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CHEMICAL AND MOLECULAR CONJUGATES III 883. Elucidating Structure-Function Relationship of Primary Amines in Cationic Polymer Gene Delivery Vehicles
Mark E. Hwang, Daniel W. Pack. Departmemt of Bioengineering, University of Illinois, UrbanaChampaign, Urbana, IL. Cationic polymers polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer present an effective means to deliver therapeutic genes to mammalian cells in a manner that is non-immunogenic, non-pathogenic, easily reproduced, and targetable. Both of these prototypical synthetic gene delivery vehicles are characterized by a high density of primary amines. The role of primary amines in PEI and PAMAM is four-fold: to 1) condense and protect cargo DNA, 2) facilitate endocytosis through electrostatic interactions at the cell surface, 3) mediate successful endocytic vesicle escape and polyplex unpackaging via hypothesized proton-sponge effects, and 4) provide easily modifiable moieties for further functionalization of these delivery vehicles. Previously, we have shown that we are able to modulate the DNA binding, pH buffering, and polyplex unpackaging properties of 25-kD PEI by acetylating a fraction of the terminal primary amine groups (Gabrielson 2006). It is hypothesized that the fifty-fold increase in gene delivery activity observed for 57% acetylated PEI relative to unmodified PEI resulted from the trade-off between reduced pH buffering capacity and enhanced polyplex unpackaging. Cationic polymer-DNA interactions, which are facilitated largely by primary amines, can be characterized by three measures of binding: 1) the ratio of polymer to DNA required to achieve a charge-neutral polyplex, 2) the degree of DNA condensation at a given N/P ratio, and 3) the ability to competitively bind DNA in the presence of other anionic species. These characteristics contribute differently to polyplex internalization at the cell surface, intracellular trafficking, and subsequent release in PEI and PAMAM delivery vehicles. In this study we acetylated both 25-kD branched PEI and generation four PAMAM dendrimers to examine the role of primary amines in the polymer-DNA interactions and intracellular barrier navigation of two different structures with similar biochemical features. We studied the ability of acetylated polymers to form polyplexes across a range of N/P ratios. Further, we evaluated their effectiveness in delivering plasmid DNA to cells and drew a mechanistic explanation for our results from the various assays that characterize polymer-DNA interactions. The results from our work indicate that the terminal, primary amines on the PAMAM dendrimer are almost entirely responsible for DNA-binding interactions within the polyplex. Partial (30%) PAMAM acetylation of these amines allowed premature DNA release from the polyplex in the extracellular environment. These primary amines also account for extremely strong DNA binding interactions. Unmodified PAMAM achieves the charge-neutral polyplex at a lower N/P ratio than PEI, and also withstands competitive binding by anionic species at concentrations twice as high as that required to dissociate PEI/DNA polyplexes. By correlating cationic polymer structure and primary amine behavior with gene delivery activity in these prototypic cationic polymers, we are able to draw insightful structure-function relationships from this study that will aid in the rational design of efficient gene delivery vehicles.
