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Hyaluronic Acid Ternary Complex for Durable Gene Delivery
CHEMICAL AND MOLECULAR CONJUGATES 153. Characterization of Lipoplex Properties and Gene Transfer Efficiency of a Novel Family of Cationic Carotenoid Lipids
Natalia Bilchuk,1 Matthew S. Adams,1 Rachel Jones,2 Alexandra M. Liberski,2,3 Christer L. Øpstad,3 Vassilia Partali,3 Hans-Richard Sliwka,3 Howard H. Lou,1 Michael D. Pungente,2 Philip L. Leopold.1 1 Stevens Institute of Technology, Hoboken, NJ; 2Weill-Cornell Medical College-Qatar, Doha, Qatar; 3Norwegian University of Science and Technology, Trondheim, Norway. Delivery of nucleic acids to cells can be facilitated by formation of complexes composed of negatively charged nucleic acids with a combination of cationic and zwitterionic lipids (lipoplexes). To help facilitate delivery of the DNA across the plasma membrane with release of the DNA in the cytoplasm, new cationic lipids structures are of ongoing interest. A novel family of carotenoid lipids composed of a positively charged head group with a rigid, 30 carbon polyene carotenoid tail and a flexible, fully saturated aliphatic tail ranging from 2 to 14 carbons in length (C30-2, -6, -12, or -14) were developed. Lipoplexes prepared from cationic carotenoid lipids at 0.5:1 to 5:1 charge ratios (positive to negative) were compared with well-characterized cationic lipids including the flexible 1,2-dioleoylsn-glycero-3-ethylphosphocholine (EPC) and the rigid, heterocyclic 3β[N-(N’N’-dimethylaminoethane) carbamoyl] cholesterol (DCChol) for complex formation, DNase protection, particle size, particle charge, gene transfer, and cytotoxicity. Cationic carotenoid family members C30-2 and C30-6 failed to produce consistent plasmid complexes. Nevertheless, C30-12 and C30-14 efficiently complexed DNA and protected it from DNase incubation at 5:1 molar ratios, yielding lipoplexes <400 nm in diameter and > 40 mV zeta potentials. At a dose of 0.38 - 0.61 µg lipid per 4 x 10^5 cells (5 hour incubation in serum free medium during transfection), cells treated with lipoplexes exhibited a reduced mitochondrial reductase activity as assayed by conversion of tetrazole to formazan (MTT assay). Remaining mitochondrial reductase activity, which ranged 60 to 95% of the value in control cells 24 hours post-transfection, achieved levels comparable to that of EPC and DC-Chol (70 to 100% compared to controls). These lipoplexes yielded gene transfer levels 10 to 30% of that achieved by EPC and DC-Chol at the same time point. Notably, the C30 family of cationic carotenoid lipids formed lipoplexes and transfered genes with greater efficiency as the aliphatic tail length increased. Therefore, the characterization of new C30 carotenoid lipids with longer aliphatic tails will be of great interest. At present, cationic carotenoid family members C30-12 and C30-14 exhibit gene transfer characteristics that are comparable to the wellestablished EPC and DC-Chol reagents confirming that optimization of this family of cationic lipids is warranted.
154. Preparation of Hydroxyapatite Nanocapsule Including DNA/PEI/Hyaluronic Acid Ternary Complex for Durable Gene Delivery Tomoko Ito,1 Makoto Otsuka.1 1 Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan.
Gene therapy has been proposed as a novel strategy for the treatment of refractory disease. Viral vectors have been widely used as carriers to deliver the therapeutic nucleic acids efficiently to the target cells. However, viral vectors have risks such as random recombination and immunogenicity. As safer alternatives, non-viral vectors such as polycations or cationic lipids have been developed to mediate the gene transfection. The DNA molecules can electrostatically associate with those cationic reagents and form small particles. The complexes usually have positive charge on their surface, which invites an adverse interaction with blood components S60
or cells. We found that hyaluronic acid (HA) could deposit onto the DNA/polycation (or cationic lipid) complexes to recharge their surface to negative. DNA/polycation/HA ternary complex was highly biocompatible, and could be targeted to the malignant cells. HA-coating also improved the transcription efficiency of the DNA complex. Moreover, we found that the DNA/PEI/HA ternary complex could be freeze-dried without any cryoprotectant maintaining their gene transfection activity. It enabled the preparation of very small DNA complex particles with relatively high concentration. Injection of those finely dispersed suspension of the small DNA/polycation/ HA complexes strongly suppressed the tumor growth in mice, and the small tumors completely disappeared. However, multiple injections of the complexes were required to achieve the satisfactory therapeutic effect, probably owing to the short duration of gene expression by such artificial vectors. On the other hand, a hydroxyapatite (HAp), which has similar inorganic components to bone and teeth, has been explored as a biodegradable and biocompatible drug-releasing devices. In this study, we developed a novel HAp nanocapsules including DNA/PEI/HA ternary complexes, and their durable gene expression was examined. Small DNA/PEI/HA complexes were prepared as reported before (Biomaterials, 31 (2010) 2912). They were added to a 1.5 times concentrated simulated body fluid (SBF) A SBF has a similar inorganic ion concentration to that of human blood plasma, and is supersaturated against hydroxyapatite. A thin apatite layer was then formed on the surface of the DNA complexes, being confirmed by X-ray diffraction and SEM-EDS analysis. The HAp nanocapsule including DNA/PEI/HA ternary complex showed evidently durable gene expression in B16 cells, and thus seems promising as a sustained-gene expression device.
