Synthetic small interfering RNA (siRNA) is a class of therapeutic entities that allow for specific silencing of target genes via RNA interference (RNAi) and comprise an enormous clinical potential for a variety of diseases, including cancer. However, efficient tissue-specific delivery of siRNA remains the major limitation in the development of RNAi-based cancer therapeutics. To achieve this, we have synthesized a series of sequence-defined oligomers, which include a cationic (oligoethanamino)amide core (for nanoparticle formation with siRNA), cysteines (as bioreversible disulfide units), and a polyethylene glycol chain (for shielding of surface charges) coupled to a terminal targeting ligand. The antifolate drug methotrexate (MTX), a well-established chemotherapeutic agent, serves as both targeting ligand and anticancer agent. The oligomers form homogeneous spherical siRNA polyplexes with a hydrodynamic diameter of approximately 6 nm. These polyplexes access KB cells by binding to the folate receptor in a MTX-dependent manner and induce efficient gene silencing activity in vitro. Impressively, in the in vivo studies, MTX-conjugated polyplexes significantly increase the intratumoral retention (168 h) of the siRNA, as compared to alanine-substituted non-targeted control polyplexes (48 h). The combination of MTX-conjugated polyplexes and eglin 5 (EG5) siRNA provides enhanced antitumoral potency with 50% of recurrence-free survival of KB tumor-bearing mice. The design of such siRNA carrier systems with a dual-functional ligand for cellular delivery and augmented tumor suppression could be a valuable strategy for translating RNAi-based cancer therapeutics to the clinics.

The synthesis of precise gene delivery vehicles by solid-supported chemistry is an effective way to establish structure–activity relationships and optimize existing transfection carriers. Sequence-defined cationic oligomers with different topologies were modified with twin disulfide-forming cysteine–arginine–cysteine (CRC) motifs. The influence of this motif versus single disulfide on the biophysical properties and biological performance of polyplexes was investigated, with pDNA and siRNA as nucleic acid cargoes. Clear differences between structures with isolated cysteines and CRC motifs were observed with respect to properties like nucleic acid binding, serum stability, response to reducing agents, and gene transfer/silencing. The main observed effect of the CRC motif was to increase polyplex stability. The consequences for nucleic acid delivery were less predictable and depended on oligomer topology. For some oligomers intrinsically forming stable polyplexes (i.e., already in the absence of CRC motif), this further stabilization resulted in a reduction or even loss in transfection efficiency. For PEGylated and targeted oligomers with intrinsically less stable polyplex structures, this modification led to a significant enhancement in transfection efficiency.

Hydroxyethyl starch (HES) has been proposed as a biodegradable polymer for shielding of DNA polyplexes, where the feasibility of this approach was shown both in vitro and in vivo. In this study, we report on the physicochemical characterization, the in vitro cytocompatibility and hemotoxicity of HES-decorated polyplexes. For this purpose, various HES molecules were coupled to a 22kDa linear polyethylenimine (LPEI22) to produce a library of nine different HES-PEI conjugates. Particle analysis using dynamic light scattering showed that, neither the molar mass of HES nor the amount of HES in the polyplexes affected the particle diameter, as it was consistently around 70-80nm. Imaging using atomic force microscopy and transmission electron microscopy showed that, both naked and HESylated polyplexes were in the same size range and had a spherical morphology. Meanwhile, the HES-mediated particle-shielding effect, manifested as reduction in the surface charge, strongly correlated with the molar mass of HES, where the charge decreased linearly with the increase in molar mass. Ethidium bromide binding assay showed that HES-PEI did not negatively affect DNA condensation at N/P ratios higher than 4. HES conjugation also showed a stabilizing effect against salt-induced particle disassembly, and particle aggregation in protein-containing media. Compatibility tests included cellular viability, as well as erythrocyte aggregation and hemolysis assays. HES-PEI conjugates showed lower cytotoxicity, no aggregation, and much lower hemolysis compared to unmodified PEI. In conclusion, these results show that the HES-PEI conjugates are promising gene delivery polymers with favorable physicochemical properties and compatibility profile.

The antifolate drug methotrexate (MTX) can serve as a dual-functional ligand in antitumoral drug delivery, inducing both a folate receptor mediated cellular uptake and an intracellular cytotoxic action. Bioactivity of MTX however changes by conjugation; the activity can be affected by the hampered intracellular conversion to more potent poly-γ-glutamyl derivatives. Therefore, in a cancer combination therapy approach for the codelivery of cytotoxic dsRNA polyinosinic-polycytidylic acid poly(I:C), a set of molecularly precise oligo(ethanamino)amides were synthesized comprising poly(ethylene glycol) conjugated MTX ligands. The conjugates differed in the number of additional glutamic acid residues to investigate the effect of different degrees of synthetic "a priori" polyglutamylation. The bioactivity of these compounds concerning dihydrofolate reductase (DHFR) inhibition, cytotoxicity, nucleic acid binding potency, cellular uptake of poly(I:C) polyplexes, and combined antifolate/poly(I:C) toxicity was investigated. Synthetic polyglutamylation had a crucial impact on several stages of efficient poly(I:C) delivery and combined MTX cytotoxicity. DHFR inhibition of the conjugates significantly increased with increasing polyglutamate chain length. The library member with highest glutamylation degree even outperformed free MTX in direct comparison. Studies in KB cells showed the corresponding enhanced cytotoxicity by polyglutamylation. Also poly(I:C) polyplexes of the glutamylated MTX variants exhibited higher cellular uptake in the folate receptor positive cell line. Finally, a synergistic combined cytotoxicity of polyglutamylated MTX ligands and complexed poly(I:C) cargo was observed in transfected KB cells. The present structure-activity relationship study of MTX-based ligands pinpoints the concept of synthetic polyglutamylation as a promising approach for optimizing bioactivity of antifolate conjugates, which might be considered as a useful tool also in context of other drug delivery systems.

