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893. Mechanism of Escape from Macropinosomes for Octaarginine Modified Nanoparticles
NON-VIRAL DELIVERY STRATEGIES
893. Mechanism of Escape from Macropinosomes for Octaarginine Modified Nanoparticles
Ayman El-Sayed.!- Ikramy A. Khalil ,I.2 Kentaro Kogure.P Hideyoshi Harashima.P }Faculty ofPharmaceutical Sciences, Hokkaido University, Sapporo . Hokkaido, Japan ; "The Core Research for Evohuional Science and Technology (CREST). Japan Science and Technology Agency (JST). To1..J1o. Japan. Background: Recently, anonviral multifunctional envelope-type nano device (MEND) was developed in our laboratory, and showed a high transfection activity comparable to that ofadenovirus III. Uptake of MEND modified with high density of octaarginine (RS-MEND) was shown to take place through macropinocytosis 121. However, the mechanism by which this RS-MEND can escape from macropinosomes remained unclear. Shedding light on this mechanism can result in more optimization ofthe currently present nonviral vectors as well as development of more efficient vectors in the future. In this study we conduct a stepwise dissection of the mechanism ofescape from macropinosome for RS-MEND in comparison to MEND modified with octalysine (KS-MEND). Methods: FACS analysis was used to determine extent ofuptake ofdifferent MENDs. The intracellular trafficking of MENDs encapsulating labeled DNA was tracked in NIH 3'1'3cells using confocal laser scanning microscopy (CLSM). Transfection activity of luciferase DNA-containing MENDs was determined in NIH 3T3 cells in presence ofvarious inhibitors. To test the ability of octaarginine to destabilize membranes, calcein leakage experiment was conducted using calcein loaded liposomes of negatively charged surface similar to that of cell membranes, To study fusion of MEND to macropinosomal membrane, FRET analysis of double labeled particles was conducted in living celIs using spectral imaging nuorescence microscopy. Results: The uptake of both RS and KS MENDs were found comparable, meanwhile transfection activity of RS-MEND was more than 16 times higher than that of KS-MEND, and 320 times higher than that of unmodified MEND. Transfection activity of KS-MEND was significantly enhanced by chloroquine meanwhile that of RS-MEND was not affected indicating superior escape of RS-MEND from macropinosomes: CLSM confirmed that RS-MEND escaped more efficiently from vesicular compartments than KS-MEND and this escape ability was significantly reduced in presence of Bafilomycin A, a specific acidification inhibitor. This was confirmed by strong inhibition of RS-MEND transfection activity in presence of Bafilomycin A or ammonium chloride. Calcein release experiment showed that RS modified liposomes released calccin more efficiently than KS modified liposomes at acidic pH. Spectral imaging proved that RS-MEND fused to macropinosomal membrane during its escape process. Conclusion: Our data highlight that compared to KS, RS modification of MEND enhances its escape efficiency from the vesicular compartments. Mechanism of escape was dependent on acidification of macropinosome and R8 modification improved fusion of MEND lipid coat to the macropinosome during its escape to cytosol. References: [1] Khalil lA, Kogure K, Harashima H, et al. (2007) Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery, Gene Ther. (In Press). [2] Khalil lA, Kogure K, Futaki S, Harashima I-I. (2006) High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression, J Bioi Chern. 281 :3544-3551.
Molecular Therapy Volume 15.Supplem ent I. M;'l"2007
Coprri ght © The American Society of G ene Th erapy
894. Novel Approach for the Nuclear Delivery of Macromolecule Via Membrane Fusion in NonDividing Cell Asako Kudo ,' Hidctaka Akita ,' Arisa Minoura ,' Kentaro Kogurc ,' Masaya Yamaguchi ,' Ikramy A. Khalil,' Tomoya Masuda,' Hideyoshi Harshirna.' 'Graduate School ofPharmaceutical Sciences . Hokkaido University, Sapporo . Japan . Background Transfection activity of non-viral vector is ratelimited by various intracellular processes. Especially, the transport of plasmid DNA from the cytoplasm into the nucleus across the nuclear membrane remains severe barrier in non-dividing cells. To date , so many challenges have been made to overcome this barrier by the conjugation ofnuclear localization signals (NLS), which aim to be recognized by importins, and permitting them to pass through the nuclear pore complex (NPC) . Here, we present a novel strategy for the nuclear delivery of plasmid DNA via NPC-independent pathway via membrane fusion. To realize this strategy, we also developed novel multi-layered envelope type gene vector (Tetralamellar Multifunctional Envelope Type Nano Devise; T-MEN D), in which pDNAIpolycation core is coated with different kinds of lipid membrane. Methods Various kinds ofliposomes labeled with NBD and Rhodamine were prepared, fusion activity between nuclear membrane and prepared liposomes were evaluated by the release of FRET after the incubation with isolated nucleus. To visualize a fusion event, the OFP-encapsulating and rhodamine-labeled liposome liposomes were incubated with isolated nucleus, and then confocal images were obtained by CLSM. T-MEND is composed of two kinds of lipid, endosomal or nuclear membrane fusogenic lipid. To investigate the transfection efficiency, T-MEND was prepared, in which pDNA core was coated with nuclear membrane-fusogenic lipid, and then additionally coated with endosome-fusogenic lipid. Tranfection activity was evaluated with JAWSII cells. a model of non-dividing cell. Fusion ofT-MEND with each organelle in JAWSII cell were proved by spectral imaging ofFRET. Results and Discussion Screening ofliposomes by means ofthe FRET analysis revealed th~t 2 kinds oflipid composition arc highly potent in fusion activity WIth nuclear membrane. After the incubation ofisolated nucleus with OFP-encapsulating and rhodamine-labeled multi lamellar vesicle (MLV) prepared with nucleus-fusogenic lipids, OFP was detected inside of nucleus whereas labeled lipids was distributed along the nuclear membrane. When the experiments were demonstrated with unilamellar vesicle (SUV) , OFP and lipid labeling was co-localized along nuclear structure, suggesting that OFP was trapped between inner and outer membrane. These data Indicated that nuclear transport of macromolecules can be achieved by encapsulating into the multilayered liposomes composed of nucleus-fusogenic lipids. Oene transfer efficiency with a T-MEND composed of nuclear and endosome-fusogenic lipid enhanced gene delivery compared to the conventional MEND in JAWSII cell. With spectral imaging, fusion of lipid envelope and nuclear membrane was confirmed in living cells. These data indicated that T-MEND was highly effective in nuclear import by membrane fusion. Conclusions Collectively, we succeeded in the regulator of intracellular trafficking and delivery of DNA into nucleus by stepwise fusion with endosomal membrane and nuclear membrane by multilayered envelope type vector.
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893. Mechanism of Escape from Macropinosomes for Octaarginine Modified Nanoparticles
Ayman El-Sayed.!- Ikramy A. Khalil ,I.2 Kentaro Kogure.P Hideyoshi Harashima.P }Faculty ofPharmaceutical Sciences, Hokkaido University, Sapporo . Hokkaido, Japan ; "The Core Research for Evohuional Science and Technology (CREST). Japan Science and Technology Agency (JST). To1..J1o. Japan. Background: Recently, anonviral multifunctional envelope-type nano device (MEND) was developed in our laboratory, and showed a high transfection activity comparable to that ofadenovirus III. Uptake of MEND modified with high density of octaarginine (RS-MEND) was shown to take place through macropinocytosis 121. However, the mechanism by which this RS-MEND can escape from macropinosomes remained unclear. Shedding light on this mechanism can result in more optimization ofthe currently present nonviral vectors as well as development of more efficient vectors in the future. In this study we conduct a stepwise dissection of the mechanism ofescape from macropinosome for RS-MEND in comparison to MEND modified with octalysine (KS-MEND). Methods: FACS analysis was used to determine extent ofuptake ofdifferent MENDs. The intracellular trafficking of MENDs encapsulating labeled DNA was tracked in NIH 3'1'3cells using confocal laser scanning microscopy (CLSM). Transfection activity of luciferase DNA-containing MENDs was determined in NIH 3T3 cells in presence ofvarious inhibitors. To test the ability of octaarginine to destabilize membranes, calcein leakage experiment was conducted using calcein loaded liposomes of negatively charged surface similar to that of cell membranes, To study fusion of MEND to macropinosomal membrane, FRET analysis of double labeled particles was conducted in living celIs using spectral imaging nuorescence microscopy. Results: The uptake of both RS and KS MENDs were found comparable, meanwhile transfection activity of RS-MEND was more than 16 times higher than that of KS-MEND, and 320 times higher than that of unmodified MEND. Transfection activity of KS-MEND was significantly enhanced by chloroquine meanwhile that of RS-MEND was not affected indicating superior escape of RS-MEND from macropinosomes: CLSM confirmed that RS-MEND escaped more efficiently from vesicular compartments than KS-MEND and this escape ability was significantly reduced in presence of Bafilomycin A, a specific acidification inhibitor. This was confirmed by strong inhibition of RS-MEND transfection activity in presence of Bafilomycin A or ammonium chloride. Calcein release experiment showed that RS modified liposomes released calccin more efficiently than KS modified liposomes at acidic pH. Spectral imaging proved that RS-MEND fused to macropinosomal membrane during its escape process. Conclusion: Our data highlight that compared to KS, RS modification of MEND enhances its escape efficiency from the vesicular compartments. Mechanism of escape was dependent on acidification of macropinosome and R8 modification improved fusion of MEND lipid coat to the macropinosome during its escape to cytosol. References: [1] Khalil lA, Kogure K, Harashima H, et al. (2007) Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery, Gene Ther. (In Press). [2] Khalil lA, Kogure K, Futaki S, Harashima I-I. (2006) High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression, J Bioi Chern. 281 :3544-3551.
Molecular Therapy Volume 15.Supplem ent I. M;'l"2007
Coprri ght © The American Society of G ene Th erapy
894. Novel Approach for the Nuclear Delivery of Macromolecule Via Membrane Fusion in NonDividing Cell Asako Kudo ,' Hidctaka Akita ,' Arisa Minoura ,' Kentaro Kogurc ,' Masaya Yamaguchi ,' Ikramy A. Khalil,' Tomoya Masuda,' Hideyoshi Harshirna.' 'Graduate School ofPharmaceutical Sciences . Hokkaido University, Sapporo . Japan . Background Transfection activity of non-viral vector is ratelimited by various intracellular processes. Especially, the transport of plasmid DNA from the cytoplasm into the nucleus across the nuclear membrane remains severe barrier in non-dividing cells. To date , so many challenges have been made to overcome this barrier by the conjugation ofnuclear localization signals (NLS), which aim to be recognized by importins, and permitting them to pass through the nuclear pore complex (NPC) . Here, we present a novel strategy for the nuclear delivery of plasmid DNA via NPC-independent pathway via membrane fusion. To realize this strategy, we also developed novel multi-layered envelope type gene vector (Tetralamellar Multifunctional Envelope Type Nano Devise; T-MEN D), in which pDNAIpolycation core is coated with different kinds of lipid membrane. Methods Various kinds ofliposomes labeled with NBD and Rhodamine were prepared, fusion activity between nuclear membrane and prepared liposomes were evaluated by the release of FRET after the incubation with isolated nucleus. To visualize a fusion event, the OFP-encapsulating and rhodamine-labeled liposome liposomes were incubated with isolated nucleus, and then confocal images were obtained by CLSM. T-MEND is composed of two kinds of lipid, endosomal or nuclear membrane fusogenic lipid. To investigate the transfection efficiency, T-MEND was prepared, in which pDNA core was coated with nuclear membrane-fusogenic lipid, and then additionally coated with endosome-fusogenic lipid. Tranfection activity was evaluated with JAWSII cells. a model of non-dividing cell. Fusion ofT-MEND with each organelle in JAWSII cell were proved by spectral imaging ofFRET. Results and Discussion Screening ofliposomes by means ofthe FRET analysis revealed th~t 2 kinds oflipid composition arc highly potent in fusion activity WIth nuclear membrane. After the incubation ofisolated nucleus with OFP-encapsulating and rhodamine-labeled multi lamellar vesicle (MLV) prepared with nucleus-fusogenic lipids, OFP was detected inside of nucleus whereas labeled lipids was distributed along the nuclear membrane. When the experiments were demonstrated with unilamellar vesicle (SUV) , OFP and lipid labeling was co-localized along nuclear structure, suggesting that OFP was trapped between inner and outer membrane. These data Indicated that nuclear transport of macromolecules can be achieved by encapsulating into the multilayered liposomes composed of nucleus-fusogenic lipids. Oene transfer efficiency with a T-MEND composed of nuclear and endosome-fusogenic lipid enhanced gene delivery compared to the conventional MEND in JAWSII cell. With spectral imaging, fusion of lipid envelope and nuclear membrane was confirmed in living cells. These data indicated that T-MEND was highly effective in nuclear import by membrane fusion. Conclusions Collectively, we succeeded in the regulator of intracellular trafficking and delivery of DNA into nucleus by stepwise fusion with endosomal membrane and nuclear membrane by multilayered envelope type vector.
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