Spray-drying of PEI-/PPI-based nanoparticles for DNA or siRNA delivery.

Spray-drying of nucleic acid-based drugs designed for gene therapy or gene knockdown is associated with many advantages including storage stability and handling as well as the possibility of pulmonary application. The encapsulation of nucleic acids in nanoparticles prior to spray-drying is one strategy for obtaining efficient formulations. This, however, strongly relies on the definition of optimal nanoparticles, excipients and spray-drying conditions. Among polymeric nanoparticles, polyethylenimine (PEI)-based complexes with or without chemical modifications have been described previously as very efficient for gene or oligonucleotide delivery. The tyrosine-modification of linear or branched low molecular weight PEIs, or of polypropylenimine (PPI) dendrimers, has led to high complex stability, improved cell uptake and transfection efficacy as well as high biocompatibility. In this study, we identify optimal spray-drying conditions for PEI-based nanoparticles containing large plasmid DNA or small siRNAs, and further explore the spray-drying of nanoparticles containing chemically modified polymers. Poly(vinyl alcohol) (PVA), but not trehalose or lactose, is particularly well-suited as excipient, retaining or even enhancing transfection efficacies compared to fresh complexes. A big mesh size is critically important as well, while the variation of the spray-drying temperature plays a minor role. Upon spray-drying, microparticles in a âˆ¼ 3.3 - 8.5 µm size range (laser granulometry) are obtained, dependent on the polymers. Upon their release from the spray-dried material, the nanoparticles show increased sizes and markedly altered zeta potentials as compared to their fresh counterparts. This may contribute to their high efficacy that is seen also after prolonged storage of the spray-dried material. We conclude that these spray-dried systems offer a great potential for the preparation of nucleic acid drug storage forms with facile reconstitution, as well as for their direct pulmonary application as dry powder.

Efficient polymeric nanoparticles for RNAi in macrophage reveal complex effects on polarization markers upon knockdown of STAT3/STAT6.

Tumor associated macrophages (TAMs) are the most abundant immune cell type in the tissue microenvironment, affecting tumor progression, metastasis and therapeutic response. Different macrophage activation ("polarization") states can be distinguished: resting (M0; non-activated), pro-inflammatory/anti-tumorigenic (M1) and anti-inflammatory/pro-tumorigenic (M2). When exploring macrophages as targets in novel cancer immunotherapy approaches, TAM repolarization from the M2 into the M1 phenotype is an intriguing strategy to block their pro-tumoral and enhance their anti-tumoral properties. In the context of RNAi-based gene knockdown of M2 promoting genes, major bottlenecks include cellular siRNA delivery and correct intracellular processing. This is particularly true in case of macrophages as a cell type well-known to be notoriously hard-to-transfect. Among polymeric nanocarriers, the cationic polymer polyethylenimine (PEI) is widely explored for delivering nucleic acids. Further advanced nanocarriers are tyrosine-modified polymers based on PEI or polypropylenimine dendrimers (PPI) for highly efficient siRNA delivery in vitro and in vivo. In this paper, we explored a panel of PEI- or PPI-based nanoparticle systems for siRNA-mediated gene knockdown efficacy in macrophages and subsequent TAM repolarization. The tyrosine-modified linear 10 kDa PEI (LP10Y) or branched 5 kDa PEI (P5Y) as well as a tyrosine-modified PPI (PPI-Y) were found most efficient for gene knockdown in macrophage cell lines or primary macrophages, independent of their polarization. Knockdown of STAT6 or STAT3 led to repolarization of M2 macrophages, as indicated by alterations in various M2 and M1 marker levels. This highly specific approach also demonstrated non-redundant functions of STAT3 and STAT6. Importantly, macrophage re-polarization from M2 to M1 upon PPI-Y/siRNA-mediated STAT6 knockdown increased tumor cell phagocytosis in a co-culture model. In conclusion, we identify certain tyrosine-modified PEI- or PPI-based nanoparticles as particularly efficient for macrophage transfection, and the specific, siRNA-mediated STAT6 knockdown as a promising approach for macrophage repolarization and enhancement of their tumor cell suppressive role.

