- Email: [email protected]
Monoolein as helper lipid for non-viral transfection in mammals
Abstracts / Journal of Controlled Release 148 (2010) e85–e101
e91
Abstract summary Adsorption and internalization of siRNA loaded dextran nanogels by blood cells were determined using flow cytometry. Positively and negatively charged nanogels with various PEGylation degrees were compared in order to find a formulation showing minimal interactions with blood.
Introduction Interaction with blood proteins is an important parameter when contemplating intravenous injection of nanoscopic gene delivery systems. However, injected nanoparticles not only encounter proteins but also a vast population of cells such as erythrocytes, thrombocytes and various leukocytes. Aiming to develop a nanoscopic gene delivery system showing minimal interactions with blood cells, siRNA loaded dextran nanogels with different charges and PEGylation degrees were incubated with citrated human blood and subsequently measured by flow cytometry. Using monoclonal antibodies, different cell populations were distinguished and their interactions with the different types of nanogels were determined.
Experimental methods 1 mg of cationic dextran methacrylate nanogels with a diameter of 180 nm and zeta potential of + 27 mV were incubated with 10 ng AlexaFluor 488-labeled dicer substrate 25/27-mer siRNA targeting EGFP (IDT, Leuven, Belgium) to obtain fluorescently labeled nanogels with a zeta potential of 25 mV. Neutrally and negatively charged nanogels were prepared by adding an additional 10 and 20 ng of unlabeled dicer substrate 25/27-mer siRNA targeting EGFP. High siRNA loading efficiency and stability were determined using fluorescence correlation spectroscopy (FCS) as described before [1]. Increasing amounts of N-hydroxysuccinimidyl activated methoxypolyethylene glycol 5000 propionic acid (NHSPEG, Sigma) were added to a nanogel dispersion to covalently attach polyethylene glycol (PEG) to primary amines on the nanogel surface. The various formulations were incubated with fresh citrated blood from healthy volunteers and incubated for 1 h. Fluorescently labeled antibodies against surface receptors of erythrocytes, thrombocytes and leukocytes were used to distinguish between cell types. All samples were prepared in triplicate and measured using a FACSCalibur flow cytometer (BD, Erembodegem, Belgium). Using trypan blue, non internalized nanogels were quenched in order to determine their internalization.
Result and discussion Both the zeta potential and the PEGylation degree of the nanogels were shown to have an important influence on the interaction with blood cells. Although nanogel adsorption and internalization by erythrocytes are almost negligible, even a limited amount of interactions may have serious repercussions as erythrocytes are the most abundant cell type in the blood (approximately 5 million per microliter of blood). Nanogel adherence to thrombocytes and lymphocytes was more pronounced and was shown to be charge dependent. The influence of PEG was minimal in these cases. Nanogel interaction with monocytes and granulocytes was charge dependent and could be further decreased by covalently PEGylating the nanogels. Monocytes and granulocytes were the only cell types to efficiently internalize nanogels. This is to be expected as monocytes, neutrophils and eosinophils are typical fagocytes. Internalization of the nanogels by these cells was also shown to be dependent on the zeta potential and the PEGylation degree of the nanogels.
Fig. 1. Nanogel adsorption to granulocytes depends on the siRNA loading (nmol siRNA/ mg nanogels) and the PEGylation degree (mg NHSPEG/mg nanogels) of the nanogels.
Fig. 2. Nanogel internalization by granulocytes depends on the siRNA loading (nmol siRNA/mg nanogels) and the PEGylation degree (mg NHSPEG/mg nanogels) of the nanogels.
Conclusion Flow cytometry has proven an interesting tool in measuring the interaction between siRNA loaded dextran nanogels and blood cells. Modification of the zeta potential and PEGylation degree of these nanogels can be utilized to develop a drug delivery system showing minimal interactions with blood cells. This method can be an interesting tool for the prediction of the interactions between blood and drug delivery systems.
