The Experts below are selected from a list of 3900 Experts worldwide ranked by ideXlab platform
Giulio Caracciolo - One of the best experts on this subject based on the ideXlab platform.
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cationic liposome dna complexes from structure to interactions with cellular membranes
European Biophysics Journal, 2012Co-Authors: Giulio Caracciolo, Heinz AmenitschAbstract:Gene-based therapeutic approaches are based upon the concept that, if a disease is caused by a mutation in a gene, then adding back the wild-type gene should restore regular function and attenuate the disease phenotype. To deliver the gene of interest, both viral and nonviral vectors are used. Viruses are efficient, but their application is impeded by detrimental side-effects. Among nonviral vectors, cationic liposomes are the most promising candidates for gene delivery. They form stable complexes with polyanionic DNA (Lipoplexes). Despite several advantages over viral vectors, the transfection efficiency (TE) of Lipoplexes is too low compared with those of engineered viral vectors. This is due to lack of knowledge about the interactions between complexes and cellular components. Rational design of efficient Lipoplexes therefore requires deeper comprehension of the interactions between the vector and the DNA as well as the cellular pathways and mechanisms involved. The importance of the lipoplex structure in biological function is revealed in the application of synchrotron small-angle X-ray scattering in combination with functional TE measurements. According to current understanding, the structure of Lipoplexes can change upon interaction with cellular membranes and such changes affect the delivery efficiency. Recently, a correlation between the mechanism of gene release from complexes, the structure, and the physical and chemical parameters of the complexes has been established. Studies aimed at correlating structure and activity of Lipoplexes are reviewed herein. This is a fundamental step towards rational design of highly efficient lipid gene vectors.
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cholesterol dependent macropinocytosis and endosomal escape control the transfection efficiency of Lipoplexes in cho living cells
Molecular Pharmaceutics, 2012Co-Authors: Francesco Cardarelli, Cristina Marchini, Daniela Pozzi, Angelo Bifone, Giulio CaraccioloAbstract:Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition and efficiency for intracellular delivery of DNA. The first formulation is made of cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic helper dioleoylphosphocholine (DOPC), while the second one is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE). Combining pharmacological and imaging approaches we show that both DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA Lipoplexes are taken up in Chinese hamster ovary (CHO) living cells mainly through fluid-phase macropinocytosis. Our results also indicate that lipoplex macropinocytosis is a cholesterol-sensitive uptake mechanism. On the other side, both clathrin-mediated and caveolae-mediated endocytosis play a minor role, if any, in the cell uptake. Colocalization of fluorescently tagged Lipoplexes and Lysosensor, a primary lysosome marker, reveals that poorly efficient DOTAP-DOPC/DNA Lipoplexes are largely degraded in the lysosomes, while efficient DC-Chol-DOPE/DNA systems can efficiently escape from endosomal compartments.
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Cholesterol-Dependent Macropinocytosis and Endosomal Escape Control the Transfection Efficiency of Lipoplexes in CHO Living Cells
2012Co-Authors: Francesco Cardarelli, Cristina Marchini, Daniela Pozzi, Angelo Bifone, Giulio CaraccioloAbstract:Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition and efficiency for intracellular delivery of DNA. The first formulation is made of cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic helper dioleoylphosphocholine (DOPC), while the second one is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE). Combining pharmacological and imaging approaches we show that both DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA Lipoplexes are taken up in Chinese hamster ovary (CHO) living cells mainly through fluid-phase macropinocytosis. Our results also indicate that lipoplex macropinocytosis is a cholesterol-sensitive uptake mechanism. On the other side, both clathrin-mediated and caveolae-mediated endocytosis play a minor role, if any, in the cell uptake. Colocalization of fluorescently tagged Lipoplexes and Lysosensor, a primary lysosome marker, reveals that poorly efficient DOTAP-DOPC/DNA Lipoplexes are largely degraded in the lysosomes, while efficient DC-Chol-DOPE/DNA systems can efficiently escape from endosomal compartments
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differential analysis of protein corona profile adsorbed onto different nonviral gene delivery systems
Analytical Biochemistry, 2011Co-Authors: Anna Laura Capriotti, Daniela Pozzi, Giulio Caracciolo, Giuseppe Caruso, Patrizia Foglia, Roberto Samperi, Aldo LaganaAbstract:A shotgun proteomics approach was used to characterize and compare the proteins that lead to the formation of a rich "protein corona" adsorbed onto the surfaces of cationic liposomes (CLs), Lipoplexes, and lipid/polycation/DNA (LPD) complexes, when they come into contact with plasma. After separation of the nanoparticle-protein complex from plasma, the protein mixture was digested, and peptides were analyzed by nanoliquid chromatography-Orbitrap LTQ-XL mass spectrometry. The number of proteins bound to Lipoplexes was double that of those identified in the corona of CLs (208 vs 105), while 77 proteins were common to both coronas. The number of proteins bound to the surface of the LPD complexes (158, 133 of which are common to Lipoplexes) is intermediate between those found in the protein corona of both CLs and Lipoplexes. About half of them were found in the protein corona of CLs. By overlapping the three formulations, it can be seen that only 12 proteins are peculiar to LPD complexes. These results may help in designing gene delivery systems capable of binding the minimum possible quantity of proteins that influence transfection negatively, binding selectively proteins capable of helping in steering in vivo the vector toward the target, and obtaining more efficient and effective gene therapy.
