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Thomas Kissel - One of the best experts on this subject based on the ideXlab platform.
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stability of sirna Polyplexes from poly ethylenimine and poly ethylenimine g poly ethylene glycol under in vivo conditions effects on pharmacokinetics and biodistribution measured by fluorescence fluctuation spectroscopy and single photon emission computed tomography spect imaging
Journal of Controlled Release, 2009Co-Authors: Olivia M Merkel, Stefaan C. De Smedt, Kevin Buyens, Niek N Sanders, Damiano Librizzi, Andreas Pfestroff, Tino Schurrat, Martin Behe, Thomas KisselAbstract:In search of optimizing siRNA delivery systems for systemic application, one critical parameter remains their stability in blood circulation. In this study, we have traced pharmacokinetics and biodistribution of each component of siRNA Polyplexes formed with polyethylenimine 25 kDa (PEI) or PEGylated PEIs by in vivo real-time gamma camera recording, SPECT imaging, and scintillation counting of blood samples and dissected organs. In vivo behavior of siRNA and polymers were compared and interpreted in the context of in vivo stability of the Polyplexes which had been measured by fluorescence fluctuation spectroscopy (FFS). Both pharmacokinetics and biodistribution of polymer-complexed siRNA were dominated by the polymer. PEGylated polymers and their siRNA Polyplexes showed significantly less uptake into liver (13.6–19.7% ID of PEGylated polymer and 9.5–10.2% ID of siRNA) and spleen compared to PEI 25 kDa (liver deposition: 36.2% ID of polymer and 14.6% ID of siRNA). With non-invasive imaging methods we were able to predict both kinetics and deposition in living animals allowing the investigation of organ distribution in real time and at different time points. FFS measurements proved stability of the applied Polyplexes under in vivo conditions which explained the different behavior of complexed from free siRNA. Despite their stability in circulation, we observed that Polyplexes dissociated upon liver passage. Therefore, siRNA/(PEG-)PEI delivery systems are not suitable for systemic administration, but instead may be useful when the first-pass effect is circumvented, which is the case in local application.
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nonviral sirna delivery to the lung investigation of peg pei Polyplexes and their in vivo performance
Molecular Pharmaceutics, 2009Co-Authors: Olivia M Merkel, Damiano Librizzi, Andreas Pfestroff, Andrea Beyerle, Thomas M Behr, Brian S Sproat, Peter J Barth, Thomas KisselAbstract:This study describes the physicobiological characterization of PEI− and PEG−PEI Polyplexes containing partially 2′-OMe modified 25/27mer dicer substrate siRNAs (DsiRNAs) and their in vivo behavior regarding biodistribution and systemic bioavailability after pulmonary application as well as their ability to knock down gene expression in the lung. Biophysical characterization included circular dichroism of siRNA in Polyplexes, condensation efficiency of polymers and in vitro stability. After in vivo application, biodistribution and kinetics of radiolabeled Polyplexes were quantified and recorded over time in three-dimensional SPECT images and by end point scintillation counting. The influence on lung tissue and on the humoral and cellular immunosystem was investigated, and finally knockdown of endogenous gene expression in the lung was determined qualitatively. While all of the polymers used in our study were proven to effectively condense siRNA, stability of the complexes depended on the PEG grafting degre...
