Mono- and Polyassociation Processes of Pentavalent Biotinylated PEI Glycopolymers for the Fabrication of Biohybrid Structures with Targeting Properties.

This study describes new mechanistic insights in the sequential polyassociation of streptavidin with biotinylated poly(ethyleneimine) glycopolymers and biotinylated PEGylated folic acid components for the preparation of biohybrid structures (BHS) for controlled targeting experiments. Characterization of the BHS revealed that during the formation and postfunctionalization of BHS, reversible dissociation and reassociation processes occur. The BHS are stable over weeks after finalizing the equilibrium-driven polyassociation process. Cellular uptake studies showed that this sequential polyassociation involving biotinylated PEGylated folic acid components does not lead to enhanced cellular uptake of the resulting BHS. In contrast, polyplexes, containing small interfering RNA and bioconjugates (1:1 molar ratio between biotinylated glycopolymer and monomeric streptavidin-lectin fusion protein), enabled us to control the targeting of tumor cells as revealed by knockdown of the tumor-associated protein survivin. Overall, this study demonstrates the high potential of (networklike) streptavidin-biotin interactions with a dynamic character in the formation of complex BHS and extracellular matrix materials.

Exploring the MIR143-UPAR Axis for the Inhibition of Human Prostate Cancer Cells In Vitro and In Vivo.

MIR143 is pathologically downregulated and may function as a tumor suppressor in prostate cancer. Likewise, the urokinase plasminogen activator receptor (UPAR) is overexpressed in prostate carcinoma, representing a negative prognostic marker and putative therapeutic target gene. In this paper, we establish UPAR as a new direct target of MIR143. Luciferase reporter gene constructs identify one of the two in silico-predicted binding sites as functionally relevant for direct MIR143 binding to the 3' UTR, and, concomitantly, transfection of MIR143 reduces UPAR protein levels in prostate carcinoma cells in vitro. Inhibitory effects on cell proliferation and colony formation, spheroid growth and integrity, and cell viability are extensively analyzed, and they are compared to direct small interfering RNA (siRNA)-mediated uPAR knockdown or combined microRNA (miRNA)-siRNA treatment. Switching to a therapeutically more relevant in vivo model, we demonstrate tumor-inhibitory effects of MIR143 replacement therapy by systemic treatment of mice bearing subcutaneous PC-3 tumor xenografts with MIR143 formulated in polymeric nanoparticles. This efficient, nanoparticle-mediated delivery of intact MIR143 mediates the marked downregulation of uPAR protein, but not mRNA levels, thus indicating translational inhibition rather than mRNA degradation. In summary, we identify UPAR as a direct target gene of MIR143, and we establish the therapeutic anti-tumor potential of nanoparticle-based MIR143 replacement in prostate cancer.

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.

Optimized polyethylenimine (PEI)-based nanoparticles for siRNA delivery, analyzed in vitro and in an ex vivo tumor tissue slice culture model.

The non-viral delivery of small RNA molecules like siRNAs still poses a major bottleneck for their successful application in vivo. This is particularly true with regard to crossing physiological barriers upon systemic administration. We have previously established polyethylenimine (PEI)-based complexes for therapeutic RNA formulation. These nanoplexes mediate full RNA protection against nucleolytic degradation, delivery to target tissues as well as cellular uptake, intracellular release and therapeutic efficacy in preclinical in vivo models. We herein present data on different polyplex modifications for the defined improvement of physicochemical and biological nanoparticle properties and for targeted delivery. (i) By non-covalent modifications of PEI polyplexes with phospholipid liposomes, ternary complexes ("lipopolyplexes") are obtained that combine the favorable features of PEI and lipid systems. Decreased cytotoxicity and highly efficient delivery of siRNA is achieved. Some lipopolyplexes also allow prolonged storage, thus providing formulations with higher stability. (ii) Novel tyrosine modifications of low molecular weight PEI offer further improvement of stability, biocompatibility, and knockdown efficacy of resulting nanoparticles. (iii) For ligand-mediated uptake, the shielding of surface charges is a critical requirement. This is achieved by PEI grafting with polyethylene glycol (PEG), prior to covalent coupling of anti-HER1 antibodies (Erbitux®) as ligand for targeted delivery and uptake. Beyond tumor cell culture, analyses are extended towards tumor slice cultures from tumor xenograft tissues which reflect more realistically the in vivo situation. The determination of siRNA-mediated knockdown of endogenous target genes, i.e., the oncogenic survival factor survivin and the oncogenic receptor tyrosine kinase HER2, reveals nanoparticle penetration and biological efficacy also under intact tissue and stroma conditions.