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surface adsorption of protein corona controls the cell internalization mechanism of dc chol dope dna Lipoplexes in serum
Biochimica et Biophysica Acta, 2010Co-Authors: Giulio Caracciolo, Daniela Pozzi, Chiara Cavaliere, Luciano Callipo, Sofia Candeloro De Sanctis, Aldo LaganaAbstract:Abstract Serum has often been reported as a barrier to efficient lipid-mediated transfection. Here we found that the transfection efficiency of DC-Chol–DOPE/DNA Lipoplexes increases in serum. To provide insight into the mechanism of lipoplex-serum interaction, several state-of-the-art methodologies have been applied. The nanostructure of DC-Chol–DOPE/DNA Lipoplexes was found to be serum-resistant as revealed by high resolution synchrotron small angle X-ray scattering, while dynamic light scattering measurements showed a marked size increase of complexes. The structural stability of DC-Chol–DOPE/DNA Lipoplexes was confirmed by electrophoresis on agarose gel demonstrating that plasmid DNA remained well protected by lipids. Proteomics experiments showed that serum proteins competed for the cationic surface of lipid membranes leading to the formation of a rich a ‘protein corona’. Combining structural results with proteomics findings, we suggest that such a protein corona can promote large aggregation of intact Lipoplexes. According to a recently proposed size-dependent mechanism of lipoplex entry within cells, protein corona-induced formation of large aggregates most likely results in a switch from a clathrin-dependent to caveolae-mediated entry pathway into the cells which is likely to be responsible for the observed transfection efficiency boost. As a consequence, we suggest that surface adsorption of protein corona can have a high biological impact on serum-resistant cationic formulations for in vitro and in vivo lipid-mediated gene delivery applications.
Daniela Pozzi - One of the best experts on this subject based on the ideXlab platform.
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Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: Interplay between nanostructure and composition
'Elsevier BV', 2014Co-Authors: Daniela Pozzi, Ma Digman, C. Marchini, F. Cardarelli, F. Salomone, Stefano Coppola, M. Montani, Elexpuru M. Zabaleta, E. Gratton, Valentina ColapicchioniAbstract:Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of Lipoplexes and lipid-protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that Lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both Lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than Lipoplexes. When cells were treated with DOTAP-DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol-DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability. © 2013 Elsevier B.V
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cholesterol dependent macropinocytosis and endosomal escape control the transfection efficiency of Lipoplexes in cho living cells
Molecular Pharmaceutics, 2012Co-Authors: Francesco Cardarelli, Cristina Marchini, Daniela Pozzi, Angelo Bifone, Giulio CaraccioloAbstract:Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition and efficiency for intracellular delivery of DNA. The first formulation is made of cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic helper dioleoylphosphocholine (DOPC), while the second one is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE). Combining pharmacological and imaging approaches we show that both DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA Lipoplexes are taken up in Chinese hamster ovary (CHO) living cells mainly through fluid-phase macropinocytosis. Our results also indicate that lipoplex macropinocytosis is a cholesterol-sensitive uptake mechanism. On the other side, both clathrin-mediated and caveolae-mediated endocytosis play a minor role, if any, in the cell uptake. Colocalization of fluorescently tagged Lipoplexes and Lysosensor, a primary lysosome marker, reveals that poorly efficient DOTAP-DOPC/DNA Lipoplexes are largely degraded in the lysosomes, while efficient DC-Chol-DOPE/DNA systems can efficiently escape from endosomal compartments.