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gene delivery using chitosan trimethyl chitosan or polyethylenglycol graft trimethyl chitosan block copolymers establishment of structure activity relationships in vitro
Journal of Controlled Release, 2008Co-Authors: Oliver Germershaus, Shirui Mao, Johannes Sitterberg, Udo Bakowsky, Thomas KisselAbstract:Chitosan, trimethyl chitosan or polyethylenglycol-graft-trimethyl chitosan/DNA complexes were characterized concerning physicochemical properties such as hydrodynamic diameter, condensation efficiency and DNA release. Furthermore, cytotoxicity of polymers and uptake- and transfection efficiency of Polyplexes were evaluated in vitro. Under conditions found in cell culture, formation of aggregates of approximately 1000 nm and strongly decreased DNA condensation efficiency was observed in the case of chitosan Polyplexes. These characteristics resulted in only 7% cellular uptake in NIH/3T3 cells and low transfection efficiencies in 4 different cell lines. By contrast, quaternization of chitosan strongly reduced aggregation tendency and pH dependency of DNA complexation. Accordingly, cellular uptake was increased 8.5-fold compared to chitosan Polyplexes resulting in up to 678-fold increased transfection efficiency in NIH/3T3 cells. Apart from reduction of the cytotoxicity, PEGylation led to improved colloidal stability of Polyplexes and significantly increased cellular uptake compared to unmodified trimethyl chitosan. These improvements resulted in a significant, up to 10-fold increase of transfection efficiency in NIH/3T3, L929 and MeWo cells compared to trimethyl chitosan. This study not only highlights the importance of investigating polyplex stability under different pH- and ionic strength conditions but also elucidates correlations between physicochemical characteristics and biological efficacy of the studied Polyplexes.
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influence of polyethylene glycol chain length on the physicochemical and biological properties of poly ethylene imine graft poly ethylene glycol block copolymer sirna Polyplexes
Bioconjugate Chemistry, 2006Co-Authors: Shirui Mao, Michael Neu, Oliver Germershaus, Olivia M Merkel, Johannes Sitterberg, Udo Bakowsky, Thomas KisselAbstract:Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio (∼50%) on the biophysical properties of the Polyplexes. Particle size, ζ potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA Polyplexes were characterized. Stability and size of siRNA Polyplexes were clearly influenced by PEI−PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)30 resulted in large (300−400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other Polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA Polyplexes against heparin displacement and RNase digestion cou...
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influence of polyethylene glycol chain length on the physicochemical and biological properties of poly ethylene imine graft poly ethylene glycol block copolymer sirna Polyplexes
Bioconjugate Chemistry, 2006Co-Authors: Shirui Mao, Michael Neu, Oliver Germershaus, Olivia M Merkel, Johannes Sitterberg, Udo Bakowsky, Thomas KisselAbstract:Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio ( approximately 50%) on the biophysical properties of the Polyplexes. Particle size, zeta potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA Polyplexes were characterized. Stability and size of siRNA Polyplexes were clearly influenced by PEI-PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)(30) resulted in large (300-400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other Polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA Polyplexes against heparin displacement and RNase digestion could be modified by PEGylation. Protection against RNase digestion was highest for PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1), while siRNA/PEI provided insufficient protection. In knockdown experiments using NIH/3T3 fibroblasts stably expressing beta-galactosidase, it was shown that PEG chain length had a significant influence on biological activity of siRNA. Polyplexes with siRNA containing PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1) yielded similar efficiencies of ca. 70% knockdown as lipofectamine controls. Confocal microscopy demonstrated enhanced cellular uptake of siRNA into cytosol by Polyplexes formation with PEI copolymers. In conclusion, both the chain length and graft density of PEG were found to strongly influence siRNA condensation and stability and hence affect the knockdown efficiency of PEI-PEG/siRNA Polyplexes.
Stanislav Rangelov - One of the best experts on this subject based on the ideXlab platform.
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poly vinyl benzyl trimethylammonium chloride homo and block copolymers complexation with dna
Journal of Physical Chemistry B, 2016Co-Authors: Emi Haladjova, Grigoris Mountrichas, Stergios Pispas, Stanislav RangelovAbstract:In this work we focus on the use of novel homo and block copolymers based on poly(vinyl benzyl trimethylammonium chloride) as gene delivery vectors. The homopolymers and block copolymers were synthesized by RAFT polymerization schemes and molecularly characterized. DNA/polymer complexes (Polyplexes) in a wide range of N/P (amino-to-phosphate groups) ratios were prepared. The ability of the novel polymers to form complexes with linear DNA was investigated by light scattering, zeta potential, and ethidium bromide fluorescence quenching measurements. The resulting Polyplexes were in the size range of 80–300 nm and their surface potential changed from negative to positive depending on the N/P ratio. The stability of Polyplexes was monitored by changes in their hydrodynamic parameters in the presence of salt. The novel vector systems were visualized by transmission electron microscopy. The influence of factors such as molar mass, content, and chemical structure of the polycationic moieties as well as presence ...