Acknowledgements Meditrans, an Integrated Project funded by the European Commission under the “nanotechnologies and nano-sciences, knowledge-based multifunctional materials and new production processes and devices” (NMP) thematic priority of the Sixth Framework Programme, contract number: NMP4-CT-2006-026668 is acknowledged. References [1] K. Raemdonck, B. Naeye, K. Buyens, R.E. Vandenbroucke, A. Hogset, J. Demeester, S. C. De Smedt, Biodegradable Dextran Nanogels for RNA Interference: Focusing on Endosomal Escape and Intracellular siRNA Delivery, Adv. Funct. Mater. 19 (2009) 1406–1415.
doi:10.1016/j.jconrel.2010.07.066
Monoolein as helper lipid for non-viral transfection in mammals A.C.N. Oliveira1, J.P. Neves Silva2, P.J.G. Coutinho2, A.A. Gomes1, O.P. Coutinho1, M.E.C.D. Real Oliveira1,⁎
e92
Abstracts / Journal of Controlled Release 148 (2010) e85–e101
1 Center of Molecular & Environmental Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal 2 Center of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal ⁎Corresponding author. E-mail: [email protected].
Abstract summary Lipoplexes composed of pDNA and DODAB/MO at different molar ratios (4:1, 2:1 and 1:1) and different cationic lipid/DNA charge ratios were investigated. The physicochemical properties of the lipoplexes (size and charge), the pDNA complexation, and the effect of heparin on pDNA release, were studied by dynamic light scattering, zeta potential, and ethidium bromide exclusion assays. The cytotoxicity, transfection efficiency and the intracellular localization of DNA were also evaluated on 293T cells. vectors for transfection. The role of the neutral lipid in liposome formulations is determinant for the efficiency of this process [1]. It was stated that the inclusion of helper lipids in the liposomal formulation facilitated the fusion of the complexes with the cell membrane, due to its propensity to form nonlamellar structures with negative curvature that are akin to membrane fusion intermediates [2,3]. In this work, we have studied the potential of monoolein (MO) as a helper lipid for cellular transfection.
Experimental methods Dynamic light scattering, zeta potential, and ethidium bromide exclusion assays were used for physicochemical characterization. LDH for cytotoxicity measurements, β-gal expression assay for transfection efficiency determination, and Fluorescein/Hoechst Epifluorescence microscopy for cell imaging.
Result and discussion It was found that the presence of MO not only increases the efficiency of pDNA complexation, but also affects the physicochemical properties of lipoplexes, which could possibly interfere with lipoplex–cell interactions. The DODAB:MO (2:1) and (4:1) formulations were capable to efficiently mediate in vitro cell transfection. These results were consistent with fluorescence microscopy studies, which illustrated that lipoplexes were able to enter into the cytosol and deliver pDNA to the nucleus. The understanding of the structure–activity relationship of MO based lipoplexes will be of primordial importance for the improvement of safe and efficient gene delivery systems.
Conclusion Monoolein-based lipoplexes have been shown to be efficient nonviral vectors for in vitro mammalian cell transfection. The complexation efficiency of DNA does not seem to be directly related with DNA release or transfection efficiency, but all are dependent on MO content.
[2] S.W. Hui, et al., The Role of Helper Lipids In Cationic Liposome-Mediated Gene Transfer, Biophys. J. 71 (1996) 590–599. [3] I.S. Zuhorn, et al., Phase Behavior Of Cationic Amphiphiles And Their Mixtures With Helper Lipids, Biophys. J. 83 (2002) 2096–2108.
doi:10.1016/j.jconrel.2010.07.067
Effects of charge density and hydrophobicity: Acetylation versus benzoylation of amino butyl SS-PAAS for gene delivery Martin Piest⁎, Johan F.J. Engbersen MIRA Institute for Biomedical Technology and Technical Medicine, Department of Biomedical Chemistry, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands ⁎Corresponding author. E-mail: [email protected]. Abstract summary High densities of positive charge in polymeric vectors not only result in efficient DNA condensation and particle uptake, but also induce membrane disruption, generally resulting in undesired high cytotoxicity. We previously developed a class of bioreducible poly(amido amine)s (SS-PAA)s that are significantly less toxic than PEI. In this paper we report the effect of different degrees of acetylation or benzoylation of pendant butylamine side groups in an SS-PAA. It was found that the introduction of the lipophilic benzoyl group results in a higher transfection efficiency.