155. Lipopolymer Mediated siRNA Delivery Strategy for Acute Myeloid Leukemia Cells
Breanne Landry,1 Hamidreza Montazeri Aliabadi,1 Hasan Uludag.1,2,3 1 Chemical and Materials Engineering, University of Alberta, Edmonton, Canada; 2Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada; 3Biomedical Engineering, University of Alberta, Edmonton, Canada. Small interfering RNA (siRNA) therapy is a viable treatment strategy for patients with acute myeloid leukemia (AML). With current drug treatments, patients often suffer relapses, are unable to handle repeat treatments due to toxic side effects and are at risk of late-effects that occur years later. siRNA can be used to specifically target aberrant protein expression in transformed cells, to reduce proliferation and induce apoptosis. However, an efficient means to deliver siRNA must be developed, as they cannot transverse cellular membranes, due to their large molecular weight and negative charge, and are biologically unstable. Carriers to facilitate intracellular delivery of siRNA were developed in this study and delivered to AML cell lines (THP-1, KG-1 and HL-60) in vitro. Polyethyleneimine 2 kDa (PEI2) was substituted with the lipids caprylic acid (CA), palmitic acid (PA), oleic acid (OA), and lineloic acid (LA). Polymer:scrambled siRNA complexes were prepared at various polymer:siRNA ratios, and incubated for 30min/RT. Binding and dissociation of the complexes were determined by semi-quantitative EMSA. Increasing the polymer:siRNA ratio during complex formation resulted in increased siRNA binding for all polymers. In the presence of serum, no apparent dissociation was observed, however dissociation of complexes was observed as a function of heparin concentration; the PEI2/PEI2lipids displayed more dissociation as compared to PEI25, in most cases. Morphology of complexes were visualized by TEM, where distinct, rounded complexes were observed (∼100 nm). Cyototoxicity of the complexes on leukemia cell lines was determined after 24h treatment. As expected, PEI25 displayed high cytotoxicity, which increased with concentration. PEI2 displayed very low cytotoxicity Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
Natalia Bilchuk,1 Matthew S. Adams,1 Rachel Jones,2 Alexandra M. Liberski,2,3 Christer L. Øpstad,3 Vassilia Partali,3 Hans-Richard Sliwka,3 Howard H. Lou,1 Michael D. Pungente,2 Philip L. Leopold.1 1 Stevens Institute of Technology, Hoboken, NJ; 2Weill-Cornell Medical College-Qatar, Doha, Qatar; 3Norwegian University of Science and Technology, Trondheim, Norway. Delivery of nucleic acids to cells can be facilitated by formation of complexes composed of negatively charged nucleic acids with a combination of cationic and zwitterionic lipids (lipoplexes). To help facilitate delivery of the DNA across the plasma membrane with release of the DNA in the cytoplasm, new cationic lipids structures are of ongoing interest. A novel family of carotenoid lipids composed of a positively charged head group with a rigid, 30 carbon polyene carotenoid tail and a flexible, fully saturated aliphatic tail ranging from 2 to 14 carbons in length (C30-2, -6, -12, or -14) were developed. Lipoplexes prepared from cationic carotenoid lipids at 0.5:1 to 5:1 charge ratios (positive to negative) were compared with well-characterized cationic lipids including the flexible 1,2-dioleoylsn-glycero-3-ethylphosphocholine (EPC) and the rigid, heterocyclic 3β[N-(N’N’-dimethylaminoethane) carbamoyl] cholesterol (DCChol) for complex formation, DNase protection, particle size, particle charge, gene transfer, and cytotoxicity. Cationic carotenoid family members C30-2 and C30-6 failed to produce consistent plasmid complexes. Nevertheless, C30-12 and C30-14 efficiently complexed DNA and protected it from DNase incubation at 5:1 molar ratios, yielding lipoplexes <400 nm in diameter and > 40 mV zeta potentials. At a dose of 0.38 - 0.61 µg lipid per 4 x 10^5 cells (5 hour incubation in serum free medium during transfection), cells treated with lipoplexes exhibited a reduced mitochondrial reductase activity as assayed by conversion of tetrazole to formazan (MTT assay). Remaining mitochondrial reductase activity, which ranged 60 to 95% of the value in control cells 24 hours post-transfection, achieved levels comparable to that of EPC and DC-Chol (70 to 100% compared to controls). These lipoplexes yielded gene transfer levels 10 to 30% of that achieved by EPC and DC-Chol at the same time point. Notably, the C30 family of cationic carotenoid lipids formed lipoplexes and transfered genes with greater efficiency as the aliphatic tail length increased. Therefore, the characterization of new C30 carotenoid lipids with longer aliphatic tails will be of great interest. At present, cationic carotenoid family members C30-12 and C30-14 exhibit gene transfer characteristics that are comparable to the wellestablished EPC and DC-Chol reagents confirming that optimization of this family of cationic lipids is warranted.
