- Email: [email protected]
1081. Targeted Nanocapsules for Liver Cell-Type Delivery of Plasmids In Vivo
CARRIER DEVELOPMENT AND THERAPEUTIC EVALUATION cooperative. This element has been included in many different gene therapy vectors including lentivirus, AAV, adenovirus etc., to stimulate heterologous cDNAs expression. In this work we have analyzed the vitro and in vivo effect of WPRE element over transgene expression using two different promoters in the context of a non-viral vector, naked DNA: an ubiquitous promoter AFR with moderate transcriptional activity, and a strong liver specific promoter AAT-EnhALb. Two different versions of the WPRE element were included in the study: the conventional one, which include 60 aminoacids of theHBV X protein aminoterminal region (named as WPRE long), and a shorter version in which only 30 aminoacids remained (named as WPRE short). For the in vitro studies, HEPG2 cells were transfected with 7.5 x 10e11 copies of plasmid expressing luciferase, with or without WPRE elements, and a renilla-luciferase expressing plasmid, using lipofectamine. Enzyme activities were measured using the DualLuciferase Reporter Assay System (Promega). In vivo, 2.5 x 10e12 copies of each plasmid were administered using hydrodynamic injection via tail vein, thus, luciferase expression was mainly detected in the liver. Luciferase expression was analyzed using a cooled CCD camera at days 1, 7 and 21. 21 days after in vivo analysis, the mice were sacrificed and luciferase expression was measured in liver extracts. DNA and RNA were extracted from the livers, and plasmid copies and luciferase mRNA was quantified using quantitative PCR and RT-PCR. Striking differences on WPRE activity were observed depending on the promoter, both in vitro and in vivo, 1 day after injection. WPRE elements significantly increased luciferase expression when gene expression was under control of AFR promoter while the expression was not significantly altered when the promoter driving luciferase expression was the liver specific AAT promoter. However, when luciferase expression was analyzed 7 or 21 days after hydrodynamic injection, WPRE element increased luciferase expression independently on which promoter was used. Moreover, significant differences were observed depending on the WPRE version. Short and long WPREs similarly increased the transgene expression from AFR promoter, while the increase was significantly higher when AAT promoter was combined with the WPRE long, than in with WPRE short. In vitro analysis of luciferase activity on liver extract corroborated the in vivo data. In conclusion, the effect of modified versions of WPRE over transgene expression depends on the promoter and has to be analysed both in vivo and in vitro in order to get a correct picture of the interaction between the promoter and expression regulatory elements.
CARRIER DEVELOPMENT AND THERAPEUTIC EVALUATION 1080. Receptor-Targeting Smart Vectors for Efficient Gene Transfer to Tumours Stephanie Grosse,1 Martin Elbs,2 John Wong,2 Alethea Tabor,2 Helen Hailes,2 Stephen Hart.1 1 Molecular Immunology Unit, Institute of Child Health, University College London, London, United Kingdom; 2Department of Chemistry, University College London, London, United Kingdom. We seek to develop novel synthetic vector formulations that can be administered systemically to target therapeutic genes to tumours. Cationic nonviral vectors are cleared rapidly from the circulation by the reticuloendothelial system as a result of binding to plasma proteins and vector aggregation. Circulation times may be extended by shielding the vectors with polyethylene glycol (PEG) moieties. However, PEGylation often leads to greatly reduced transfection efficiency due to excessive vector stability. The aim of this project was to develop novel formulations of PEGylated vectors which may be administered intravenously (i. v.), and persist in the circulation S414
and target tumour-associated receptors. The vector has been further modified to disassemble within the cell in response to the intracellular environment to achieve high transfection efficiency. Such virus-like vector formulations are often referred to as “smart vectors”. The vector developed is a lipopolyplex-class formulation based on an integrin-targeting peptide with an oligolysine nucleic acidbinding element, and a cationic lipid that enhances integrin-targeted transfection by promoting endosomal release. New cleavable peptides (CP) were designed containing CP motifs located between the DNAbinding and the integrin-targeting domains. Peptide cleavage should enhance transfection by promoting the trafficking of DNA to the cytoplasm and so to the nucleus. Cleavable PEGylated lipids (PEGCL) were also synthesised for improved in vivo stability in blood, enhancing intracellular complex dissociation and endosomal escape. The cleavage studies showed PEG-CL were efficiently hydrolysed at acid pH using