Monitoring integrity and localization of modified single-stranded RNA oligonucleotides using ultrasensitive fluorescence methods

Short single-stranded oligonucleotides represent a class of promising therapeutics with diverse application areas. Antisense oligonucleotides, for example, can interfere with various processes involved in mRNA processing through complementary base pairing. Also RNA interference can be regulated by antagomirs, single-stranded siRNA and single-stranded microRNA mimics. The increased susceptibility to nucleolytic degradation of unpaired RNAs can be counteracted by chemical modification of the sugar phosphate backbone. In order to understand the dynamics of such single-stranded RNAs, we investigated their fate after exposure to cellular environment by several fluorescence spectroscopy techniques. First, we elucidated the degradation of four differently modified, dual-dye labeled short RNA oligonucleotides in HeLa cell extracts by fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy and Forster resonance energy transfer. We observed that the integrity of the oligonucleotide sequence correlates with the extent of chemical modifications. Furthermore, the data showed that nucleolytic degradation can only be distinguished from unspecific effects like aggregation, association with cellular proteins, or intramolecular dynamics when considering multiple measurement and analysis approaches. We also investigated the localization and integrity of the four modified oligonucleotides in cultured HeLa cells using fluorescence lifetime imaging microscopy. No intracellular accumulation could be observed for unmodified oligonucleotides, while completely stabilized oligonucleotides showed strong accumulation within HeLa cells with no changes in fluorescence lifetime over 24 h. The integrity and accumulation of partly modified oligonucleotides was in accordance with their extent of modification. In highly fluorescent cells, the oligonucleotides were transported to the nucleus. The lifetime of the RNA in the cells could be explained by a balance between release of the oligonucleotides from endosomes, degradation by RNases and subsequent depletion from the cells

Formulation development of lyophilized, long-term stable siRNA/oligoaminoamide polyplexes

Polyplexes based on precise oligoaminoamides exhibited promising results in non-viral siRNA delivery. However, one serious limitation is insufficient stability of polyplexes in liquid, which raises the demand for lyophilized, long-term stable formulations. Two different siRNA/oligoaminoamide polyplexes were prepared. Freeze-thaw experiments were performed, in order to test various formulations containing sucrose, trehalose, lactosucrose, and hydroxypropyl-β-cyclodextrin for their cryoprotective potential and to investigate the influence of the oligoaminoamide structure on particle stability. Selected formulations were lyophilized and tested for storage stability up to 6 months. Moreover, reconstitution of the lyophilisates in reduced volume as a technique to prepare higher concentration formulations was studied. Samples were analyzed for particle size, gene silencing, cytotoxicity, turbidity, subvisible particles, osmolarity, residual moisture content, glass transition temperature, and morphology. Depending on the oligoaminoamide, siRNA polyplexes maintained particle size and gene silencing efficiency in the absence or presence of low amounts (7%) of stabilizers after freeze-thawing, lyophilization, and reconstitution. Particle stability was highly dependent on the oligoaminoamide used, but independent of the presence of cysteines that form intra-particular disulfide bridges. In contrast to all other excipients, hydroxypropyl-β-cyclodextrin did not provide sufficient stability. For lyophilized 5%/10% sucrose and 7% lactosucrose formulations, long-term stability was demonstrated at 40 °C with retained particle size, retained gene silencing activity, unchanged turbidity values, low numbers of subvisible particles, low residual moisture level, and sufficiently high glass transition temperature. Hence, this work is a promising approach in order to provide long-term stable siRNA polyplex formulations that are ready to use after a simple reconstitution step

Comparison of four different particle sizing methods for siRNA polyplex characterization

The ability to reliably determine the size of siRNA polyplexes is the key for the rational design of particles and their formulation, as well as, their safe application in vivo. At the moment, no standard technique for size measurements is available. Each method has different underlying principles and hence may give different results. Here, four different analytical methods were evaluated for their suitability to analyze the characteristics of homogeneous and heterogeneous siRNA polyplexes: dynamic light scattering (DLS), atomic force microscopy (AFM), nanoparticle trafficking analysis (NTA), and fluorescence correlation spectroscopy (FCS). Three different siRNA polyplex compositions generated with different, precise, and hydrophobically modified oligoaminoamides were used in this study. All of the evaluated methods were suitable for analysis of medium sized, homogeneous siRNA polyplexes (∼120 nm). Small particles (<40 nm) could not be tracked with NTA, but with the other three methods. Heterogeneous polyplexes were generally difficult to analyze. Only by visualization with AFM, the heterogeneity of those polyplexes was observable. FCS was the only method suitable for measuring polyplex stability in 90% fetal bovine serum. Physico-chemical characteristics of polyplexes are important quality criterions for successful in vivo application and future formulation development. Therefore, a comprehensive analysis by more than one method is of particular importance

Solid-phase-assisted synthesis of targeting peptide-PEG-oligo(ethane amino)amides for receptor-mediated gene delivery

In the forthcoming era of cancer gene therapy, efforts will be devoted to the development of new efficient and non-toxic gene delivery vectors. In this regard, the use of Fmoc/Boc-protected oligo(ethane amino)acids as building blocks for solid-phase-supported assembly represents a novel promising approach towards fully controlled syntheses of effective gene vectors. Here we report on the synthesis of defined polymers containing the following: (i) a plasmid DNA (pDNA) binding domain of eight succinoyl-tetraethylenpentamine (Stp) units and two terminal cysteine residues; (ii) a central polyethylene glycol (PEG) chain (with twenty-four oxyethylene units) for shielding; and (iii) specific peptides for targeting towards cancer cells. Peptides B6 and c(RGDfK), which bind transferrin receptor and avß3 integrin, respectively, were chosen because of the high expression of these receptors in many tumoral cells. This study shows the feasibility of designing these kinds of fully controlled vectors and their success for targeted pDNA-based gene transfer.Peer Reviewe

Application of Thermoresponsive PNIPAAM‑b‑PAMPTMA Diblock Copolymers in siRNA Delivery

Gene knockdown has emerged as an important tool for cancer gene therapy as well as for viral infections and dominantly inherited genetic disorders. The generation of suitable siRNA delivery systems poses some challenges, namely, to avoid nuclease degradation, to surpass the cytoplasmic membrane, and to release the nucleic acids into the cytosol. Aiming at evaluating the ability of thermoresponsive block copolymers formed by units of N-isopropylacrylamide and of (3- acrylamidopropyl)trimethylammonium chloride to efficiently deliver siRNAs, an extensive study was performed with four different copolymers using a human fibrosarcoma cell line as cell model. The silencing ability and cytotoxicity of the generated copolymer-based siRNA delivery systems were found to be dependent on the cloud point of the polymer, which corresponds to the transition temperature at which the aggregation or precipitation of the polymer molecules becomes thermodynamically more favorable than their solubilization. In the present study, a system capable of delivering siRNAs efficiently, specifically and without presenting relevant cytotoxicity, even in the presence of serum, was developed. Confocal fluorescence experiments showed that the ability of the generated systems to silence the target gene is related to some extent to nucleic acid internalization, being also dependent on polymer/siRNA dissociation at 37 °C. Thus, a delicate balance between nucleic acid internalization and intracellular release must be met in order to reach an ideal knockdown efficiency. The special features and potential for manipulation of the N-isopropylacrylamide-based copolymers make them suitable materials for the design and synthesis of new and promising siRNA delivery systems.This work was supported by the Portuguese Foundation for Science and Technology and FEDER/COMPETE (research grants PTDC/QUI-BIQ/103001/2008, PTDC/DTP-FTO/ 0265/2012, and Pest-C/SAU/LA0001/2013-2014) and by the Norwegian Research Council, Project Number 190403. A.M.C., C.M.M, and A.L.C. are recipients of fellowships from the Portuguese Foundation for Science and Technology (SFRH/BD/63288/2009, SFRH/BD/79077/2011, and SFRH/BPD/46228/2008, respectively)