The Experts below are selected from a list of 11229 Experts worldwide ranked by ideXlab platform
Cyrus R Safinya - One of the best experts on this subject based on the ideXlab platform.
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competition of charge mediated and specific binding by peptide tagged Cationic Liposome dna nanoparticles in vitro and in vivo
Biomaterials, 2018Co-Authors: Emily Wonder, Kai K Ewert, Ramsey N Majzoub, Venkata Ramana Kotamraju, Tambet Teesalu, Lorena Simongracia, Pablo Scodeller, Cyrus R SafinyaAbstract:Abstract Cationic Liposome–nucleic acid (CL–NA) complexes, which form spontaneously, are a highly modular gene delivery system. These complexes can be sterically stabilized via PEGylation [PEG: poly (ethylene glycol)] into nanoparticles (NPs) and targeted to specific tissues and cell types via the conjugation of an affinity ligand. However, there are currently no guidelines on how to effectively navigate the large space of compositional parameters that modulate the specific and nonspecific binding interactions of peptide-targeted NPs with cells. Such guidelines are desirable to accelerate the optimization of formulations with novel peptides. Using PEG-lipids functionalized with a library of prototypical tumor-homing peptides, we varied the peptide density and other parameters (binding motif, peptide charge, CL/DNA charge ratio) to study their effect on the binding and uptake of the corresponding NPs. We used flow cytometry to quantitatively assess binding as well as internalization of NPs by cultured cancer cells. Surprisingly, full peptide coverage resulted in less binding and internalization than intermediate coverage, with the optimum coverage varying between cell lines. In, addition, our data revealed that great care must be taken to prevent nonspecific electrostatic interactions from interfering with the desired specific binding and internalization. Importantly, such considerations must take into account the charge of the peptide ligand as well as the membrane charge density and the CL/DNA charge ratio. To test our guidelines, we evaluated the in vivo tumor selectivity of selected NP formulations in a mouse model of peritoneally disseminated human gastric cancer. Intraperitoneally administered peptide-tagged CL–DNA NPs showed tumor binding, minimal accumulation in healthy control tissues, and preferential penetration of smaller tumor nodules, a highly clinically relevant target known to drive recurrence of the peritoneal cancer.
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synthesis of linear and cyclic peptide peg lipids for stabilization and targeting of Cationic Liposome dna complexes
Bioorganic & Medicinal Chemistry Letters, 2016Co-Authors: Kai K Ewert, Ramsey N Majzoub, Venkata Ramana Kotamraju, Victoria M Steffes, Emily Wonder, Tambet Teesalu, Erkki Ruoslahti, Cyrus R SafinyaAbstract:Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for Cationic Liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a Cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles.
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patterned threadlike micelles and dna tethered nanoparticles a structural study of pegylated Cationic Liposome dna assemblies
Langmuir, 2015Co-Authors: Ramsey N Majzoub, Kai K Ewert, Erica L Jacovetty, Bridget Carragher, Clinton S Potter, Cyrus R SafinyaAbstract:The self-assembly of oppositely charged biomacromolecules has been extensively studied due to its pertinence in the design of functional nanomaterials. Using cryo electron microscopy (cryo-EM), optical light scattering, and fluorescence microscopy, we investigated the structure and phase behavior of PEGylated (PEG: poly(ethylene glycol)) Cationic Liposome–DNA nanoparticles (CL–DNA NPs) as a function of DNA length, topology (linear and circular), and ρchg (the molar charge ratio of Cationic lipid to anionic DNA). Although all NPs studied exhibited lamellar internal nanostructure, NPs formed with short (∼2 kbps), linear, polydisperse DNA were defect-rich and contained smaller domains. Unexpectedly, we found distinctly different equilibrium structures away from the isoelectric point. At ρchg > 1, in the excess Cationic lipid regime, threadlike micelles rich in PEG-lipid were found to coexist with NPs, Cationic Liposomes, and spherical micelles. At high concentrations these PEGylated threadlike micelles forme...