TLC, and CP were cleaved in the presence of endosomal proteases using HPLC. The size of lipopolyplexes comprising PEG-CL in physiological salt solution was 394±14 nm by light scattering methods, and stable for at least one hour, which was considerably smaller (p<0.05) than complexes made of unPEGylated lipids (1388±198 nm after one hour). The smaller particles generated with PEG-CL were less efficient in in vitro transfections unless a centrifugation (1500 rpm, 5 min) was performed to promote their sedimentation and cell contact. After centrifugation, in murine neuroblastoma cell line (Neuro2A), transfection efficiency with lipopolyplexes containing PEG-CL, CP and a plasmid encoding the luciferase gene was 2 times more efficient than with Lipofectin and non-CP (p<0.05). The transfection efficiency of PEG-CL/CP lipopolyplexes using a plasmid encoding the GFP protein, was about 40% in Neuro2A and mouse fibroblast (AJ3.1) cells, whereas only 20% of these cells were transfected using Lipofectin and non-CP (p<0.05). Finally, in a murine neuroblastoma model, i. v. administration of these integrin-targeted PEG-CL/CP lipopolyplexes showed a higher level of luciferase expression in tumour (52142±7121 RLU/mg) than in lung (2347±577 RLU/mg) or in liver (1198±58 RLU/mg) (p<0.05). In conclusion, we have developed a novel, targeted, synthetic, smart vector formulation that offers exciting prospects for tumourspecific therapeutic gene transfer.
1081. Targeted Nanocapsules for Liver Cell-Type Delivery of Plasmids In Vivo Betsy T. Kren,1 Gretchen M. Unger,2 Mark T. Reding,1 Clifford J. Steer.1 1 Department of Medicine, University of Minnesota, Minneapolis, MN; 2GeneSegues, Inc., Chaska, MN. In contrast to the efficient delivery afforded by viral vectors, the use of nonviral vectors for gene therapy has been hindered by the lack of adequate in vivo delivery systems. Although hydrodynamic push has been used extensively for hepatic plasmid delivery, this method delivers DNA nonselectively to liver cell types. One approach for increasing delivery to a specific cell type is by targeting receptor(s) that are either unique or highly expressed by that cell. Hepatocytes (heps) express aisaloglycoprotein receptors (ASGPr), and liver sinusoidal endothelial cells (LSECs) express hyaluronan receptors (HAr) in high abundance providing ideal targets for ligandmediated receptor uptake. The aim of this study was to determine if liver cell type specific delivery of DNA could be achieved in vivo using asialoorosomucoid (ASOR) targeting to the hep ASGPr and HA for the LSEC HAr. Using a novel dispersion atomization method (Mol Cell Biochem 274:77, 2005) that forms sub 50 nm capsules with the receptor ligand noncovalently bound to the capsule coating, a red fluorescent protein (DsRed2) reporter plasmid was encapsulated using either ASOR for hep or HA for LSEC uptake. Eight week (wk) old C57/BL6 mice received 100 µ g of the encapsulated Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
CARRIER DEVELOPMENT AND THERAPEUTIC EVALUATION plasmid targeted using ASOR or HA via tail vein injection and were sacrificed 1 wk post-injection. Liver, spleen, kidneys, lung, heart and brain were excised and processed for histology and protein extracts. Immunohistochemical identification of the LSECs in cryosections was done using anti-CD14 antibody (Ab), a marker specific for the discontinuous endothelial cells in the liver and a Cy5 labeled secondary Ab for confocal microscopy. The merged confocal micrographs of the DsRed2 and Cy5 fluorescence demonstrated colocalization of DsRed2 expression and the LSEC specific CD14 marker when HA was used as the targeting ligand. In contrast, there was no detectable colocalization when ASOR was used for targeting DsRed2 to heps. The presence of the DsRed2 protein was confirmed by western blot (WB) analysis of total liver protein extracts using a polyclonal anti-DsRed2 Ab and detection by ECL. There was no detectable DsRed2 expression in the other major organs. Transgenic hemophilia A mice were administered HA nanocapsules containing a Sleeping Beauty (SB) transposon (Tn) expressing B-domain deleted coagulation factor (F) VIII using a plasmid that codelivers an expression cassette for the SB transposase required for genomic Tn insertion. The mice were bled 2 and 5 wks post injection and plasma activated partial thromboplastin time (aPTT) determined. The treated mice had aPTTs of 25.5 ± 3.1 (2 wks) and 26 ± 1.9 sec (5 wks) which were not significantly different from the wild type (wt) aPTT of 23.5 ± 1.3 sec. In contrast, untreated hemophilia A mice had aPTTs of 46.7 ± 3.5 sec (P < 0.001 from treated and wt mice). In conclusion, ASOR and HA targeted nanocapsules can deliver plasmids in vivo to heps or LSECs, respectively. SB-Tns in HA nanocapsules targeted to LSECs provided expression of a clinically relevant gene product, FVIII that improved the phenotype of hemophilia A transgenic mice.