Introduction Therapeutic genes can be delivered using polymeric vectors. A cationic polymer can condense a therapeutic gene into small nanosized particles and deliver these into cells to cure various kinds of metabolic diseases. It is known that protonated amine functions in a polymer facilitate condensation of DNA into small and highly positively charged polyplexes, which can adhere strongly to the negatively charged cell membrane. A high charge density may be favorable for endosomal escape, but the trade-off is that polymers with high levels of permanent charges are frequently toxic. Previously, we have investigated a broad range of biodegradable disulfide-containing cationic poly(amido amine)s (SS-PAA) for gene delivery and obtained indications that the amount of primary amine groups in the side chains of these polymers is related with cytotoxicity [1–5]. In this study we have systematically varied the relative amount of primary amines and acetylated or benzoylated amines in SS-PAA polymers derived from cystamine bisacrylamide and diaminobutane by acetylation and benzoylation of the pending primary amino group to different degrees. With this approach we were able to vary both the charge density and hydrophobicity of the polymers.
Acknowledgements The Portuguese Foundation for Science and Technology (FCT) for the financial support to the Center of Physics and Center of Molecular & Environmental Biology and funding through projects PTDC/QUI/ 69795/2006 and SFRH/BD/46968/2009 are acknowledged. References [1] P.L. Felgner, et al., Lipofection Procedure—A Highly, Efficient, Lipid-mediated DNAtransfection Procedure. Proceedings of the National Academy of Sciences U.S.A. 84 (1987) 7413–7417.
Scheme 1. i) Michael addition polymerization of cystamine bisacrylamide with N-BOC 1,4-diaminobutane; ii) deprotection with HCl (g); iiia) functionalization with acetic anhydride; iiib) functionalization with benzoyl chloride.
e91
Abstract summary Adsorption and internalization of siRNA loaded dextran nanogels by blood cells were determined using flow cytometry. Positively and negatively charged nanogels with various PEGylation degrees were compared in order to find a formulation showing minimal interactions with blood.
Introduction Interaction with blood proteins is an important parameter when contemplating intravenous injection of nanoscopic gene delivery systems. However, injected nanoparticles not only encounter proteins but also a vast population of cells such as erythrocytes, thrombocytes and various leukocytes. Aiming to develop a nanoscopic gene delivery system showing minimal interactions with blood cells, siRNA loaded dextran nanogels with different charges and PEGylation degrees were incubated with citrated human blood and subsequently measured by flow cytometry. Using monoclonal antibodies, different cell populations were distinguished and their interactions with the different types of nanogels were determined.
Experimental methods 1 mg of cationic dextran methacrylate nanogels with a diameter of 180 nm and zeta potential of + 27 mV were incubated with 10 ng AlexaFluor 488-labeled dicer substrate 25/27-mer siRNA targeting EGFP (IDT, Leuven, Belgium) to obtain fluorescently labeled nanogels with a zeta potential of 25 mV. Neutrally and negatively charged nanogels were prepared by adding an additional 10 and 20 ng of unlabeled dicer substrate 25/27-mer siRNA targeting EGFP. High siRNA loading efficiency and stability were determined using fluorescence correlation spectroscopy (FCS) as described before [1]. Increasing amounts of N-hydroxysuccinimidyl activated methoxypolyethylene glycol 5000 propionic acid (NHSPEG, Sigma) were added to a nanogel dispersion to covalently attach polyethylene glycol (PEG) to primary amines on the nanogel surface. The various formulations were incubated with fresh citrated blood from healthy volunteers and incubated for 1 h. Fluorescently labeled antibodies against surface receptors of erythrocytes, thrombocytes and leukocytes were used to distinguish between cell types. All samples were prepared in triplicate and measured using a FACSCalibur flow cytometer (BD, Erembodegem, Belgium). Using trypan blue, non internalized nanogels were quenched in order to determine their internalization.