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Cationic Liposome nucleic acid complexes for gene delivery and gene silencing
New Journal of Chemistry, 2014Co-Authors: Cyrus R Safinya, Kai K Ewert, Ramsey N Majzoub, Cecilia LealAbstract:Cationic Liposomes (CLs) are studied worldwide as carriers of DNA and short interfering RNA (siRNA) for gene delivery and gene silencing, and related clinical trials are ongoing. Optimization of transfection efficiency and silencing efficiency by Cationic Liposome carriers requires a comprehensive understanding of the structures of CL–nucleic acid complexes and the nature of their interactions with cell membranes as well as events leading to release of active nucleic acids within the cytoplasm. Synchrotron X-ray scattering has revealed that CL–nucleic acid complexes spontaneously assemble into distinct liquid crystalline phases including the lamellar, inverse hexagonal, hexagonal, and gyroid cubic phases, and fluorescence microscopy has revealed CL–DNA pathways and interactions with cells. The combining of custom synthesis with characterization techniques and gene expression and silencing assays has begun to unveil structure–function relations in vitro. As a recent example, this review will briefly describe experiments with surface-functionalized PEGylated CL–DNA nanoparticles. The functionalization, which is achieved through custom synthesis, is intended to address and overcome cell targeting and endosomal escape barriers to nucleic acid delivery faced by PEGylated nanoparticles designed for in vivo applications.
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uptake and transfection efficiency of pegylated Cationic Liposome dna complexes with and without rgd tagging
Biomaterials, 2014Co-Authors: Ramsey N Majzoub, Kai K Ewert, Erica L Jacovetty, Bridget Carragher, Clinton S Potter, Bruno F B Silva, Chialing Chan, Keng S Liang, Cyrus R SafinyaAbstract:Steric stabilization of Cationic Liposome-DNA (CL-DNA) complexes is required for in vivo applications such as gene therapy. PEGylation (PEG: poly(ethylene glycol)) of CL-DNA complexes by addition of PEG2000-lipids yields sterically stabilized nanoparticles but strongly reduces their gene delivery efficacy. PEGylation-induced weakening of the electrostatic binding of CL-DNA nanoparticles to cells (leading to reduced uptake) has been considered as a possible cause, but experimental results have been ambiguous. Using quantitative live-cell imaging in vitro, we have investigated cell attachment and uptake of PEGylated CL-DNA nanoparticles with and without a custom synthesized RGD-peptide grafted to the distal ends of PEG2000-lipids. The RGD-tagged nanoparticles exhibit strongly increased cellular attachment as well as uptake compared to nanoparticles without grafted peptide. Transfection efficiency of RGD-tagged PEGylated CL-DNA NPs increases by about an order of magnitude between NPs with low and high membrane charge density (σM; the average charge per unit area of the membrane; controlled by the molar ratio of Cationic to neutral lipid), even though imaging data show that uptake of RGD-tagged particles is only slightly enhanced by high σM. This suggests that endosomal escape and, as a result, transfection efficiency of RGD-tagged NPs is facilitated by high σM. We present a model describing the interactions between PEGylated CL-DNA nanoparticles and the anionic cell membrane which shows how the PEG grafting density and membrane charge density affect adhesion of nanoparticles to the cell surface.
Leaf Huang - One of the best experts on this subject based on the ideXlab platform.
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reactive oxygen species play a central role in the activity of Cationic Liposome based cancer vaccine
Journal of Controlled Release, 2008Co-Authors: Weili Yan, Weihsu Chen, Leaf HuangAbstract:Recently, we developed a simple and potent therapeutic Liposome cancer vaccine consisting of a peptide antigen and a Cationic lipid. The molecular mechanism of the adjuvanticity of Cationic Liposome was studied and described in the current report. First, Cationic DOTAP Liposome, but not the neutral Liposome DOPC, was shown to generate reactive oxygen species (ROS) in mouse bone marrow-derived dendritic cells (BMDC). ROS generation by DOTAP was required for ERK and p38 activation and downstream chemokine/cytokine induction. Furthermore, ROS were shown to be involved in the expression of the co-stimulatory molecules CD86/CD80 induced by DOTAP. However, as the DOTAP concentration increased from 50 to 800 μM, the apoptotic marker Annexin V and ROS double positive cells increased, suggesting that high dose of DOTAP-generated ROS causes cell apoptosis. In vivo, optimal amount of ROS in the draining lymph nodes (DLN) and anti-tumor (HPV positive TC-1 tumor) activity induced by E7 peptide (antigen derived from E7 oncoprotein of human papillomavirus (HPV) type 16) formulated in 100 nmol DOTAP were attenuated by incorporating DOPC in the formulation, suggesting that ROS are essential for the vaccine induced anti-tumor activity. Moreover, 600 nmol DOTAP/E7 generated huge amount of ROS in the DLN and showed no activity of tumor regression. Interestingly, 600 nmol DOTAP/E7-induced ROS were tuned down to the same level induced by 100 nmol DOTAP/E7 by adding DOPC in the formulation and this formulation showed tumor regression activity. In conclusion, DOTAP is an active DC stimulator resulting in the activation of ERK and p38 and induction of chemokines, cytokines and co-stimulatory molecules mediated by appropriate amount of ROS. Our data elucidated an important mechanism of adjuvant activity of Cationic Liposome and could facilitate rational design of synthetic lipid based adjuvants and vaccine formulation.