1082. Delivery of Anti-Oxidative Enzyme Genes Protects Against Ischemia/Reperfusion-Induced Liver Injury in Mice Song-Qing He,1 Yan-Hong Zhang,1 Senthil K. Venugopal,1 Christopher W. Dicus,2 Richard V. Perez,3 Mark A. Zern,1 Michael H. Nantz,2 Jian Wu.1 1 Transplant Research Institute, University of California, Davis Medical Center, Sacramento, CA; 2Department of Chemistry, University of California-Davis, Davis, CA; 3Department of Surgery, University of California, Davis Medical Center, Sacramento, CA. BACKGROUND: Hepatic ischemic/reperfusion (I/R) injury is characterized by the generation of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide. The aim of this study is to investigate whether anti-oxidative gene delivery by our polycationic liposomes will be an effective approach for prevention of the injury. METHODS: Polycationic liposome-mediated extracellular superoxide dismutase (EC-SOD) or/and catalase gene delivery by portal vein injection was undertaken one day prior to warm I/R injury in mice. The effects of the gene delivery were determined 6 hours after starting reperfusion. RESULTS: Polycationic liposome-mediated anti-oxidative gene delivery led to marked hepatic transgene expression. There was a 56-fold increase in human EC-SOD gene expression in the liver 30 hours after injection of EC-SOD lipoplexes as shown by real time RT-PCR; Western blot analysis demonstrated a 10-fold increase in catalase protein levels in the liver as compared to controls. Liver SOD and catalase activity increased 16.6-fold and 9-fold, respectively. The overexpression of these two anti-oxidative genes significantly suppressed the subsequent I/R-induced acute liver injury as reflected by marked
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
reduction of serum ALT levels (520±55 and 643±24 vs. 1217±193 units/ml, p<0.01). The I/R procedure significantly increased liver superoxide anions (699±83 vs. 20±13 RLU/sec/mg, p<0.01) as detected by a chemoluminescent method with a superoxide tracer, MCLA. These levels were markedly reduced to a nearly normal level (24±7 RLU/sec/mg) by the prior delivery of the EC-SOD gene. In addition, there were reduced liver malondialdehyde levels, and restored glutathione levels, in mice receiving either EC-SOD or catalase gene delivery. Moreover, the enhanced activation of nuclear factor-κB and AP-1 in hepatic I/R injury was minimized by the anti-oxidative gene delivery. The control lipoplexes did not affect the extent of liver injury and oxidant stress levels in this I/R model. The combination of EC-SOD and catalase lipoplex injection (in half amount of each plasmid) resulted in a slight improvement in hepatic I/R injury when compared to either alone. CONCLUSIONS: The findings demonstrate that polycationic liposome-mediated EC-SOD or catalase gene delivery led to high levels of the transgene activity in the liver, and markedly attenuated liver I/R injury. The protection is associated with enhanced anti-oxidative effects in the liver. To our knowledge, this is the first successful demonstration that polycationic liposome-mediated EC-SOD and/or catalase gene delivery ameliorates warm I/R-associated liver injury in mice. This approach may become a potential novel therapy to improve graft function and survival after liver transplantation.