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Cholesterol-Dependent Macropinocytosis and Endosomal Escape Control the Transfection Efficiency of Lipoplexes in CHO Living Cells
2012Co-Authors: Francesco Cardarelli, Cristina Marchini, Daniela Pozzi, Angelo Bifone, Giulio CaraccioloAbstract:Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition and efficiency for intracellular delivery of DNA. The first formulation is made of cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic helper dioleoylphosphocholine (DOPC), while the second one is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE). Combining pharmacological and imaging approaches we show that both DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA Lipoplexes are taken up in Chinese hamster ovary (CHO) living cells mainly through fluid-phase macropinocytosis. Our results also indicate that lipoplex macropinocytosis is a cholesterol-sensitive uptake mechanism. On the other side, both clathrin-mediated and caveolae-mediated endocytosis play a minor role, if any, in the cell uptake. Colocalization of fluorescently tagged Lipoplexes and Lysosensor, a primary lysosome marker, reveals that poorly efficient DOTAP-DOPC/DNA Lipoplexes are largely degraded in the lysosomes, while efficient DC-Chol-DOPE/DNA systems can efficiently escape from endosomal compartments
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differential analysis of protein corona profile adsorbed onto different nonviral gene delivery systems
Analytical Biochemistry, 2011Co-Authors: Anna Laura Capriotti, Daniela Pozzi, Giulio Caracciolo, Giuseppe Caruso, Patrizia Foglia, Roberto Samperi, Aldo LaganaAbstract:A shotgun proteomics approach was used to characterize and compare the proteins that lead to the formation of a rich "protein corona" adsorbed onto the surfaces of cationic liposomes (CLs), Lipoplexes, and lipid/polycation/DNA (LPD) complexes, when they come into contact with plasma. After separation of the nanoparticle-protein complex from plasma, the protein mixture was digested, and peptides were analyzed by nanoliquid chromatography-Orbitrap LTQ-XL mass spectrometry. The number of proteins bound to Lipoplexes was double that of those identified in the corona of CLs (208 vs 105), while 77 proteins were common to both coronas. The number of proteins bound to the surface of the LPD complexes (158, 133 of which are common to Lipoplexes) is intermediate between those found in the protein corona of both CLs and Lipoplexes. About half of them were found in the protein corona of CLs. By overlapping the three formulations, it can be seen that only 12 proteins are peculiar to LPD complexes. These results may help in designing gene delivery systems capable of binding the minimum possible quantity of proteins that influence transfection negatively, binding selectively proteins capable of helping in steering in vivo the vector toward the target, and obtaining more efficient and effective gene therapy.
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surface adsorption of protein corona controls the cell internalization mechanism of dc chol dope dna Lipoplexes in serum
Biochimica et Biophysica Acta, 2010Co-Authors: Giulio Caracciolo, Daniela Pozzi, Chiara Cavaliere, Luciano Callipo, Sofia Candeloro De Sanctis, Aldo LaganaAbstract:Abstract Serum has often been reported as a barrier to efficient lipid-mediated transfection. Here we found that the transfection efficiency of DC-Chol–DOPE/DNA Lipoplexes increases in serum. To provide insight into the mechanism of lipoplex-serum interaction, several state-of-the-art methodologies have been applied. The nanostructure of DC-Chol–DOPE/DNA Lipoplexes was found to be serum-resistant as revealed by high resolution synchrotron small angle X-ray scattering, while dynamic light scattering measurements showed a marked size increase of complexes. The structural stability of DC-Chol–DOPE/DNA Lipoplexes was confirmed by electrophoresis on agarose gel demonstrating that plasmid DNA remained well protected by lipids. Proteomics experiments showed that serum proteins competed for the cationic surface of lipid membranes leading to the formation of a rich a ‘protein corona’. Combining structural results with proteomics findings, we suggest that such a protein corona can promote large aggregation of intact Lipoplexes. According to a recently proposed size-dependent mechanism of lipoplex entry within cells, protein corona-induced formation of large aggregates most likely results in a switch from a clathrin-dependent to caveolae-mediated entry pathway into the cells which is likely to be responsible for the observed transfection efficiency boost. As a consequence, we suggest that surface adsorption of protein corona can have a high biological impact on serum-resistant cationic formulations for in vitro and in vivo lipid-mediated gene delivery applications.