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mechanism of adjuvant activity of Cationic Liposome phosphorylation of a map kinase erk and induction of chemokines
Molecular Immunology, 2007Co-Authors: Weili Yan, Weihsu Chen, Leaf HuangAbstract:Cationic Liposome has been effectively used as a delivery system for DNA and protein vaccines. Recently, we discovered that strong anti-tumor immunity could be generated when a peptide antigen (E7) was incorporated into 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) Cationic Liposome. Therefore, DOTAP Liposome exhibits not only efficient delivery capacity, but also a potent adjuvant activity. In this report, the molecular mechanism of the adjuvanticity was studied both in vitro and in vivo. Microarray of mRNA analysis demonstrated that several chemokine genes are up-regulated by DOTAP Liposome, including CCL2, CCL3 and CCL4, upon treatment of dendritic cells (DC) with DOTAP Liposomes. CCL2 induction was mediated through extracellular-signal-regulated kinase (ERK) pathway, demonstrated by specific inhibitors of ERK pathway and siRNA approaches. Furthermore, DOTAP-induced CCL2 expression is negatively regulated by the p38 pathway. Consistently, ERK activation by DOTAP is also negatively regulated by p38. Moreover, PI-3 kinase was shown to be involved in both activation of ERK and induction of CCL2 by DOTAP. DOTAP- induced CCL2 release was also confirmed in the draining lymph nodes. More importantly, inhibition of ERK pathway completely abolishes the CCL2 accumulation in the draining lymph nodes and attenuates anti-tumor activity of DOTAP/E7. In conclusion, DOTAP is an active lipid stimulator for DC resulting in ERK activation and CC chemokine induction. Our data elucidated one important mechanism of adjuvant activity of Cationic Liposome and could facilitate rational design of synthetic lipid based adjuvants.
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mechanistic studies of sequential injection of Cationic Liposome and plasmid dna
Molecular Therapy, 2006Co-Authors: Jing Shi Zhang, Feng Liu, Yadi Tan, Christine C Conwell, Leaf HuangAbstract:Abstract We previously reported that sequential injection of Cationic Liposome and plasmid DNA leads to notably reduced inflammatory toxicity and improved transfection in the lung (Y. Tan et al ., 2001, Mol. Ther. 3, 673–682). The purpose of the current study was to explore the mechanism involved in sequential injection. It was observed that sequential injection resulted in dramatically lower DNA uptake by the liver and higher DNA levels in the lung than the lipoplex injection. In vitro experiments with macrophage cells further showed that sequential addition of Liposomes and DNA could diminish the cellular uptake of DNA by these cells. The contributions of serum to the enhanced bioactivity and decreased toxicity were examined by injecting mice with samples of premixed Liposome with serum and then DNA (LSD sample), and the resulting activities were compared to those obtained with injection of lipoplex–serum mixtures (LDS sample). LSD yielded 80% lower TNF-α levels and over 10-fold higher transfection than lipoplex, which is consistent with the reported findings with sequential injection. In contrast, LDS resulted in the same TNF-α levels and comparable transfection with lipoplex. Thus, the results suggest that the primary interaction of serum with Liposome is a critical factor contributing to the superior activity and reduced toxicity of sequential injection. Studies on the interaction between mouse serum, Liposomes, and DNA showed that DNA could bind negatively charged Liposome–serum complex to form a ternary complex, which has a density similar to that of the ternary complex formed between lipoplex with serum. Further in vitro tests showed that LSD and LDS were similar in particle size and protein content, but different in protein composition as observed by 2-D gel electrophoresis. In addition, DNA in LSD was more readily displaced by dextran sulfate, an anionic polymer, than in LDS. The above findings suggest that the inhibition of opsonin protein binding on the particle surface with the sequential injection may contribute to the reduced macrophage uptake and cytokine induction and that the high ability of DNA release from the particles formed after sequential injection may contribute to the improved lung gene transfection.
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sequential injection of Cationic Liposome and plasmid dna effectively transfects the lung with minimal inflammatory toxicity
Molecular Therapy, 2001Co-Authors: Yadi Tan, Feng Liu, Leaf HuangAbstract:A major hurdle to lipoplex-based systemic gene delivery is acute inflammatory toxicity. In this study, a safe, simple, and effective alternative to lipoplex administration, specifically, sequential injection of Cationic Liposome and plasmid DNA, was evaluated. When plasmid DNA was injected into the tail vein of mice 2–5 min after the injection of Cationic Liposomes, 50–80% lower levels of proinflammatory cytokines, including TNF-α, IL-12, and IFN-γ, were observed compared to lipoplex injection. The sequential injection technique yielded a two- to fivefold higher level of transgene expression in the lung and was more effective in repeated dosing than lipoplex. Other types of lipoplex-associated toxicities, such as neutropenia, lymphopenia, thrombocytopenia, and complement depletion, were also significantly reduced with sequential injection. The reduction in cytokine release was observed with several different Liposome formulations and appeared to be a general phenomenon.
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Time-dependent maturation of Cationic Liposome-DNA complex for serum resistance.
Gene therapy, 1998Co-Authors: Leaf HuangAbstract:Following our previous finding that increasing charge ratio (+/-) can overcome the serum sensitivity of Cationic Liposome-DNA complex, in the present studies we report that serum sensitivity of Cationic Liposome-DNA complex could also be overcome by prolonging the incubation time of the complex. Such time-dependent maturation of the complex was Cationic lipid-dependent; complexes containing monovalent Cationic lipids, eg DC-chol, DOTAP and lipofectin, matured with time, but those containing multivalent lipid, eg lipofectamine, do not. High charge ratio high concentration and high temperature accelerated the process of maturation. The serum sensitivity of cellular uptake of DNA was also dependent on the incubation time. The matured Cationic Liposome-DNA complexes are homogeneous particles with a mean diameter of 170 to 400 nm, depending on the Cationic lipid in the complex.
Kai K Ewert - One of the best experts on this subject based on the ideXlab platform.
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competition of charge mediated and specific binding by peptide tagged Cationic Liposome dna nanoparticles in vitro and in vivo
Biomaterials, 2018Co-Authors: Emily Wonder, Kai K Ewert, Ramsey N Majzoub, Venkata Ramana Kotamraju, Tambet Teesalu, Lorena Simongracia, Pablo Scodeller, Cyrus R SafinyaAbstract:Abstract Cationic Liposome–nucleic acid (CL–NA) complexes, which form spontaneously, are a highly modular gene delivery system. These complexes can be sterically stabilized via PEGylation [PEG: poly (ethylene glycol)] into nanoparticles (NPs) and targeted to specific tissues and cell types via the conjugation of an affinity ligand. However, there are currently no guidelines on how to effectively navigate the large space of compositional parameters that modulate the specific and nonspecific binding interactions of peptide-targeted NPs with cells. Such guidelines are desirable to accelerate the optimization of formulations with novel peptides. Using PEG-lipids functionalized with a library of prototypical tumor-homing peptides, we varied the peptide density and other parameters (binding motif, peptide charge, CL/DNA charge ratio) to study their effect on the binding and uptake of the corresponding NPs. We used flow cytometry to quantitatively assess binding as well as internalization of NPs by cultured cancer cells. Surprisingly, full peptide coverage resulted in less binding and internalization than intermediate coverage, with the optimum coverage varying between cell lines. In, addition, our data revealed that great care must be taken to prevent nonspecific electrostatic interactions from interfering with the desired specific binding and internalization. Importantly, such considerations must take into account the charge of the peptide ligand as well as the membrane charge density and the CL/DNA charge ratio. To test our guidelines, we evaluated the in vivo tumor selectivity of selected NP formulations in a mouse model of peritoneally disseminated human gastric cancer. Intraperitoneally administered peptide-tagged CL–DNA NPs showed tumor binding, minimal accumulation in healthy control tissues, and preferential penetration of smaller tumor nodules, a highly clinically relevant target known to drive recurrence of the peritoneal cancer.