154. Preparation of Hydroxyapatite Nanocapsule Including DNA/PEI/Hyaluronic Acid Ternary Complex for Durable Gene Delivery Tomoko Ito,1 Makoto Otsuka.1 1 Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan.
Gene therapy has been proposed as a novel strategy for the treatment of refractory disease. Viral vectors have been widely used as carriers to deliver the therapeutic nucleic acids efficiently to the target cells. However, viral vectors have risks such as random recombination and immunogenicity. As safer alternatives, non-viral vectors such as polycations or cationic lipids have been developed to mediate the gene transfection. The DNA molecules can electrostatically associate with those cationic reagents and form small particles. The complexes usually have positive charge on their surface, which invites an adverse interaction with blood components S60
or cells. We found that hyaluronic acid (HA) could deposit onto the DNA/polycation (or cationic lipid) complexes to recharge their surface to negative. DNA/polycation/HA ternary complex was highly biocompatible, and could be targeted to the malignant cells. HA-coating also improved the transcription efficiency of the DNA complex. Moreover, we found that the DNA/PEI/HA ternary complex could be freeze-dried without any cryoprotectant maintaining their gene transfection activity. It enabled the preparation of very small DNA complex particles with relatively high concentration. Injection of those finely dispersed suspension of the small DNA/polycation/ HA complexes strongly suppressed the tumor growth in mice, and the small tumors completely disappeared. However, multiple injections of the complexes were required to achieve the satisfactory therapeutic effect, probably owing to the short duration of gene expression by such artificial vectors. On the other hand, a hydroxyapatite (HAp), which has similar inorganic components to bone and teeth, has been explored as a biodegradable and biocompatible drug-releasing devices. In this study, we developed a novel HAp nanocapsules including DNA/PEI/HA ternary complexes, and their durable gene expression was examined. Small DNA/PEI/HA complexes were prepared as reported before (Biomaterials, 31 (2010) 2912). They were added to a 1.5 times concentrated simulated body fluid (SBF) A SBF has a similar inorganic ion concentration to that of human blood plasma, and is supersaturated against hydroxyapatite. A thin apatite layer was then formed on the surface of the DNA complexes, being confirmed by X-ray diffraction and SEM-EDS analysis. The HAp nanocapsule including DNA/PEI/HA ternary complex showed evidently durable gene expression in B16 cells, and thus seems promising as a sustained-gene expression device.
155. Lipopolymer Mediated siRNA Delivery Strategy for Acute Myeloid Leukemia Cells
Breanne Landry,1 Hamidreza Montazeri Aliabadi,1 Hasan Uludag.1,2,3 1 Chemical and Materials Engineering, University of Alberta, Edmonton, Canada; 2Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada; 3Biomedical Engineering, University of Alberta, Edmonton, Canada. Small interfering RNA (siRNA) therapy is a viable treatment strategy for patients with acute myeloid leukemia (AML). With current drug treatments, patients often suffer relapses, are unable to handle repeat treatments due to toxic side effects and are at risk of late-effects that occur years later. siRNA can be used to specifically target aberrant protein expression in transformed cells, to reduce proliferation and induce apoptosis. However, an efficient means to deliver siRNA must be developed, as they cannot transverse cellular membranes, due to their large molecular weight and negative charge, and are biologically unstable. Carriers to facilitate intracellular delivery of siRNA were developed in this study and delivered to AML cell lines (THP-1, KG-1 and HL-60) in vitro. Polyethyleneimine 2 kDa (PEI2) was substituted with the lipids caprylic acid (CA), palmitic acid (PA), oleic acid (OA), and lineloic acid (LA). Polymer:scrambled siRNA complexes were prepared at various polymer:siRNA ratios, and incubated for 30min/RT. Binding and dissociation of the complexes were determined by semi-quantitative EMSA. Increasing the polymer:siRNA ratio during complex formation resulted in increased siRNA binding for all polymers. In the presence of serum, no apparent dissociation was observed, however dissociation of complexes was observed as a function of heparin concentration; the PEI2/PEI2lipids displayed more dissociation as compared to PEI25, in most cases. Morphology of complexes were visualized by TEM, where distinct, rounded complexes were observed (∼100 nm). Cyototoxicity of the complexes on leukemia cell lines was determined after 24h treatment. As expected, PEI25 displayed high cytotoxicity, which increased with concentration. PEI2 displayed very low cytotoxicity Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy