Sabrina Höbel

Maltose- and maltotriose-modified, hyperbranched poly(ethylene imine)s (OM-PEIs): Physicochemical and biological properties of DNA and siRNA complexes

Polycationic non-viral polymers are widely employed as delivery platforms of plasmid DNA, or of small interfering RNAs (siRNAs) for the induction of RNA interference (RNAi). Among those, poly(ethylene imine)s (PEIs) take a prominent position due to their relatively high efficacy; however, their biodistribution profiles upon systemic delivery and their toxicity pose limitations which can be addressed by the introduction of PEI modifications. In this paper, we systematically analyse physicochemical and biological properties of DNA and siRNA complexes prepared from a set of maltose-, maltotriose- or maltoheptaose-modified hyperbranched PEIs (termed (oligo-)maltose-modified PEIs; OM-PEIs). We show that pH-dependent charge densities of the OM-PEIs correlate with the structure and degree of grafting, and the length of the oligomaltose. Decreased zeta potentials of OM-PEI-based complexes and changes in the thermodynamics of DNA complex formation are observed, while the complex sizes are largely unaffected by maltose grafting and the presence of serum proteins. Furthermore, although complexation efficacies of siRNAs are not altered, complex stabilities are markedly increased in OM-PEI complexes. DNA complex uptake and transfection kinetics are slowed down upon maltose-grafting of the PEI which can be attributed to decreased zeta potentials, and alterations in the uptake mechanisms (clathrin-dependent/clathrin-independent endocytosis) are observed. Independent of the maltose architecture, DNA and siRNA complexes based on maltose-grafted PEI show considerably lower cytotoxicity as compared to PEI complexes. While maltose grafting generally leads to reduced in vitro transfection efficacies, this effect is less profound in some OM-PEI/siRNA complexes as compared to OM-PEI/DNA complexes. Importantly, upon their systemic application in vivo, OM-PEI/siRNA complexes show marked differences in the siRNA biodistribution profile with e.g. substantially decreased siRNA levels in the liver and increased siRNA levels in the muscle. Taken together, we demonstrate that OM-PEI complexes show structure-dependent physicochemical and biological properties and may represent promising, tailor-made platforms for the delivery of siRNAs, particularly for in vivo applications.

Polyethylenimine/small interfering RNA-mediated knockdown of vascular endothelial growth factor in vivo exerts anti-tumor effects synergistically with Bevacizumab

RNA interference is a powerful method for the knockdown of pathologically relevant genes. The in vivo delivery of siRNAs, preferably through systemic, nonviral administration, poses the major challenge in the therapeutic application of RNAi. Small interfering RNA (siRNA) complexation with polyethylenimines (PEI) may represent a promising strategy for siRNA‐based therapies and, recently, the novel branched PEI F25‐LMW has been introduced in vitro. Vascular endothelial growth factor (VEGF) is frequently overexpressed in tumors and promotes tumor growth, angiogenesis and metastasis and thus represents an attractive target gene in tumor therapy.

Polyethylenimine PEI F25-LMW allows the long-term storage of frozen complexes as fully active reagents in siRNA-mediated gene targeting and DNA delivery.

BACKGROUND Polyethylenimines (PEIs) are synthetic, charged polymers which function as transfection reagents based on their ability to compact DNA into complexes. Recently, PEI-mediated delivery of nucleic acids has been extended towards small interfering RNAs (siRNAs) which are instrumental in the induction of RNA interference (RNAi). Since RNAi represents a powerful method for specific gene silencing, the PEI-based delivery of siRNAs is a promising tool for novel putative therapeutic strategies. AIM For therapeutic use, major requirements are the development of formulations which (i) are sufficiently stable in the presence of serum, and which can be (ii) easily and reproducibly manufactured and (iii) stored for a prolonged time with full retention of their integrity and bioactivity. In this paper, we explore the potential of PEI F25-LMW, a low-molecular weight PEI with superior transfection efficacy and low toxicity, towards these goals. RESULTS We have systematically analyzed and determined optimal DNA and siRNA complexation conditions with regard to various parameters including buffer concentration, ionic strength, pH and incubation time. As opposed to 22kDa linear PEI (L-PEI), the low-molecular weight (4-10kDa) PEI F25-LMW performs DNA transfection and siRNA gene targeting with identical efficacies in the presence of serum, thus emphasizing its usefulness in vivo. Furthermore, in contrast to other polyethylenimines, PEI F25-LMW-based DNA or siRNA complexes allow freeze/thawing and frozen storage for several months. Their activity is fully retained without requiring specific buffer conditions or the addition of any lyoprotectant. Physicochemical analysis and atomic force microscopy reveal a distinct size pattern with the presence of two complex subgroups and show that frozen PEI F25-LMW complexes remain stable with little increase in complex size, no changes regarding their zeta potential and cytotoxicity, and full retention of nucleic acid protection. CONCLUSIONS Frozen PEI F25-LMW-based complexes represent efficient and stable ready-to-use formulations of DNA- or siRNA-based gene therapy products.