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synthesis of linear and cyclic peptide peg lipids for stabilization and targeting of Cationic Liposome dna complexes
Bioorganic & Medicinal Chemistry Letters, 2016Co-Authors: Kai K Ewert, Ramsey N Majzoub, Venkata Ramana Kotamraju, Victoria M Steffes, Emily Wonder, Tambet Teesalu, Erkki Ruoslahti, Cyrus R SafinyaAbstract:Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for Cationic Liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a Cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles.
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patterned threadlike micelles and dna tethered nanoparticles a structural study of pegylated Cationic Liposome dna assemblies
Langmuir, 2015Co-Authors: Ramsey N Majzoub, Kai K Ewert, Erica L Jacovetty, Bridget Carragher, Clinton S Potter, Cyrus R SafinyaAbstract:The self-assembly of oppositely charged biomacromolecules has been extensively studied due to its pertinence in the design of functional nanomaterials. Using cryo electron microscopy (cryo-EM), optical light scattering, and fluorescence microscopy, we investigated the structure and phase behavior of PEGylated (PEG: poly(ethylene glycol)) Cationic Liposome–DNA nanoparticles (CL–DNA NPs) as a function of DNA length, topology (linear and circular), and ρchg (the molar charge ratio of Cationic lipid to anionic DNA). Although all NPs studied exhibited lamellar internal nanostructure, NPs formed with short (∼2 kbps), linear, polydisperse DNA were defect-rich and contained smaller domains. Unexpectedly, we found distinctly different equilibrium structures away from the isoelectric point. At ρchg > 1, in the excess Cationic lipid regime, threadlike micelles rich in PEG-lipid were found to coexist with NPs, Cationic Liposomes, and spherical micelles. At high concentrations these PEGylated threadlike micelles forme...
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Cationic Liposome nucleic acid complexes for gene delivery and gene silencing
New Journal of Chemistry, 2014Co-Authors: Cyrus R Safinya, Kai K Ewert, Ramsey N Majzoub, Cecilia LealAbstract:Cationic Liposomes (CLs) are studied worldwide as carriers of DNA and short interfering RNA (siRNA) for gene delivery and gene silencing, and related clinical trials are ongoing. Optimization of transfection efficiency and silencing efficiency by Cationic Liposome carriers requires a comprehensive understanding of the structures of CL–nucleic acid complexes and the nature of their interactions with cell membranes as well as events leading to release of active nucleic acids within the cytoplasm. Synchrotron X-ray scattering has revealed that CL–nucleic acid complexes spontaneously assemble into distinct liquid crystalline phases including the lamellar, inverse hexagonal, hexagonal, and gyroid cubic phases, and fluorescence microscopy has revealed CL–DNA pathways and interactions with cells. The combining of custom synthesis with characterization techniques and gene expression and silencing assays has begun to unveil structure–function relations in vitro. As a recent example, this review will briefly describe experiments with surface-functionalized PEGylated CL–DNA nanoparticles. The functionalization, which is achieved through custom synthesis, is intended to address and overcome cell targeting and endosomal escape barriers to nucleic acid delivery faced by PEGylated nanoparticles designed for in vivo applications.
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uptake and transfection efficiency of pegylated Cationic Liposome dna complexes with and without rgd tagging
Biomaterials, 2014Co-Authors: Ramsey N Majzoub, Kai K Ewert, Erica L Jacovetty, Bridget Carragher, Clinton S Potter, Bruno F B Silva, Chialing Chan, Keng S Liang, Cyrus R SafinyaAbstract:Steric stabilization of Cationic Liposome-DNA (CL-DNA) complexes is required for in vivo applications such as gene therapy. PEGylation (PEG: poly(ethylene glycol)) of CL-DNA complexes by addition of PEG2000-lipids yields sterically stabilized nanoparticles but strongly reduces their gene delivery efficacy. PEGylation-induced weakening of the electrostatic binding of CL-DNA nanoparticles to cells (leading to reduced uptake) has been considered as a possible cause, but experimental results have been ambiguous. Using quantitative live-cell imaging in vitro, we have investigated cell attachment and uptake of PEGylated CL-DNA nanoparticles with and without a custom synthesized RGD-peptide grafted to the distal ends of PEG2000-lipids. The RGD-tagged nanoparticles exhibit strongly increased cellular attachment as well as uptake compared to nanoparticles without grafted peptide. Transfection efficiency of RGD-tagged PEGylated CL-DNA NPs increases by about an order of magnitude between NPs with low and high membrane charge density (σM; the average charge per unit area of the membrane; controlled by the molar ratio of Cationic to neutral lipid), even though imaging data show that uptake of RGD-tagged particles is only slightly enhanced by high σM. This suggests that endosomal escape and, as a result, transfection efficiency of RGD-tagged NPs is facilitated by high σM. We present a model describing the interactions between PEGylated CL-DNA nanoparticles and the anionic cell membrane which shows how the PEG grafting density and membrane charge density affect adhesion of nanoparticles to the cell surface.
Ramsey N Majzoub - One of the best experts on this subject based on the ideXlab platform.
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competition of charge mediated and specific binding by peptide tagged Cationic Liposome dna nanoparticles in vitro and in vivo
Biomaterials, 2018Co-Authors: Emily Wonder, Kai K Ewert, Ramsey N Majzoub, Venkata Ramana Kotamraju, Tambet Teesalu, Lorena Simongracia, Pablo Scodeller, Cyrus R SafinyaAbstract:Abstract Cationic Liposome–nucleic acid (CL–NA) complexes, which form spontaneously, are a highly modular gene delivery system. These complexes can be sterically stabilized via PEGylation [PEG: poly (ethylene glycol)] into nanoparticles (NPs) and targeted to specific tissues and cell types via the conjugation of an affinity ligand. However, there are currently no guidelines on how to effectively navigate the large space of compositional parameters that modulate the specific and nonspecific binding interactions of peptide-targeted NPs with cells. Such guidelines are desirable to accelerate the optimization of formulations with novel peptides. Using PEG-lipids functionalized with a library of prototypical tumor-homing peptides, we varied the peptide density and other parameters (binding motif, peptide charge, CL/DNA charge ratio) to study their effect on the binding and uptake of the corresponding NPs. We used flow cytometry to quantitatively assess binding as well as internalization of NPs by cultured cancer cells. Surprisingly, full peptide coverage resulted in less binding and internalization than intermediate coverage, with the optimum coverage varying between cell lines. In, addition, our data revealed that great care must be taken to prevent nonspecific electrostatic interactions from interfering with the desired specific binding and internalization. Importantly, such considerations must take into account the charge of the peptide ligand as well as the membrane charge density and the CL/DNA charge ratio. To test our guidelines, we evaluated the in vivo tumor selectivity of selected NP formulations in a mouse model of peritoneally disseminated human gastric cancer. Intraperitoneally administered peptide-tagged CL–DNA NPs showed tumor binding, minimal accumulation in healthy control tissues, and preferential penetration of smaller tumor nodules, a highly clinically relevant target known to drive recurrence of the peritoneal cancer.
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synthesis of linear and cyclic peptide peg lipids for stabilization and targeting of Cationic Liposome dna complexes
Bioorganic & Medicinal Chemistry Letters, 2016Co-Authors: Kai K Ewert, Ramsey N Majzoub, Venkata Ramana Kotamraju, Victoria M Steffes, Emily Wonder, Tambet Teesalu, Erkki Ruoslahti, Cyrus R SafinyaAbstract:Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for Cationic Liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a Cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles.
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patterned threadlike micelles and dna tethered nanoparticles a structural study of pegylated Cationic Liposome dna assemblies
Langmuir, 2015Co-Authors: Ramsey N Majzoub, Kai K Ewert, Erica L Jacovetty, Bridget Carragher, Clinton S Potter, Cyrus R SafinyaAbstract:The self-assembly of oppositely charged biomacromolecules has been extensively studied due to its pertinence in the design of functional nanomaterials. Using cryo electron microscopy (cryo-EM), optical light scattering, and fluorescence microscopy, we investigated the structure and phase behavior of PEGylated (PEG: poly(ethylene glycol)) Cationic Liposome–DNA nanoparticles (CL–DNA NPs) as a function of DNA length, topology (linear and circular), and ρchg (the molar charge ratio of Cationic lipid to anionic DNA). Although all NPs studied exhibited lamellar internal nanostructure, NPs formed with short (∼2 kbps), linear, polydisperse DNA were defect-rich and contained smaller domains. Unexpectedly, we found distinctly different equilibrium structures away from the isoelectric point. At ρchg > 1, in the excess Cationic lipid regime, threadlike micelles rich in PEG-lipid were found to coexist with NPs, Cationic Liposomes, and spherical micelles. At high concentrations these PEGylated threadlike micelles forme...
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Cationic Liposome nucleic acid complexes for gene delivery and gene silencing
New Journal of Chemistry, 2014Co-Authors: Cyrus R Safinya, Kai K Ewert, Ramsey N Majzoub, Cecilia LealAbstract:Cationic Liposomes (CLs) are studied worldwide as carriers of DNA and short interfering RNA (siRNA) for gene delivery and gene silencing, and related clinical trials are ongoing. Optimization of transfection efficiency and silencing efficiency by Cationic Liposome carriers requires a comprehensive understanding of the structures of CL–nucleic acid complexes and the nature of their interactions with cell membranes as well as events leading to release of active nucleic acids within the cytoplasm. Synchrotron X-ray scattering has revealed that CL–nucleic acid complexes spontaneously assemble into distinct liquid crystalline phases including the lamellar, inverse hexagonal, hexagonal, and gyroid cubic phases, and fluorescence microscopy has revealed CL–DNA pathways and interactions with cells. The combining of custom synthesis with characterization techniques and gene expression and silencing assays has begun to unveil structure–function relations in vitro. As a recent example, this review will briefly describe experiments with surface-functionalized PEGylated CL–DNA nanoparticles. The functionalization, which is achieved through custom synthesis, is intended to address and overcome cell targeting and endosomal escape barriers to nucleic acid delivery faced by PEGylated nanoparticles designed for in vivo applications.
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uptake and transfection efficiency of pegylated Cationic Liposome dna complexes with and without rgd tagging
Biomaterials, 2014Co-Authors: Ramsey N Majzoub, Kai K Ewert, Erica L Jacovetty, Bridget Carragher, Clinton S Potter, Bruno F B Silva, Chialing Chan, Keng S Liang, Cyrus R SafinyaAbstract:Steric stabilization of Cationic Liposome-DNA (CL-DNA) complexes is required for in vivo applications such as gene therapy. PEGylation (PEG: poly(ethylene glycol)) of CL-DNA complexes by addition of PEG2000-lipids yields sterically stabilized nanoparticles but strongly reduces their gene delivery efficacy. PEGylation-induced weakening of the electrostatic binding of CL-DNA nanoparticles to cells (leading to reduced uptake) has been considered as a possible cause, but experimental results have been ambiguous. Using quantitative live-cell imaging in vitro, we have investigated cell attachment and uptake of PEGylated CL-DNA nanoparticles with and without a custom synthesized RGD-peptide grafted to the distal ends of PEG2000-lipids. The RGD-tagged nanoparticles exhibit strongly increased cellular attachment as well as uptake compared to nanoparticles without grafted peptide. Transfection efficiency of RGD-tagged PEGylated CL-DNA NPs increases by about an order of magnitude between NPs with low and high membrane charge density (σM; the average charge per unit area of the membrane; controlled by the molar ratio of Cationic to neutral lipid), even though imaging data show that uptake of RGD-tagged particles is only slightly enhanced by high σM. This suggests that endosomal escape and, as a result, transfection efficiency of RGD-tagged NPs is facilitated by high σM. We present a model describing the interactions between PEGylated CL-DNA nanoparticles and the anionic cell membrane which shows how the PEG grafting density and membrane charge density affect adhesion of nanoparticles to the cell surface.
Tadaaki Ohgi - One of the best experts on this subject based on the ideXlab platform.
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liver target delivery of small interfering rna to the hcv gene by lactosylated Cationic Liposome
Journal of Hepatology, 2007Co-Authors: Tsunamasa Watanabe, Takuya Umehara, Fumihiko Yasui, Shinichiro Nakagawa, Junichi Yano, Tadaaki Ohgi, Satoru Sonoke, Kenichi Satoh, Kazuaki Inoue, Makoto YoshibaAbstract:Background/Aims RNA interference has considerable therapeutic potential, particularly for anti-viral therapy. We previously reported that hepatitis C virus (HCV)-directed small interfering RNA (siRNA; siE) efficiently inhibits HCV replication, using HCV replicon cells. To employ the siRNA as a therapeutic strategy, we attempted in vivo silencing of intrahepatic HCV gene expression by siE using a novel Cationic Liposome. Methods The Liposomes consisted of conjugated lactose residues, based on the speculation that lactose residues would effectively deliver siRNA to the liver via a liver specific receptor. The lactosylated Cationic Liposome 5 (CL-LA5) that contained the most lactose residues introduced the most siRNA into a human hepatoma cell line, which then inhibited replication of HCV replicons. Results In mice, the siRNA/CL-LA5 complexes accumulated primarily in the liver and were widespread throughout the hepatic parenchymal cells. Moreover, siE/CL-LA5 specifically and dose-dependently suppressed intrahepatic HCV expression in transgenic mice without an interferon response. Conclusions The present results indicate that the CL-LA5 we developed is a good vehicle to lead siRNA to the liver. Hence, CL-LA5 will be helpful for siRNA therapy targeting liver diseases, especially hepatitis C.
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antitumor activity of small interfering rna Cationic Liposome complex in mouse models of cancer
Clinical Cancer Research, 2004Co-Authors: Junichi Yano, Shinichiro Nakagawa, Kazuko Hirabayashi, Tohru Yamaguchi, Masaki Nogawa, Isao Kashimori, Haruna Naito, Hidetoshi Kitagawa, Kouichi Ishiyama, Tadaaki OhgiAbstract:Purpose: The RNA interference effect is an alternative to antisense DNA as an experimental method of down-regulating a specific target protein. Although the RNA interference effect, which is mediated by small interfering RNA (siRNA) or micro-RNA, has potential application to human therapy, the hydrodynamic method usually used for rapid administration of oligonucleotides is unsuitable for use in humans. In this study, we have investigated the antitumor activity of a synthetic siRNA, B717, which is sequence specific for the human bcl-2 oncogene, complexed with a novel Cationic Liposome, LIC-101. Experimental Design: In a mouse model of liver metastasis, we administered B717/LIC-101 by bolus intravenous injection, adjusting the rate and volume of administration to what is feasible in human therapy. In a mouse model bearing prostate cancer in which the cells were inoculated under the skin, B717/LIC-101 was administered subcutaneously around the tumor. Results: The B717/LIC-101 complex inhibited the expression of bcl-2 protein and the growth of tumor cell lines in vitro in a sequence-specific manner in the concentration range of 3 to 100 nmol/L. Furthermore, the complex had a strong antitumor activity when administered intravenously in the mouse model of liver metastasis. B717 (siRNA) was shown to be delivered to tumor cells in the mouse liver, but only when complexed with LIC-101. The complex also inhibited tumor cell growth in the mouse model bearing prostate cancer. Conclusions: By combining siRNA with our Cationic Liposome, we overcame the difficulty of administering siRNA to animals in ways that can be applied in human therapy. Although our siRNA/Liposome complex is not yet in clinical trials, it is expected to provide a novel siRNA therapy for cancer patients.
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inhibition of cancer cell growth by polyinosinic polycytidylic acid Cationic Liposome complex a new biological activity
Cancer Research, 1999Co-Authors: Kazuko Hirabayashi, Junichi Yano, Tadaaki Ohgi, Tohru Yamaguchi, Kouichi Ishiyama, Toshihiko Inoue, Kazuaki Tanigawara, Gerald E Smyth, Kiyoshi Kimura, Tatsuro IrimuraAbstract:A complex of polyinosinic-polycytidylic acid [poly(I)•poly(C)] and Cationic Liposome (LIC) inhibited the growth of many tumor cell lines at low concentration in vitro , but poly(I)•poly(C) alone had no such antiproliferative effect. The IC 50 values of LIC against the tumor cells ranged from 0.1 to 1000 ng/ml. LIC had strong cytotoxic effects on malignant cells of epithelial and fibroblastic origin from various tissues and was also effective against Adriamycin-resistant tumor cells. LIC did not significantly affect the growth of lymphoma cells, leukemia cells, normal diploid fibroblasts, or primary liver cells at concentrations up to 10 μg/ml. The mechanism of the antiproliferative effect of LIC against malignant cells was the induction of apoptosis. LIC induced the fragmentation of nuclear DNA and the degradation of rRNA in tumor cells. The DNA fragmentation occurred within 1–5 h after the addition of LIC, and both the fragmentation and the inhibition of cancer-cell growth were suppressed by a nuclease inhibitor. In contrast, caspase inhibitors did not affect the antiproliferative activity of LIC. These results suggest that LIC induced apoptosis in malignant cells through the direct activation of nucleases and not through the activation of caspases. LIC reduced the incidence and the size of metastatic liver-cancer tumors in two different mouse metastatic liver-cancer models using human colon carcinoma cells. Histochemical analysis revealed that the KM12-HX cells in the tumor nodules were undergoing apoptosis; therefore, LIC also induced the apoptosis of tumor cells in vivo . In these animal models, LIC caused no observed changes in normal hepatocytes.