Result and discussion Both the zeta potential and the PEGylation degree of the nanogels were shown to have an important influence on the interaction with blood cells. Although nanogel adsorption and internalization by erythrocytes are almost negligible, even a limited amount of interactions may have serious repercussions as erythrocytes are the most abundant cell type in the blood (approximately 5 million per microliter of blood). Nanogel adherence to thrombocytes and lymphocytes was more pronounced and was shown to be charge dependent. The influence of PEG was minimal in these cases. Nanogel interaction with monocytes and granulocytes was charge dependent and could be further decreased by covalently PEGylating the nanogels. Monocytes and granulocytes were the only cell types to efficiently internalize nanogels. This is to be expected as monocytes, neutrophils and eosinophils are typical fagocytes. Internalization of the nanogels by these cells was also shown to be dependent on the zeta potential and the PEGylation degree of the nanogels.
Fig. 1. Nanogel adsorption to granulocytes depends on the siRNA loading (nmol siRNA/ mg nanogels) and the PEGylation degree (mg NHSPEG/mg nanogels) of the nanogels.
Fig. 2. Nanogel internalization by granulocytes depends on the siRNA loading (nmol siRNA/mg nanogels) and the PEGylation degree (mg NHSPEG/mg nanogels) of the nanogels.
Conclusion Flow cytometry has proven an interesting tool in measuring the interaction between siRNA loaded dextran nanogels and blood cells. Modification of the zeta potential and PEGylation degree of these nanogels can be utilized to develop a drug delivery system showing minimal interactions with blood cells. This method can be an interesting tool for the prediction of the interactions between blood and drug delivery systems.
Acknowledgements Meditrans, an Integrated Project funded by the European Commission under the “nanotechnologies and nano-sciences, knowledge-based multifunctional materials and new production processes and devices” (NMP) thematic priority of the Sixth Framework Programme, contract number: NMP4-CT-2006-026668 is acknowledged. References [1] K. Raemdonck, B. Naeye, K. Buyens, R.E. Vandenbroucke, A. Hogset, J. Demeester, S. C. De Smedt, Biodegradable Dextran Nanogels for RNA Interference: Focusing on Endosomal Escape and Intracellular siRNA Delivery, Adv. Funct. Mater. 19 (2009) 1406–1415.
doi:10.1016/j.jconrel.2010.07.066
Monoolein as helper lipid for non-viral transfection in mammals A.C.N. Oliveira1, J.P. Neves Silva2, P.J.G. Coutinho2, A.A. Gomes1, O.P. Coutinho1, M.E.C.D. Real Oliveira1,⁎
e92
Abstracts / Journal of Controlled Release 148 (2010) e85–e101
1 Center of Molecular & Environmental Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal 2 Center of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal ⁎Corresponding author. E-mail: [email protected].
Abstract summary Lipoplexes composed of pDNA and DODAB/MO at different molar ratios (4:1, 2:1 and 1:1) and different cationic lipid/DNA charge ratios were investigated. The physicochemical properties of the lipoplexes (size and charge), the pDNA complexation, and the effect of heparin on pDNA release, were studied by dynamic light scattering, zeta potential, and ethidium bromide exclusion assays. The cytotoxicity, transfection efficiency and the intracellular localization of DNA were also evaluated on 293T cells. vectors for transfection. The role of the neutral lipid in liposome formulations is determinant for the efficiency of this process [1]. It was stated that the inclusion of helper lipids in the liposomal formulation facilitated the fusion of the complexes with the cell membrane, due to its propensity to form nonlamellar structures with negative curvature that are akin to membrane fusion intermediates [2,3]. In this work, we have studied the potential of monoolein (MO) as a helper lipid for cellular transfection.