884. A Novel Class of Rigid, Cationic Nucleic Acid Transfection Reagents Based on Carotenoid Lipids
Michael D. Pungente,1 Christer L. Oepstad,2 Vassila Partali,2 Richard Sliwka,2 Sharen Joseph,1 Dhanya Vijayakumar,1 Ka Ning Yip,3 Anne M. Doody,3 Philip L. Leopold.4 1 Weill Cornell Medical College - Qatar, Doha, Qatar; 2Norwegian University of Science and Technology, Trondheim, Norway; 3 Cornell University, Ithaca, NY; 4Stevens Institute of Technology, Hoboken, NJ. Cationic lipids have long been valued for their ability to form complexes with anionic nucleic acids as well as their ability to convey the lipid-nucleic acid complex across the plasma membrane and into the cytosol. The most common formulations include cationic lipids with flexible saturated or mono-unsaturated hydrocarbon chains. One aspect of non-viral vector development that is critical for optimization involves the balance between complex formation and complex dissolution. To address this issue, we hypothesized that replacement of flexible hydrocarbon tails with rigid hydrocarbon tails would act as a barrier to nuclease-mediated degradation of encapsulated DNA while retaining the ability to release nucleic acids within the cell. To test this hypothesis, we employed three members of a novel class of cationic phospholipids carrying one highly unsaturated chain based on a carotenoid structure and a second, shorter saturated hydrocarbon chain with a length of either 2, 6, or 12 carbons. The lipids were formulated with a co-lipid (DOPE, a zwitterion that carries two long mono-unsaturated tails) and were compared to a canonical cationic lipid (DC-Chol, a modified cholesterol with a short saturated tail). Particle size and zeta potential analysis demonstrated that the C2, C6, and C12 carotenoid lipids formed small particles when mixed with DOPE with sizes of 282.0, 128.0, and 115.7 nm, respectively, with zeta potentials of 2.9, 34.3, and 47.3 mV, respectively. Using the same formulation conditions, lipid-DNA particles using DCChol and DOPE as a co-lipid were found to have a particle size of 278.5 nm and a zeta potential of 31.4 mV. All three lipids created complexes with DNA as demonstrated by gel retardation assays with complete complex formation achieved at a molar charge ratio of 5 (lipid:DNA). The binding affinities of the lipids for DNA were qualitatively assessed in a SDS-competition/lipid binding assay. The carotenoid series showed a gradation of lipid binding in decreasing strength from C2 to C12, and all were weaker than DC-Chol. In DNA degradation assays using either serum or DNAse I, DC-Chol gave the best protection followed by C2, C6, and then C12. In gene transfer experiments using an expression plasmid encoding betagalactosidase, the reverse relationship was observed. C12-mediated gene expression was comparable to DC-Chol-mediated expression while exceeding C6-mediated expression by >5-fold and C2-mediated expression by >50-fold. These data establish this group of first generation rigid carotenoid lipids as promising gene transfer reagents and begin the process of developing a structure-function relationship to predict gene transfer activity.
885. Preparation of Radiolabeled Plasmid DNA and Nanoparticles: A Step towards Determination of Biodistribution Kinetics by SPECT/CT Imaging
Rajesh R. Patil,1 Gary Huang,2 Xuan Jiang,1 Yuzhao Liu,1 Dara Kraitchman,2 Hai Quan Mao.1 1 Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD; 2Department of Radiology, Johns Hopkins University, Baltimore, MD. INTRODUCTION. Insufficient characterization of transport kinetics of gene-loaded nanoparticles has hindered development of safe and efficient gene carriers. The objective of this project is to develop a radiolabeling method to characterize in vivo transport Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
S337
CHEMICAL AND MOLECULAR CONJUGATES III kinetics of DNA nanoparticles using single photon emission computed tomography (SPECT)/computerized tomography (CT) technique. SPECT/CT imaging allows for direct fusion of X-ray morphologic information and radionuclide functional imaging information, offering high temporal and spatial resolution. Here we have developed a method to label plasmid DNA with 99mTc, and prepared nanoparticles with 99mTc-labeled plasmid. This method will ensure monitoring of biodistribution kinetics of therapeutic payload, in contrast to reported methods on labeling carrier polymers. METHODS. Amino groups were first introduced to plasmid DNA using Mirus Label IT reagent, and then derivatized by reacting with 6-hydrazinonicotinamide (HyNIC), which is a bifunctional chelating ligand for 99mTc. The pDNA was then labeled with 99mTc using triacine as coligand and stannous chloride as a reducing agent. We prepared nanoparticles with 99mTc-DNA using chitosan and PEG5K-g-chitosan. Biophysical properties and transfection efficiency of labeled nanoparticles were characterized. These radiolabeled nanoparticles were administered to Wistar rats (n =4), and their blood clearance and organ distribution profile was monitored using gamma scintigraphy. RESULTS. The HyNIC-modified pDNA retained same ability to form nanoparticles with chitosan and PEG-chitosan, as depicted by gel electrophoresis and particle size analysis. High (〉90%) and stable (3 h) radiolabeling efficiency of 99mTc was obtained with minimum radiocolloid formation, resulting in high specific labeling, equivalent to 0.1 mCi/ µg of pDNA, crucial for SPECT/CT imaging. The blood clearance and biodistribution analysis revealed that naked DNA was cleared up rapidly from blood; chitosan/DNA nanoparticles prolonged the circulation time of nanoparticles/plasmid DNA; whereas the PEG chitosan/DNA nanoparticles yielded the longest circulation time following intravenous infusion.