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Poly(vinyl benzyl trimethylammonium chloride) Homo and Block Copolymers Complexation with DNA
2016Co-Authors: Emi Haladjova, Grigoris Mountrichas, Stergios Pispas, Stanislav RangelovAbstract:In this work we focus on the use of novel homo and block copolymers based on poly(vinyl benzyl trimethylammonium chloride) as gene delivery vectors. The homopolymers and block copolymers were synthesized by RAFT polymerization schemes and molecularly characterized. DNA/polymer complexes (Polyplexes) in a wide range of N/P (amino-to-phosphate groups) ratios were prepared. The ability of the novel polymers to form complexes with linear DNA was investigated by light scattering, zeta potential, and ethidium bromide fluorescence quenching measurements. The resulting Polyplexes were in the size range of 80–300 nm and their surface potential changed from negative to positive depending on the N/P ratio. The stability of Polyplexes was monitored by changes in their hydrodynamic parameters in the presence of salt. The novel vector systems were visualized by transmission electron microscopy. The influence of factors such as molar mass, content, and chemical structure of the polycationic moieties as well as presence of a hydrophilic poly[oligo(ethylene glycol) methacrylate] block on the structure and stability of the Polyplexes, kinetics of their formation, and effectiveness of the (co)polymers to shrink and pack DNA was discussed
Johan F J Engbersen - One of the best experts on this subject based on the ideXlab platform.
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Dynamic Colocalization Microscopy To Characterize Intracellular Trafficking of Nanomedicines
2016Co-Authors: Dries Vercauteren, Johan F J Engbersen, Hendrik Deschout, Katrien Remaut, Arwyn T. Jones, Jo Demeester, Stefaan C. De Smedt, Kevin BraeckmansAbstract:To gain a better understanding of intracellular processing of nanomedicines, we employed quantitative live-cell fluorescence colocalization microscopy to study endosomal trafficking of Polyplexes in retinal pigment epithelium cells. A new, dynamic colocalization algorithm was developed, based on particle tracking and trajectory correlation, allowing for spatiotemporal characterization of internalized Polyplexes in comparison with endosomal compartments labeled with EGFP constructs. This revealed early trafficking of the Polyplexes specifically to Rab5- and flotillin-2-positive vesicles and subsequent delivery to Rab7 and LAMP1-labeled late endolysosomes where the major fraction of the Polyplexes remains entrapped for days, suggesting the functional loss of these nanomedicines. Colocalization of Polyplexes with the autophagy marker LC3 suggests for the first time that the process of xenophagy could play an important role in the persistent endosomal entrapment of nanomedicines
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coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy
Journal of Controlled Release, 2015Co-Authors: Thomas Martens, Johan F J Engbersen, Hendrik Deschout, Katrien Remaut, Jo Demeester, Stefaan C. De Smedt, Wim E Hennink, Mies J Van Steenbergen, Kevin BraeckmansAbstract:Retinal gene therapy could potentially affect the lives of millions of people suffering from blinding disorders. Yet, one of the major hurdles remains the delivery of therapeutic nucleic acids to the retinal target cells. Due to the different barriers that need to be overcome in case of topical or systemic administration, intravitreal injection is an attractive alternative administration route for large macromolecular therapeutics. Here it is essential that the therapeutics do not aggregate and remain mobile in the vitreous humor in order to reach the retina. In this study, we have evaluated the use of hyaluronic acid (HA) as an electrostatic coating for nonviral polymeric gene nanomedicines, p(CBA-ABOL)/pDNA complexes, to provide them with an anionic hydrophilic surface for improved intravitreal mobility. Uncoated Polyplexes had a Z-averaged diameter of 108 nm and a zeta potential of + 29 mV. We evaluated Polyplexes coated with HA of different molecular weights (22 kDa, 137 kDa and 2700 kDa) in terms of size, surface charge and complexation efficiency and noticed their zeta potentials became anionic at 4-fold molar excess of HA-monomers compared to cationic monomers, resulting in submicron ternary Polyplexes. Next, we used a previously optimized ex vivo model based on excised bovine eyes