[en] This study aims at developing chitosan-based nanoparticles suitable for an intravenous administration of small interfering RNA (siRNA) able to achieve (i) high gene silencing without cytotoxicity and (ii) stability in biological media including blood. Therefore, the influence of chitosan/tripolyphosphate ratio, chitosan physicochemical properties, PEGylation of chitosan as well as the addition of an endosomal disrupting agent and a negatively charged polymer was assessed. The gene silencing activity and cytotoxicity were evaluated on B16 melanoma cells expressing luciferase. We monitored the integrity and the size behavior of siRNA nanoparticles in human plasma using fluorescence fluctuation spectroscopy and single particle tracking respectively. The presence of PEGylated chitosan and poly(ethylene imine) was essential for high levels of gene silencing in vitro. Chitosan nanoparticles immediately released siRNA in plasma while the inclusion of hyaluronic acid and high amount of poly(ethylene glycol) in the formulation improved the stability of the particles. The developed formulations of PEGylated chitosan-based nanoparticles that achieve high gene silencing in vitro, low cytotoxicity and high stability in plasma could be promising for intravenous delivery of siRNA.
Research Center/Unit :
Center for Education and Research on Macromolecules (CERM)
Disciplines :
Materials science & engineering Chemistry
Author, co-author :
Ragelle, Héloïse; Université catholique de Louvain (UCL), Brussels > Louvain Drug Research Institute (LDRI) > Pharmaceutics and Drug Delivery
Riva, Raphaël ; Université de Liège - ULiège > Department of Chemistry > Center for Education and Research on Macromolecules (CERM)
Vandermeulen, G.; Université catholique de Louvain (UCL), Brussels > Louvain Drug Research Institute (LDRI) > Pharmaceutics and Drug Delivery
Naeye, B.; Ghent University > Faculty of Pharmaceutical Sciences > Laboratory of General Biochemistry and Physical Pharmacy
Pourcelle, Vincent; Université Catholique de Louvain (UCL) > Institute of Condensed Matter and Nanosciences > Molecules, Solids and Reactivity
Le Duff, C. S.; Université Catholique de Louvain (UCL) > Institute of Condensed Matter and Nanosciences > Laboratory of General Biochemistry and Physical Pharmacy
D'Haese, C.; Université Catholique de Louvain (UCL) > Institute of Condensed Matter and Nanosciences > Bio & Soft Matter
Nysten, B.; Université Catholique de Louvain (UCL) > Institute of Condensed Matter and Nanosciences > Bio & Soft Matter
Braeckmans, K.; Ghent University > Center for Nano- and Biophotonics and Faculty of Pharmaceutical Sciences > Laboratory of General Biochemistry and Physical Pharmacy
De Smedt, S. C.; Ghent University > Faculty of Pharmaceutical Sciences > Laboratory of General Biochemistry and Physical Pharmacy
Jérôme, Christine ; Université de Liège - ULiège > Department of Chemistry > Center for Education and Research on Macromolecules (CERM)
Préat, Véronique; Université catholique de Louvain (UCL), Brussels > Louvain Drug Research Institute (LDRI) > Pharmaceutics and Drug Delivery
Language :
English
Title :
Chitosan nanoparticles for siRNA delivery: Optimizing formulation to increase stability and efficiency
A. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, and C.C. Mello Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Nature 391 1998 806 811
F. Petrocca, and J. Lieberman Promise and challenge of RNA interference-based therapy for cancer J. Clin. Oncol. 29 2011 747 754
J. Tabernero, G.I. Shapiro, P.M. Lorusso, A. Cervantes, G.K. Schwartz, G.J. Weiss, L. Paz-Ares, D.C. Cho, J.R. Infante, M. Alsina, M.M. Gounder, R. Falzone, J. Harrop, A.C. White, I. Toudjarska, D. Bumcrot, R.E. Meyers, G. Hinkle, N. Svrzikapa, R.M. Hutabarat, V.A. Clausen, J. Cehelsky, S.V. Nochur, C. Gamba-Vitalo, A.K. Vaishnaw, D.W. Sah, J.A. Gollob, and H.A. Burris III First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement Cancer Discov. 3 2013 406 417
M.A. Rahman, A.R. Amin, X. Wang, J.E. Zuckerman, C.H. Choi, B. Zhou, D. Wang, S. Nannapaneni, L. Koenig, Z. Chen, Z.G. Chen, Y. Yen, M.E. Davis, and D.M. Shin Systemic delivery of siRNA nanoparticles targeting RRM2 suppresses head and neck tumor growth J. Control. Release 159 2012 384 392
K. Raemdonck, T.F. Martens, K. Braeckmans, J. Demeester, and S.C. De Smedt Polysaccharide-based nucleic acid nanoformulations Adv. Drug Deliv. Rev. 65 2013 1123 1147
B. Ballarin-Gonzalez, and K.A. Howard Polycation-based nanoparticle delivery of RNAi therapeutics: adverse effects and solutions Adv. Drug Deliv. Rev. 64 2012 1717 1729
J. Nguyen, and F.C. Szoka Nucleic acid delivery: the missing pieces of the puzzle? Acc. Chem. Res. 45 2012 1153 1162
K.A. Howard, U.L. Rahbek, X. Liu, C.K. Damgaard, S.Z. Glud, M.O. Andersen, M.B. Hovgaard, A. Schmitz, J.R. Nyengaard, F. Besenbacher, and J. Kjems RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system Mol. Ther. 14 2006 476 484
H. Katas, and H.O. Alpar Development and characterisation of chitosan nanoparticles for siRNA delivery J. Control. Release 115 2006 216 225
J. Malmo, H. Sorgard, K.M. Varum, and S.P. Strand siRNA delivery with chitosan nanoparticles: molecular properties favoring efficient gene silencing J. Control. Release 158 2012 261 268
J.C. Fernandes, X. Qiu, F.M. Winnik, M. Benderdour, X. Zhang, K. Dai, and Q. Shi Low molecular weight chitosan conjugated with folate for siRNA delivery in vitro: optimization studies Int. J. Nanomedicine 7 2012 5833 5845
P. Holzerny, B. Ajdini, W. Heusermann, K. Bruno, M. Schuleit, L. Meinel, and M. Keller Biophysical properties of chitosan/siRNA polyplexes: profiling the polymer/siRNA interactions and bioactivity J. Control. Release 157 2012 297 304
M. Jean, M. Alameh, J.D. De, M. Thibault, M. Lavertu, V. Darras, M. Nelea, M.D. Buschmann, and A. Merzouki Chitosan-based therapeutic nanoparticles for combination gene therapy and gene silencing of in vitro cell lines relevant to type 2 diabetes Eur. J. Pharm. Sci. 45 2012 138 149
K.A. Howard Delivery of RNA interference therapeutics using polycation-based nanoparticles Adv. Drug Deliv. Rev. 61 2009 710 720
I. Nawroth, J. Alsner, B.W. Deleuran, F. Dagnaes-Hansen, C. Yang, M.R. Horsman, J. Overgaard, K.A. Howard, J. Kjems, and S. Gao Peritoneal macrophages mediated delivery of chitosan/siRNA nanoparticle to the lesion site in a murine radiation-induced fibrosis model Acta Oncol. 52 2013 1730 1738
H.D. Han, L.S. Mangala, J.W. Lee, M.M. Shahzad, H.S. Kim, D. Shen, E.J. Nam, E.M. Mora, R.L. Stone, C. Lu, S.J. Lee, J.W. Roh, A.M. Nick, G. Lopez-Berestein, and A.K. Sood Targeted gene silencing using RGD-labeled chitosan nanoparticles Clin. Cancer Res. 16 2010 3910 3922
