. (2002).

Ulrich-Vinther, M. (2007). Gene therapy methods in bone and joint disorders. Evaluation of

Vaishnaw, A.K., Gollob, J., Gamba-Vitalo, C., Hutabarat, R., Sah, D., Meyers, R., de, F.T. &

Vandenberghe, L.H. & Wilson, J.M. (2007). AAV as An Immunogen. *Curr. Gene Ther.,* 7, 325-

Wang, J., Lu, Z., Wientjes, M.G. & Au, J.L. (2010). Delivery of siRNA Therapeutics: Barriers

Whittaker, G.R. & Helenius, A. (1998). Nuclear import and export of viruses and virus

Wong, K., Sun, G., Zhang, X., Dai, H., Liu, Y., He, C. & Leong, K.W. (2006). PEI-g-chitosan, a

Varma, A.J.; Deshpande, S.V. & Kennedy, J.F. (2004). Metal complexation by chitosan and its

Varurn, K.M.; Myhr, M.M.; Hjerde, R.J.N. & Smidsrod, O. (1997). In vitro degradation rates

Xia, W., Liu, W., Cui, L., Liu, Y., Zhong, W., Liu, D., Wu, J., Chua, K. & Cao, Y. (2004). Tissue

Yang, P.T., Hoang, L., Jia, W.W. & Skarsgard, E.D. (2010). In Utero Gene Delivery Using

Yip, V.L.Y. & Withers, S.G. (2004). Nature's many mechanisms for the degradation of

Yoo, H.S., Lee, J.E., Chung, H., Kwon, I.C.& Jeong, S.Y. (2005). Self-assembled nanoparticles

Yu, B., Zhao, X., Lee, L.J. & Lee, R.J. (2009). Targeted delivery systems for oligonucleotide

Yu, Y.Y.; Wang, Z.; Cai, L.; Wang, G.; Yang, X.; Wan, X.P.; Xu, X.H.; Li, Y. & Gao R. (2010).

derivatives: a review. *Carbohydr. Polym*., 55, 77-93, ISSN 0144-8617

*Mater Res B Appl Biomater.,* 15:71(2), 373-80, ISSN 1552-4973

oligosaccharides. *Org. Biol. Chem.*, 2, 2707–2713.

therapeutics. *AAPS. J.,* 11, 195-203, ISSN 1550-7416

*Release*, 103, 235-243, ISSN 0168-3659

novel gene delivery system with transfection efficiency comparable to polyethylenimine in vitro and after liver administration in vivo. *Bioconjug. Chem.,*

of partially *N*-acetylated chitosans in human serum, *Carbohydr. Res*., 299, 99- 101,

engineering of cartilage with the use of chitosan-gelatin complex scaffolds. *J Biomed* 

Chitosan-DNA Nanoparticles in Mice. *J. Surg. Res.,* 2010 Jun 12. [Epub ahead of

containing hydrophobically modified glycol chitosan for gene delivery. *J. Control* 

Synthesis and characterization of methoxy poly(ethylene glycol)-O-chitosanpolyethylenimine for gene delivery. *Carbohydr. Polym.* 81: 269-274, ISSN 0144-8617

arthritis. *Arthritis Rheum.,* 46, 1947-1955, ISSN 1529-0131

and Carriers. *AAPS. J.,* 12(4):492-503, ISSN 1550-7416

genomes. *Virology,* 246, 1-23, ISSN 0042-6822

8995

ISSN 1745-3674

333, ISSN 1566-5232

17, 152-158, ISSN 1043-1802

ISSN 0144-8617

print], ISSN 0022-4804

1758-907X

of ultrasonic degradation of chitosan. *J. Appl. Polym. Sci*. 90, 3526-3531, ISSN 0021-

Folate-targeted imaging of activated macrophages in rats with adjuvant-induced

the adeno-associated virus vector in experimental models of articular cartilage disorders, periprosthetic osteolysis and bone healing. *Acta Orthop Suppl,* 78, 1-64,

Maraganore, J. (2010). A status report on RNAi therapeutics. *Silence.,* 1, 14, ISSN


Sinha, V.R.; Singla, A.K.; Wadhawan, S.; Kaushik, R.; Kumria, R.; Bansal, K. & Dhawan, S.

Sinnott, M.L. (1990). Catalytic mechanism of enzymic glycosyl transfer. *Chem. Rev.*, 90, 1171–

Suda, T. & Liu, D. (2007). Hydrodynamic gene delivery: its principles and applications. *Mol.* 

Sudimack, J. & Lee, R.J. (2000). Targeted drug delivery via the folate receptor. *Adv. Drug* 

Suh, J., Wirtz, D. & Hanes, J. (2003). Efficient active transport of gene nanocarriers to the cell nucleus. *Proc. Natl. Acad. Sci. U. S. A*, 100, 3878-3882. ISSN 2227 8424 Sun, S., Liu, W., Cheng, N., Zhang, B., Cao, Z., Yao, K., Liang, D., Zuo, A., Guo, G. & Zhang,

Sun, B.; Zhao, R.L.; Kong, F.Q.; Ren, Y.P.; Zuo, A.J.; Liang, D.C.; Zhang, J.Y. (2010).

Tahara, K., Sakai, T., Yamamoto, H., Takeuchi, H. & Kawashima, Y. (2008). Establishing

Tan, W.B., Jiang, S. & Zhang,Y. (2007). Quantum-dot based nanoparticles for targeted

Tang, E.S.K.; Huang, M. & Lim, L.Y. (2003). Ultrasonication of chitosan and chitosan

Techaarpornkul, S., Wongkupasert, S., Opanasopit, P., Apirakaramwong, A., Nunthanid, J.

Terbojevich, M.; Cosani, A. & Muzzarelli, R.A.A. (1996). Molecular parameters of chitosans

Tiera, M.J.; Qiu, X.P.; Bechaouch, S.; Shi, Q.; Fernandes, J.C. & Winnik, F.M. (2006).

Tolaimate, A.; Desbrieres, J.; Rhazia, M. & Alagui, A. (2003). Contribution to the preparation

Tong, H., Qin, S., Fernandes, J.C., Li, L., Dai, K. & Zhang, X. (2009). Progress and prospects

chitosan from squid chitin. *Polymer,* 41, 2463–2469, ISSN 0032-3861

for gene transfection. *Bioconjug. Chem.,* 16, 972-980, ISSN 1043-1802

CS/DNA complex. *Int. J. Pharm.,* 397: 206-210, ISSN 0378-5173

nanoparticles. *Int. J. Pharm*., 265,103–114, ISSN 0378-5173

J. (2005). A thermoresponsive chitosan-NIPAAm/vinyl laurate copolymer vector

Phosphorylatable short peptide conjugation for facilitating transfection efficacy of

chitosan coated PLGA nanosphere platform loaded with wide variety of nucleic acid by complexation with cationic compound for gene delivery. *Int. J. Pharm.,* 354,

silencing of HER2/neu gene via RNA interference. *Biomaterials,* 28, 1565-1571, ISSN

& Ruktanonchai, U. (2010). Chitosan-mediated siRNA delivery in vitro: effect of polymer molecular weight, concentration and salt forms. *AAPS. PharmSciTech.,* 11,

depolymerized with the aid of papain, *Carbohydr. Polym.,* 29*,* 63-68, ISSN 0144-8617

Synthesis and Characterization of Phosphorylcholine-Substituted Chitosans Soluble in Physiological pH Conditions. *Biomacromolecules*, 7, 3151-3156, ISSN 1525-7797 Tolaimate, A.; Desbrieres, J.; Rhazi, M.; Alagui, A.; Vincendon, M. & Vottero, P. (2000). On

the influence of deacetylation process on the physicochemical characteristics of

of chitins and chitosans with controlled physico-chemical properties, *Polymer,* 44,

of chitosan and its derivatives as non-viral gene vectors in gene therapy. *Curr. Gene* 

33, ISSN 0378-5173

1202, ISSN 0009-2665

210-216, ISSN 0378-5173

64-72, ISSN 1530-9932

7939–7952. ISSN 0032-3861

*Ther.,* 9, 495-502, ISSN 1566-5232

1043-1802

*Ther.,* 15, 2063-2069, ISSN 1525-0016

*Deliv. Rev.,* 41, 147-162, ISSN 0169-409X

(2004). Chitosan microspheres as a potential carrier for drugs. *Int. J. Pharm. 274,* 1-


**20** 

*Germany*

*Helmholtz Zentrum, München* 

**Toxicity of Polymeric-Based Non-Viral Vector** 

**Systems for Pulmonary siRNA Application** 

*1Comprehensive Pneumology Center, Institute of Lung Biology and Disease* 

*2Department of Pharmaceutics and Biopharmacy, Philipps-University Marburg* 

Nanomedicine has the potential of clinical benefit by combination of engineering technologies and materials (Schatzlein, 2006). Development of nanometre scaled therapeutics which provides new and improved properties by specifically targeting the site of action and causing low level of side effects would be a big challenge to treat patients with severe and live-threatening diseases like cancer. Gene therapy provides a new way to treat patients and a lot of effort is made to improve the clinical benefit. But current gene therapy is still experimental and has not proven success in the clinics. Nevertheless there is a need for new approaches to treat "undruggable" disease sites and there are some clinical trials

RNA interference (RNAi), the Nobel Prize winning mechanism for gene silencing (Fire *et al.*, 1998), raises nowadays increasing attention of many researchers as a new way to treat lifethreatening diseases like cancer (Akhtar, 2006) or other genetic disorders like cystic fibrosis (Griesenbach and Alton, 2009) or viral infection as respiratory syncytial virus (RSV) (Ge *et al.*, 2004) and as an in vitro research tool to investigate mechanisms which are involved in those diseases. Small interfering RNA (siRNA) duplexes of 19-23 base pairs could trigger sequence specific gene silencing in mammalian cells (Caplen *et al.*, 2001; Elbashir *et al.*, 2001; Hannon and Rossi, 2004; Meister *et al.*, 2004; Mello and Conte, 2004). The siRNAs are double stranded molecules, consisting of a guide strand that is perfectly complementary to a target mRNA and a passenger strand. Core components of this siRNA-mediated posttranscriptional silencing include the RNAse III enzyme Dicer and its co-factor transactivating response RNA-binding protein (TRBP) along with the Argonaute family of proteins, in particular Argonaute 2 (Ago 2) (Meister *et al.*, 2004), which is the catalytic engine of the RNA induced silencing complex (RISC). Dicer converts dsRNA into 21-25 nucleotide duplexes with 3' 2nt overhangs. The siRNA is incorporated into one or more of the Argonaute proteins in RISC for sequence specific target degradation or translational inhibition (Tuschl *et al.*, 1999). In general, perfect or near perfect base pairing between the siRNA guide strand and the target mRNA is required for Ago2 cleavage to occur. In

ongoing which using RNA inference (RNAi) as therapeutic mechanism (Table 1).

**1. Introduction** 

**2. Gene silencing by siRNAs** 

**2.1 RNA interference** 

Andrea Beyerle1,2, Thomas Kissel2 and Tobias Stoeger1,2