CONCLUSION. A 99mTc-radiolabeling method for pDNA was successfully developed and optimized. The method was highly efficient, reproducible; and labeled plasmid and nanoparticles were highly stable. We have demonstrated that PEG chitosan/DNA nanoparticles were more stable than chitosan/DNA nanoparticles, likely as a result of reduced uptake by reticuloendothelial system. This radiolabeling method can also be used to tag a wide array of pDNA-based gene delivery systems. The SPECT/CT imaging study using these 99mTc-labeled nanoparticles is currently under way.
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886. Chitosan as a Carrier for Corneal Gene Transfer
Eytan A. Klausner,1 Zhong Zhang,1 Robert L. Chapman,1 Richard F. Multack,2 Michael V. Volin.3 1 Department of Pharmaceutical Sciences, Midwestern University Chicago College of Pharmacy, Downers Grove, IL; 2Department of Surgery, Midwestern University Chicago College of Osteopathic Medicine, Downers Grove, IL; 3Department of Microbiology and Immunology, Midwestern University Chicago College of Osteopathic Medicine, Downers Grove, IL. Corneal gene therapy can potentially correct corneal diseases, which are second only to cataract as the leading cause of vision loss. The majority of common gene vectors have been tested in the cornea through various modes of administration using different animal models. Positive results have been obtained in preclinical studies for prevention and treatment of corneal graft rejection, neovascularization, haze and herpetic stromal keratitis. However, in order to translate corneal gene delivery systems to pharmaceutical products there is a need to widen and deepen the scope of research in the aspect of delivery, which is still the biggest single obstacle for gene therapy. Chitosan is a biocompatible, biodegradable and nontoxic biopolymer that was well tolerated when applied to the eye as a tear substitute or a drug carrier. It has been extensively studied for gene therapy. However, the transfection efficiency of chitosan-DNA nanoparticles was inconsistent between different studies. In order to enhance transgene expression there is a need for additional studies to characterize the effect of pharmaceutical parameters, such as molecular weight and ratio of nitrogen atoms from the polycation to phosphate groups from the DNA (N/P ratio), on transfection efficiency. We have designed chitosan-DNA nanoparticles using low molecular weight chitosan with N/P ratios of 10 to 60, and high molecular weight chitosan with N/P ratios of 2 to 10. Mean sizes of nanoparticles of low and high molecular weight were 95 and 150 nm, respectively. Mean zeta potentials of nanoparticles with low and high molecular weight were 46 to 55 millivolts and 42 to 85 millivolts, respectively. Studies in cell cultures showed that transgene expression was greater in COS-7 cells than in corneal endothelial cells. In both cell lines chitosan-DNA nanoparticles based on low-molecular-weight chitosan led to greater luciferase gene expression than nanoparticles based on high-molecular-weight chitosan. A gel retardation assay showed complete DNA encapsulation in all formulations. A 9-fold concentration of the formulations achieved using a combination of vacuum and mild heat (Vacufuge) led to a substantial increase in the size of several nanoparticle formulations: Low-molecular-weight chitosan with N/P ratio of 60 and high-molecular-weight chitosan with N/P ratio of 7 and 10. Injection of a select formulation of chitosanDNA nanoparticles to the corneal stroma of rats yielded detectable luciferase gene expression. These studies lay the foundations for evaluating chitosan-DNA nanoparticles for corneal gene therapy. At the moment the potential of chitosan-DNA nanoparticles to improve the therapeutic treatment of acquired and inherited corneal diseases is immense. In addition, an optimized formulation of chitosan-DNA nanoparticles may find clinical benefits for treatment of ocular diseases outside of the cornea, and for gene therapy applications in general.