approach to synthesize POEGMA conjugates from the C-terminus of arecombinant anti-diabetic peptide drug Exendin. Our preliminary datademonstrate that the Exendin–POEGMA conjugates retained thereceptor-activation activity of Exendin and showed significantlyimproved control of blood glucose level in a db/db mouse model. Inconclusion, we report a novel strategy to directly grow stealth polymerconjugates at the N- or C-terminus of proteins with significantlyimproved pharmacology.

Scheme 1. In situ growth of stealth polymer conjugates from the N- or C-terminal ends ofproteins.

Keywords: Polymer–protein conjugate, Polymer engineering, Proteinengineering, Drug delivery

References[1] W. Gao, W. Liu, T. Christensen, M.R. Zalutsky, A. Chilkoti, In situ growth of a

PEG-like polymer from the C-terminus of an intein fusion protein improvespharmacokinetics and tumor accumulation, Proc. Natl. Acad. Sci. U. S. A. 107(2010) 16432–16437.

[2] W. Gao, W. Liu, J.A. Mackay, M.R. Zalusky, E.J. Toone, A. Chilkoti, In situgrowth of a stoichiometric PEG-like conjugate at a protein's N-terminus withsignificantly improved pharmacokinetics, Proc. Natl. Acad. Sci. U. S. A. 106 (2009)15231–15236.

doi:10.1016/j.jconrel.2013.08.279

Modification of PEI with cyclodextrin as a tool for betterunderstanding the major barriers for DNA delivery

Wenyu Li, Lu Yang, Youxiang Wang⁎

MOE Key Laboratory of Macromolecular Synthesis and Functionalization,Department of Polymer Science and Engineering, Zhejiang University,Hangzhou 310027, ChinaE-mail address: yx_wang@zju.edu.cn (Y. Wang).

Cyclodextrin (CD) as a kind of polysaccharide has been widely usedto modify polycations due to their low cytotoxicity, low immunogenic-ity and improved transfection efficiency [1]. It was found that PEI(25 kDa)with a CD-grafting level (2.5%, named PEI-CD15)was favorablefor caveolae-mediated endocytosis with improved transfection effi-ciency compared to that of PEI [2]. However, our recent researchindicated that PEI with a higher CD-grafting level (7.1%, PEI-CD41) couldnot condense DNA into small nanoparticles at pH 7.4. The significantdifference caused by the small change of CD-grafting level wassurprising and investigated in detail. It was assumed that the reasonfor these differences was based on the hydrogen bond interactionsbetween CD and PEI. To confirm this, ferrocene modified PEG (PEG-Fc)was synthesized and used to obstruct the hydrogen bond interactions. Itwas found that PEI-CD41/PEG-Fc supramolecular polymer couldcondense DNA into small-sized particles both at pH 7.4 and 6.0. Onthe other hand, by changing the pH from 7.4 to 6.0, the hydrogen bondinteractions between PEI and CD might reduce. Therefore, PEI-CD41/DNA polyplexes were obtained with a diameter of about 140 nm.However, the PEI-CD41/pDNA and PEGylated polyplexes with compara-ble size showed lowgene expression. Subsequently, several critical extra-and intracellular barriers including extracellular stability, endosomalescape for cytoplasmic release and gene entry into the nucleus werethoroughly investigated. The results indicated that complex disassemblyfor gene release and entry into the nucleuswas themost critical factor for

gene expression (Fig. 1). This report provides good proof for betterunderstanding the barriers for DNA delivery and designing non-viralvectors with efficient gene expression.

Fig. 1. CLSM images of cells exposed to PEI-CD15 (a), PEI-CD41 (b) and PEI-CD41/PEG-Fc (c)complexedwith FITC-DNA (green) for 12 h incubation. DAPIwas used to stain the cell nuclei(blue).

Keywords: Gene delivery, Polyethylenimine, Cyclodextrins, Barriers

References[1] Y. Ping, C.D. Liu, Z.X. Zhang, K.L. Liu, J.H. Chen, J. Li, Chitosan-graft-(PEI-beta-

cyclodextrin) copolymers and their supramolecular PEGylation for DNA andsiRNA delivery, Biomaterials 32 (2011) 8328–8341.

[2] W.Y. Li, L.N. Chen, Z.X. Huang, X.F. Wu, Y.F. Zhang, Q.L. Hu, Y.X. Wang, Theinfluence of cyclodextrin modification on cellular uptake and transfectionefficiency of polyplexes, Org. Biomol. Chem. (2011) 7799–7806.

doi:10.1016/j.jconrel.2013.08.280

Investigation of siRNA loading rate and cell internalization ofchitosan nanoparticles

DemengZhanga,b, Xiudong Liuc,⁎, YanYanga,b,WeitingYua, XiaojunMaa,⁎aLaboratory of Biomedical Material Engineering, Dalian Institute ofChemical Physics, Chinese Academy of Sciences, Dalian 116023, ChinabGraduate School of the Chinese Academy of Sciences, Beijing 100039,ChinacCollege of Environment and Chemical Engineering, Dalian University,Dalian 116622, ChinaE-mail addresses: liuxd@dicp.ac.cn (X. Liu), maxj@dicp.ac.cn (X. Ma).

Chitosan as one of the polymeric siRNA vectors aroused greatinterest in the past few years for its low toxicity, good biocompat-ibility and biodegradability, excellent transcellular transport ability,and easy modification [1]. It has been recognized that N/P ratio,molecule weight (Mw) and degree of deacetylation (DD) of chitosanhave a major impact on gene silencing efficiency [2].

To get insight in the relation between the preparation parametersand the final gene silencing efficiency of chitosan/siRNA nanoparticles(NPs), the loading rate (LR) of siRNA and endocytosis rate (ER) ofNPs were determined. Chitosan/siRNA NPs formulated at differentparameters were nanoscale sized (100–300 nm) with positive zeta-potentials of 10–25 mV. When the N/P ratio was over 10, the LR ofsiRNA reached a stable level above 80% (Fig. 1), which was hardlyaffected by other parameters. However, the ER of chitosan/siRNA NPsincubated with B16-F10 cells for 5 h ranged from approximately 20%to 80% demonstrating the possible obstacles for high gene silencingefficiency.

Abstracts / Journal of Controlled Release 172 (2013) e98–e124 e117