Olivia M. Merkel

Stability of siRNA polyplexes from poly(ethylenimine) and poly(ethylenimine)-g-poly(ethylene glycol) under in vivo conditions: Effects on pharmacokinetics and biodistribution measured by Fluorescence Fluctuation Spectroscopy and Single Photon Emission Computed Tomography (SPECT) imaging

In search of optimizing siRNA delivery systems for systemic application, one critical parameter remains their stability in blood circulation. In this study, we have traced pharmacokinetics and biodistribution of each component of siRNA polyplexes formed with polyethylenimine 25 kDa (PEI) or PEGylated PEIs by in vivo real-time gamma camera recording, SPECT imaging, and scintillation counting of blood samples and dissected organs. In vivo behavior of siRNA and polymers were compared and interpreted in the context of in vivo stability of the polyplexes which had been measured by fluorescence fluctuation spectroscopy (FFS). Both pharmacokinetics and biodistribution of polymer-complexed siRNA were dominated by the polymer. PEGylated polymers and their siRNA polyplexes showed significantly less uptake into liver (13.6-19.7% ID of PEGylated polymer and 9.5-10.2% ID of siRNA) and spleen compared to PEI 25 kDa (liver deposition: 36.2% ID of polymer and 14.6% ID of siRNA). With non-invasive imaging methods we were able to predict both kinetics and deposition in living animals allowing the investigation of organ distribution in real time and at different time points. FFS measurements proved stability of the applied polyplexes under in vivo conditions which explained the different behavior of complexed from free siRNA. Despite their stability in circulation, we observed that polyplexes dissociated upon liver passage. Therefore, siRNA/(PEG-)PEI delivery systems are not suitable for systemic administration, but instead may be useful when the first-pass effect is circumvented, which is the case in local application.

In vivo pharmacokinetics, tissue distribution and underlying mechanisms of various PEI(–PEG)/siRNA complexes

BACKGROUND RNA interference (RNAi) represents a novel therapeutic strategy allowing the knockdown of any pathologically relevant target gene. Since it relies on the action of small interfering RNAs (siRNAs), the in vivo delivery of siRNAs is instrumental. Polyethylenimines (PEIs) and PEGylated PEIs have been shown previously to complex siRNAs, thus mediating siRNA protection against nucleolytic degradation, cellular uptake and intracellular release. PURPOSE The present study determines in vivo pharmacokinetics, tissue distribution/efficacy of siRNA delivery and adverse effects of a broad panel of PEI(-PEG)-based siRNA complexes. The aim is to systematically evaluate the effects of different degrees and patterns of PEGylation in PEI-PEG copolymers on the in vivo behavior of PEI(-PEG)/siRNA complexes in mice. RESULTS Upon i.v. injection of radioactively labeled, PEI(-PEG) complexed siRNAs, marked differences in the pharmacokinetics and biodistribution of the complexes are observed, with the fate of the PEI(-PEG)/siRNA complexes being mainly dependent on the degree of uptake in liver, spleen, lung and kidney. Thus, the role of these tissues is investigated in greater detail using representative PEI(-PEG)/siRNA complexes. The induction of erythrocyte aggregation and hemorrhage is dependent on the degree and pattern of PEGylation as well as on the PEI/siRNA (N/P) ratio, and represents one important effect in the lung. Furthermore, siRNA uptake in liver and spleen, but not in lung or kidney, is mediated by macrophage and is dependent on macrophage activity. In the kidney PEI(-PEG)/siRNA uptake is mostly passive and reflects the total stability of the complexes. CONCLUSION Liver, lung, spleen and kidney are the major players determining the in vivo biodistribution of PEI(-PEG)/siRNA complexes. Beyond their physicochemical and in vitro bioactivity characteristics, PEI(-PEG)/siRNA complexes show marked differences in vivo which can be explained by distinct effects in different tissues. Based on these data, our study also identifies which PEGylated PEIs are promising tools for in vivo siRNA delivery in future therapeutic studies and which major determinants require further investigation.

Triazine dendrimers as nonviral vectors for in vitro and in vivo RNAi: the effects of peripheral groups and core structure on biological activity.

A family of triazine dendrimers, differing in their core flexibility, generation number, and surface functionality, was prepared and evaluated for its ability to accomplish RNAi. The dendriplexes were analyzed with respect to their physicochemical and biological properties, including condensation of siRNA, complex size, surface charge, cellular uptake and subcellular distribution, their potential for reporter gene knockdown in HeLa/Luc cells, and ultimately their stability, biodistribution, pharmacokinetics and intracellular uptake in mice after intravenous (iv) administration. The structure of the backbone was found to significantly influence siRNA transfection efficiency, with rigid, second generation dendrimers displaying higher gene knockdown than the flexible analogues while maintaining less off-target effects than Lipofectamine. Additionally, among the rigid, second generation dendrimers, those with either arginine-like exteriors or peripheries containing hydrophobic functionalities mediated the most effective gene knockdown, thus showing that dendrimer surface groups also affect transfection efficiency. Moreover, these two most effective dendriplexes were stable in circulation upon intravenous administration and showed passive targeting to the lung. Both dendriplex formulations were taken up into the alveolar epithelium, making them promising candidates for RNAi in the lung. The ability to correlate the effects of triazine dendrimer core scaffolds, generation number, and surface functionality with siRNA transfection efficiency yields valuable information for further modifying this nonviral delivery system and stresses the importance of only loosely correlating effective gene delivery vectors with siRNA transfection agents.

PEGylation affects cytotoxicity and cell-compatibility of poly(ethylene imine) for lung application: structure-function relationships.

Poly(ethylene imine) (PEI) has widely been used as non-viral gene carrier due to its capability to form stable complexes by electrostatic interactions with nucleic acids. To reduce cytotoxicity of PEI, several studies have addressed modified PEIs such as block or graft copolymers containing cationic and hydrophilic non-ionic components. Copolymers of PEI and hydrophilic poly(ethylene glycol) (PEG) with various molecular weights and graft densities were shown to exhibit decreased cytotoxicity and potential for DNA and siRNA delivery. In this study, we evaluated the cytotoxicity and cell-compatibility of different PEGylated PEI polymers in two murine lung cell lines. We found that the degree of PEGylation correlated with both cytotoxicity and oxidative stress, but not with proinflammatory effects. AB type copolymers with long PEG blocks caused high membrane damage and significantly decreased the metabolic activity of lung cells. In addition, they significantly increased the release of two lipid mediators such as 8-isoprostanes (8-IP) and prostaglandin E(2) (PGE(2)) in a dose-dependent manner. In contrast, the cytokine profiles which indicated high levels of acute-phase cytokines such as TNF-alpha, IL-6, and G-CSF did not follow any clear structure-function relationship. In conclusion, we found that modification of PEI 25 kDa with high degree of PEGylation and low PEG chain length reduced cytotoxic and oxidative stress response in lung cells, while the proinflammatory potential remained unaffected. A degree of substitution in the range of 10 to 30 and PEG-chain lengths up to 2000 Da seem to be beneficial and merit further investigations.

In vitro and in vivo complement activation and related anaphylactic effects associated with polyethylenimine and polyethylenimine-graft-poly(ethylene glycol) block copolymers

Complement activation by polymeric gene and drug delivery systems has been overlooked in the past. As more reports appear in the literature concerning immunogenicity of polymers and their impact on gene expression patterns, it is important to address possible immune side effects of polymers, namely complement activation. Therefore, in this study the activity of low and high molecular weight poly(ethylene imine) and two PEGylated derivatives to induce complement activation were investigated in human serum. These in vitro results revealed that PEI 25 kDa caused significant and concentration dependent complement activation, whereas none of the other polymers induced such effects at their IC(50) concentrations determined by MTT-assays. To verify these in vitro results, additionally, studies were carried out in a swine model after intravenous administration, showing complement activation-related pseudoallergy (CARPA), reflected in symptoms of transient cardiopulmonary distress. Injections of PEI 25 kDa or PEI(25k)-PEG(2k)(10) at a dose of 0.05 and 0.1 mg/kg caused strong reactivity, while PEI 5 kDa and with PEI(25k)-PEG(20k)(1) were also reactogenic at 0.1 mg/kg. It was found that PEI 25 kDa caused both self- and cross-tolerance, whereas the PEG-PEIs were neither self- nor cross-reactively tachyphylactic. As a result of this study, it was shown that PEGylation of polycations with PEG of 20 kDa or higher molecular weight may be favorable. However, potential safety concerns in the development of PEI-based polymeric carriers for drugs and nucleic acids and their translation from bench to bedside need to be taken into consideration for human application.

Comparative in vivo study of poly(ethylene imine)/siRNA complexes for pulmonary delivery in mice

Pulmonary siRNA delivery offers a new way to treat various lung diseases. Poly(ethylene imines) (PEIs) are promising cationic nanocarriers and various modifications are still under investigations to improve their cytotoxicity and efficacy for siRNA delivery. In this study, we analyzed two different types of PEI-based nanocomplexes in mice after intratracheal administration regarding their toxicity and efficacy in the lungs. Ubiquitously enhanced green fluorescent protein (EGFP) expressing transgenic and BALB/c mice were intratracheally instilled with 35μg siRNA complexed with the different types of PEI nanocarriers. Lung toxicity and inflammation were investigated after 24h, 3d and 7d treatment and knockdown of EGFP expression was analyzed by flow cytometry and fluorescence microscopy five days post instillation. Three different polyplexes caused more than 60% knockdown of EGFP expression, but only the fatty acid modified low molecular weight PEI 8.3kDa (C16-C18-EO25)1.4 specifically reduced EGFP expression in CD45+ leucocytes (25±12%) and CD11b-/CD11c+ lung macrophages (36±14%). Hydrophobic and hydrophilic PEG modifications on PEI caused severe inflammatory response and elevated levels of IgM in broncho-alveolar fluid (BALF). Thus, the PEG modification reduced cytotoxicity, but elevated the immune response and proinflammatory effects. Further investigations of the proinflammatory and immunomodulatory effects of the PEI-modified carriers are necessary to clarify the highly unspecific knockdown effects in the lung in more detail. Nevertheless, the more hydrophobic modification of PEI based non-viral vector system appeared to be a promising approach for improved siRNA therapeutics offering successful pulmonary siRNA delivery.

In vivo SPECT and real-time gamma camera imaging of biodistribution and pharmacokinetics of siRNA delivery using an optimized radiolabeling and purification procedure.

Single photon emission computed tomography (SPECT) imaging provides a three-dimensional method for exactly locating gamma emitters in a noninvasive procedure under in vivo conditions. For characterization of siRNA delivery systems, molecular imaging techniques are extremely helpful to follow biodistribution under in experimental animal studies. Quantification of biodistribution of siRNA and nonviral delivery systems using this technique requires efficient methods to stably label siRNA with a gamma emitter (e.g., 111In or 99mTc) and to purify labeled material from excesses of radiolabel or linkers. In the following study, we have optimized labeling and purification of siRNA, which was then applied as free siRNA or after complexation with polyethylenimine (PEI) 25 kDa for in vivo real-time gamma camera and SPECT imaging. Quantification of scintillation counts in regions of interest(ROIs) was compared to conventional scintillation counting of dissected organs, and the data acquired by imaging was shown to corroborate that of scintillation counting. This optimization and proof of principle study demonstrates that biodistribution and pharmacokinetics of siRNA and the corresponding polyplexes can be determined using SPECT, leading to comparable results as conventional methodology.

Nonviral Pulmonary Delivery of siRNA

RNA interference (RNAi) is an important part of the cells defenses against viruses and other foreign genes. Moreover, the biotechnological exploitation of RNAi offers therapeutic potential for a range of diseases for which drugs are currently unavailable. Unfortunately, the small interfering RNAs (siRNAs) that are central to RNAi in the cytoplasm are readily degradable by ubiquitous nucleases, are inefficiently targeted to desired organs and cell types, and are excreted quickly upon systemic injection. As a result, local administration techniques have been favored over the past few years, resulting in great success in the treatment of viral infections and other respiratory disorders. Because there are several advantages of pulmonary delivery over systemic administration, two of the four siRNA drugs currently in phase II clinical trials are delivered intranasally or by inhalation. The air-blood barrier, however, has only limited permeability toward large, hydrophilic biopharmaceuticals such as nucleic acids; in addition, the lung imposes intrinsic hurdles to efficient siRNA delivery. Thus, appropriate formulations and delivery devices are very much needed. Although many different formulations have been optimized for in vitro siRNA delivery to lung cells, only a few have been reported successful in vivo. In this Account, we discuss both obstacles to pulmonary siRNA delivery and the success stories that have been achieved thus far. The optimal pulmonary delivery vehicle should be neither cytotoxic nor immunogenic, should protect the payload from degradation by nucleases during the delivery process, and should mediate the intracellular uptake of siRNA. Further requirements include the improvement of the pharmacokinetics and lung distribution profiles of siRNA, the extension of lung retention times (through reduced recognition by macrophages), and the incorporation of reversible or stimuli-responsive binding of siRNA to allow for efficient release of the siRNAs at the target site. In addition, the ideal carrier would be biodegradable (to address difficulties with repeated administration for the treatment of chronic diseases) and would contain targeting moieties to enhance uptake by specific cell types. None of the currently available polymer- and lipid-based formulations meet every one of these requirements, but we introduce here several promising new approaches, including a biodegradable, nonimmunogenic polyester. We also discuss imaging techniques for following the biodistribution according to the administration route. This tracking is crucial for better understanding the translocation and clearance of nanoformulated siRNA subsequent to pulmonary delivery. In the literature, the success of pulmonary siRNA delivery is evaluated solely by relief from or prophylaxis against a disease; side effects are not studied in detail. It also remains unclear which cell types in the lung eventually take up siRNA. These are critical issues for the translational use of pulmonary siRNA formulations; accordingly, we present a flow cytometry technique that can be utilized to differentiate transfected cell populations in a mouse model that expresses transgenic enhanced green fluorescence protein (EGFP). This technique, in which different cell types are identified on the basis of their surface antigen expression, may eventually help in the development of safer carriers with minimized side effects in nontargeted tissues.

Amphiphilic biodegradable PEG-PCL-PEI triblock copolymers for FRET-capable in vitro and in vivo delivery of siRNA and quantum dots.

Amphiphilic triblock copolymers represent a versatile delivery platform capable of co-delivery of nucleic acids, drugs, and/or dyes. Multifunctional cationic triblock copolymers based on poly(ethylene glycol), poly-ε-caprolactone, and polyethylene imine, designed for the delivery of siRNA, were evaluated in vitro and in vivo. Moreover, a nucleic acid-unpacking-sensitive imaging technique based on quantum dot-mediated fluorescence resonance energy transfer (QD-FRET) was established. Cell uptake in vitro was measured by flow cytometry, whereas transfection efficiencies of nanocarriers with different hydrophilic block lengths were determined in vitro and in vivo by quantitative real-time PCR. Furthermore, after the proof of concept was demonstrated by fluorescence spectroscopy/microscopy, a prototype FRET pair was established by co-loading QDs and fluorescently labeled siRNA. The hydrophobic copolymer mediated a 5-fold higher cellular uptake and good knockdown efficiency (61 ± 5% in vitro, 55 ± 18% in vivo) compared to its hydrophilic counterpart (13 ± 6% in vitro, 30 ± 17% in vivo), which exhibited poor performance. FRET was demonstrated by UV-induced emission of the acceptor dye. Upon complex dissociation, which was simulated by the addition of heparin, a dose-dependent decrease in FRET efficiency was observed. We believe that in vitro/in vivo correlation of the structure and function of polymeric nanocarriers as well as sensitive imaging functionality for mechanistic investigations are prerequisites for a more rational design of amphiphilic gene carriers.

Enhancing in vivo circulation and siRNA delivery with biodegradable polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) copolymers

The purpose of this study was to enhance the in vivo blood circulation time and siRNA delivery efficiency of biodegradable copolymers polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) (hy-PEI-g-PCL-b-PEG) by introducing high graft densities of PCL-PEG chains. SYBR(®) Gold and heparin assays indicated improved stability of siRNA/copolymer-complexes with a graft density of 5. At N/P 1, only 40% siRNA condensation was achieved with non-grafted polymer, but 95% siRNA was condensed with copolymer PEI25k-(PCL570-PEG5k)(5). Intracellular uptake studies with confocal laser scanning microscopy and flow cytometry showed that the cellular uptake was increased with graft density, and copolymer PEI25k-(PCL570-PEG5k)(5) was able to deliver siRNA much more efficiently into the cytosol than into the nucleus. The in vitro knockdown effect of siRNA/hyPEI-g-PCL-b-PEG was also significantly improved with increasing graft density, and the most potent copolymer PEI25k-(PCL570-PEG5k)(5) knocked down 84.43% of the GAPDH expression. Complexes of both the copolymers with graft density 3 and 5 circulated much longer than unmodified PEI25 kDa and free siRNA, leading to a longer elimination half-life, a slower clearance and a three- or fourfold increase of the AUC compared to free siRNA, respectively. We demonstrated that the graft density of the amphiphilic chains can enhance the siRNA delivery efficiency and blood circulation, which highlights the development of safe and efficient non-viral polymeric siRNA nanocarriers that are especially stable and provide longer circulation in vivo.