Y.K. Kim, A. Minai-Tehrani, J.H. Lee, C.S. Cho, M.H. Cho, and H.L. Jiang Therapeutic efficiency of folated poly(ethylene glycol)-chitosan-graft- polyethylenimine-Pdcd4 complexes in H-ras12V mice with liver cancer Int. J. Nanomedicine 8 2013 1489 1498
V. Dehousse, N. Garbacki, S. Jaspart, D. Castagne, G. Piel, A. Colige, and B. Evrard Comparison of chitosan/siRNA and trimethylchitosan/siRNA complexes behaviour in vitro Int. J. Biol. Macromol. 46 2010 342 349
L. Casettari, D. Vllasaliu, G. Mantovani, S.M. Howdle, S. Stolnik, and L. Illum Effect of PEGylation on the toxicity and permeability enhancement of chitosan Biomacromolecules 11 2010 2825 3218
P.P. Lv, Y.F. Ma, R. Yu, H. Yue, D.Z. Ni, W. Wei, and G.H. Ma Targeted delivery of insoluble cargo (paclitaxel) by PEGylated chitosan nanoparticles grafted with Arg-Gly-Asp (RGD) Mol. Pharm. 9 2012 1736 1747
M. Malhotra, C. Lane, C. Tomaro-Duchesneau, S. Saha, and S. Prakash A novel method for synthesizing PEGylated chitosan nanoparticles: strategy, preparation, and in vitro analysis Int. J. Nanomedicine 6 2011 485 494
A. Akinc, M. Thomas, A.M. Klibanov, and R. Langer Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis J. Gene Med. 7 2005 657 663
R.V. Benjaminsen, M.A. Mattebjerg, J.R. Henriksen, S.M. Moghimi, and T.L. Andresen The possible "proton sponge" effect of polyethylenimine (PEI) does not include change in lysosomal pH Mol. Ther. 21 2013 149 157
H.Q. Mao, K. Roy, V.L. Troung-Le, K.A. Janes, K.Y. Lin, Y. Wang, J.T. August, and K.W. Leong Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency J. Control. Release 70 2001 399 421
A.K. Varkouhi, R.J. Verheul, R.M. Schiffelers, T. Lammers, G. Storm, and W.E. Hennink Gene silencing activity of siRNA polyplexes based on thiolated N,N,N-trimethylated chitosan Bioconjug. Chem. 21 2010 2339 2346
Y. Luo, X. Zhai, C. Ma, P. Sun, Z. Fu, W. Liu, and J. Xu An inhalable beta(2)-adrenoceptor ligand-directed guanidinylated chitosan carrier for targeted delivery of siRNA to lung J. Control. Release 162 2012 28 36
D. Jere, H.L. Jiang, Y.K. Kim, R. Arote, Y.J. Choi, C.H. Yun, M.H. Cho, and C.S. Cho Chitosan-graft-polyethylenimine for Akt1 siRNA delivery to lung cancer cells Int. J. Pharm. 378 2009 194 200
F. Kong, G. Liu, B. Sun, S. Zhou, A. Zuo, R. Zhao, and D. Liang Phosphorylatable short peptide conjugated low molecular weight chitosan for efficient siRNA delivery and target gene silencing Int. J. Pharm. 422 2012 445 453
S. Al-Qadi, M. Alatorre-Meda, E.M. Zaghloul, P. Taboada, and C. Remunan-Lopez Chitosan-hyaluronic acid nanoparticles for gene silencing: the role of hyaluronic acid on the nanoparticles' formation and activity Colloids Surf. B: Biointerfaces 103C 2012 615 623
M. Rinaudo, M. Milas, and D.P. Le Characterization of chitosan. Influence of ionic strength and degree of acetylation on chain expansion Int. J. Biol. Macromol. 15 1993 281 285
D.P. Cioca, Y. Aoki, and K. Kiyosawa RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines Cancer Gene Ther. 10 2003 125 133
N.J. Caplen, S. Parrish, F. Imani, A. Fire, and R.A. Morgan Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems Proc. Natl. Acad. Sci. U. S. A. 98 2001 9742 9747