The effect of novel tyrosine-modified polyethyleneimines on human albumin structure - Thermodynamic and spectroscopic study.

The interaction of proteins with nanoparticle components are crucial for the evaluation of nanoparticle function, toxicity and biodistribution. Polyethyleneimines (PEIs) with defined tyrosine modifications are a class of novel polymers designed for improved siRNA delivery. Their interactions with biomacromolecules are still poorly described. This paper analyzes the interaction of different tyrosine-modified PEIs with human serum albumin as the most abundant serum protein. The ability of tyrosine modified, linear or branched PEIs to bind human serum albumin (HSA) was analyzed and further characterized. The interaction with hydrophobic parts of protein were studied using 1- nilinonaphthalene-8-sulfonic acid (ANS) and changes in the HSA secondary structure were evaluated using circular dichroism (CD). Complex formation and sizes were studied by transmission electron microscopy (TEM) and dynamic light scattering methods (DLS). We demonstrate that tyrosine modified PEIs are able to bind human serum albumin. Based on thermodynamic studies, van der Waals interaction, H-bonding and hydrophobic interactions are determined as main molecular forces involved in complex formation. Analysis of secondary structures revealed that the polymers decreased α-helix content, while increasing levels of randomly folded structures. Complex formation was confirmed by TEM and DLS. These findings are crucial for understanding polymer-protein interactions and the properties of nanoparticles.

Analysis of polymeric nanoparticle properties for siRNA/DNA delivery in a tumor xenograft tissue slice air-liquid interface model.

BACKGROUND: Classical two-dimensional (2D) cell culture as a drug or nanoparticle test system only poorly recapitulates in vivo conditions. Animal studies are costly, ethically controversial, and preclude large-scale testing. METHODS AND RESULTS: We established a three-dimensional (3D) tissue slice air-liquid interface (ALI) culture model for nanoparticle testing. We developed an optimized procedure for the reproducible generation of large sets of tissue slices from tumor xenografts that retain their tissue architecture. When used for the analysis of nanoparticles based on chemically modified polyethylenimines (PEIs) to deliver siRNA or DNA, differences in transfection efficacy and cytotoxicity between nanoparticles were observed more clearly than in 2D cell culture. While nanoparticle efficacies between cell culture and the tissue slice model overall correlated, the tissue slice model also identified particularly suitable candidates whose efficacy was underestimated in 2D cell culture and had already been shown in previous in vivo studies. CONCLUSION: The ex vivo 3D tissue slice ALI culture model is a powerful system that allows the effective evaluation of biological nanoparticle efficacy and biocompatibility in an intact tissue environment. It is comparably inexpensive, time-saving, and follows the 3R principle, while allowing the identification of critical nanoparticle properties and optimal candidates for in vivo applications.

Amphiphilic Anionic Oligomer-Stabilized Calcium Phosphate Nanoparticles with Prospects in siRNA Delivery via Convection-Enhanced Delivery.

Convection-enhanced delivery (CED) has been introduced as a concept in cancer treatment to generate high local concentrations of anticancer therapeutics and overcome the limited diffusional distribution, e.g., in the brain. RNA interference provides interesting therapeutic options to fight cancer cells but requires nanoparticulate (NP) carriers with a size below 100 nm as well as a low zeta potential for CED application. In this study, we investigated calcium phosphate NPs (CaP-NPs) as siRNA carriers for CED application. Since CaP-NPs tend to aggregate, we introduced a new terpolymer (o14PEGMA(1:1:2.5) NH3) for stabilization of CaP-NPs intended for delivery of siRNA to brain cancer cells. This small terpolymer provides PEG chains for steric stabilization, and a fat alcohol to improve interfacial activity, as well as maleic anhydrides that allow for both labeling and high affinity to Ca(II) in the hydrolyzed state. In a systematic approach, we varied the Ca/P ratio as well as the terpolymer concentration and successfully stabilized NPs with the desired properties. Labeling of the terpolymer with the fluorescent dye Cy5 revealed the terpolymer's high affinity to CaP. Importantly, we also determined a high efficiency of siRNA binding to the NPs that caused very effective survivin siRNA silencing in F98 rat brain cancer cells. Cytotoxicity investigations with a standard cell line resulted in minor and transient effects; no adverse effects were observed in organotypic brain slice cultures. However, more specific cytotoxicity investigations are required. This study provides a systematic and mechanistic analysis characterizing the effects of the first oligomer of a new class of stabilizers for siRNA-loaded CaP-NPs.