and fluorescence single particle tracking (fSPT) microscopy to evaluate mobility in intact vitreous humor. It was confirmed that HA-coated Polyplexes had good mobility in bovine vitreous humor, similar to Polyplexes functionalized with polyethylene glycol (PEG), except for those coated with high molecular weight HA (2700 kDa). However, contrary to PEGylated Polyplexes, HA-coated Polyplexes were efficiently taken up in vitro in ARPE-19 cells, despite their negative charge, indicating uptake via CD44-receptor mediated endocytosis. Furthermore, the HA-Polyplexes were able to induce GFP expression in this in vitro cell line without apparent cytotoxicity, where coating with low molecular weight HA (22 kDa) was shown to induce the highest expression. Taken together our experiments show that HA-coating of nonviral gene complexes is an interesting approach towards retinal gene therapy by intravitreal administration. To our knowledge, this is the first time electrostatic HA-coating of Polyplexes with different molecular weights has been evaluated in terms of their suitability for intravitreal delivery of therapeutic nucleic acids towards the retina
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pegylated bioreducible poly amido amine s for non viral gene delivery
Materials Science and Engineering: C, 2011Co-Authors: C Lin, Johan F J EngbersenAbstract:A facile method for PEGylated bioreducible poly(amido amine)s is described by a one-pot Michael-type addition polymerization of N, N′-cystaminebisacrylamide (CBA) with a mixture of 4-amino-1-butanol (ABOL) and mono-tert-butoxycarbonyl (Boc) PEG diamine. By this approach, two Boc-amino-PEGylated p(CBA-ABOL) copolymers were obtained with the PEG/ABOL composition ratio of 1/10 (1a) and 1/6 (2a), respectively. These copolymers were characterized by 1H NMR and gel permeation chromatography. The PEGylated copolymers 1a, and its deprotected analog 1b with a terminal amino group at the PEG chain, were further evaluated as gene delivery vectors. The copolymers 1a and 1b condense DNA into nano-scaled PEGylated Polyplexes (< 250 nm) with near neutral (2–5 mV, 1a) or slightly positive (9–13 mV, 1b) surface charge which remain stable in 150 mM buffer solution over 24 h. UnPEGylated Polyplexes from p(CBA-ABOL), however, are relatively less stable and increase in size to more than 1 μm. The PEGylated Polyplexes showed very low cytotoxicity in MCF-7 and NIH 3T3 cells and induced appreciable transfection efficiencies in the presence of 10% serum, although that are lower than those of p(CBA-ABOL) lacking PEG. The lower transfection efficiency of the PEGylated p(CBA-ABOL) Polyplexes is discussed regarding the effect of PEGylation on endosomal escape of the PEGylated Polyplexes
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flotillin dependent endocytosis and a phagocytosis like mechanism for cellular internalization of disulfide based poly amido amine dna Polyplexes
Biomaterials, 2011Co-Authors: Dries Vercauteren, Johan F J Engbersen, Stefaan C. De Smedt, Martin Piest, Leonardus J Van Der Aa, Monerah Al Soraj, Arwyn Tomos Jones, Kevin BraeckmansAbstract:Extensive research is currently performed on designing safe and efficient non-viral carriers for gene delivery. To increase their efficiency, it is essential to have a thorough understanding of the mechanisms involved in cellular attachment, internalization and intracellular processing in target cells. In this work, we studied in vitro the cellular dynamics of Polyplexes, composed of a newly developed bioreducible poly(amido amine) carrier, formed by polyaddition of N,N-cystamine bisacrylamide and 1-amino-4-butanol (p(CBA-ABOL)) on retinal pigment epithelium (RPE) cells, which are attractive targets for ocular gene therapy. We show that these net cationic p(CBA-ABOL)/DNA Polyplexes require a charge-mediated attachment to the sulfate groups of cell surface heparan sulfate proteoglycans in order to be efficiently internalized. Secondly, we assessed the involvement of defined endocytic pathways in the internalization of the Polyplexes in ARPE-19 cells by using a combination of endocytic inhibitors, RNAi depletion of endocytic proteins and live cell fluorescence colocalization microscopy. We found that the p(CBA-ABOL) Polyplexes enter RPE cells both via flotillin-dependent endocytosis and a PAK1 dependent phagocytosis-like mechanism. The capacity of Polyplexes to transfect cells was, however, primarily dependent on a flotillin-1-dependent endocytosis pathway.
