No abstract

This study describes the physicobiological characterization of PEI- and PEG-PEI polyplexes containing partially 2'-OMe modified 25/27mer dicer substrate siRNAs (DsiRNAs) and their in vivo behavior regarding biodistribution and systemic bioavailability after pulmonary application as well as their ability to knock down gene expression in the lung. Biophysical characterization included circular dichroism of siRNA in polyplexes, condensation efficiency of polymers and in vitro stability. After in vivo application, biodistribution and kinetics of radiolabeled polyplexes were quantified and recorded over time in three-dimensional SPECT images and by end point scintillation counting. The influence on lung tissue and on the humoral and cellular immunosystem was investigated, and finally knockdown of endogenous gene expression in the lung was determined qualitatively. While all of the polymers used in our study were proven to effectively condense siRNA, stability of the complexes depended on the PEG grafting degree. Interestingly, PEI 25 kDa, which showed the least interaction with mucin or surfactant in vitro, performed poorly in vivo. Our nuclear imaging approach enabled us to follow biodistribution of the instilled nanocarriers over time and indicated that PEGylated nanocarriers are more suitable for lung application. While moderate proinflammatory effects were attributed to PEI25k-PEG(2k)(10) nanocarriers, none of the treatments caused histological abnormalities. Our preliminary in vivo knockdown experiment suggests that PEG-PEI/siRNA complexes are promising nanomedicines for pulmonary siRNA delivery. These results encouraged us to further investigate possible adverse effects and to quantify in vivo gene silencing in the lung after intratracheal instillation of PEG-PEI/siRNA complexes.

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

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.

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Efficient delivery of functional nucleic acids into specific cells or tissues is still a challenge for gene therapy and largely depends on targeted delivery strategies. The folate receptor (FR) is known to be overexpressed extracellularly on a variety of human cancers and is therefore an outstanding gate for tumor-targeted Trojan horse-like delivery of therapeutics. In this study, an amphiphilic and biodegradable ternary copolymer conjugated with folate as ligand, polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol)-folate (PEI-PCL-PEG-Fol) was synthesized and evaluated for targeted siRNA delivery via folate-FR recognition. The amphiphilic character of similar polymers was shown previously to support endosomal release of endocytosed nanocarriers and to promote formation of long circulating micelles. The obtained PEI-PCL-PEG-Fol exhibited less cytotoxicity in comparison with the corresponding ternary copolymer without folate (PEI-PCL-PEG) and with unmodified PEI25kDa. Stable micelle-like polyplexes with hydrodynamic diameters about 100 nm were found to have a zeta potential of +8.6 mV, which was lower than that of micelleplexes without folate-conjugation (+13-16 mV). Nonetheless, increased cellular uptake and in vitro gene knockdown of PEI-PCL-PEG-Fol/siRNA micelleplexes were observed in SKOV-3 cells, an FR overexpressing cell line, in comparison with the nonfolate-conjugated ones. Moreover, PEI-PCL-PEG-Fol/siRNA micelleplexes exhibited excellent stability in vivo during the analysis of 120 min and a longer circulation half life than hyPEI25kDa/siRNA polyplexes. Most interestingly, the targeted delivery system yielded 17% deposition of the i.v. injected siRNA per gram in the tumor after 24 h due to the effective folate targeting and the prolonged circulation.

Ironically, population aging which is considered a public health success has been accompanied by a myriad of new health challenges, which include neurodegenerative disorders (NDDs), the incidence of which increases proportionally to age. Among them, Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most common, with the misfolding and the aggregation of proteins being common and causal in the pathogenesis of both diseases. AD is characterized by the presence of hyperphosphorylated τ protein (tau), which is the main component of neurofibrillary tangles (NFTs), and senile plaques the main component of which is β-amyloid peptide aggregates (Aβ). The neuropathological hallmark of PD is α-synuclein aggregates (α-syn), which are present as insoluble fibrils, the primary structural component of Lewy body (LB) and neurites (LN). An increasing number of non-invasive PET examinations have been used for AD, to monitor the pathological progress (hallmarks) of disease. Notwithstanding, still the need for the development of novel detection tools for other proteinopathies still remains. This review, although not exhaustively, looks at the timeline of the development of existing tracers used in the imaging of Aβ and important moments that led to the development of these tracers.

This study aimed to identify suitable siRNA delivery systems based on flexible generation 2–4 triazine dendrimers by correlating physico-chemical and biological in vitro and in vivo properties of the complexes with thermodynamic parameters calculated using molecular modeling. The siRNA binding properties of the dendrimers and PEI 25 kDa were simulated, binding and stability were measured in SYBR Gold assays, and hydrodynamic diameters, zeta potentials, and cytotoxicity were quantified. These parameters were compared with cellular uptake of the complexes and their ability to mediate RNAi. Radiolabeled complexes were administered intravenously, and pharmacokinetic profiles and biodistribution of these polyplexes were assessed both invasively and non-invasively. All flexible triazine dendrimers formed thermodynamically more stable complexes than PEI. While PEI and the generation 4 dendrimer interacted more superficially with siRNA, generation 2 and 3 virtually coalesced with siRNA, forming a tightly intertwined structure. These dendriplexes were therefore more efficiently charge-neutralized than PEI complexes, reducing agglomeration. This behavior was confirmed by results of hydrodynamic diameters (72.0 nm – 153.5 nm) and zeta potentials (4.9 mV – 21.8 mV in 10 mM HEPES) of the dendriplexes in comparison to PEI complexes (312.8 nm – 480.0 nm and 13.7 mV 17.4 mV in 10 mM HEPES). All dendrimers, even generation 3 and 4, were less toxic than PEI. All dendriplexes were efficiently endocytosed and showed significant and specific luciferase knockdown in HeLa/Luc cells. Scintillation counting confirmed that the generation 2 triazine complexes showed more than twofold prolonged circulation times as a result of their good thermodynamic stability. Conversely, generation 3 complexes dissociated in vivo, and generation 4 complexes were captured by the reticulo-endothelial system due to their increased surface charge. Molecular modeling proves very valuable for rationalizing experimental parameters based on the dendrimers’ structural properties. Non-invasive molecular imaging predicted the in vivo fate of the complexes. Therefore, both techniques effectively promote the rapid development of safe and efficient siRNA formulations that are stable in vivo.