Unmodified and tyrosine-modified polyethylenimines as potential carriers for siRNA: Biophysical characterization and toxicity.

Polyethylenimines (PEIs) are being explored as efficient non-viral nanocarriers for nucleic acid delivery in vitro and in vivo. To address limitations regarding PEI efficacy and biocompatibility, modifications of the chemical structure of linear and branched PEIs have been introduced, including grafting with tyrosine. The aim has been to compare linear and branched polyethylenimines of a wider range of different molecular mass with their tyrosine-modified derivatives. To do so, physico-chemical and biological properties of the polymers were investigated. Even in the absence of a negatively charged nucleic acid counterpart, PEIs form particle structures with defined size and surface potential. Tyrosine modification of PEI led to significantly reduced toxicity, while simultaneously increasing interaction with cellular membranes. All the effects were also dependent on the PEI molecular weight and structure (i.e., linear vs. branched). Especially in the case of linear PEIs, the improved membrane interaction also translated into slightly enhanced hemolysis, whereas their genotoxic potential was essentially abolished. Due to the improvement of properties critical for nano-vector efficacy and biocompatibility, our data demonstrate that tyrosine-modified PEIs are very promising and safe nanocarriers for the delivery of small RNAs, like siRNAs and miRNAs.

A new player in the game: treatment with antagomiR-21a-5p significantly attenuates histological and echocardiographic effects of experimental autoimmune myocarditis.

AIMS: Myocarditis is associated with formidable symptoms and increased risk of adverse outcomes. Current approaches mostly rely on symptomatic treatments, warranting novel concepts for clinical practice. The aim of this study was to investigate the microRNA (miRNA) expression profile of Balb/c mice with experimental autoimmune myocarditis (EAM), choose a representative miRNA to antagonize after review of available literature and test its effects on myocardial inflammation in vitro and in vivo. METHODS AND RESULTS: Phase 1: EAM was induced in 12 male Balb/c mice, 10 animals served as controls. After sacrifice, next-generation sequencing (NGS) of the miRNA expression profile was performed. Based on these results, H9C2 cells and human ventricular cardiac fibroblasts exposed to lipopolysaccharide (LPS) were treated with the selected candidate antagomiR-21a-5p. Phase 2: EAM was induced in 48 animals. Thereof, 24 animals were either treated with antagomiR-21a-5p or negative control oligonucleotide in a nanoparticle formulation. Transthoracic echocardiography (TTE) was performed on Days 0, 7, 14, and 21. Histopathological examination was performed after sacrifice. Phase 1: EAM resulted in a significant up-regulation of 27 miRNAs, including miR-21a-5p (log2FC: 2.23, adj. P = 0.0026). Transfection with antagomiR-21a-5p resulted in a significant reduction of TNFα, IL-6, and collagen I in vitro. Phase 2: Treatment with antagomiR-21a-5p, formulated in polymeric nanoparticles for systemic injection, significantly attenuated myocardial inflammation (P = 0.001) and fibrosis (P = 0.013), as well as myocardial 'hypertrophy' on TTE. CONCLUSIONS: Silencing of miR-21a-5p results in a significant reduction of the expression of pro-inflammatory cytokines in vitro, as well as a significant attenuation of inflammation, fibrosis and echocardiographic effects of EAM in vivo.

Non-viral siRNA transfection of primary mesenchymal stromal cells (MSCs): Assessment of tyrosine-modified PEI and PPI efficacy and biocompatibility.

Mesenchymal stromal cells (MSCs) are multipotent cells derived from different sources and able to differentiate into distinct cell lineages. For their possible biomedical application, the "tuning" of MSCs also involves the specific knockdown of defined target genes. A major limitation, however, is the notoriously low transfection efficacy especially of primary MSCs. In this paper, we systemically analyze a large set of tyrosine-modified linear or branched low molecular weight polyethylenimines (PEIs) of different sizes, as well as the tyrosine-modified polypropylenimine dendrimer PPI-G4, for their capacity of non-viral siRNA transfection into umbilical cord-derived MSCs from two different donors. Knockdown efficacies are determined on the molecular level and confirmed in functional assays. Beyond the determination of cell viabilities, acute cytotoxicity, induction of apoptosis/necrosis and mitochondrial membrane alterations are also studied. On the molecular level, caspase activation, ROS induction and genotoxic effects are analyzed. Major differences are observed between the various tyrosine-modified PEIs, with some candidates showing high knockdown efficacy and biocompatibility. PPI-G4-Y dendrimers, however, are identified as most efficient for siRNA transfection into MSCs. PPI-G4-Y/siRNA nanoparticles lead to particularly high gene knockdown, without cytotoxic and genotoxic effects on the cellular and molecular level, and are thus particularly well-suited for the tuning of MSCs.

Comparison of tyrosine-modified low molecular weight branched and linear polyethylenimines for siRNA delivery.

Polyethylenimines (PEIs) have been previously introduced for siRNA delivery. In particular, in the case of higher molecular weight PEIs, this is associated with toxicity, while low molecular weight PEIs are often insufficient for siRNA complexation. The tyrosine-modification of PEIs has been shown to enhance PEI efficacy and biocompatibility. This paper evaluates a set of tyrosine-modified low molecular weight linear or branched polyethylenimines as efficient carriers of siRNA. Complexation efficacies and biophysical complex properties were analyzed by zeta potential, dynamic light scattering and circular dichroism measurements as well as gel electrophoresis. Biological knockdown was studied in 2 D cell culture and 3 D ex vivo tissue slice air-liquid interface culture. The results demonstrate that siRNAs were able to form stable complexes with all tested polymers. Complexation was able to protect siRNA from degradation by RNase and to mediate target gene knockdown, as determined on the mRNA level and in PC3-Luc3/EGFP and HCT116-Luc3/EGFP expressing reporter cells on the protein level, using flow cytometry and confocal microscopy. The direct comparison of the studied polymers revealed differences in biological efficacies. Moreover, the tyrosine-modified PEIs showed high biocompatibility, as determined by LDH release and mitochondria integrity (J-aggregate assay) as well as caspase 3/7 (apoptosis) and H2O2 levels (ROS). In 3 D tissue slices, complexes based on LP10Y proved to be most efficient, by combining tissue penetration with efficient gene expression knockdown.

Targeted RNAi of BIRC5/Survivin Using Antibody-Conjugated Poly(Propylene Imine)-Based Polyplexes Inhibits Growth of PSCA-Positive Tumors.

Delivery of siRNAs for the treatment of tumors critically depends on the development of efficient nucleic acid carrier systems. The complexation of dendritic polymers (dendrimers) results in nanoparticles, called dendriplexes, that protect siRNA from degradation and mediate non-specific cellular uptake of siRNA. However, large siRNA doses are required for in vivo use due to accumulation of the nanoparticles in sinks such as the lung, liver, and spleen. This suggests the exploration of targeted nanoparticles for enhancing tumor cell specificity and achieving higher siRNA levels in tumors. In this work, we report on the targeted delivery of a therapeutic siRNA specific for BIRC5/Survivin in vitro and in vivo to tumor cells expressing the surface marker prostate stem cell antigen (PSCA). For this, polyplexes consisting of single-chain antibody fragments specific for PSCA conjugated to siRNA/maltose-modified poly(propylene imine) dendriplexes were used. These polyplexes were endocytosed by PSCA-positive 293TPSCA/ffLuc and PC3PSCA cells and caused knockdown of reporter gene firefly luciferase and Survivin expression, respectively. In a therapeutic study in PC3PSCA xenograft-bearing mice, significant anti-tumor effects were observed upon systemic administration of the targeted polyplexes. This indicates superior anti-tumor efficacy when employing targeted delivery of Survivin-specific siRNA, based on the additive effects of siRNA-mediated Survivin knockdown in combination with scFv-mediated PSCA inhibition.

Tyrosine-modified linear PEIs for highly efficacious and biocompatible siRNA delivery in vitro and in vivo.

Therapeutic gene silencing by RNA interference relies on the safe and efficient in vivo delivery of small interfering RNAs (siRNAs). Polyethylenimines are among the most studied cationic polymers for gene delivery. For several reasons including superior tolerability, small linear PEIs would be preferable over branched PEIs, but they show poor siRNA complexation. Their chemical modification for siRNA formulation has not been extensively explored so far. We generated a set of small linear PEIs bearing tyrosine modifications (LPxY), leading to substantially enhanced siRNA delivery and knockdown efficacy in vitro in various cell lines, including hard-to-transfect cells. The tyrosine-modified linear 10 kDa PEI (LP10Y) is particularly powerful, associated with favorable physicochemical properties and very high biocompatibility. Systemically administered LP10Y/siRNA complexes reveal antitumor effects in mouse xenograft and patient-derived xenograft (PDX) models, and their direct application into the brain achieves therapeutic inhibition of orthotopic glioma xenografts. LP10Y is particularly interesting for therapeutic siRNA delivery.

The combined disulfide cross-linking and tyrosine-modification of very low molecular weight linear PEI synergistically enhances transfection efficacies and improves biocompatibility.

Efficient and non-toxic DNA delivery is still a major limiting factor for non-viral gene therapy. Among the large diversity of non-viral vectors, the cationic polymer polyethylenimine (PEI) plays a prominent role in nucleic acid delivery. Since higher molecular weight of PEI is beneficial for transfection efficacy, but also leads to higher cytotoxicity, the biodegradable cross-linking of low-molecular PEIs, e.g. through disulfide-groups, has been introduced. Another promising strategy is the chemical modification of PEI, for example with amino acids like tyrosine. In the case of small RNA molecules, this PEI grafting has been found to enhance transfection efficacies and improve biocompatibility. In this paper, we report on the combination of these two strategies for improving DNA delivery: the (i) cross-linking of very small 2 kDa PEI ("P2") molecules through biodegradable disulfide-groups ("SS"), in combination with (ii) tyrosine-modification ("Y"). We demonstrate a surprisingly substantial, synergistic enhancement of transfection efficacies of these SSP2Y/DNA complexes over their non- or mono-modified polymer counterparts, accompanied by high biocompatibility as well as favorable physicochemical and biological properties. Beyond various cell lines, high biological activity of the SSP2Y-based complexes is also seen in an ex vivo tissue slice model, more closely mimicking in vivo conditions. The particularly high transfection efficacy SSP2Y/DNA complexes in 2D and 3D models, based on their optimized complex stability and DNA release, as well as their high biocompatibility thus provides the basis for their further exploration for therapeutic application.

miR24-3p activity after delivery into pancreatic carcinoma cell lines exerts profound tumor-inhibitory effects through distinct pathways of apoptosis and autophagy induction.

Pancreatic cancer is among the most detrimental tumors, with novel treatment options urgently needed. The pathological downregulation of a miRNA in tumors can lead to the overexpression of oncogenes, thus suggesting miRNA replacement as novel strategy in cancer therapy. While the role of miR24 in cancer, including pancreatic carcinoma, has been described as ambiguous, it may hold great promise and deserves further studies. Here, we comprehensively analyze the effects of miR24-3p replacement in a set of pancreatic carcinoma cell lines. Transfection of miR24-3p mimics leads to profound cell inhibition in various 2D and 3D cell assays, based on the induction of apoptosis, autophagy and ROS. Comprehensive analyses of miR24-3p effects on the molecular level reveal the transcriptional regulation of several important oncogenes and oncogenic pathways. Based on these findings, miRNA replacement therapy was preclinically explored by treating tumor xenograft-bearing mice with miR24-3p mimics formulated in polymeric nanoparticles. The obtained tumor inhibition was associated with the induction of apoptosis and necrosis. Taken together, we identify miR24-3p as powerful tumor-inhibitory miRNA for replacement therapy, and describe a complex network of oncogenic pathways affected by miR24.

Nanoparticle-complexed antimiRs for inhibiting tumor growth and metastasis in prostate carcinoma and melanoma.

BACKGROUND: MiRNAs act as negative regulators of gene expression through target mRNA degradation or inhibition of its translation. In cancer, several miRNAs are upregulated and play crucial roles in tumorigenesis, making the inhibition of these oncomiRs an interesting therapeutic approach. This can be achieved by directly complementary single-stranded anti-miRNA oligonucleotides (antimiRs). A major bottleneck in antimiR therapy, however, is their efficient delivery. The nanoparticle formation with polyethylenimine (PEI) may be particularly promising, based on the PEI's ability to electrostatically interact with oligonucleotides. This leads to their protection and supports delivery. In the present study, we explore for the first time PEI for antimiR formulation and delivery. We use the branched low molecular weight PEI F25-LMW for the complexation of different antimiRs, and analyse tumor- and metastasis-inhibitory effects of PEI/antimiR complexes in different tumor models. RESULTS: In prostate carcinoma, transfection of antimiRs against miR-375 and miR-141 leads to tumor cell inhibition in 2D- and 3D-models. More importantly, an in vivo tumor therapy study in prostate carcinoma xenografts reveals anti-tumor effects of the PEI/antimiR complexes. In advanced melanoma and metastasis, we identify by a microRNA screen miR-150 as a particularly relevant oncomiR candidate, and validate this result in vitro and in vivo. Again, the systemic application of PEI/antimiR complexes inhibiting this miRNA, or the previously described antimiR-638, leads to profound tumor growth inhibition. These effects are associated with the upregulation of direct miRNA target genes. In a melanoma metastasis mouse model, anti-metastatic effects of PEI/antimiR treatment are observed as well. CONCLUSIONS: We thus describe PEI-based complexes as efficient platform for antimiR therapy, as determined in two different tumor entities using in vivo models of tumor growth or metastasis. Our study also highlights the therapeutic relevance of miR-375, miR-141, miR-150 and miR-638 as target miRNAs for antimiR-mediated inhibition.

Tyrosine-Modification of Polypropylenimine (PPI) and Polyethylenimine (PEI) Strongly Improves Efficacy of siRNA-Mediated Gene Knockdown.

The delivery of small interfering RNAs (siRNA) is an efficient method for gene silencing through the induction of RNA interference (RNAi). It critically relies, however, on efficient vehicles for siRNA formulation, for transfection in vitro as well as for their potential use in vivo. While polyethylenimines (PEIs) are among the most studied cationic polymers for nucleic acid delivery including small RNA molecules, polypropylenimines (PPIs) have been explored to a lesser extent. Previous studies have shown the benefit of the modification of small PEIs by tyrosine grafting which are featured in this paper. Additionally, we have now extended this approach towards PPIs, presenting tyrosine-modified PPIs (named PPI-Y) for the first time. In this study, we describe the marked improvement of PPI upon its tyrosine modification, leading to enhanced siRNA complexation, complex stability, siRNA delivery, knockdown efficacy and biocompatibility. Results of PPI-Y/siRNA complexes are also compared with data based on tyrosine-modified linear or branched PEIs (LPxY or PxY). Taken together, this establishes tyrosine-modified PPIs or PEIs as particularly promising polymeric systems for siRNA formulation and delivery.

Synthetic circular miR-21 RNA decoys enhance tumor suppressor expression and impair tumor growth in mice.

Naturally occurring circular RNAs efficiently impair miRNA functions. Synthetic circular RNAs may thus serve as potent agents for miRNA inhibition. Their therapeutic effect critically relies on (i) the identification of optimal miRNA targets, (ii) the optimization of decoy structures and (iii) the development of efficient formulations for their use as drugs. In this study, we extensively explored the functional relevance of miR-21-5p in cancer cells. Analyses of cancer transcriptomes reveal that miR-21-5p is the by far most abundant miRNA in human cancers. Deletion of the MIR21 locus in cancer-derived cells identifies several direct and indirect miR-21-5p targets, including major tumor suppressors with prognostic value across cancers. To impair miR-21-5p activities, we evaluate synthetic, circular RNA decoys containing four repetitive binding elements. In cancer cells, these decoys efficiently elevate tumor suppressor expression and impair tumor cell vitality. For their in vivo delivery, we for the first time evaluate the formulation of decoys in polyethylenimine (PEI)-based nanoparticles. We demonstrate that PEI/decoy nanoparticles lead to a significant inhibition of tumor growth in a lung adenocarcinoma xenograft mouse model via the upregulation of tumor suppressor expression. These findings introduce nanoparticle-delivered circular miRNA decoys as a powerful potential therapeutic strategy in cancer treatment.

Extracellular vesicle (ECV)-modified polyethylenimine (PEI) complexes for enhanced siRNA delivery in vitro and in vivo.

Extracellular vesicles (ECVs) are secreted cell-derived membrane particles involved in intercellular signaling and cell-cell communication. By transporting various bio-macromolecules, ECVs and in particular exosomes are relevant in various (patho-) physiological processes. ECVs are also released by cancer cells and can confer pro-tumorigenic effects. Their target cell tropism, effects on proliferation rates, natural stability in blood and immunotolerance makes ECVs particularly interesting as delivery vehicles. Polyethylenimines (PEIs) are linear or branched polymers which are capable of forming non-covalent complexes with small RNA molecules including siRNAs or antimiRs, for their delivery in vitro and in vivo. This study explores for the first time the combination of PEI-based nanoparticles with naturally occurring ECVs from different cell lines, for the delivery of small RNAs. ECV-modified PEI/siRNA complexes are analyzed by electron microscopy vs. ECV or complex alone. On the functional side, we demonstrate increased knockdown efficacy and storage stability of PEI/siRNA complexes upon their modification with ECVs. This is paralleled by enhanced tumor cell-inhibition by ECV-modified PEI/siRNA complexes targeting Survivin. Pre-treatment with various inhibitors of cellular internalization reveals alterations in cellular uptake mechanisms and biological activities of PEI/siRNA complexes upon their ECV modification. Extending our studies towards PEI-complexed antimiRs against miR-155 or miR-1246, dose-dependent cellular and molecular effects are enhanced in ECV-modified complexes, based on the de-repression of direct miRNA target genes. Differences between ECVs from different cell lines are observed regarding their capacity of enhancing PEI/siRNA efficacies, independent of the target cell line for transfection. Finally, an in vivo therapy study in mice bearing s.c. PC3 prostate carcinoma xenografts reveals marked inhibition of tumor growth upon treatment with ECVPC3-modified PEI/siSurvivin complexes, based on profound target gene knockdown. We conclude that ECV-modification enhances the activity of PEI-based complexes, by altering pivotal physicochemical and biological nanoparticle properties.

Polymeric Nanoparticles Based on Tyrosine-Modified, Low Molecular Weight Polyethylenimines for siRNA Delivery.

A major hurdle for exploring RNA interference (RNAi) in a therapeutic setting is still the issue of in vivo delivery of small RNA molecules (siRNAs). The chemical modification of polyethylenimines (PEIs) offers a particularly attractive avenue towards the development of more efficient non-viral delivery systems. Here, we explore tyrosine-modified polyethylenimines with low or very low molecular weight (P2Y, P5Y, P10Y) for siRNA delivery. In comparison to their respective parent PEI, they reveal considerably increased knockdown efficacies and very low cytotoxicity upon tyrosine modification, as determined in different reporter and wildtype cell lines. The delivery of siRNAs targeting the anti-apoptotic oncogene survivin or the serine/threonine-protein kinase PLK1 (polo-like kinase 1; PLK-1) oncogene reveals strong inhibitory effects in vitro. In a therapeutic in vivo setting, profound anti-tumor effects in a prostate carcinoma xenograft mouse model are observed upon systemic application of complexes for survivin or PLK1 knockdown, in the absence of in vivo toxicity. We thus demonstrate the tyrosine-modification of (very) low molecular weight PEIs for generating efficient nanocarriers for siRNA delivery in vitro and in vivo, present data on their physicochemical and biological properties, and show their efficacy as siRNA therapeutic in vivo, in the absence of adverse effects.

Therapeutic Targeting of Stat3 Using Lipopolyplex Nanoparticle-Formulated siRNA in a Syngeneic Orthotopic Mouse Glioma Model.

Glioblastoma (GBM), WHO grade IV, is the most aggressive primary brain tumor in adults. The median survival time using standard therapy is only 12⁻15 months with a 5-year survival rate of around 5%. Thus, new and effective treatment modalities are of significant importance. Signal transducer and activator of transcription 3 (Stat3) is a key signaling protein driving major hallmarks of cancer and represents a promising target for the development of targeted glioblastoma therapies. Here we present data showing that the therapeutic application of siRNAs, formulated in nanoscale lipopolyplexes (LPP) based on polyethylenimine (PEI) and the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), represents a promising new approach to target Stat3 in glioma. We demonstrate that the LPP-mediated delivery of siRNA mediates efficient knockdown of Stat3, suppresses Stat3 activity and limits cell growth in murine (Tu2449) and human (U87, Mz18) glioma cells in vitro. In a therapeutic setting, intracranial application of the siRNA-containing LPP leads to knockdown of STAT3 target gene expression, decreased tumor growth and significantly prolonged survival in Tu2449 glioma-bearing mice compared to negative control-treated animals. This is a proof-of-concept study introducing PEI-based lipopolyplexes as an efficient strategy for therapeutically targeting oncoproteins with otherwise limited druggability.

Influence of nanoparticle-mediated transfection on proliferation of primary immune cells in vitro and in vivo.

INTRODUCTION: One of the main obstacles in the widespread application of gene therapeutic approaches is the necessity for efficient and safe transfection methods. For the introduction of small oligonucleotide gene therapeutics into a target cell, nanoparticle-based methods have been shown to be highly effective and safe. While immune cells are a most interesting target for gene therapy, transfection might influence basic immune functions such as cytokine expression and proliferation, and thus positively or negatively affect therapeutic intervention. Therefore, we investigated the effects of nanoparticle-mediated transfection such as polyethylenimine (PEI) or magnetic beads on immune cell proliferation. METHODS: Human adherent and non-adherent PBMCs were transfected by various methods (e.g. PEI, Lipofectamine® 2000, magnetofection) and stimulated. Proliferation was measured by lymphocyte transformation test (LTT). Cell cycle stages as well as expression of proliferation relevant genes were analyzed. Additionally, the impact of nanoparticles was investigated in vivo in a murine model of the severe systemic immune disease GvHD (graft versus host disease). RESULTS: The proliferation of primary immune cells was influenced by nanoparticle-mediated transfection. In particular in the case of magnetic beads, proliferation inhibition coincided with short-term cell cycle arrest and reduced expression of genes relevant for immune cell proliferation. Notably, proliferation inhibition translated into beneficial effects in a murine GvHD model with animals treated with PEI-nanoparticles showing increased survival (pPEI = 0.002) most likely due to reduced inflammation. CONCLUSION: This study shows for the first time that nanoparticles utilized for gene therapeutic transfection are able to alter proliferation of immune cells and that this effect depends on the type of nanoparticle. For magnetic beads, this was accompanied by temporary cell cycle arrest. Notably, in GvHD this nonspecific anti-proliferative effect might contribute to reduced inflammation and increased survival.