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Mark E. Hwang, Daniel W. Pack. Departmemt of Bioengineering, University of Illinois, UrbanaChampaign, Urbana, IL. Cationic polymers polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer present an effective means to deliver therapeutic genes to mammalian cells in a manner that is non-immunogenic, non-pathogenic, easily reproduced, and targetable. Both of these prototypical synthetic gene delivery vehicles are characterized by a high density of primary amines. The role of primary amines in PEI and PAMAM is four-fold: to 1) condense and protect cargo DNA, 2) facilitate endocytosis through electrostatic interactions at the cell surface, 3) mediate successful endocytic vesicle escape and polyplex unpackaging via hypothesized proton-sponge effects, and 4) provide easily modifiable moieties for further functionalization of these delivery vehicles. Previously, we have shown that we are able to modulate the DNA binding, pH buffering, and polyplex unpackaging properties of 25-kD PEI by acetylating a fraction of the terminal primary amine groups (Gabrielson 2006). It is hypothesized that the fifty-fold increase in gene delivery activity observed for 57% acetylated PEI relative to unmodified PEI resulted from the trade-off between reduced pH buffering capacity and enhanced polyplex unpackaging. Cationic polymer-DNA interactions, which are facilitated largely by primary amines, can be characterized by three measures of binding: 1) the ratio of polymer to DNA required to achieve a charge-neutral polyplex, 2) the degree of DNA condensation at a given N/P ratio, and 3) the ability to competitively bind DNA in the presence of other anionic species. These characteristics contribute differently to polyplex internalization at the cell surface, intracellular trafficking, and subsequent release in PEI and PAMAM delivery vehicles. In this study we acetylated both 25-kD branched PEI and generation four PAMAM dendrimers to examine the role of primary amines in the polymer-DNA interactions and intracellular barrier navigation of two different structures with similar biochemical features. We studied the ability of acetylated polymers to form polyplexes across a range of N/P ratios. Further, we evaluated their effectiveness in delivering plasmid DNA to cells and drew a mechanistic explanation for our results from the various assays that characterize polymer-DNA interactions. The results from our work indicate that the terminal, primary amines on the PAMAM dendrimer are almost entirely responsible for DNA-binding interactions within the polyplex. Partial (30%) PAMAM acetylation of these amines allowed premature DNA release from the polyplex in the extracellular environment. These primary amines also account for extremely strong DNA binding interactions. Unmodified PAMAM achieves the charge-neutral polyplex at a lower N/P ratio than PEI, and also withstands competitive binding by anionic species at concentrations twice as high as that required to dissociate PEI/DNA polyplexes. By correlating cationic polymer structure and primary amine behavior with gene delivery activity in these prototypic cationic polymers, we are able to draw insightful structure-function relationships from this study that will aid in the rational design of efficient gene delivery vehicles.