K. Buyens, B. Lucas, K. Raemdonck, K. Braeckmans, J. Vercammen, J. Hendrix, Y. Engelborghs, S.C. De Smedt, and N.N. Sanders A fast and sensitive method for measuring the integrity of siRNA-carrier complexes in full human serum J. Control. Release 126 2008 67 76
K. Braeckmans, K. Buyens, W. Bouquet, C. Vervaet, P. Joye, F. De Vos, L. Plawinski, L. Doeuvre, E. Angles-Cano, N.N. Sanders, J. Demeester, and S.C. De Smedt Sizing nanomatter in biological fluids by fluorescence single particle tracking Nano Lett. 10 2010 4435 4442
L. Casettari, D. Vllasaliu, G. Mantovani, S.M. Howdle, S. Stolnik, and L. Illum Effect of PEGylation on the toxicity and permeability enhancement of chitosan Biomacromolecules 11 2010 2854 2865
P. Holzerny, B. Ajdini, W. Heusermann, K. Bruno, M. Schuleit, L. Meinel, and M. Keller Biophysical properties of chitosan/siRNA polyplexes: profiling the polymer/siRNA interactions and bioactivity J. Control. Release 157 2012 337 345
S.K. Tripathi, R. Goyal, P. Kumar, and K.C. Gupta Linear polyethylenimine-graft-chitosan copolymers as efficient DNA/siRNA delivery vectors in vitro and in vivo Nanomedicine 8 3 2011 337 345
M. Ravina, E. Cubillo, D. Olmeda, R. Novoa-Carballal, E. Fernandez-Megia, R. Riguera, A. Sanchez, A. Cano, and M.J. Alonso Hyaluronic acid/chitosan-g-poly(ethylene glycol) nanoparticles for gene therapy: an application for pDNA and siRNA delivery Pharm. Res. 27 2010 2544 2555
S. Mao, W. Sun, and T. Kissel Chitosan-based formulations for delivery of DNA and siRNA Adv. Drug Deliv. Rev. 62 2010 12 27
V. Dehousse, N. Garbacki, A. Colige, and B. Evrard Development of pH-responsive nanocarriers using trimethylchitosans and methacrylic acid copolymer for siRNA delivery Biomaterials 31 2010 1839 1849
H. Ragelle, G. Vandermeulen, and V. Preat Chitosan-based siRNA delivery systems J. Control. Release 172 2013 207 218
I. Richard, M. Thibault, C.G. De, M.D. Buschmann, and M. Lavertu Ionization behavior of chitosan and chitosan-DNA polyplexes indicate that chitosan has a similar capability to induce a proton-sponge effect as PEI Biomacromolecules 14 2013 1732 1740
A.M. Portis, G. Carballo, G.L. Baker, C. Chan, and S.P. Walton Confocal microscopy for the analysis of siRNA delivery by polymeric nanoparticles Microsc. Res. Tech. 73 2010 878 885
T. Miller, A. Hill, S. Uezguen, M. Weigandt, and A. Goepferich Analysis of immediate stress mechanisms upon injection of polymeric micelles and related colloidal drug carriers: implications on drug targeting Biomacromolecules 13 2012 1707 1718
K. Buyens, M. Meyer, E. Wagner, J. Demeester, S.C. De Smedt, and N.N. Sanders Monitoring the disassembly of siRNA polyplexes in serum is crucial for predicting their biological efficacy J. Control. Release 141 2010 38 41
W.A. Dufay, H. Hillaireau, T.L. Nascimento, S. Arpicco, M. Taverna, S. Ribes, M. Bourge, V. Nicolas, A. Bochot, C. Vauthier, N. Tsapis, and E. Fattal Hyaluronic acid-bearing lipoplexes: physico-chemical characterization and in vitro targeting of the CD44 receptor J. Control. Release 162 2012 545 552
R.A. D'Sa, P.J. Dickinson, J. Raj, B.K. Pierscionek, and B.J. Meenan Inhibition of lens epithelial cell growth via immobilisation of hyaluronic acid on atmospheric pressure plasma modified polystyrene Soft Matter 7 2010 608 617
