Polyelectrolyte complex formation at defined interfaces and structure formation in solution studied by analytical ultracentrifugation

The interactions and structural formation of polyelectrolytes in solution and environmental stimuli-responsive water-soluble polymers are subjects of intense academic research, development, and application. An increasing number of novel materials are being created by synthesizing new molecules but also by assembly of existing molecules via intermolecular interactions. Such materials have found practical applications in cell and drug immobilization, environment protection, or sophisticated analytics and sensors. The reproducible synthesis, well-defined assembly, and full understanding of the molecular impact on the application behaviour require knowledge of structure-property relationships. These needs have motivated the selection of the research performed in this thesis. To establish such relationships and to gain more insight on the molecular level, powerful characterization methods must be employed. In this context, analytical ultracentrifugation was used. On the one hand, conventional analytical ultracentrifugation techniques were applied to complicated molecular characterization. On the other hand, analytical ultracentrifugation principles were extended to problems not studied before with this technique. Specifically, the following problems were addressed with polyelectrolytes as primary polymeric substances: Online study of hydrogel formation by interaction of oppositely charged polyelectrolytes using a modified synthetic boundary experiment Characterization of polydisperse branched high molar mass charged copolymers Molecular assembly of di-block copolymers containing one polyelectrolyte block Temperature-induced concentration dependent phase transition in aqueous solution. Overall, results were obtained from analytical ultracentrifugation experiments which were not accessible by other characterization techniques. Systematic online studies of hydrogel formation at the interface of aqueous solutions of oppositely charged polyelectrolytes revealed the impact of chemical structure, concentration, molar mass, and pH on the network quality of three combinations of polyanions and polycations: sodium alginate/oligochitosan, sodium alginate/poly(L-lysine), and sodium cellulose sulfate/poly(diallyldimethyl ammonium chloride). These combinations are of practical interest. The results are expected to contribute to the optimization of network properties for specific applications in medicine, biotechnology, and pharmacy. High molar mass polyelectrolytes, which were polydispersed with regard to molar mass, charge density, and chain architecture were characterized by hydrodynamic methods, particularly analytical ultracentrifugation. By combining synthetic boundary and sedimentation velocity experiments at various speeds, the degree of homogeneity of the various distributions of the samples were identified. The correlation of molecular characteristics and application properties corresponded to predictions from molecular simulations. Sedimentation velocity studies revealed concentration- and temperature-dependent aggregation of stimuli-responsive pre-associated polymers. The monotonic increase of the sedimentation coefficient with concentration and temperature confirmed pre-association prior to the phase separation. The aggregation process was proven to be completely reversible. Moreover, absorbance concentration profiles identified a temperature-dependent portion of polymer not participating in the aggregation process. Sedimentation equilibrium experiments allowed the estimation of the influence of concentration on the molar mass.

Gene Delivery with Hyperbranched Polylysine

Zuzana Kadlecova

The delivery of exogenous nucleic acids into mammalian cells is a valuable technique for basic research studies on the expression, regulation and function of genes and proteins. At the same time, gene delivery to cultivated mammalian cells has become fundamentally important as a technology for the production of therapeutic proteins for preclinical and clinical applications. Alongside with the applications of gene delivery in biotechnology, gene transfer emerged in the early 1990s as a potential therapeutical method in which a functional copy of the defective gene is introduced to provide the missing function. Polymeric cations are widely used as nonviral transfection agents. The efficiency of these vectors, however, is lower than that of viral vectors and has until now precluded their extensive use in applications where stable and high level gene expression is required. Therefore a better understanding of the parameters that govern transfection efficiency of polycationic vectors is crucial for rational design of novel gene carriers. Thus, the main motivation behind this research was to investigate the role of the architecture and molecular weight of the polycation on the efficiency of transfection. The efficiency of transfection is determined by the cytotoxicity of the gene carrier, its reversible complexation with DNA, and proper intracellular trafficking of the resulting complex. For this purpose the 17 member library was synthesized, containing linear (LPL), hyperbranched (HBPL) and dendritic polylysine (DPL) analogues covering a broad range of molecular weights. HBPL, a new class of cationic polymers prepared by polycondensation of L-lysine, was developed to explore the influence of very high molecular weights and randomly branched structures on cytotoxicity, transfection efficiency, and internalization. The thesis is divided into four chapters. Chapter 1 reviews the current state-of-the-art on gene carriers and the future prospects of the field. Additionally, the current use of polycationic gene carriers for the production of recombinant proteins (r-proteins) via a large-scale transient gene expression (TGE) is discussed. The main goal of the Chapter 2 was to determine the influence of the architecture and molecular weight of the polycations on their in vitro biocompatibility. The extent of acute cell death after short exposure was found to be molecular weight dependent. The acute cytotoxicity of high molecular weight polycations was triggered by the permeabilization of the cell membrane. At high concentrations of small molecular weight polycations, membrane destabilization was caused by increased osmotic pressure. In contrast, delayed cell death was correlated to the collapse of mitochondrial transmembrane potential, triggering caspase-dependent apoptosis after intracellular accumulation of the polycation. The onset and extent of the delayed mode of cell death was shown to be dependent on the molecular weight and degree of branching of the polylysine analogues. Based on these results, the differential long term cytotoxicity reflects the resistance of branched polylysine analogues to proteolytic degradation. Thus the degradability and cumulative cytotoxicty can be predetermined by fine-tuning the frequency of branching points in the architecture of peptidic polycation. Chapter 3 demonstrates the evolution of transfection activity with the molecular weight and degree of branching of polylysine analogues. In summary, the results show that under identical cell culture and transfection conditions, the most critical parameter for high gene expression is the structure of the gene carrier. HBPL is the most efficient gene carrier in comparison to LPL and DPL and results in gene expression levels comparable to polyethyleneimine (PEI). Physico-chemical characterization of complexes revealed that those derived from plasmid DNA and HBPL contained a high quantity of the free polycation in comparison to those formed by LPL and DPL. The presence of the free polycation in the course of endocytosis of the complex may provide an explanation for the high transfection efficiency of HBPL. Chapter 4 describes a feasibility study aimed at proving whether HBPL is potentially applicable to large-scale r-protein production via TGE. Gene delivery efficiency and yield of r-protein were evaluated in serum-free, suspension-adapted CHO-DG44 cells under conditions mimicking the industrial manufacture of r-protein by TGE. High molecular weight HBPL mediates transfection and r-protein expression at levels comparable to the ones obtained with PEI. The main advantage of HBPL is its partial enzymatic degradability, potentially decreasing its cumulative cytotoxicity in the course of TGE. Also, HBPL efficiently transfects cell lines and primary cells in the presence of serum, thus broadening the potential applications of HBPL as a novel gene carrier for basic research applications in molecular and cellular biology. In conclusion, it is possible to obtain an efficient gene carrier by simple rearrangement of the topology of poly-L-lysine.

EPFL2011

Development of Accurate and Efficient Electronic Structure Approaches for the Description of Organic Systems

Riccardo Petraglia

The overarching objective of this thesis is extending and adapting the set of computational tools available for describing molecular precursors of organic semiconductors. The research presented within develops adhering to three principle goals: (1) provide accurate energies and geometries for large-scale assemblies at an affordable computational cost; (2) provide tools that thoroughly explore and map free energy landscapes and emphasize the importance of this comprehensive mapping; (3) improve density functional theory (DFT) descriptions of precursors to charge carrier molecules which are currently quite challenging. The Self Consistent-Charge Density Functional Tight Binding (SCC-DFTB), particularly the most recent variant (DFTB3), provides an excellent balance between accuracy and efficiency needed to study large molecules and to perform molecular dynamic simulations. As DFTB approximates the DFT Hamil- tonian it suffers from the same principle shortcomings, including neglecting dispersion interactions. Such interactions are crucial for establishing the proper relative orientations of organic molecules in aggregate, a property of fundamental importance for elucidating conduction properties. Building upon our laboratories experience with the dDsC dispersion correction, an a posteriori pairwise dispersion correction that depends upon Mulliken charges, dDMC, was developed specifically for DFTB. During the course of this work, a caveat in the DFTB parameterization of sulfur was identified that caused sulfur containing molecules to exhibit strong non-covalent binding, even in the absence of a dispersion correction. From a computational perspective, detecting all conformations present in a chemical space and recognizing those structures that are the most chemically relevant represents a significant challenge. To address this problem, we have borrowed a technique usually used for molecular mechanics simulation and combined it with DFTB (REMD@DFTB3). Using this technique allows through exploration of various chemical systems at a quantum level in which bond breaking and formation is possible, thereby allowing description that surpass the typically employed static picture. Although the DFTB approach is unquestionable practical, certain situations require more sophisticated and accurate treatments. Radical cation dimers are, for instance, illustrative as models for organic charge carrier molecules, yet their computational description possesses inherent challenges. Although accurate, post-HF methods are too expensive for routine use, while DFT approaches suffer from their typical shortcomings (self-interaction error, lack of dispersion interactions, missing static correlation). To overcome these problems, a new density functional, ÏB97X-dDsC, was developed in which the parameters of the dDsC correction and those of ÏB97X were fitted together. Preliminary examination of the overall performance of ÏB97X-dDsC is very promising.

EPFL2016

Flowing Gas Experiments Reveal Mechanistic Details of Interfacial Reactions on a Molecular Level at Knudsen Flow Conditions

Riccardo Iannarelli, Christian Ludwig, Michel Rossi

Knudsen flow experiments and its interpretation in terms of adsorption/desorption kinetics as well as quantitative uptake on substrates of interest is presented together with the description of critical design parameters of the Knudsen Flow Reactor (KFR). Hitherto focused almost exclusively on the uptake phase exposing a virgin substrate to interacting gases, we now extend the experiment and its interpretation to the desorption phase at ambient temperature. We present analytical expressions for different experimental situations in terms of adsorption (ka), desorption (kd) and effusion (ke) rate constants. The measurement of kd leads to surface residence times (1/kd) obtained under the same experimental conditions as the uptake (ka) that results in the determination of the Langmuir equilibrium constant KL = ka/kd. We emphasize the interaction of semivolatile organic probe gases and small polar molecules with amorphous carbon and mineral dust materials at ambient temperatures. The latter leads to a molecular characterization scheme based on the use of up to ten different reactive probe gases. After saturation of the uptake of each probe gas this results in a reactivity map of the interface. Several examples are used to underline the broad applicability of the technique such as the silver/air (Ag) interface and the reactivity of TiO2 materials towards uptake of CO2 and CH3OH. Following characterization of several types of amorphous carbon a model incorporating several structural elements in agreement with the reactive gas titration is proposed. For instance, an interface that is at the same time weakly basic and strongly reducing is composed of pyrones and hydroquinones whose simultaneous occurrence leads to stable free radicals that may play a role in atmospheric chemistry (EPFR). The question is raised what makes an interface hydrophobic in terms of surface functional groups when interacting with small polar molecules such as H2O(D2O), HCl, NO2 and NH2OH. Multidiagnostic studies of heterogeneous reactions are enabled using stirred-flow reactors (SRF) that are a logical extension of the KFR approach thus relaxing the Knudsen flow requirements. Previous work using SRF on low-temperature substrates such as H2O ices is highlighted that may be of interest to the exoplanetary and space sciences community.

2022