Polyethylenimine (PEI)/siRNA-Mediated Gene Knockdown In Vitro and In Vivo

Since its discovery about 10 years ago, RNA interference (RNAi) has become an almost standard method for the knockdown of any target gene of interest. It is mediated by small interfering RNAs (siRNAs), which trigger a catalytic mechanism for mRNA degradation. Consequently, the delivery of intact siRNA is of critical importance for the induction of RNAi. Due to the physicochemical and biological properties of siRNAs, resulting in high instability and poor cellular uptake, siRNA modifications and pharmaceutical formulations have been used to enhance RNAi efficacy. This is particularly relevant for the in vivo delivery of siRNAs, which still poses a major hurdle for the experimental or therapeutic application of RNAi.Polyethylenimines (PEIs) are water-soluble, linear, or branched synthetic polymers of variable length with protonable amino groups in every third position. We have shown that certain PEIs are able to form noncovalent complexes with siRNAs, which mediate their protection against nucleolytic degradation as well as enhance their cellular uptake and intracellular release. In this chapter, the preparation and use of PEI/siRNA complexes for various in vitro and in vivo applications are described. Examples for conducting gene targeting experiments and the analysis of knockdown efficacies are given.

Colloidal stability of nano-sized particles in the peritoneal fluid: Towards optimizing drug delivery systems for intraperitoneal therapy

Intraperitoneal (IP) administration of nano-sized delivery vehicles containing small interfering RNA (siRNA) has recently gained attention as an alternative route for the efficient treatment of peritoneal carcinomatosis. The colloidal stability of nanomatter following IP administration has, however, not been thoroughly investigated yet. Here, enabled by advanced microscopy methods such as single particle tracking and fluorescence correlation spectroscopy, we follow the aggregation and cargo release of nano-scaled systems directly in peritoneal fluids from healthy mice and ascites fluid from a patient diagnosed with peritoneal carcinomatosis. The colloidal stability in the peritoneal fluids was systematically studied as a function of the charge (positive or negative) and poly(ethylene glycol) (PEG) degree of liposomes and polystyrene nanoparticles, and compared to human serum. Our data demonstrate strong aggregation of cationic and anionic nanoparticles in the peritoneal fluids, while only slight aggregation was observed for the PEGylated ones. PEGylated liposomes, however, lead to a fast and premature release of siRNA cargo in the peritoneal fluids. Based on our observations, we reflect on how to tailor improved delivery systems for IP therapy.

Biocompatibility and efficacy of oligomaltose-grafted poly(ethylene imine)s (OM-PEIs) for in vivo gene delivery.

Polycationic polymers like poly(ethylene imine)s (PEIs) are extensively explored for the nonviral transfer of DNA or small RNAs (siRNAs). To enhance biocompatibility and alter pharmacokinetic properties, hyperbranched PEI was recently grafted with the nonligand oligosaccharides maltose or maltotriose at various degrees in a systematic study to yield (oligo-)maltose PEIs (OM-PEIs). In this paper, we investigate the in vivo biocompatibility and efficacy of a whole set of (OM-)PEIs and the corresponding (OM-)PEI-based DNA or siRNA complexes upon systemic (intravenous, i.v.) administration in mice. We determine the overall survival and animal welfare, hepatotoxicity, immune stimulation, erythrocyte aggregation, and the efficacy of DNA delivery in vivo. Higher-degree oligomaltose-grafting of PEI substantially decreases weight loss, abolishes lethality upon repeated treatment with the free polymers or with complexes, and abrogates hepatotoxicity, as determined by serum levels of liver enzymes. Immunostimulatory effects (TNF-α, IFN-γ) and erythrocyte aggregation are mainly observed upon treatment with partially maltotriose-grafted PEI or PEI-based complexes and are largely abolished upon higher-degree grafting. In vivo transfection experiments in mice bearing subcutaneous (s.c.) tumor xenografts reveal a strong dependence of reporter gene expression in a given organ on the mode of complex administration (i.v. vs intraperitoneal injection) and the OM-PEI architecture, with high-level maltose-grafted PEI (PEI-(2-Mal)) being most efficient for DNA delivery. We conclude that distinct differences between different patterns of maltose- or maltotriose-grafting are observed with regard to both biocompatibility and in vivo efficacy and identify optimal oligomaltose-PEIs for therapeutic applications.

The neural crest transcription factor Brn3a is expressed in melanoma and required for cell cycle progression and survival

Pigment cells and neuronal cells both are derived from the neural crest. Here, we describe the Pit‐Oct‐Unc (POU) domain transcription factor Brn3a, normally involved in neuronal development, to be frequently expressed in melanoma, but not in melanocytes and nevi. RNAi‐mediated silencing of Brn3a strongly reduced the viability of melanoma cell lines and decreased tumour growth in vivo. In melanoma cell lines, inhibition of Brn3a caused DNA double‐strand breaks as evidenced by Mre11/Rad50‐containing nuclear foci. Activated DNA damage signalling caused stabilization of the tumour suppressor p53, which resulted in cell cycle arrest and apoptosis. When Brn3a was ectopically expressed in primary melanocytes and fibroblasts, anchorage‐independent growth was increased. In tumourigenic melanocytes and fibroblasts, Brn3a accelerated tumour growth in vivo. Furthermore, Brn3a cooperated with proliferation pathways such as oncogenic BRAF, by reducing oncogene‐induced senescence in non‐malignant melanocytes. Together, these results identify Brn3a as a new factor in melanoma that is essential for melanoma cell survival and that promotes melanocytic transformation and tumourigenesis.

Anti-tumor effects of fibroblast growth factor-binding protein (FGF-BP) knockdown in colon carcinoma

BackgroundFibroblast growth factors FGF-1 and FGF-2 are often upregulated in tumors, but tightly bound to heparan sulphate proteoglycans of the extracellular matrix (ECM). One mechanism of their bioactivation relies on the FGF-binding protein (FGF-BP) which, upon reversible binding to FGF-1 or -2, leads to their release from the ECM. FGF-BP increases tumorigenicity and is highly expressed in tumors like colon carcinoma. In this paper, we analyse cellular and molecular consequences of RNAi-mediated FGF-BP knockdown in colon carcinoma, and explore the therapeutic effects of the nanoparticle-mediated delivery of small interfering RNAs (siRNAs) for FGF-BP targeting.ResultsEmploying stable RNAi cells, we establish a dose-dependence of cell proliferation on FGF-BP expression levels. Decreased proliferation is mirrored by alterations in cell cycle distribution and upregulation of p21, which is relevant for mediating FGF-BP effects. While inhibition of proliferation is mainly associated with reduced Akt and increased GSK3β activation, antibody array-based analyses also reveal other alterations in MAPK signalling. Additionally, we demonstrate induction of apoptosis, mediated through caspase-3/7 activation, and alterations in redox status upon FGF-BP knockdown. These effects are based on the upregulation of Bad, Bax and HIF-1α, and the downregulation of catalase. In a therapeutic FGF-BP knockdown approach based on RNAi, we employ polymer-based nanoparticles for the in vivo delivery of siRNAs into established wildtype colon carcinoma xenografts. We show that the systemic treatment of mice leads to the inhibition of tumor growth based on FGF-BP knockdown.ConclusionsFGF-BP is integrated in a complex network of cytoprotective effects, and represents a promising therapeutic target for RNAi-based knockdown approaches.

A Novel Method for the Assessment of Targeted PEI-Based Nanoparticle Binding Based on a Static Surface Plasmon Resonance System

The delivery of nucleic acids is a major hurdle in gene therapy or therapeutic gene knockdown, and the development of intelligent and safe nanoparticles as carrier systems is thus under intense investigation. The introduction of ligands for their targeted delivery is of major interest. Here, we describe a novel approach for the analysis of the binding properties of antibody-functionalized nanoparticles, using surface plasmon resonance (SPR) in a static cuvette system. By chemical coupling of the Epidermal Growth Factor Receptor (EGFR)-specific antibody cetuximab to poly(ethylene imine) (PEI) via a PEG-spacer and subsequent DNA or siRNA complexation, we generated targeted nanoplexes with low surface charge. Antibody-mediated uptake into EGFR overexpressing cells was observed. SPR measurements with use of a novel, protein A-based sandwich system for the immobilization of the target receptor in its correct steric orientation allowed the analysis of the specific PEI-PEG-cetuximab binding to EGFR and the determination of binding affinities. Importantly, our cuvette-based SPR assay system was also suitable for the monitoring of ligand-mediated nanoparticle binding, without convection or shear stress. We conclude that our SPR sandwich system allows the precise analysis of the binding of ligand-functionalized nanoparticles in real-time, and we thus establish SPR for the in vitro evaluation of ligand modifications for generating targeted nanoparticles.

Polyethylenimine Nanoparticle-Mediated siRNA Delivery to Reduce α-Synuclein Expression in a Model of Parkinson’s Disease

RNA interference (RNAi)-based strategies that mediate the specific knockdown of target genes by administration of small interfering RNAs (siRNAs) could be applied for treatment of presently incurable neurodegenerative diseases such as Parkinson’s disease. However, inefficient delivery of siRNA into neurons hampers in vivo application of RNAi. We have previously established the 4–12 kDa branched polyethylenimine (PEI) F25-LMW with superior transfection efficacy for delivery of siRNA in vivo. Here, we present that siRNA complexed with this PEI extensively distributes across the CNS down to the lumbar spinal cord after a single intracerebroventricular infusion. siRNA against α-synuclein (SNCA), a pre-synaptic protein that aggregates in Parkinson’s disease, was complexed with PEI F25-LMW and injected into the lateral ventricle of mice overexpressing human wild-type SNCA (Thy1-aSyn mice). Five days after the single injection of 0.75 μg PEI/siRNA, SNCA mRNA expression in the striatum was reduced by 65%, accompanied by reduction of SNCA protein by ∼50%. Mice did not show signs of toxicity or adverse effects. Moreover, ependymocytes and brain parenchyma were completely preserved and free of immune cell invasion, astrogliosis, or microglial activation. Our results support the efficacy and safety of PEI nanoparticle-mediated delivery of siRNA to the brain for therapeutic intervention.