884. A Novel Class of Rigid, Cationic Nucleic Acid Transfection Reagents Based on Carotenoid Lipids
Michael D. Pungente,1 Christer L. Oepstad,2 Vassila Partali,2 Richard Sliwka,2 Sharen Joseph,1 Dhanya Vijayakumar,1 Ka Ning Yip,3 Anne M. Doody,3 Philip L. Leopold.4 1 Weill Cornell Medical College - Qatar, Doha, Qatar; 2Norwegian University of Science and Technology, Trondheim, Norway; 3 Cornell University, Ithaca, NY; 4Stevens Institute of Technology, Hoboken, NJ. Cationic lipids have long been valued for their ability to form complexes with anionic nucleic acids as well as their ability to convey the lipid-nucleic acid complex across the plasma membrane and into the cytosol. The most common formulations include cationic lipids with flexible saturated or mono-unsaturated hydrocarbon chains. One aspect of non-viral vector development that is critical for optimization involves the balance between complex formation and complex dissolution. To address this issue, we hypothesized that replacement of flexible hydrocarbon tails with rigid hydrocarbon tails would act as a barrier to nuclease-mediated degradation of encapsulated DNA while retaining the ability to release nucleic acids within the cell. To test this hypothesis, we employed three members of a novel class of cationic phospholipids carrying one highly unsaturated chain based on a carotenoid structure and a second, shorter saturated hydrocarbon chain with a length of either 2, 6, or 12 carbons. The lipids were formulated with a co-lipid (DOPE, a zwitterion that carries two long mono-unsaturated tails) and were compared to a canonical cationic lipid (DC-Chol, a modified cholesterol with a short saturated tail). Particle size and zeta potential analysis demonstrated that the C2, C6, and C12 carotenoid lipids formed small particles when mixed with DOPE with sizes of 282.0, 128.0, and 115.7 nm, respectively, with zeta potentials of 2.9, 34.3, and 47.3 mV, respectively. Using the same formulation conditions, lipid-DNA particles using DCChol and DOPE as a co-lipid were found to have a particle size of 278.5 nm and a zeta potential of 31.4 mV. All three lipids created complexes with DNA as demonstrated by gel retardation assays with complete complex formation achieved at a molar charge ratio of 5 (lipid:DNA). The binding affinities of the lipids for DNA were qualitatively assessed in a SDS-competition/lipid binding assay. The carotenoid series showed a gradation of lipid binding in decreasing strength from C2 to C12, and all were weaker than DC-Chol. In DNA degradation assays using either serum or DNAse I, DC-Chol gave the best protection followed by C2, C6, and then C12. In gene transfer experiments using an expression plasmid encoding betagalactosidase, the reverse relationship was observed. C12-mediated gene expression was comparable to DC-Chol-mediated expression while exceeding C6-mediated expression by >5-fold and C2-mediated expression by >50-fold. These data establish this group of first generation rigid carotenoid lipids as promising gene transfer reagents and begin the process of developing a structure-function relationship to predict gene transfer activity.
885. Preparation of Radiolabeled Plasmid DNA and Nanoparticles: A Step towards Determination of Biodistribution Kinetics by SPECT/CT Imaging
Rajesh R. Patil,1 Gary Huang,2 Xuan Jiang,1 Yuzhao Liu,1 Dara Kraitchman,2 Hai Quan Mao.1 1 Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD; 2Department of Radiology, Johns Hopkins University, Baltimore, MD. INTRODUCTION. Insufficient characterization of transport kinetics of gene-loaded nanoparticles has hindered development of safe and efficient gene carriers. The objective of this project is to develop a radiolabeling method to characterize in vivo transport Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
S337
CHEMICAL AND MOLECULAR CONJUGATES III kinetics of DNA nanoparticles using single photon emission computed tomography (SPECT)/computerized tomography (CT) technique. SPECT/CT imaging allows for direct fusion of X-ray morphologic information and radionuclide functional imaging information, offering high temporal and spatial resolution. Here we have developed a method to label plasmid DNA with 99mTc, and prepared nanoparticles with 99mTc-labeled plasmid. This method will ensure monitoring of biodistribution kinetics of therapeutic payload, in contrast to reported methods on labeling carrier polymers. METHODS. Amino groups were first introduced to plasmid DNA using Mirus Label IT reagent, and then derivatized by reacting with 6-hydrazinonicotinamide (HyNIC), which is a bifunctional chelating ligand for 99mTc. The pDNA was then labeled with 99mTc using triacine as coligand and stannous chloride as a reducing agent. We prepared nanoparticles with 99mTc-DNA using chitosan and PEG5K-g-chitosan. Biophysical properties and transfection efficiency of labeled nanoparticles were characterized. These radiolabeled nanoparticles were administered to Wistar rats (n =4), and their blood clearance and organ distribution profile was monitored using gamma scintigraphy. RESULTS. The HyNIC-modified pDNA retained same ability to form nanoparticles with chitosan and PEG-chitosan, as depicted by gel electrophoresis and particle size analysis. High (〉90%) and stable (3 h) radiolabeling efficiency of 99mTc was obtained with minimum radiocolloid formation, resulting in high specific labeling, equivalent to 0.1 mCi/ µg of pDNA, crucial for SPECT/CT imaging. The blood clearance and biodistribution analysis revealed that naked DNA was cleared up rapidly from blood; chitosan/DNA nanoparticles prolonged the circulation time of nanoparticles/plasmid DNA; whereas the PEG chitosan/DNA nanoparticles yielded the longest circulation time following intravenous infusion.