1083. Crosslinked PEI Polyplexes for LightTriggered DNA Release Rachel G. Handwerger,1 Moon-Suk Kim,2 Scott L. Diamond.1,3 Bioengineering, University of Pennsylvania, Philadelphia, PA; 2 Medicinal Science, Korea Research Institute of Chemical Technology, Yuseong, Daejeon, Korea; 3Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA. 1
Viruses protect their genome and after endosome escape can disassemble to release their genetic contents. These two functions of protection and triggered-release are competing functionalities that have been difficult to engineer into nonviral transfection reagents. In order to mimic viral condensation of DNA, packaging and protection, and triggered release, we have designed a novel photolabile monomer, P25M, for plasmid DNA (pDNA) delivery. P25M was synthesized via a 6-step reaction scheme and consists of three functional domains: 1) a cationic domain of 25 kDa polyethylenimine (PEI) to electrostatically condense the pDNA; 2) a polymerizable domain (a methacrylamide moiety) for crosslinking the pDNA within the polyplex; and 3) a photolabile nitrobenzyl domain for triggered release by 365 nm light (Fig. 1). The formation of P25M was verified through 1H NMR. In addition to establishing the chemical structure, the functionality of each domain was verified. HPLC studies showed that upon exposure to 365 nm light (time = 4 min, distance = 20 cm), P25M (retention time = 0.99 min) was cleaved into two products (retention time = 0.34 and 1.85 min). Similar results were also found after P25M had been polymerized with VA-044, an azo-initiator. Agarose gels confirmed that P25M, essentially a modified PEI, condensed pDNA when the N/P ratio was ≥ 4. Dynamic light scattering confirmed that these polyplexes were suitable for in vitro transfection (polyplex diameter < 500 nm) both before and after polymerization with VA-044 and overnight incubation in 150 mM NaCl. Finally, heparin challenge with agarose gels confirmed that once polymerized, the polyplexes retained the pDNA. This was true for three different polymerizing agents, VA-044, ammonium persulfate, and Grubb’s 2nd generation catalyst. Unpolymerized polyplexes were unable to resist pDNA release in the heparin
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CARRIER DEVELOPMENT AND THERAPEUTIC EVALUATION 1080. Receptor-Targeting Smart Vectors for Efficient Gene Transfer to Tumours Stephanie Grosse,1 Martin Elbs,2 John Wong,2 Alethea Tabor,2 Helen Hailes,2 Stephen Hart.1 1 Molecular Immunology Unit, Institute of Child Health, University College London, London, United Kingdom; 2Department of Chemistry, University College London, London, United Kingdom. We seek to develop novel synthetic vector formulations that can be administered systemically to target therapeutic genes to tumours. Cationic nonviral vectors are cleared rapidly from the circulation by the reticuloendothelial system as a result of binding to plasma proteins and vector aggregation. Circulation times may be extended by shielding the vectors with polyethylene glycol (PEG) moieties. However, PEGylation often leads to greatly reduced transfection efficiency due to excessive vector stability. The aim of this project was to develop novel formulations of PEGylated vectors which may be administered intravenously (i. v.), and persist in the circulation S414
and target tumour-associated receptors. The vector has been further modified to disassemble within the cell in response to the intracellular environment to achieve high transfection efficiency. Such virus-like vector formulations are often referred to as “smart vectors”. The vector developed is a lipopolyplex-class formulation based on an integrin-targeting peptide with an oligolysine nucleic acidbinding element, and a cationic lipid that enhances integrin-targeted transfection by promoting endosomal release. New cleavable peptides (CP) were designed containing CP motifs located between the DNAbinding and the integrin-targeting domains. Peptide cleavage should enhance transfection by promoting the trafficking of DNA to the cytoplasm and so to the nucleus. Cleavable PEGylated lipids (PEGCL) were also synthesised for improved in vivo stability in blood, enhancing intracellular complex dissociation and endosomal escape. The cleavage studies showed PEG-CL were efficiently hydrolysed at acid pH using TLC, and CP were cleaved in the presence of endosomal proteases using HPLC. The size of lipopolyplexes comprising PEG-CL in physiological salt solution was 394±14 nm by light scattering methods, and stable for at least one hour, which was considerably smaller (p<0.05) than complexes made of unPEGylated lipids (1388±198 nm after one hour). The smaller particles generated with PEG-CL were less efficient in in vitro transfections unless a centrifugation (1500 rpm, 5 min) was performed to promote their sedimentation and cell contact. After centrifugation, in murine neuroblastoma cell line (Neuro2A), transfection efficiency with lipopolyplexes containing PEG-CL, CP and a plasmid encoding the luciferase gene was 2 times more efficient than with Lipofectin and non-CP (p<0.05). The transfection efficiency of PEG-CL/CP lipopolyplexes using a plasmid encoding the GFP protein, was about 40% in Neuro2A and mouse fibroblast (AJ3.1) cells, whereas only 20% of these cells were transfected using Lipofectin and non-CP (p<0.05). Finally, in a murine neuroblastoma model, i. v. administration of these integrin-targeted PEG-CL/CP lipopolyplexes showed a higher level of luciferase expression in tumour (52142±7121 RLU/mg) than in lung (2347±577 RLU/mg) or in liver (1198±58 RLU/mg) (p<0.05). In conclusion, we have developed a novel, targeted, synthetic, smart vector formulation that offers exciting prospects for tumourspecific therapeutic gene transfer.
