Renate Liebl

Breaking up the correlation between efficacy and toxicity for nonviral gene delivery

Nonviral nucleic acid delivery to cells and tissues is considered a standard tool in life science research. However, although an ideal delivery system should have high efficacy and minimal toxicity, existing materials fall short, most of them being either too toxic or little effective. We hypothesized that disulfide cross-linked low-molecular-weight (MW) linear poly(ethylene imine) (MW <4.6 kDa) would overcome this limitation. Investigations with these materials revealed that the extracellular high MW provided outstandingly high transfection efficacies (up to 69.62 ± 4.18% in HEK cells). We confirmed that the intracellular reductive degradation produced mainly nontoxic fragments (cell survival 98.69 ± 4.79%). When we compared the polymers in >1,400 individual experiments to seven commercial transfection reagents in seven different cell lines, we found highly superior transfection efficacies and substantially lower toxicities. This renders reductive degradation a highly promising tool for the design of new transfection materials.

Layer-by-Layer Assembled Gold Nanoparticles for siRNA Delivery

Although uptake into cells is highly complex and regulated, heterogeneous particle collectives are usually employed to deliver small interfering RNA (siRNA) to cells. Within these collectives, it is difficult to accurately identify the active species, and a decrease in efficacy is inherent to such preparations. Here, we demonstrate the manufacture of uniform nanoparticles with the deposition of siRNA on gold in a layer-by-layer approach, and we further report on the cellular delivery and siRNA activity as functions of surface properties.

Gene delivery with low molecular weight linear polyethylenimines.

Linear polyethylenimine (LPEI) with a molecular weight (MW) of 22 kDa has been described as having a superior ability to induce gene transfer compared to its branched form. However, the transfection efficiency of the polymer cannot be enhanced beyond a certain limit due to cytotoxicity. We explored the potential of utilizing LPEIs with MWs ranging from 1.0 to 9.5 kDa to overcome this limitation.

Layer‐by‐Layer Coated Gold Nanoparticles: Size‐Dependent Delivery of DNA into Cells

Because nanoparticles are finding uses in myriad biomedical applications, including the delivery of nucleic acids, a detailed knowledge of their interaction with the biological system is of utmost importance. Here the size-dependent uptake of gold nanoparticles (AuNPs) (20, 30, 50 and 80 nm), coated with a layer-by-layer approach with nucleic acid and poly(ethylene imine) (PEI), into a variety of mammalian cell lines is studied. In contrast to other studies, the optimal particle diameter for cellular uptake is determined but also the number of therapeutic cargo molecules per cell. It is found that 20 nm AuNPs, with diameters of about 32 nm after the coating process and about 88 nm including the protein corona after incubation in cell culture medium, yield the highest number of nanoparticles and therapeutic DNA molecules per cell. Interestingly, PEI, which is known for its toxicity, can be applied at significantly higher concentrations than its IC(50) value, most likely because it is tightly bound to the AuNP surface and/or covered by a protein corona. These results are important for the future design of nanomaterials for the delivery of nucleic acids in two ways. They demonstrate that changes in the nanoparticle size can lead to significant differences in the number of therapeutic molecules delivered per cell, and they reveal that the toxicity of polyelectrolytes can be modulated by an appropriate binding to the nanoparticle surface.

A library of strictly linear poly(ethylene glycol)–poly(ethylene imine) diblock copolymers to perform structure–function relationship of non-viral gene carriers

A library of 39 strictly linear poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) diblock copolymers was synthesized for the delivery of plasmid DNA using PEG of 2, 5, or 10 kDa in combination with linear PEI with a molecular weight (MW) ranging from 1.5 to 10.8 kDa. In contrast to other approaches, the copolymers demonstrated a clear separation between the hydrophilic PEG and the nucleic acid condensing PEI moieties. Hence, the hypothesis was that PEG may not sterically counteract the interaction between the nucleic acid and PEI and that consequently, the copolymers are perfectly suited to build small and stable polyplexes. Analysis of the polyplexes revealed structure-function relationships and the general guideline was that the PEG domain had a greater influence on the physicochemical properties of the polyplexes than PEI. A PEG content higher than 50% led to small (<150 nm), nearly neutral polyplexes with favorable stability. The transfection efficacy of these polyplexes was significantly reduced compared to the PEI homopolymer, but was restored by the application of the corresponding degradable copolymer, which involved a redox triggerable PEG domain. In conclusion, valuable design criteria for the optimization of gene delivery carriers, which is only possible through the screening of such a large library, were gained.

RNA “Traffic Lights”: An Analytical Tool to Monitor siRNA Integrity

The combination of thiazole orange and thiazole red as an internal energy transfer-based fluorophore pair in oligonucleotides provides an outstanding analytical tool to follow DNA/RNA hybridization through a distinct fluorescence color change from red to green. Herein, we demonstrate that this concept can be applied to small interfering RNA (siRNA) to monitor RNA integrity in living cells in real time with a remarkable dynamic range and excellent contrast ratios in cellular media. Furthermore, we show that our siRNA-sensors still possess their gene silencing function toward the knockdown of enhanced green fluorescent protein in CHO-K1 cells.

Oligolayer-Coated Nanoparticles: Impact of Surface Topography at the Nanobio Interface

Layer-by-layer coating of nanoparticles with a layer number in the single-digit range has gained increasing attention in the field of nanomedicinal research. However, the impact of using various polyelectrolytes on oligolayer formation and, more importantly, their influence on the interaction with the biological system has not often been considered in the past. Hence, we investigated the polyelectrolyte deposition profiles and resulting surface topographies of up to three polyelectrolyte layers on a flat gold sensor surface using three different polycations, namely, poly(ethylene imine) (PEI), poly(allylamine hydrochloride) (PAH), and poly(diallylammonium chloride) (PD), each in combination with poly(styrenesulfonate) (PSS). Surface plasmon resonance spectroscopy and atomic force microscopy revealed that the PEI/PSS pair in particular showed a so-called overshoot phenomenon, which is associated with partial polyelectrolyte desorption from the surface. This is also reflected by a significant increase in the surface roughness. Then, after having transferred the oligolayer assembly onto nanoparticles of ∼32 nm, we realized that quite similar surface topographies must have emerged on a curved gold surface. A major finding was that the extent of surface roughness contributes significantly to the fashion by which the oligolayer-coated nanoparticles interact with serum proteins and associate with cells. For example, for the PEI/PSS system, both the surface roughness and protein adsorption increased by a factor of ∼12 from the second to third coating layer and, at the same time, the cell association massively decreased to only one-third. Our study shows that surface roughness, along with other particle properties such as size, shape, zeta potential, and hydrophobicity, is another decisive factor for nanoparticles in a biological context, which has indeed been discussed previously but has not to date been investigated for oligolayers.

Influence of PEGylation on nanoparticle mobility in different models of the extracellular matrix

Nanoparticle transport inside the extracellular matrix (ECM) is a crucial factor affecting the therapeutic success. In this work, two in vitro ECM models - a neutrally charged collagen I network with an effective pore size of 0.47μm and Matrigel, a basement membrane matrix with strong negative charge and effective pore size of 0.14μm - were assessed for barrier function in the context of diffusing nanoparticles. Nanoparticles with a size of 120nm were coated with poly(ethylene glycol) (PEG) of different molecular weights - 2, 5 and 20kDa - over a range of gradually increasing coating densities - precisely 0.2, 2, 8 and 20PEG/nm2. The PEG corona was imaged in its native state without any drying process by atomic force microscopy, revealing that the experimentally determined arrangement of PEG at the surface did not match with what was theoretically expected. In a systematic investigation of nanoparticle mobility via fluorescence recovery after photobleaching, increasing both PEG MW and PEGylation density gradually improved diffusion properties predominately in collagen. Due to its smaller pore size and electrostatic obstruction, diffusion coefficients were about ten times lower in Matrigel than in the collagen network and an extension of the PEG MW and density did not necessarily lead to better diffusing particles. Consequently, collagen gels were revealed to be a poor model for nanoparticle mobility assessment, as neither their pore size nor their electrostatic properties reflect the expected in vivo conditions. In Matrigel, diffusion proceeded according to a sigmoidal increase with gradually increasing PEG densities showing threshold zeta potentials of 11.6mV (PEG2kDa) and 13.8mV (PEG5kDa), below which particles were regarded as mobile. Irrespective of the molecular weight particles with a PEGylation density lower than 2PEG/nm2 were defined as immobile and those with a PEG coverage of more than 8PEG/nm2 as mobile.