Experimental methods Dynamic light scattering, zeta potential, and ethidium bromide exclusion assays were used for physicochemical characterization. LDH for cytotoxicity measurements, β-gal expression assay for transfection efficiency determination, and Fluorescein/Hoechst Epifluorescence microscopy for cell imaging.
Result and discussion It was found that the presence of MO not only increases the efficiency of pDNA complexation, but also affects the physicochemical properties of lipoplexes, which could possibly interfere with lipoplex–cell interactions. The DODAB:MO (2:1) and (4:1) formulations were capable to efficiently mediate in vitro cell transfection. These results were consistent with fluorescence microscopy studies, which illustrated that lipoplexes were able to enter into the cytosol and deliver pDNA to the nucleus. The understanding of the structure–activity relationship of MO based lipoplexes will be of primordial importance for the improvement of safe and efficient gene delivery systems.
Conclusion Monoolein-based lipoplexes have been shown to be efficient nonviral vectors for in vitro mammalian cell transfection. The complexation efficiency of DNA does not seem to be directly related with DNA release or transfection efficiency, but all are dependent on MO content.
[2] S.W. Hui, et al., The Role of Helper Lipids In Cationic Liposome-Mediated Gene Transfer, Biophys. J. 71 (1996) 590–599. [3] I.S. Zuhorn, et al., Phase Behavior Of Cationic Amphiphiles And Their Mixtures With Helper Lipids, Biophys. J. 83 (2002) 2096–2108.
doi:10.1016/j.jconrel.2010.07.067
Effects of charge density and hydrophobicity: Acetylation versus benzoylation of amino butyl SS-PAAS for gene delivery Martin Piest⁎, Johan F.J. Engbersen MIRA Institute for Biomedical Technology and Technical Medicine, Department of Biomedical Chemistry, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands ⁎Corresponding author. E-mail: [email protected]. Abstract summary High densities of positive charge in polymeric vectors not only result in efficient DNA condensation and particle uptake, but also induce membrane disruption, generally resulting in undesired high cytotoxicity. We previously developed a class of bioreducible poly(amido amine)s (SS-PAA)s that are significantly less toxic than PEI. In this paper we report the effect of different degrees of acetylation or benzoylation of pendant butylamine side groups in an SS-PAA. It was found that the introduction of the lipophilic benzoyl group results in a higher transfection efficiency.
Introduction Therapeutic genes can be delivered using polymeric vectors. A cationic polymer can condense a therapeutic gene into small nanosized particles and deliver these into cells to cure various kinds of metabolic diseases. It is known that protonated amine functions in a polymer facilitate condensation of DNA into small and highly positively charged polyplexes, which can adhere strongly to the negatively charged cell membrane. A high charge density may be favorable for endosomal escape, but the trade-off is that polymers with high levels of permanent charges are frequently toxic. Previously, we have investigated a broad range of biodegradable disulfide-containing cationic poly(amido amine)s (SS-PAA) for gene delivery and obtained indications that the amount of primary amine groups in the side chains of these polymers is related with cytotoxicity [1–5]. In this study we have systematically varied the relative amount of primary amines and acetylated or benzoylated amines in SS-PAA polymers derived from cystamine bisacrylamide and diaminobutane by acetylation and benzoylation of the pending primary amino group to different degrees. With this approach we were able to vary both the charge density and hydrophobicity of the polymers.
Acknowledgements The Portuguese Foundation for Science and Technology (FCT) for the financial support to the Center of Physics and Center of Molecular & Environmental Biology and funding through projects PTDC/QUI/ 69795/2006 and SFRH/BD/46968/2009 are acknowledged. References [1] P.L. Felgner, et al., Lipofection Procedure—A Highly, Efficient, Lipid-mediated DNAtransfection Procedure. Proceedings of the National Academy of Sciences U.S.A. 84 (1987) 7413–7417.
Scheme 1. i) Michael addition polymerization of cystamine bisacrylamide with N-BOC 1,4-diaminobutane; ii) deprotection with HCl (g); iiia) functionalization with acetic anhydride; iiib) functionalization with benzoyl chloride.