1081. Targeted Nanocapsules for Liver Cell-Type Delivery of Plasmids In Vivo Betsy T. Kren,1 Gretchen M. Unger,2 Mark T. Reding,1 Clifford J. Steer.1 1 Department of Medicine, University of Minnesota, Minneapolis, MN; 2GeneSegues, Inc., Chaska, MN. In contrast to the efficient delivery afforded by viral vectors, the use of nonviral vectors for gene therapy has been hindered by the lack of adequate in vivo delivery systems. Although hydrodynamic push has been used extensively for hepatic plasmid delivery, this method delivers DNA nonselectively to liver cell types. One approach for increasing delivery to a specific cell type is by targeting receptor(s) that are either unique or highly expressed by that cell. Hepatocytes (heps) express aisaloglycoprotein receptors (ASGPr), and liver sinusoidal endothelial cells (LSECs) express hyaluronan receptors (HAr) in high abundance providing ideal targets for ligandmediated receptor uptake. The aim of this study was to determine if liver cell type specific delivery of DNA could be achieved in vivo using asialoorosomucoid (ASOR) targeting to the hep ASGPr and HA for the LSEC HAr. Using a novel dispersion atomization method (Mol Cell Biochem 274:77, 2005) that forms sub 50 nm capsules with the receptor ligand noncovalently bound to the capsule coating, a red fluorescent protein (DsRed2) reporter plasmid was encapsulated using either ASOR for hep or HA for LSEC uptake. Eight week (wk) old C57/BL6 mice received 100 µ g of the encapsulated Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
CARRIER DEVELOPMENT AND THERAPEUTIC EVALUATION plasmid targeted using ASOR or HA via tail vein injection and were sacrificed 1 wk post-injection. Liver, spleen, kidneys, lung, heart and brain were excised and processed for histology and protein extracts. Immunohistochemical identification of the LSECs in cryosections was done using anti-CD14 antibody (Ab), a marker specific for the discontinuous endothelial cells in the liver and a Cy5 labeled secondary Ab for confocal microscopy. The merged confocal micrographs of the DsRed2 and Cy5 fluorescence demonstrated colocalization of DsRed2 expression and the LSEC specific CD14 marker when HA was used as the targeting ligand. In contrast, there was no detectable colocalization when ASOR was used for targeting DsRed2 to heps. The presence of the DsRed2 protein was confirmed by western blot (WB) analysis of total liver protein extracts using a polyclonal anti-DsRed2 Ab and detection by ECL. There was no detectable DsRed2 expression in the other major organs. Transgenic hemophilia A mice were administered HA nanocapsules containing a Sleeping Beauty (SB) transposon (Tn) expressing B-domain deleted coagulation factor (F) VIII using a plasmid that codelivers an expression cassette for the SB transposase required for genomic Tn insertion. The mice were bled 2 and 5 wks post injection and plasma activated partial thromboplastin time (aPTT) determined. The treated mice had aPTTs of 25.5 ± 3.1 (2 wks) and 26 ± 1.9 sec (5 wks) which were not significantly different from the wild type (wt) aPTT of 23.5 ± 1.3 sec. In contrast, untreated hemophilia A mice had aPTTs of 46.7 ± 3.5 sec (P < 0.001 from treated and wt mice). In conclusion, ASOR and HA targeted nanocapsules can deliver plasmids in vivo to heps or LSECs, respectively. SB-Tns in HA nanocapsules targeted to LSECs provided expression of a clinically relevant gene product, FVIII that improved the phenotype of hemophilia A transgenic mice.
