Article

Detailed cellular assessment of albumin-bound oligonucleotides: Increased stability and lower non-specific cell uptake

Article

Detailed cellular assessment of albumin-bound oligonucleotides: Increased stability and lower non-specific cell uptake

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Abstract

Conjugation of lipid moieties to nucleic-acid therapeutics increases their interaction with cellular membranes, enhances their uptake and influences in vivo distribution. Once injected in biological fluids, such modifications trigger the binding of various serum proteins, which in turn play a major role in determining the fate of oligonucleotides. Yet, the role played by each of these proteins, more than 300 in serum, remains to be elucidated. Albumin, the most abundant circulating protein is an attractive candidate to study, as it was previously used to enhance the therapeutic effect of various drugs. Herein, we present a thorough fluorescent-based methodology to study the effect of strong and specific albumin-binding on the fate and cellular uptake of DNA oligonucleotides. We synthesized a library of molecules that exhibit non-covalent binding to albumin, with affinities ranging from high (nanomolar) to none. Our results revealed that strong albumin binding can be used as a strategy to reduce degradation of oligonucleotides in physiological conditions caused by enzymes (nucleases), to reduce uptake and degradation by immune cells (macrophages) and to prevent non-specific uptake by cells. We believe that introducing protein-binding domains in oligonucleotides can be used as a strategy to control the fate of oligonucleotides in physiological environments. While our study focuses on albumin, we believe that such systematic studies, which elucidate the role of serum proteins systematically, will ultimately provide a toolbox to engineer the next-generation of therapeutic oligonucleotides, overcoming many of the barriers encountered by these therapeutics, such as stability, immunogenicity and off-target effects.

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The search for understanding the interactions of nanosized materials with living organisms is leading to the rapid development of key applications, including improved drug delivery by targeting nanoparticles, and resolution of the potential threat of nanotechnological devices to organisms and the environment. Unless they are specifically designed to avoid it, nanoparticles in contact with biological fluids are rapidly covered by a selected group of biomolecules to form a corona that interacts with biological systems. Here we review the basic concept of the nanoparticle corona and its structure and composition, and highlight how the properties of the corona may be linked to its biological impacts. We conclude with a critical assessment of the key problems that need to be resolved in the near future.
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Antisense oligonucleotides (AONs) are a class of compounds with high therapeutic potential. One of the challenges facing this platform is the development of effective techniques to achieve cellular delivery. AON conjugates, in which traditional AONs are attached to certain biomolecules, can exhibit improved intracellular bioavailability in the absence of delivery systems. In this study, the lipophilic moieties docosahexaenoic acid, cholesterol, and docosanoic acid (DSA) were conjugated to various phosphorothioated DNA and chemically-modified 2'-fluoro-arabinonucleic acid AONs via an amino-hexanol-linker added to the 5'-end of the molecule. The gene silencing potential of these compounds was evaluated in vitro in the absence or presence of a transfecting agent (polyion complex micelle). Incubation with sub-micromolar concentration of DSA-conjugates could, in the absence of serum proteins, downregulate more than 60% of the targeted mRNA under carrier-free and carrier-loaded delivery methods. Gene silencing activity of carrier-free DSA-conjugates was, however, decreased in a dose-dependent fashion by adding albumin in the transfection medium. Supplementing the medium with free fatty acid prevented the interaction of the DSA-conjugate with albumin, and restored its silencing activity. These findings suggest that strategies aiming at preventing the association of hydrophobized AONs to serum proteins at the site of action may improve their activity.
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Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of a certain color and brightness but instead have their own "personalities" regarding spectroscopic parameters, redox properties, size, water solubility, photostability, and several other factors. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single-molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single-molecule biophysical studies that was later overcome using Trolox through a reducing/oxidizing system but a boon for super-resolution imaging owing to the controllable photoswitching. Understanding photophysics is critical in the design and interpretation of single-molecule experiments.
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A potential barrier to progression of siRNA therapeutics to the clinic is the ability of these agents to cross the vascular endothelium to reach target cells. This study aimed to bypass the endothelial barrier by harnessing the extravasation capability of the serum protein albumin to allow siRNA to reach cardiomyocytes. A strategy for conjugating siRNA to albumin in vivo was developed that involved activating 3'-amine, 2'-O-methyl, phosphorothioate modified siRNA with succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to yield maleimide-functionalized siRNA ("activated siRNA"); this thiol-reactive species can then irreversibly link to the single surface-exposed cysteine residue of endogenous albumin following intravenous administration. An IGF-I-receptor (IGF-IR) siRNA sequence which was effective in vitro was used to test the ability of the siRNA-albumin conjugate to bypass the endothelial barrier in Balb/C mice and produce silencing. In situ conjugation of maleimide-functionalized siRNA to albumin in mouse serum occurred within minutes of addition, and the resulting conjugate was found to be nuclease stable by SDS-PAGE analysis. In Sprague-Dawley rats, activated siRNA showed a significantly enhanced elimination half-life (75.9 ± 18.2 min) compared to unactivated siRNA (5.1 ± 0.2 min). Intravenous injection of this activated siRNA (1 mg/kg daily for four days) significantly reduced left ventricle IGF-IR mRNA to 64.1 ± 4.1% of that in vehicle-treated animals (mean ± SEM), while the control siRNA (unconjugated) had no effect (n = 4, P > 0.05). Imaging of microvessels from mice treated with fluorescein-labeled activated siRNA showed clear evidence of extravasation and cellular uptake in capillary endothelial cells, cardiomyocytes and vascular smooth muscle cells for mice treated with the activated siRNA but not mice treated with the unactivated siRNA. siRNA-albumin constructs are therefore capable of extravasation to the myocardium resulting in silencing in this otherwise silencing-resistant organ.
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Mammalian cells have been shown to internalize oligonucleotide-functionalized gold nanoparticles (DNA-Au NPs or siRNA-Au NPs) without the aid of auxiliary transfection agents and use them to initiate an antisense or RNAi response. Previous studies have shown that the dense monolayer of oligonucleotides on the nanoparticle leads to the adsorption of serum proteins and facilitates cellular uptake. Here, we show that serum proteins generally act to inhibit cellular uptake of DNA-Au NPs. We identify the pathway for DNA-Au NP entry in HeLa cells. Biochemical analyses indicate that DNA-Au NPs are taken up by a process involving receptor-mediated endocytosis. Evidence shows that DNA-Au NP entry is primarily mediated by scavenger receptors, a class of pattern-recognition receptors. This uptake mechanism appears to be conserved across species, as blocking the same receptors in mouse cells also disrupted DNA-Au NP entry. Polyvalent nanoparticles functionalized with siRNA are shown to enter through the same pathway. Thus, scavenger receptors are required for cellular uptake of polyvalent oligonucleotide functionalized nanoparticles.
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Recent evidence suggests that bacterial DNA activates immune responses. Here we showed that TNF-alpha mRNA was induced in bone marrow-derived macrophages and the macrophage cell line RAW 264 by plasmid DNA, but not by DNaseI-digested plasmid, plasmid methylated on CpG dinucleotides, or by vertebrate genomic DNA, which is naturally largely methylated on these sequences. Synthetic polynucleotides poly d(I-C) and poly I x poly C also induced TNF-alpha. IL-1 beta and plasminogen activator inhibitor-2 mRNAs were induced by plasmid DNA, and IFN-gamma-pretreated macrophages responded to DNA with induction of inducible nitric oxide synthase. The HIV-1 long terminal repeat was activated by exogenous DNA in a manner similar to TNF-alpha, and was also activated by a CpG-containing oligonucleotide. Transcription factor nuclear factor-kappa B (NF-kappa B) is involved in regulation of the HIV-1 long terminal repeat and many inflammatory response genes. NF-kappa B binding activity was increased by plasmid DNA. An important question is whether these effects involve DNA binding to a cell surface receptor that signals to the interior, or whether internalization is necessary. Here we found that plasmid was taken up by RAW 264 cells and remained sufficiently intact to code for luciferase protein. Results suggest that DNA is taken up by macrophages and characteristic bacterial DNA sequences, which include an unmethylated CpG sequence, activate a signaling cascade leading to activation of NF-kappa B and inflammatory gene induction. Relevance to DNA vaccination, gene therapy, antisense, and transfection studies is discussed.
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A data anomaly was observed that affected the uniformity and reproducibility of fluorescent signal across DNA microarrays. Results from experimental sets designed to identify potential causes (from microarray production to array scanning) indicated that the anomaly was linked to a batch process; further work allowed us to localize the effect to the posthybridization array stringency washes. Ozone levels were monitored and highly correlated with the batch effect. Controlled exposures of microarrays to ozone confirmed this factor as the root cause, and we present data that show susceptibility of a class of cyanine dyes (e.g., Cy5, Alexa 647) to ozone levels as low as 5-10 ppb for periods as short as 10-30 s. Other cyanine dyes (e.g., Cy3, Alexa 555) were not significantly affected until higher ozone levels (> 100 ppb). To address this environmental effect, laboratory ozone levels should be kept below 2 ppb (e.g., with filters in HVAC) to achieve high quality microarray data.
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After apoptosis or necrosis, macrophages clear dead cells by phagocytosis. Although this process is efficient, circulating nucleosomes can occur in certain diseases, presumably reflecting either increased production or impaired clearance. To investigate the generation of blood nucleosomes, graded numbers of apoptotic and necrotic cells were administered to healthy mice, and levels of blood nucleosomes and DNA were determined. Using Jurkat cells as a model, nucleosomes and DNA were detected in the blood after the administration of 108 apoptotic or necrotic cells per mouse by the intraperitoneal route. The kinetics of the response were similar for both types of cells. The role of macrophages was assessed by eliminating these cells with clodronate liposomes or silica. Although clodronate treatment alone produced a peak level of blood DNA, the subsequent administration of dead cells caused no change in DNA levels. In contrast, silica treatment alone did not elicit a blood DNA response, though this treatment limited the rise in DNA from administered cells. Molecular studies showed that the blood DNA following the administration of apoptotic or necrotic cells arose from the mouse and the Jurkat cells, and its size distribution was consistent with apoptosis. Together, these findings suggest that the generation of blood nucleosomes depends on macrophages, with apoptosis a concomitant of a high burden of dead and dying cells.
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Modulation of pH-responsive cyanine dye pK(a) values via heteroatom substitution allows for design of fluorescent reporters that are tuned for potential imaging of biologically relevant acidic environments.