743 Analysis of Intracellular Trafficking of MPC Coated Multifunctional Envelope Type Nano Device (MPC MEND) in Isolated Hepatocyte Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The[.]
CHEMICAL AND MOLECULAR CONJUGATES III brillary acidic protein (GFAP) promoter was inserted into pRNAT H1.1 for HSC-specic TGF-β1 gene silencing to avoid nonspecic inhibition of TGF-β1 expression to avoid nonspecic inhibition in other liver cells, or other cells outside the nervous system Two of the most potent shRNAs were rst inserted into a pSilencer 1.0 vector to co-express and target two different regions of TGF-β1 mRNA, which was found to efciently inhibit collagen and TGF-β1 gene expression in HSC-T6 cell lines These two shRNA sequences were also inserted into the GFAP-pRNAT vector to achieve both high effectiveness and cell-specic gene silencing We are now testing whether these shRNA vectors will reduce liver injury and brosis when expressed in common bile duct ligated (CBDL) mice Mel28mut or HMGA2β) spheres are shown Figure shows that microinjected 100 nm spheres initially spread in the cytoplasm Upon cell division, the nanospheres accumulated in a specic perinuclear region Nuclear enclosure was never observed Possibly the spheres end up in the golgi apparatus which prevents nuclear inclusion Therefore, reaching the nucleoplasm seems to be very difcult Also the Xenopus nuclear envelope assembly assay does not seem to be representative for the situation in living cells Chemical and Molecular Conjugates III 742 Nuclear Enclosure of Nanospheres in Articial Nuclei and in Living Cells Nathalie Symens, Katrien Remaut, Jo Demeester, Stefaan C De Smedt Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Ghent, Belgium AIM Little is known on the nuclear inclusion of nanoparticles during cell division We were interested to what extent nanospheres get enclosed in articial Xenopus nuclei and nuclei of dividing cells We investigated the inuence of size and charge of the nanospheres and if the enclosure was enhanced by chromatin binding peptides MATERIAL AND METHODS Non-targeted positively charged, poly-ethyleenglycol (PEG)ylated and negatively charged green fluorescent polystyrene nanospheres (Molecular Probes®) of 100, 200 or 500 nm were used The 100 nm nanospheres were also modified with Mel-28 (GPSKPRGRPPKHKAKT), mutated Mel-28 (GPSKPGGGPPGHKAKT) or HMGA2β (SPKRPRGRPKGSKNKS), containing an AT-hook or a mutated AT-hook (targeted nanospheres) Articial nuclei are obtained with the ‘Xenopus nuclear envelope assembly assay’ The enclosure of the nanospheres in the articial nuclei and upon microinjection is visualised with confocal microscopy RESULTS Figure 1A shows an artical nucleus obtained in the Xenopus nuclear envelope assembly assay Figure 1B shows the percentage of nuclei containing 0, 1-5, 6-10 or >10 nanospheres The enclosure in articial nuclei is limited, with the positively charged spheres being better then the negatively charged and the PEG-ylated ones, likely due to aspecic interactions with the netto negatively charged chromatin Apart from the charge, also size matters: spheres of 200 nm and 100 nm are better enclosed than the 500 nm ones Figure 1B also shows that the enclosure of 100 nm targeted spheres is indeed higher than for the non-targeted ones and those modied with the mutated AT-hook Fig1 A) Confocal image of artificial Xenopus nuclei Blue: chromatin; orange: membranes; green: non-targeted spheres (100 nm, positive) B) Percentage of nuclei containing 0, 1-5, 6-10 and >10 spheres Non-targeted of different size (100, 200 or 500 nm) and ζ-potential (positive, pegylated or negative) and targeted (Mel28, Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy Fig2 Microinjection of HeLa cells with non-targeted nanospheres (100 nm, positive, green), co-injected with 70 kDa TRITC-dextran (orange) 743 Analysis of Intracellular Trafcking of MPC-Coated Multifunctional Envelope-Type Nano Device (MPC-MEND) in Isolated Hepatocyte Masami Ukawa,1 Hidetaka Akita,1 Tomoya Masuda,1 Yasuhiro Hayashi,1 Tomohiro Konno,2 Kazuhiko Ishihara,2 Hideyoshi Harashima.1 Hokkaido University, Sapporo, Japan; 2The University of Tokyo, Tokyo, Japan Liver plays an important role in homeostasis of body and detoxification by its extensive activities of biosynthesis and metabolism Liver-targeting gene delivery would be one of the promising strategies for a cure of these intractable diseases We developed a nuclear delivery system where we referred to as multifunctional envelope-type nano device (MEND) For an in vivo application, serum-resistant cationic lipids were used as envelope lipid composition Plasmid DNA (pDNA) was