Tumor tissue slice cultures as a platform for analyzing tissue-penetration and biological activities of nanoparticles.

The success of therapeutic nanoparticles depends, among others, on their ability to penetrate a tissue for actually reaching the target cells, and their efficient cellular uptake in the context of intact tissue and stroma. Various nanoparticle modifications have been implemented for altering physicochemical and biological properties. Their analysis, however, so far mainly relies on cell culture experiments which only poorly reflect the in vivo situation, or is based on in vivo experiments that are often complicated by whole-body pharmacokinetics and are rather tedious especially when analyzing larger nanoparticle sets. For the more precise analysis of nanoparticle properties at their desired site of action, efficient ex vivo systems closely mimicking in vivo tissue properties are needed. In this paper, we describe the setup of organotypic tumor tissue slice cultures for the analysis of tissue-penetrating properties and biological activities of nanoparticles. As a model system, we employ 350µm thick slice cultures from different tumor xenograft tissues, and analyze modified or non-modified polyethylenimine (PEI) complexes as well as their lipopolyplex derivatives for siRNA delivery. The described conditions for tissue slice preparation and culture ensure excellent tissue preservation for at least 14days, thus allowing for prolonged experimentation and analysis. When using fluorescently labeled siRNA for complex visualization, fluorescence microscopy of cryo-sectioned tissue slices reveals different degrees of nanoparticle tissue penetration, dependent on their surface charge. More importantly, the determination of siRNA-mediated knockdown efficacies of an endogenous target gene, the oncogenic survival factor Survivin, reveals the possibility to accurately assess biological nanoparticle activities in situ, i.e. in living cells in their original environment. Taken together, we establish tumor (xenograft) tissue slices for the accurate and facile ex vivo assessment of important biological nanoparticle properties. Beyond the quantitative analysis of nanoparticle tissue-penetration, the excellent tissue preservation and cell viability also allows for the evaluation of biological activities.

Antigen specific immune response in Chlamydia muridarum genital infection is dependent on murine microRNAs-155 and -182.

Anti-chlamydial immunity involves efficient presentation of antigens (Ag) to effector cells resulting in Ag-specific immune responses. There is limited information on inherent underlying mechanisms regulating these events. Previous studies from our laboratory have established that select microRNAs (miRs) function as molecular regulators of immunity in Chlamydia muridarum (Cm) genital infection. In this report, we investigated immune cell type-specific miRs, i.e. miR-155 and -182, and the role in Ag-specific immunity. We observed significant up-regulation of miR-155 in C57BL/6 bone marrow derived dendritic cells (BMDC), and miR-182 in splenic Ag-specific CD4+ T-cells. Using mimics and inhibitors, we determined that miR-155 contributed to BMDC activation following Cm infection. Co-cultures of miR-155 over-expressed in BMDC and miR-182 over-expressed in Ag-specific CD4+ T-cells, or miR-155-/- BMDC with miR-182 inhibitor treated Ag-specific CD4+ T-cells, resulted in IFN-γ production comparable to Ag-specific CD4+ T-cells isolated from Cm infected mice. Additionally, miR-182 was significantly up-regulated in intranasally vaccinated mice protected against Cm infection. In vivo depletion of miR-182 resulted in reduction in Ag-specific IFN-γ and genital pathology in Cm infected mice. To the best of our knowledge, this is the first study to report an interaction of miR-155 (in Cm infected DC) and miR-182 (in CD4+ T-cell) resulting in Ag specific immune responses against genital Cm.

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.

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.

The chorioallantoic membrane assay is a promising ex vivo model system for the study of vascular anomalies.

UNLABELLED: The present feasibility study evaluated the chorioallantoic membrane (CAM) assay established in cancer and angiogenesis research as a tool for the study of vascular anomalies (VAs) in the head and neck area, since the lack of appropriate model systems poses a major obstacle in VA research. MATERIALS AND METHODS: VA tissues from three patients, two with an arteriovenous and one with a lymphatic malformation, were analyzed and evaluated in the CAM assay. RESULTS: The arteriovenous malformations induced a potent angiogenic reaction, resulting in new vessel growth and reperfusion by chicken embryo blood, which was comparable in extent with the positive vascular endothelial growth factor control. An angiogenic reaction, although less pronounced, was also observed in the single-tested lymphatic malformation. CONCLUSION: Our observations indicate the CAM assay to be a suitable model system for the study of VAs, as well as to show how treatment with pro- and antiangiogenic drugs affects VA growth patterns. The CAM assay has the potential to become a valuable tool for VA studies.

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.

Polymer-based delivery of RNA-based therapeutics in ovarian cancer.

RNA interference (RNAi) is a naturally occurring, powerful mechanism for gene silencing, based on the cleavage of a given target mRNA. It relies on small interfering RNAs (siRNAs) in the cell. Being similar in structure, microRNAs (miRNAs) are important regulators of gene expression which mainly act by blocking mRNA translation. In cancer, certain miRNAs have been found to be pathologically downregulated. The therapeutic application of siRNAs or miRNAs for the induction of RNAi or miRNA replacement, respectively, relies on their efficient delivery through a non-viral formulation. Complexation of siRNAs/miRNAs in polymeric nanoparticles based on polyethylenimines (PEIs) offers protection against degradation, delivery to the target site, cellular uptake, and intracellular release. This chapter provides protocols for therapeutic gene silencing and miRNA replacement therapy, based on PEI complexes for in vitro and in vivo use.

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.

Polyethylenimines for siRNA and miRNA delivery in vivo.

The discovery of RNA interference (RNAi) as a naturally occurring mechanism for gene knockdown has attracted considerable attention toward the use of small interfering RNAs (siRNAs) for therapeutic purposes. Likewise, microRNAs (miRNAs) have emerged as important cellular regulators of gene expression, and their pathological underexpression allows for novel therapeutic strategies ('miRNA replacement therapy'). To address issues related to the instability, charge, and molecular weight of small RNA molecules, nanoparticle formulations have been explored for their in vivo application. Polyethylenimines (PEIs) are positively charged, linear, or branched polymers that are able to form nanoscale complexes with small RNAs, leading to RNA protection, cellular delivery, and intracellular release. This review highlights the important properties of various PEIs with regard to their use for in vivo RNA delivery. PEI modifications for increased efficacy, altered pharmacokinetic properties, improved biocompatibility and, upon covalent coupling of ligands, targeted delivery are described. An overview of various modified PEIs and a comprehensive list of representative studies using PEI-based siRNA or miRNA delivery in vivo are given.

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

BACKGROUND: Fibroblast 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. RESULTS: Employing 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 GSK3ß 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. CONCLUSIONS: FGF-BP is integrated in a complex network of cytoprotective effects, and represents a promising therapeutic target for RNAi-based knockdown approaches.

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.

Targeted CRM197-PEG-PEI/siRNA Complexes for Therapeutic RNAi in Glioblastoma.

RNA interference (RNAi) allows the specific knockdown of tumor relevant genes. To induce RNAi, the delivery of small interfering RNAs (siRNAs) is of crucial importance. This is particularly challenging for their therapeutic applications in vivo. Low molecular weight branched polyethylenimine (PEI) is safe and efficient for nucleic acid delivery including small RNA molecules, based on its ability to electrostatically complex siRNA molecules, thereby protecting them from nuclease degradation. The nanoscale PEI/siRNA complexes are endocytosed by cells prior to intracellular complex release from the lysosome and cytoplasmic release of the siRNAs from the complexes. Chemical modification and ligand decoration of the complexes aim at introducing target tissue specificity and further increased efficacy of PEI-mediated siRNA delivery. CRM197 is a mutated, non-toxic diphtheria toxin (DT) that binds to the membrane-bound precursor of HB-EGF-like growth factor/diphtheria toxin receptor highly expressed in glioblastoma cells. Likewise, the growth factor pleiotrophin (PTN/HB-GAM/HARP) is overexpressed in glioblastoma and is rate limiting for tumor growth, thus representing an attractive target gene for therapeutic knockdown approaches. PEGylation of PEI was performed to reduce the surface charge, and by CRM197 coupling we prepared a modified PEI for siRNA delivery into glioblastoma cells. The novel PEI conjugates were analyzed for their complexation efficiency and optimal mixing ratios, and complexes were physicochemically characterized regarding stability, size and zeta potential. The biological activity of the complexes was confirmed in cell culture by reporter gene knockdown. For the therapeutic treatment of subcutaneous human gliobastoma xenografts in athymic nude mice, we systemically injected the modified PEI/siRNA complexes targeting PTN. Antitumor effects based on PTN knockdown demonstrated the advantage of tumor-targeted CRM197-PEG-PEI/siRNA over untargeted PEG-PEI polyplexes. Thus, we establish targeted CRM197-PEG-PEI-based complexes for siRNA delivery in vivo, and show therapeutic effects of CRM197-PEG-PEI/siRNA-mediated knockdown of PTN.