Cristina Marchini - One of the best experts on this subject based on the ideXlab platform.
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cholesterol dependent macropinocytosis and endosomal escape control the transfection efficiency of Lipoplexes in cho living cells
Molecular Pharmaceutics, 2012Co-Authors: Francesco Cardarelli, Cristina Marchini, Daniela Pozzi, Angelo Bifone, Giulio CaraccioloAbstract:Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition and efficiency for intracellular delivery of DNA. The first formulation is made of cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic helper dioleoylphosphocholine (DOPC), while the second one is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE). Combining pharmacological and imaging approaches we show that both DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA Lipoplexes are taken up in Chinese hamster ovary (CHO) living cells mainly through fluid-phase macropinocytosis. Our results also indicate that lipoplex macropinocytosis is a cholesterol-sensitive uptake mechanism. On the other side, both clathrin-mediated and caveolae-mediated endocytosis play a minor role, if any, in the cell uptake. Colocalization of fluorescently tagged Lipoplexes and Lysosensor, a primary lysosome marker, reveals that poorly efficient DOTAP-DOPC/DNA Lipoplexes are largely degraded in the lysosomes, while efficient DC-Chol-DOPE/DNA systems can efficiently escape from endosomal compartments.
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Cholesterol-Dependent Macropinocytosis and Endosomal Escape Control the Transfection Efficiency of Lipoplexes in CHO Living Cells
2012Co-Authors: Francesco Cardarelli, Cristina Marchini, Daniela Pozzi, Angelo Bifone, Giulio CaraccioloAbstract:Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition and efficiency for intracellular delivery of DNA. The first formulation is made of cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic helper dioleoylphosphocholine (DOPC), while the second one is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE). Combining pharmacological and imaging approaches we show that both DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA Lipoplexes are taken up in Chinese hamster ovary (CHO) living cells mainly through fluid-phase macropinocytosis. Our results also indicate that lipoplex macropinocytosis is a cholesterol-sensitive uptake mechanism. On the other side, both clathrin-mediated and caveolae-mediated endocytosis play a minor role, if any, in the cell uptake. Colocalization of fluorescently tagged Lipoplexes and Lysosensor, a primary lysosome marker, reveals that poorly efficient DOTAP-DOPC/DNA Lipoplexes are largely degraded in the lysosomes, while efficient DC-Chol-DOPE/DNA systems can efficiently escape from endosomal compartments
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role of temperature independent lipoplex cell membrane interactions in the efficiency boost of multicomponent Lipoplexes
Cancer Gene Therapy, 2011Co-Authors: Cristina Marchini, Maura Montani, Augusto Amici, Cinzia Alfonsi, Candeloro S De Sanctis, Ma Digman, Susana A Sanchez, Heinz AmenitschAbstract:Multicomponent Lipoplexes have recently emerged as especially promising transfection candidates, as they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Previously, we investigated a number of chemical-physical properties of DNA-lipid complexes that were proposed to affect transfection efficiency (TE) of Lipoplexes, such as nanoscale structure, size, surface potential, DNA-protection ability and DNA release from complexes upon interaction with cellular lipids. Although some minor differences between multicomponent and binary Lipoplexes were found, they did not correlate clearly with efficiency. Instead, here we show that a marked difference between the cell internalization mechanism of binary and multicomponent Lipoplexes does exist. Multicomponent Lipoplexes significantly transfect cells at 4 °C, when endocytosis does not take place suggesting that they can enter cells via a temperature-independent mechanism. Confocal fluorescence microscopy experiments showed the existence of a correlation between endosomal escape and TE. Multicomponent Lipoplexes exhibited a distinctive ability of endosomal escape and release DNA into the nucleus, whereas, poorly efficient binary Lipoplexes exhibited minor, if any, endosomal rupture ability and remained confined in perinuclear late endosomes. Stopped-flow mixing measurements showed that the fusion rates of multicomponent cationic liposomes with anionic vesicles, used as model systems of cell membranes, were definitely shorter than those of binary liposomes. As either lipoplex uptake and endosomal escape involve fusion between lipoplex and cellular membranes, we suggest that a mechanism of lipoplex-cellular membrane interaction, driven by lipid mixing between cationic and anionic cellular lipids, does explain the TE boost of multicomponent Lipoplexes.