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novel poly amido amine s with bioreducible disulfide linkages in their diamino units structure effects and in vitro gene transfer properties
Journal of Controlled Release, 2008Co-Authors: Martin Piest, C Lin, Wim E Hennink, Martin C Lok, Jan Feijen, Miguel A Mateostimoneda, Johan F J EngbersenAbstract:A series of novel water-soluble, bioreducible poly(amido amine)s (SS-PAAs) was synthesized by Michael addition polymerization of N,N′-dimethylcystamine (DMC) with various bisacrylamides. DMC-based SS-PAAs show high buffer capacities in the pH range 5.1–7.4, facilitating endosomal escape. The polymers form nanoscaled ( + 20 mV) Polyplexes which are relatively stable, but rapidly disintegrate in the presence of 2.5 mM DTT. These Polyplexes have transfection efficiencies up to 4 times higher than bPEI in COS-7 cells, with low cytotoxicity.
Ernst Wagner - One of the best experts on this subject based on the ideXlab platform.
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minicircle versus plasmid dna delivery by receptor targeted Polyplexes
Human Gene Therapy, 2017Co-Authors: Ana Krhac Levacic, Ernst Wagner, Stephan Morys, Susanne Kempter, Ulrich LacheltAbstract:Due to its minimal size and lack of bacterial backbone sequences, minicircle (MC) DNA presents a promising alternative to plasmid DNA (pDNA) for non-viral gene delivery in terms of biosafety and improved gene transfer. Here, luciferase pDNA (pCMV-luc) and analogous MC DNA (MC07.CMV-luc) were formulated into Polyplexes with c-Met targeted, PEG-shielded sequence-defined oligoaminoamides, or linear PEI (linPEI) as standard transfection agent. Distinct physicochemical and biological characteristics were observed for Polyplexes formed with either pDNA or MC DNA as vectors. The carriers were found to dominate the shape of Polyplexes, whereas the DNA type was decisive for the nanoparticle size. c-Met-targeted, tyrosine trimer-containing Polyplexes were optimized into compacted rod structures with a size of 65–100 nm for pDNA and 35–40 nm for MC. Notably, these MC Polyplexes display a lack of cell cycle dependence of transfection and a ∼200-fold enhanced gene transfer efficiency in c-Met-positive DU145 prostate c...
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the effect of molar mass and degree of hydroxyethylation on the controlled shielding and deshielding of hydroxyethyl starch coated Polyplexes
Biomaterials, 2013Co-Authors: Matthaus Noga, Ernst Wagner, Daniel Edinger, Raphaela Klager, Seraphine V Wegner, Joachim P Spatz, Gerhard Winter, Ahmed BesheerAbstract:PEGylation is currently the gold-standard in shielding cationic DNA-Polyplexes against non-specific interaction with blood components. However, it reduces cellular uptake and transfection, in what is known as the "PEG-dilemma". In an approach to solve this problem we developed hydroxyethyl starch (HES)-shielded Polyplexes which get deshielded under the action of alpha amylase (AA). In this study, the effect of molar mass and degree of hydroxyethylation on the shielding and deshielding of the Polyplexes as well as their in vivo performance were investigated. For this purpose, a battery of HES-polyethylenimine (PEI) conjugates was synthesized, and their rate and extent of biodegradation were investigated using asymmetric flow-field flow fractionation (AF4) and quartz-crystal microbalance with dissipation (QCM-D). Additionally, the transfection efficiency of the Polyplexes was tested in Neuro2A cells and tumor-bearing mice. AF4 and QCM results show a rapid degradation for HES with lower degrees of hydroxyethylation. Meanwhile, in vitro transfection experiments showed a better shielding for higher HES molar masses, as well as deshielding with a significant boost in transfection upon addition of AA. Finally, in vivo experiments showed that the biodegradable HES markedly reduced the non-specific lung transcription of the Polyplexes, but maintained gene expression in the tumor, contrary to the non-degradable HES and PEG controls, which reduced both tumor and lung expression. This study shows that by controlling the molecular characteristics of HES it is possible to engineer the shielding and deshielding properties of the Polyplexes for more efficient gene delivery.
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controlled shielding and deshielding of gene delivery Polyplexes using hydroxyethyl starch hes and alpha amylase
Journal of Controlled Release, 2012Co-Authors: Matthaus Noga, Ernst Wagner, Daniel Edinger, Gerhard Winter, Wolfgang Rodl, Ahmed BesheerAbstract:Abstract The non-viral delivery of nucleic acids faces many extracellular and intracellular hurdles on the way from injection site to the site of action. Among these, aggregation in the blood stream and rapid elimination by the mononuclear phagocytic system (MPS) represent strong obstacles towards successful development of these promising therapeutic modalities. Even the state-of-the-art solutions using PEGylation show low transfection efficiency due to limited uptake and hindered endosomal escape. Engineering the carriers with sheddable coats reduces aggregation and phagocytosis due to the effective shielding, while the controlled deshielding at the desired site of action enhances the uptake and intracellular release. This work reports for the first time the use of hydroxyethyl starch (HES) for the controlled shielding/deshielding of Polyplexes. HES, with different molar masses, was grafted to polyethylenimine (PEI) and characterized using 1 H NMR, colorimetric copper-assay, and SEC. HES–PEI conjugates were used to generate Polyplexes with the luciferase-expressing plasmid DNA pCMVluc, and were characterized by DLS and zeta potential measurements. Deshielding was tested in vitro by zeta potential measurements and, erythrocyte aggregation assay upon addition of α-amylase (AA) to the HES-decorated particles. The addition of AA led to gradual increase in the zeta potential of the nanoparticles over 0.5 to 1 h and to a higher aggregation tendency for erythrocytes due to the degradation of the HES-coat and exposure of the Polyplexes' positive charge. In vitro transfection experiments were conducted in 2 cell-lines ± AA in the culture medium. The amylase-treated HES-decorated complexes showed up to 2 orders of magnitude higher transfection levels compared to the untreated HES-shielded particles, while AA had no effect on the transfection of PEG-coated or uncoated Polyplexes. Finally, flow cytometry showed that the addition of AA increased the amount of delivered DNA per cell for the HES-shielded Polyplexes. This study shows that decorating nanoparticles with HES can be a promising tool for the controlled shielding/deshielding of Polyplexes.
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the establishment of an up scaled micro mixer method allows the standardized and reproducible preparation of well defined plasmid lpei Polyplexes
European Journal of Pharmaceutics and Biopharmaceutics, 2011Co-Authors: Julia Christina Kasper, Ernst Wagner, Manfred Ogris, David Schaffert, Wolfgang FriessAbstract:Polyplexes based on linear polyethylenimine (LPEI) and plasmid DNA are known as efficient non-viral gene delivery systems. However, the requirement for freshly prepared complexes prior to administration due to their instability in aqueous suspension poses the risk of batch-to-batch variations. Therefore, the aim of the study was the establishment of a reproducible and up-scalable method for the preparation of well-defined Polyplexes. Polyplexes consisting of pCMVLuc plasmid and 22 kDa linear polyethylenimine (LPEI) were prepared by classical pipetting or with a micro-mixer method using different mixing speeds and plasmid DNA concentrations (20-400 μg/mL). The z-average diameter of the Polyplexes was measured by dynamic light scattering. Metabolic activity and transfection efficiency was evaluated on murine neuroblastoma cells after transfection with Polyplexes. When varying mixing speeds of the micro-mixer, polyplex size (59-197 nm) and polydispersity index (0.05-0.19) could be directly controlled. The z-average diameter (65-170 nm) and polydispersity index (0.05-0.22) of the Polyplexes increased with increasing plasmid DNA concentration (20-400 μg/mL). The established up-scaled micro-mixer method allows the standardized and reproducible preparation of well-defined, transfection-competent plasmid/LPEI Polyplexes with high reproducibility.
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monitoring the disassembly of sirna Polyplexes in serum is crucial for predicting their biological efficacy
Journal of Controlled Release, 2010Co-Authors: Kevin Buyens, Stefaan C. De Smedt, Ernst Wagner, Martin Meyer, J Demeester, Niek N SandersAbstract:To exploit the full therapeutic potential of short interfering RNA (siRNA), efficient delivery vehicles are needed as siRNA fails to enter cells spontaneously. Such carriers should also protect siRNA against degradation while it is on its way to the cytosol of the target cells. Cationic polymers are widely investigated as siRNA carriers. Cationic polymers and siRNA self-assemble into siRNA Polyplexes which have been shown to silence genes in cell cultures. While siRNA Polyplexes will become exposed to full blood after intravenous injection, in vitro gene knockdown is mostly evaluated in serum free media or media containing only a few percent of serum. Little knowledge is currently available on the stability of siRNA Polyplexes in blood, while there are no methods available which allow a quantitative measurement of the disassembly of nucleic acid containing nanoparticles in such complex biological media. This paper shows that fluorescence fluctuation spectroscopy allows us to quantitatively monitor the disassembly of siRNA containing nanoparticles in full serum. It further shows that the gene silencing efficacy of siRNA Polyplexes in serum containing media can be very well explained by their disassembling behavior in these media. Our findings are important for the further development of siRNA Polyplexes and also other nanoparticulate nucleic acid delivery systems.
Olivia M Merkel - One of the best experts on this subject based on the ideXlab platform.
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stability of sirna Polyplexes from poly ethylenimine and poly ethylenimine g poly ethylene glycol under in vivo conditions effects on pharmacokinetics and biodistribution measured by fluorescence fluctuation spectroscopy and single photon emission computed tomography spect imaging
Journal of Controlled Release, 2009Co-Authors: Olivia M Merkel, Stefaan C. De Smedt, Kevin Buyens, Niek N Sanders, Damiano Librizzi, Andreas Pfestroff, Tino Schurrat, Martin Behe, Thomas KisselAbstract:In search of optimizing siRNA delivery systems for systemic application, one critical parameter remains their stability in blood circulation. In this study, we have traced pharmacokinetics and biodistribution of each component of siRNA Polyplexes formed with polyethylenimine 25 kDa (PEI) or PEGylated PEIs by in vivo real-time gamma camera recording, SPECT imaging, and scintillation counting of blood samples and dissected organs. In vivo behavior of siRNA and polymers were compared and interpreted in the context of in vivo stability of the Polyplexes which had been measured by fluorescence fluctuation spectroscopy (FFS). Both pharmacokinetics and biodistribution of polymer-complexed siRNA were dominated by the polymer. PEGylated polymers and their siRNA Polyplexes showed significantly less uptake into liver (13.6–19.7% ID of PEGylated polymer and 9.5–10.2% ID of siRNA) and spleen compared to PEI 25 kDa (liver deposition: 36.2% ID of polymer and 14.6% ID of siRNA). With non-invasive imaging methods we were able to predict both kinetics and deposition in living animals allowing the investigation of organ distribution in real time and at different time points. FFS measurements proved stability of the applied Polyplexes under in vivo conditions which explained the different behavior of complexed from free siRNA. Despite their stability in circulation, we observed that Polyplexes dissociated upon liver passage. Therefore, siRNA/(PEG-)PEI delivery systems are not suitable for systemic administration, but instead may be useful when the first-pass effect is circumvented, which is the case in local application.
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nonviral sirna delivery to the lung investigation of peg pei Polyplexes and their in vivo performance
Molecular Pharmaceutics, 2009Co-Authors: Olivia M Merkel, Damiano Librizzi, Andreas Pfestroff, Andrea Beyerle, Thomas M Behr, Brian S Sproat, Peter J Barth, Thomas KisselAbstract:This study describes the physicobiological characterization of PEI− and PEG−PEI Polyplexes containing partially 2′-OMe modified 25/27mer dicer substrate siRNAs (DsiRNAs) and their in vivo behavior regarding biodistribution and systemic bioavailability after pulmonary application as well as their ability to knock down gene expression in the lung. Biophysical characterization included circular dichroism of siRNA in Polyplexes, condensation efficiency of polymers and in vitro stability. After in vivo application, biodistribution and kinetics of radiolabeled Polyplexes were quantified and recorded over time in three-dimensional SPECT images and by end point scintillation counting. The influence on lung tissue and on the humoral and cellular immunosystem was investigated, and finally knockdown of endogenous gene expression in the lung was determined qualitatively. While all of the polymers used in our study were proven to effectively condense siRNA, stability of the complexes depended on the PEG grafting degre...
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influence of polyethylene glycol chain length on the physicochemical and biological properties of poly ethylene imine graft poly ethylene glycol block copolymer sirna Polyplexes
Bioconjugate Chemistry, 2006Co-Authors: Shirui Mao, Michael Neu, Oliver Germershaus, Olivia M Merkel, Johannes Sitterberg, Udo Bakowsky, Thomas KisselAbstract:Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio (∼50%) on the biophysical properties of the Polyplexes. Particle size, ζ potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA Polyplexes were characterized. Stability and size of siRNA Polyplexes were clearly influenced by PEI−PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)30 resulted in large (300−400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other Polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA Polyplexes against heparin displacement and RNase digestion cou...
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influence of polyethylene glycol chain length on the physicochemical and biological properties of poly ethylene imine graft poly ethylene glycol block copolymer sirna Polyplexes
Bioconjugate Chemistry, 2006Co-Authors: Shirui Mao, Michael Neu, Oliver Germershaus, Olivia M Merkel, Johannes Sitterberg, Udo Bakowsky, Thomas KisselAbstract:Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio ( approximately 50%) on the biophysical properties of the Polyplexes. Particle size, zeta potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA Polyplexes were characterized. Stability and size of siRNA Polyplexes were clearly influenced by PEI-PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)(30) resulted in large (300-400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other Polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA Polyplexes against heparin displacement and RNase digestion could be modified by PEGylation. Protection against RNase digestion was highest for PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1), while siRNA/PEI provided insufficient protection. In knockdown experiments using NIH/3T3 fibroblasts stably expressing beta-galactosidase, it was shown that PEG chain length had a significant influence on biological activity of siRNA. Polyplexes with siRNA containing PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1) yielded similar efficiencies of ca. 70% knockdown as lipofectamine controls. Confocal microscopy demonstrated enhanced cellular uptake of siRNA into cytosol by Polyplexes formation with PEI copolymers. In conclusion, both the chain length and graft density of PEG were found to strongly influence siRNA condensation and stability and hence affect the knockdown efficiency of PEI-PEG/siRNA Polyplexes.