Liposome-polyethylenimine complexes for enhanced DNA and siRNA delivery.

The efficient delivery of nucleic acids into cells is critical for successful gene therapy or gene knockdown. Polyethylenimines (PEIs) are positively charged polymers which complex and deliver DNA for gene transfection or small interfering RNAs (siRNAs) for the induction of RNA interference (RNAi), and mediate their endosomal release. Likewise, various liposomes act as transfection reagents, with some lipids showing increased endocytosis and influencing endosomal escape. This study combines the favourable properties of PEI and lipid systems for DNA and siRNA delivery. Various lipids and lipid combinations, which cover a broad range of physicochemical properties and form optimal liposomes, are assessed. By addition of the liposomes to pre-formed polyplexes, based on the low molecular weight PEI F25-LMW, we establish liposome-PEI complexes (lipopolyplexes) and characterise them in comparison to their 'parent' polyplexes and liposomes regarding size, shape and zeta-potential. Furthermore, while the lipidation of polyplexes generally decreases their toxicity, our studies on DNA transfection and siRNA-mediated knockdown also establish certain lipopolyplexes based on rigid, negatively charged lipids as particularly efficient vehicles for nucleic acid delivery, further improving DNA transfection. The analysis of their mechanism and kinetics of cellular uptake confirms that the biological properties of lipopolyplexes are mainly determined by the liposome shell. We conclude that certain lipopolyplexes show improved biological properties over PEI complexes, thus representing potentially attractive non-viral vectors for gene therapy and RNAi.

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.

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

BACKGROUND: 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. METHODS: In subcutaneous tumor xenograft mouse models, we established the therapeutic efficacy and safety of PEI F25-LMW/siRNA-mediated knockdown of VEGF. In biodistribution and siRNA quantification studies, we optimized administration strategies and, employing chemically modified siRNAs, compared the anti-tumorigenic efficacies of: (i) PEI/siRNA-mediated VEGF targeting; (ii) treatment with the monoclonal anti-VEGF antibody Bevacizumab (Avastin); and (iii) a combination of both. RESULTS: Efficient siRNA delivery is observed upon systemic administration, with the biodistribution being dependent on the mode of injection. Toxicity studies reveal no hepatotoxicity, proinflammatory cytokine induction or other side-effects of PEI F25-LMW/siRNA complexes or polyethylenimine, and tumor analyses show efficient VEGF knockdown upon siRNA delivery, leading to reduced tumor cell proliferation and angiogenesis. The determination of anti-tumor effects reveals that, in pancreas carcinoma xenografts, single treatment with PEI/siRNA complexes or Bevacizumab is already highly efficacious, whereas, in prostate carcinoma, synergistic effects of both treatments are observed. CONCLUSIONS: PEI F25-LMW/siRNA complexes, which can be stored frozen as opposed to many other carriers, represent an efficient, safe and promising avenue in anti-tumor therapy, and PEI/siRNA-mediated, therapeutic VEGF knockdown exerts anti-tumor effects.

In vitro cytotoxic and immunomodulatory profiling of low molecular weight polyethylenimines for pulmonary application.

Polyethylenimines (PEI) are potent non-viral nucleic acid delivery vehicles used for gene delivery and RNA interference (RNAi). For non-invasive pulmonary RNAi therapy the respiratory tissue is an attractive application route, but offers particularly unwanted side-effects like cytotoxicity as well as inflammatory and immune responses. In the current study, we determined the most crucial issues of pulmonary applications for two low molecular weight PEIs in comparison to the well-known lung toxic crystalline silica. Cytotoxic effects and inflammatory responses were evaluated in three murine pulmonary target cell lines, the alveolar epithelial (LA4), the alveolar macrophage (MH-S) and the macrophage-monocyte-like (RAW 264.7) cell line. For both PEIs, cytotoxicity was detected most prominently in the alveolar epithelial cells and only at high doses. Cytokine responses, in contrast were observed already at low PEI concentrations and could be divided into three groups, induced (i) by free PEI (IL-6, TNF-alpha, G-CSF), (ii) by PEI/siRNA complexes (CCL2, -5, CXCL1, -10), or (iii) unaffected by either treatment (IL-2, -4,-7, -9, and CCL3). We conclude that even for the respiratory tissue both PEIs represent powerful siRNA delivery tools with reduced cytotoxicity and minor proinflammatory potency. However, in relation to response levels observed upon crystalline silica exposures, some PEI induced proapoptotic and proinflammatory responses might not be considered completely harmless, therefore further in vivo investigations are advisable.