1082. Delivery of Anti-Oxidative Enzyme Genes Protects Against Ischemia/Reperfusion-Induced Liver Injury in Mice Song-Qing He,1 Yan-Hong Zhang,1 Senthil K. Venugopal,1 Christopher W. Dicus,2 Richard V. Perez,3 Mark A. Zern,1 Michael H. Nantz,2 Jian Wu.1 1 Transplant Research Institute, University of California, Davis Medical Center, Sacramento, CA; 2Department of Chemistry, University of California-Davis, Davis, CA; 3Department of Surgery, University of California, Davis Medical Center, Sacramento, CA. BACKGROUND: Hepatic ischemic/reperfusion (I/R) injury is characterized by the generation of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide. The aim of this study is to investigate whether anti-oxidative gene delivery by our polycationic liposomes will be an effective approach for prevention of the injury. METHODS: Polycationic liposome-mediated extracellular superoxide dismutase (EC-SOD) or/and catalase gene delivery by portal vein injection was undertaken one day prior to warm I/R injury in mice. The effects of the gene delivery were determined 6 hours after starting reperfusion. RESULTS: Polycationic liposome-mediated anti-oxidative gene delivery led to marked hepatic transgene expression. There was a 56-fold increase in human EC-SOD gene expression in the liver 30 hours after injection of EC-SOD lipoplexes as shown by real time RT-PCR; Western blot analysis demonstrated a 10-fold increase in catalase protein levels in the liver as compared to controls. Liver SOD and catalase activity increased 16.6-fold and 9-fold, respectively. The overexpression of these two anti-oxidative genes significantly suppressed the subsequent I/R-induced acute liver injury as reflected by marked
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
reduction of serum ALT levels (520±55 and 643±24 vs. 1217±193 units/ml, p<0.01). The I/R procedure significantly increased liver superoxide anions (699±83 vs. 20±13 RLU/sec/mg, p<0.01) as detected by a chemoluminescent method with a superoxide tracer, MCLA. These levels were markedly reduced to a nearly normal level (24±7 RLU/sec/mg) by the prior delivery of the EC-SOD gene. In addition, there were reduced liver malondialdehyde levels, and restored glutathione levels, in mice receiving either EC-SOD or catalase gene delivery. Moreover, the enhanced activation of nuclear factor-κB and AP-1 in hepatic I/R injury was minimized by the anti-oxidative gene delivery. The control lipoplexes did not affect the extent of liver injury and oxidant stress levels in this I/R model. The combination of EC-SOD and catalase lipoplex injection (in half amount of each plasmid) resulted in a slight improvement in hepatic I/R injury when compared to either alone. CONCLUSIONS: The findings demonstrate that polycationic liposome-mediated EC-SOD or catalase gene delivery led to high levels of the transgene activity in the liver, and markedly attenuated liver I/R injury. The protection is associated with enhanced anti-oxidative effects in the liver. To our knowledge, this is the first successful demonstration that polycationic liposome-mediated EC-SOD and/or catalase gene delivery ameliorates warm I/R-associated liver injury in mice. This approach may become a potential novel therapy to improve graft function and survival after liver transplantation.
1083. Crosslinked PEI Polyplexes for LightTriggered DNA Release Rachel G. Handwerger,1 Moon-Suk Kim,2 Scott L. Diamond.1,3 Bioengineering, University of Pennsylvania, Philadelphia, PA; 2 Medicinal Science, Korea Research Institute of Chemical Technology, Yuseong, Daejeon, Korea; 3Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA. 1
Viruses protect their genome and after endosome escape can disassemble to release their genetic contents. These two functions of protection and triggered-release are competing functionalities that have been difficult to engineer into nonviral transfection reagents. In order to mimic viral condensation of DNA, packaging and protection, and triggered release, we have designed a novel photolabile monomer, P25M, for plasmid DNA (pDNA) delivery. P25M was synthesized via a 6-step reaction scheme and consists of three functional domains: 1) a cationic domain of 25 kDa polyethylenimine (PEI) to electrostatically condense the pDNA; 2) a polymerizable domain (a methacrylamide moiety) for crosslinking the pDNA within the polyplex; and 3) a photolabile nitrobenzyl domain for triggered release by 365 nm light (Fig. 1). The formation of P25M was verified through 1H NMR. In addition to establishing the chemical structure, the functionality of each domain was verified. HPLC studies showed that upon exposure to 365 nm light (time = 4 min, distance = 20 cm), P25M (retention time = 0.99 min) was cleaved into two products (retention time = 0.34 and 1.85 min). Similar results were also found after P25M had been polymerized with VA-044, an azo-initiator. Agarose gels confirmed that P25M, essentially a modified PEI, condensed pDNA when the N/P ratio was ≥ 4. Dynamic light scattering confirmed that these polyplexes were suitable for in vitro transfection (polyplex diameter < 500 nm) both before and after polymerization with VA-044 and overnight incubation in 150 mM NaCl. Finally, heparin challenge with agarose gels confirmed that once polymerized, the polyplexes retained the pDNA. This was true for three different polymerizing agents, VA-044, ammonium persulfate, and Grubb’s 2nd generation catalyst. Unpolymerized polyplexes were unable to resist pDNA release in the heparin
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