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tailoring lipoplex composition to the lipid composition of plasma membrane a trojan horse for cell entry
Langmuir, 2010Co-Authors: Cristina Marchini, Heinz Amenitsch, Maura Montani, Augusto Amici, Cinzia Alfonsi, Sofia Candeloro De SanctisAbstract:The first interaction between Lipoplexes and cells is charge-mediated and not specific. Endocytosis is considered to be the main pathway for lipoplex entry. Upon interaction between Lipoplexes and the plasma membrane, intermixing between lipoplex and membrane lipids is necessary for efficient endocytosis. Here we study the mechanism of the different endocytic pathways in lipid-mediated gene delivery. We show that DC−Chol−DOPE/DNA Lipoplexes preferentially use a raft-mediated endocytosis, while DOTAP−DOPC/DNA systems are mainly internalized by not specific fluid phase macropinocitosys. On the other hand, most efficient multicomponent Lipoplexes, incorporating different lipid species in their lipid bilayer, can use multiple endocytic pathways to enter cells. Our data demonstrate that efficiency of endocytosis is regulated by shape coupling between lipoplex and membrane lipids. We suggest that such a shape-dependent coupling regulates efficient formation of endocytic vesicles thus determining the success of ...
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toward the rational design of lipid gene vectors shape coupling between lipoplex and anionic cellular lipids controls the phase evolution of Lipoplexes and the efficiency of dna release
ACS Applied Materials & Interfaces, 2009Co-Authors: Ruggero Caminiti, Heinz Amenitsch, Cristina Marchini, Maura Montani, Sofia Candeloro De Sanctis, Augusto AmiciAbstract:A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of Lipoplexes upon interaction with anionic cellular lipids, resulting in DNA release. At the early stages of interaction, we found a universal behavior of lipoplex/anionic lipid (AL) mixtures: the lipoplex structure is slightly perturbed, while the one-dimensional DNA lattice between cationic membranes is largely diluted by ALs. This finding is in excellent agreement with previous suggestions on the mechanism of DNA unbinding from Lipoplexes by ALs. Upon further interaction, the propensity of a given lipoplex structure to be solubilized by anionic cellular lipids strongly depends on the shape coupling between lipoplex and ALs. Furthermore, we investigated the effect of the membrane charge density and a general correlation resulted: the higher the membrane charge density of anionic membranes, the higher their ability to solubilize the structure of Lipoplexes and to promote DNA releas...
Heinz Amenitsch - One of the best experts on this subject based on the ideXlab platform.
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cationic liposome dna complexes from structure to interactions with cellular membranes
European Biophysics Journal, 2012Co-Authors: Giulio Caracciolo, Heinz AmenitschAbstract:Gene-based therapeutic approaches are based upon the concept that, if a disease is caused by a mutation in a gene, then adding back the wild-type gene should restore regular function and attenuate the disease phenotype. To deliver the gene of interest, both viral and nonviral vectors are used. Viruses are efficient, but their application is impeded by detrimental side-effects. Among nonviral vectors, cationic liposomes are the most promising candidates for gene delivery. They form stable complexes with polyanionic DNA (Lipoplexes). Despite several advantages over viral vectors, the transfection efficiency (TE) of Lipoplexes is too low compared with those of engineered viral vectors. This is due to lack of knowledge about the interactions between complexes and cellular components. Rational design of efficient Lipoplexes therefore requires deeper comprehension of the interactions between the vector and the DNA as well as the cellular pathways and mechanisms involved. The importance of the lipoplex structure in biological function is revealed in the application of synchrotron small-angle X-ray scattering in combination with functional TE measurements. According to current understanding, the structure of Lipoplexes can change upon interaction with cellular membranes and such changes affect the delivery efficiency. Recently, a correlation between the mechanism of gene release from complexes, the structure, and the physical and chemical parameters of the complexes has been established. Studies aimed at correlating structure and activity of Lipoplexes are reviewed herein. This is a fundamental step towards rational design of highly efficient lipid gene vectors.