CONCLUSION. A 99mTc-radiolabeling method for pDNA was successfully developed and optimized. The method was highly efficient, reproducible; and labeled plasmid and nanoparticles were highly stable. We have demonstrated that PEG chitosan/DNA nanoparticles were more stable than chitosan/DNA nanoparticles, likely as a result of reduced uptake by reticuloendothelial system. This radiolabeling method can also be used to tag a wide array of pDNA-based gene delivery systems. The SPECT/CT imaging study using these 99mTc-labeled nanoparticles is currently under way.
S338
886. Chitosan as a Carrier for Corneal Gene Transfer
Eytan A. Klausner,1 Zhong Zhang,1 Robert L. Chapman,1 Richard F. Multack,2 Michael V. Volin.3 1 Department of Pharmaceutical Sciences, Midwestern University Chicago College of Pharmacy, Downers Grove, IL; 2Department of Surgery, Midwestern University Chicago College of Osteopathic Medicine, Downers Grove, IL; 3Department of Microbiology and Immunology, Midwestern University Chicago College of Osteopathic Medicine, Downers Grove, IL. Corneal gene therapy can potentially correct corneal diseases, which are second only to cataract as the leading cause of vision loss. The majority of common gene vectors have been tested in the cornea through various modes of administration using different animal models. Positive results have been obtained in preclinical studies for prevention and treatment of corneal graft rejection, neovascularization, haze and herpetic stromal keratitis. However, in order to translate corneal gene delivery systems to pharmaceutical products there is a need to widen and deepen the scope of research in the aspect of delivery, which is still the biggest single obstacle for gene therapy. Chitosan is a biocompatible, biodegradable and nontoxic biopolymer that was well tolerated when applied to the eye as a tear substitute or a drug carrier. It has been extensively studied for gene therapy. However, the transfection efficiency of chitosan-DNA nanoparticles was inconsistent between different studies. In order to enhance transgene expression there is a need for additional studies to characterize the effect of pharmaceutical parameters, such as molecular weight and ratio of nitrogen atoms from the polycation to phosphate groups from the DNA (N/P ratio), on transfection efficiency. We have designed chitosan-DNA nanoparticles using low molecular weight chitosan with N/P ratios of 10 to 60, and high molecular weight chitosan with N/P ratios of 2 to 10. Mean sizes of nanoparticles of low and high molecular weight were 95 and 150 nm, respectively. Mean zeta potentials of nanoparticles with low and high molecular weight were 46 to 55 millivolts and 42 to 85 millivolts, respectively. Studies in cell cultures showed that transgene expression was greater in COS-7 cells than in corneal endothelial cells. In both cell lines chitosan-DNA nanoparticles based on low-molecular-weight chitosan led to greater luciferase gene expression than nanoparticles based on high-molecular-weight chitosan. A gel retardation assay showed complete DNA encapsulation in all formulations. A 9-fold concentration of the formulations achieved using a combination of vacuum and mild heat (Vacufuge) led to a substantial increase in the size of several nanoparticle formulations: Low-molecular-weight chitosan with N/P ratio of 60 and high-molecular-weight chitosan with N/P ratio of 7 and 10. Injection of a select formulation of chitosanDNA nanoparticles to the corneal stroma of rats yielded detectable luciferase gene expression. These studies lay the foundations for evaluating chitosan-DNA nanoparticles for corneal gene therapy. At the moment the potential of chitosan-DNA nanoparticles to improve the therapeutic treatment of acquired and inherited corneal diseases is immense. In addition, an optimized formulation of chitosan-DNA nanoparticles may find clinical benefits for treatment of ocular diseases outside of the cornea, and for gene therapy applications in general.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy