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.

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.

Targeted Transposition of Minicircle DNA Using Single-Chain Antibody Conjugated Cyclodextrin-Modified Poly (Propylene Imine) Nanocarriers.

Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play a prominent role in nucleic acid delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise ß-cyclodextrin-modified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53-deficient HCT116p53-/-/PSCA cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities.

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.

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.

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.

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.

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.