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role of temperature independent lipoplex cell membrane interactions in the efficiency boost of multicomponent Lipoplexes
Cancer Gene Therapy, 2011Co-Authors: Cristina Marchini, Maura Montani, Augusto Amici, Cinzia Alfonsi, Candeloro S De Sanctis, Ma Digman, Susana A Sanchez, Heinz AmenitschAbstract:Multicomponent Lipoplexes have recently emerged as especially promising transfection candidates, as they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Previously, we investigated a number of chemical-physical properties of DNA-lipid complexes that were proposed to affect transfection efficiency (TE) of Lipoplexes, such as nanoscale structure, size, surface potential, DNA-protection ability and DNA release from complexes upon interaction with cellular lipids. Although some minor differences between multicomponent and binary Lipoplexes were found, they did not correlate clearly with efficiency. Instead, here we show that a marked difference between the cell internalization mechanism of binary and multicomponent Lipoplexes does exist. Multicomponent Lipoplexes significantly transfect cells at 4 °C, when endocytosis does not take place suggesting that they can enter cells via a temperature-independent mechanism. Confocal fluorescence microscopy experiments showed the existence of a correlation between endosomal escape and TE. Multicomponent Lipoplexes exhibited a distinctive ability of endosomal escape and release DNA into the nucleus, whereas, poorly efficient binary Lipoplexes exhibited minor, if any, endosomal rupture ability and remained confined in perinuclear late endosomes. Stopped-flow mixing measurements showed that the fusion rates of multicomponent cationic liposomes with anionic vesicles, used as model systems of cell membranes, were definitely shorter than those of binary liposomes. As either lipoplex uptake and endosomal escape involve fusion between lipoplex and cellular membranes, we suggest that a mechanism of lipoplex-cellular membrane interaction, driven by lipid mixing between cationic and anionic cellular lipids, does explain the TE boost of multicomponent Lipoplexes.
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tailoring lipoplex composition to the lipid composition of plasma membrane a trojan horse for cell entry
Langmuir, 2010Co-Authors: Cristina Marchini, Heinz Amenitsch, Maura Montani, Augusto Amici, Cinzia Alfonsi, Sofia Candeloro De SanctisAbstract:The first interaction between Lipoplexes and cells is charge-mediated and not specific. Endocytosis is considered to be the main pathway for lipoplex entry. Upon interaction between Lipoplexes and the plasma membrane, intermixing between lipoplex and membrane lipids is necessary for efficient endocytosis. Here we study the mechanism of the different endocytic pathways in lipid-mediated gene delivery. We show that DC−Chol−DOPE/DNA Lipoplexes preferentially use a raft-mediated endocytosis, while DOTAP−DOPC/DNA systems are mainly internalized by not specific fluid phase macropinocitosys. On the other hand, most efficient multicomponent Lipoplexes, incorporating different lipid species in their lipid bilayer, can use multiple endocytic pathways to enter cells. Our data demonstrate that efficiency of endocytosis is regulated by shape coupling between lipoplex and membrane lipids. We suggest that such a shape-dependent coupling regulates efficient formation of endocytic vesicles thus determining the success of ...
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toward the rational design of lipid gene vectors shape coupling between lipoplex and anionic cellular lipids controls the phase evolution of Lipoplexes and the efficiency of dna release
ACS Applied Materials & Interfaces, 2009Co-Authors: Ruggero Caminiti, Heinz Amenitsch, Cristina Marchini, Maura Montani, Sofia Candeloro De Sanctis, Augusto AmiciAbstract:A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of Lipoplexes upon interaction with anionic cellular lipids, resulting in DNA release. At the early stages of interaction, we found a universal behavior of lipoplex/anionic lipid (AL) mixtures: the lipoplex structure is slightly perturbed, while the one-dimensional DNA lattice between cationic membranes is largely diluted by ALs. This finding is in excellent agreement with previous suggestions on the mechanism of DNA unbinding from Lipoplexes by ALs. Upon further interaction, the propensity of a given lipoplex structure to be solubilized by anionic cellular lipids strongly depends on the shape coupling between lipoplex and ALs. Furthermore, we investigated the effect of the membrane charge density and a general correlation resulted: the higher the membrane charge density of anionic membranes, the higher their ability to solubilize the structure of Lipoplexes and to promote DNA releas...
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structural stability against disintegration by anionic lipids rationalizes the efficiency of cationic liposome dna complexes
Langmuir, 2007Co-Authors: Cristina Marchini, Heinz Amenitsch, Ruggero Caminiti, Maura Montani, Augusto AmiciAbstract:Reported here is the correlation between the transfection efficiency of cationic liposome/DNA complexes (Lipoplexes) and the structural evolution that they undergo when interacting with anionic membrane lipids. Multicomponent Lipoplexes, incorporating from three to six lipid species simultaneously, presented a much higher transfection efficiency than binary Lipoplexes, which are more commonly used for gene-delivery purposes. The discovery that a high transfection efficiency can be achieved by employing multicomponent complexes at a lower-than-ever-before membrane charge density of Lipoplexes was of primary significance. Synchrotron small-angle X-ray diffraction (SAXD) experiments showed that anionic liposomes made of dioleoylphosphatidylglycerol (DOPG) disintegrated the lamellar phase of Lipoplexes. DNA unbinding was measured by electrophoresis on agarose gels. Most importantly, structural changes induced by anionic lipids strictly depended on the lipid composition of Lipoplexes. We found evidence of the ...
Aldo Lagana - One of the best experts on this subject based on the ideXlab platform.
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differential analysis of protein corona profile adsorbed onto different nonviral gene delivery systems
Analytical Biochemistry, 2011Co-Authors: Anna Laura Capriotti, Daniela Pozzi, Giulio Caracciolo, Giuseppe Caruso, Patrizia Foglia, Roberto Samperi, Aldo LaganaAbstract:A shotgun proteomics approach was used to characterize and compare the proteins that lead to the formation of a rich "protein corona" adsorbed onto the surfaces of cationic liposomes (CLs), Lipoplexes, and lipid/polycation/DNA (LPD) complexes, when they come into contact with plasma. After separation of the nanoparticle-protein complex from plasma, the protein mixture was digested, and peptides were analyzed by nanoliquid chromatography-Orbitrap LTQ-XL mass spectrometry. The number of proteins bound to Lipoplexes was double that of those identified in the corona of CLs (208 vs 105), while 77 proteins were common to both coronas. The number of proteins bound to the surface of the LPD complexes (158, 133 of which are common to Lipoplexes) is intermediate between those found in the protein corona of both CLs and Lipoplexes. About half of them were found in the protein corona of CLs. By overlapping the three formulations, it can be seen that only 12 proteins are peculiar to LPD complexes. These results may help in designing gene delivery systems capable of binding the minimum possible quantity of proteins that influence transfection negatively, binding selectively proteins capable of helping in steering in vivo the vector toward the target, and obtaining more efficient and effective gene therapy.
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surface adsorption of protein corona controls the cell internalization mechanism of dc chol dope dna Lipoplexes in serum
Biochimica et Biophysica Acta, 2010Co-Authors: Giulio Caracciolo, Daniela Pozzi, Chiara Cavaliere, Luciano Callipo, Sofia Candeloro De Sanctis, Aldo LaganaAbstract:Abstract Serum has often been reported as a barrier to efficient lipid-mediated transfection. Here we found that the transfection efficiency of DC-Chol–DOPE/DNA Lipoplexes increases in serum. To provide insight into the mechanism of lipoplex-serum interaction, several state-of-the-art methodologies have been applied. The nanostructure of DC-Chol–DOPE/DNA Lipoplexes was found to be serum-resistant as revealed by high resolution synchrotron small angle X-ray scattering, while dynamic light scattering measurements showed a marked size increase of complexes. The structural stability of DC-Chol–DOPE/DNA Lipoplexes was confirmed by electrophoresis on agarose gel demonstrating that plasmid DNA remained well protected by lipids. Proteomics experiments showed that serum proteins competed for the cationic surface of lipid membranes leading to the formation of a rich a ‘protein corona’. Combining structural results with proteomics findings, we suggest that such a protein corona can promote large aggregation of intact Lipoplexes. According to a recently proposed size-dependent mechanism of lipoplex entry within cells, protein corona-induced formation of large aggregates most likely results in a switch from a clathrin-dependent to caveolae-mediated entry pathway into the cells which is likely to be responsible for the observed transfection efficiency boost. As a consequence, we suggest that surface adsorption of protein corona can have a high biological impact on serum-resistant cationic formulations for in vitro and in vivo lipid-mediated gene delivery applications.