condensed with protamine at +/- ratio to form negatively charged core particle To improve an endosomal release, pH-dependently fusogenic peptide (GALA) was modied on the envelope by incorporation of cholesteryl-modied peptide into a lipid lm (GALA-MEND) Here, we found that coating with butylated 2-Methacryloyloxyethyl PhosphorylCholine (MPC) drastically increased the hepatic gene expression in vivo after intraveneous administration (MPC/GALA-MEND) Thus, intracellular trafcking of pDNA was compared among MEND, GALA-MEND and MPC/GALA-MEND in primary hepatocyte in vitro to clarify the mechanism Step-wise argumentation of transgene expression by modication with GALA and MPC was reproduced also in vitro studies using primary hepatocyte Intracellular imaging showed that MPC polymers were dominantly trapped in endosome/lysosome compartment These data indicate that MPC may not directly affect the subsequent nuclear delivery processes Moreover, MENDs could escape from endosomes comparably Thus it is too simple to conclude that high gene expression in MPC/GALA-MEND is attributed to the altered intracellular trafcking The transgene expression efciency in postnuclear transfer processes (TEnuc), which is dened as transfection activity divided by the amount of nucleus-delivered pDNA was elevated in logarithmic scale in the rank order of MPC/GALAMEND > GALA-MEND > MEND Imaging of dually labeled MENDs (rhodamine in pDNA and NBD in lipid envelope) shows that intracellular dissociation of pDNA is enhanced in the rank S289 CHEMICAL AND MOLECULAR CONJUGATES III order of MPC/GALA-MEND > GALA-MEND > MEND Since modication of MPC decrease the Z-potential of the particle, MPC may assist the effective display of GALA, a peptide composed of a negatively charged peptide Correctively, the high transfection activity and TEnuc in MPC/GALA-MEND can be explained by assuming that MPC may assist the intracellular dissociation by stimulating the GALA-mediated membrane fusion process between lipid envelope and endosome As a consequence intracellular translation process may be improved by avoiding the extensive interaction between mRNA and cationic lipid components 744 Photo-Activated Release of DNA from Liposome Complexes Caroline M LaManna,1 Jiazuo H Feng,1 Hrvoje Lusic,1 Mark W Grinstaff.1 Biomedical Engineering and Chemistry, Boston University, Boston, MA To improve gene transfection activity using nonviral vectors (i.e cationic amphiphiles, dendrimers, and polymers), synthetic carriers are being engineered to overcome the extra- and intracellular barriers of the transfection pathway Though cationic amphiphiles electrostatically interact with DNA and protect DNA as it moves into the cell, it is also necessary to release the DNA to facilitate gene transcription We have previously developed charge-reversal amphiphiles which have a net change in charge from positive to negative, thus resulting in DNA release This charge-reversal was dependent on enzymatic cleavage by esterases In this work we propose the use of light to control the charge-reversal effect of our new amphiphiles, in order to improve gene transfection efciency We have developed peptide based liposomes which bind DNA and disassemble upon photolysis By using dynamic light scattering we have analyzed the liposomes before and after photolysis Also, an ethidium bromide exclusion uorescence quenching assay has been utilized to characterize the resulting liposome-DNA complex (lipoplexes) Our results demonstrate that UV irradiation can efciently cleave the photo-active terminal groups on our amphiphiles, which affords liposome destabilization and DNA release from the supramolecular structure Use of these functional amphiphiles represents a new approach to gene delivery 745 A Novel Cationic Lipophosphoramide with Di-Unsaturated Lipid Chains: Synthesis, PhysicoChemical Properties, and Transfection Activities Tony Le Gall,1,3 Damien Loiseau,2 Erwan Picquet,2 Nathalie Carmoy,1,3 Jean Jacques Yaouanc,2 Laure Burel Deschamps,3 Pascal Delépine,1,3 Philippe Giamarchi,2 Paul Alain Jaffrès,2 Pierre Lehn,1,3 Tristan Montier.1,3 Gene Therapy Team, INSERM U613, Brest, France; 2Laboratoire CEMCA, CNRS UMR 6521, Brest, France; 3IBiSA SynNanoVect platform, IFR 148 ScInBIoS, Brest, France Cationic lipophosphoramidates constitute a class of cationic lipids we have previously reported to be efcient for gene transfection Here, we synthesized and studied a novel lipophosphoramidate derivative characterized by an arsonium headgroup linked, via a phosphoramidate linker, to an unconventional lipidic moiety consisting of two di-unsaturated linoleic chains Physicochemical studies allowed us to comparatively evaluate the specic uidity and fusogenicity properties of the liposomes formed Although corresponding lipoplexes exhibited signicant but relatively modest in vitro transfection efciencies, they showed a remarkably efcient and reproducible ability to transfect mouse lung, with in vivo transfection levels higher than those observed with a mono-unsaturated analogue previously described Thus, these results demonstrate that this diunsaturated cationic lipophosphoramidate constitutes an efcient S290 and versatile nonviral-vector for gene transfection They also invite further evaluations of the transfection activity, especially in vivo, of gene delivery systems incorporating the lipid reported herein and/or other lipids bearing poly-unsaturated chains 746 Modifying DNA Nanoparticles with Biotin for Versatile Ligand Substitution Wenchao Sun,1 Pamela B Davis.1 Department of Biochemistry and Department of Pediatrics, Case Western Reserve University, Cleveland, OH DNA nanoparticles (DNA NPs), formulated by compacting DNA plasmids with polylysine-poly(ethylene glycol) (PEG) block copolymer, have been successfully used to transfect multiple cell types in vitro and in vivo To further enhance the gene transfer efciency and control the tropism of the vector in vivo, targeting of DNA NPs with ligands is attempted in this study To preserve the overall structure of the NP and ensure the availability of functional ligands on the surface, adding the ligands to the free end of the PEG chain in a postDNA compaction fashion is preferred The noncovalent biotin-avidin interaction was used to link ligands with the DNA NPs, because this strategy avoids the problems encountered in covalent conjugation methods, including lack of suitable reactive groups on the polymer, unstable intermediates, and inefcient coupling and purication In a one step reaction, a 30-mer of polylysine with an N-terminal cysteine (CK30) was coupled to heterobifunctional PEG, maleimidePEG-biotin, to produce a triblock copolymer biotin-PEG-CK30 which was puried by ion exchange chromatography The intactness of biotin was conrmed by 1H-NMR The conjugation was also tested by transferring the polymer from an acid-urea gel to a membrane and blotting the membrane with peroxidase-conjugated streptavidin Luminescent signal was detected only from biotin-PEG-CK30, but not from PEG-CK30, free biotin or a mixture of free biotin and PEGCK30 Biotin-PEG-CK30 was then used to compact DNA plasmids into biotin-DNA NPs which showed identical colloidal and morphological properties compared to non-targeted DNA NPs in sedimentation assay and electron microscopy Two assays were carried out to test whether functional biotin was available for binding In the rst assay, DNA NPs were incubated with NeutrAvidin immobilized on agarose beads DNA was pulled down by the beads only when the DNA NPs were biotinylated Blocking the beads with free biotin or pretreating the biotin-DNA NPs with NeutrAvidin signicantly reduced the amount of DNA pulled down In the second assay, the binding of uorophore-conjugated streptavidin to the biotin-DNA NPs was tested The biotin-DNA NPs were immobilized on the NeutrAvidin beads, which enabled a subsequent washing step and visualization by uorescence microscopy Beads were uorescently decorated only when incubated with biotin-DNA NPs in the presence of the uorophore-streptavidin conjugate, but not with plain DNA NPs or free biotin-PEG-CK30, which further demonstrated that the biotin on the DNA NPs were accessible and fully functional After initial testing with biotin targeting alone, future studies will focus on developing a ligand-NeutrAvidin fusion protein Since each NeutrAvidin is composed of four subunits, each fused to a ligand, a clustering effect will be achieved Whether ligand clustering will have benets for gene delivery will be tested Once established, this strategy is widely applicable to conjugation of different types of ligands (peptide, protein or uorescent labels) to DNA NPs Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ... CONJUGATES III order of MPC/ GALA-MEND > GALA-MEND > MEND Since modication of MPC decrease the Z-potential of the particle, MPC may assist the effective display of GALA, a peptide composed of a negatively... activity and TEnuc in MPC/ GALA-MEND can be explained by assuming that MPC may assist the intracellular dissociation by stimulating the GALA-mediated membrane fusion process between lipid envelope and... invite further evaluations of the transfection activity, especially in vivo, of gene delivery systems incorporating the lipid reported herein and/or other lipids bearing poly-unsaturated chains