Two antitumoral siRNAs (directed against target genes Eg5 and Ran) complexed with one of three sequence-defined cationic oligomers were compared in gene silencing in vitro and antitumoral in vivo efficacy upon intratumoral injection. Two lipo-oligomers (T-shape 49, i-shape 229) and the three-arm oligomer 386 were chosen because of their high efficiency in previous marker gene silencing screens. The oligomers showed very similar target-specific gene knockdown in murine neuroblastoma cells. Silencing persisted only for a short period (maximum on day 1 at mRNA and day 2 at protein level) triggering siRNA specific in vitro tumor cell killing. The fastest onset of protein knockdown and strongest antitumoral effect was mediated by oligomer 386. Tumor growth reduction in vivo was evaluated in the subcutaneous Neuro2A mouse model. Intratumoral injections of either Eg5 or Ran siRNA/oligomer 49 polyplexes led to reduced tumor growth and prolonged survival of mice compared to control siRNA and buffer treatment. Target knockdown was evidenced in tumors by mitotic Aster formation for Eg5 knockdown and apoptotic TUNEL stain for Ran knockdown. Ran siRNA displayed better antitumoral efficacy and was chosen for in vivo comparison of the oligomers. A very clear order of antitumoral activity (oligomer 386 > 49 > 229) was observed. In summary, the similar in vitro gene silencing efficiencies on mRNA level by the tested oligomers did not correlate with the observed therapeutic effects in vivo. Oligomer 386 with the fastest onset of protein knockdown and best in vitro cell killing mediated the best in vivo antitumoral efficacy.

Despite their great potential, gene delivery polyplexes have a number of limitations, including their tendency for aggregation in vivo or upon storage. In previous studies, we could show that hydroxyethyl starch (HES)-decoration of polyplexes reduces aggregation in vitro and in vivo. The current study investigates the ability of HES-decoration to improve the stability of polyplexes upon storage as frozen-liquid or lyophilizate, and uses naked polyplexes or PEGylated ones as controls. For this purpose, freeze-thaw (FT) experiments of the polyplexes were conducted in the presence of standard excipients (glucose, sucrose or trehalose). Dynamic light scattering (DLS) measurements showed that HES-decoration imparted better stability when glucose was used, while both HES and PEG were effective in inhibiting aggregation in the presence of trehalose or sucrose. In contrast, the lyophilized HES-coated polyplexes were more stable than the PEGylated ones as shown by DLS, even after storage for 10 weeks at an elevated temperature. Evaluation of the gene transfer efficiency of the stored samples showed no negative effect of storage, except for the lyophilized naked polyplexes. In general, this study shows that, while both HES- or PEG-coats could prevent aggregation under frozen-liquid storage, the HES-coat resulted in superior protective effect upon lyophilization, with possible advantages for in vivo application. In summary, our developed HES-coats provided effective cryo- and lyoprotection to the DNA polyplexes.

Nine sequence-defined, polycationic oligomers were synthesized containing motifs of three consecutive tyrosines (Y3) as stabilizing components for pDNA and siRNA polyplex assembly. For pDNA, a combination of terminal oligotyrosines and cysteines was necessary and sufficient for stable polyplex formation. Stable siRNA binding required a combination of terminal cysteines and oligotyrosines, as well as a central hydrophobic modification (oligotyrosines or fatty acids). The phenolic group within the aromatic amino acids of Y3 containing oligomers further increased the endosomal buffer capacity. As a result, the new class of oligotyrosine containing oligomers was efficient in pDNA and siRNA transfection, in most cases superior to a previously established cysteine-containing, dioleic acid modified oligomer without the Y3 motif. Additionally, increased serum stability of the new oligomers with terminal Y3 motifs was demonstrated by gel shift and fluorescence correlations spectroscopy (FCS). In vivo stability and biodistribution was monitored by intravenous administration of chemically stabilized Cy7 siRNA either as free form, or complexed with the nine Y3 containing oligomers or control oligomers. Oligomer 332, with the overall most beneficial in vitro and in vivo characteristics, was applied in RAN siRNA polyplexes for intratumoral treatment of neuroblastoma-bearing mice. This resulted in significantly reduced tumor growth compared to animal treated with control siRNA polyplexes.

PEGylation is currently the gold-standard in shielding cationic DNA-polyplexes against non-specific interaction with blood components. However, it reduces cellular uptake and transfection, in what is known as the "PEG-dilemma". In an approach to solve this problem we developed hydroxyethyl starch (HES)-shielded polyplexes which get deshielded under the action of alpha amylase (AA). In this study, the effect of molar mass and degree of hydroxyethylation on the shielding and deshielding of the polyplexes as well as their in vivo performance were investigated. For this purpose, a battery of HES-polyethylenimine (PEI) conjugates was synthesized, and their rate and extent of biodegradation were investigated using asymmetric flow-field flow fractionation (AF4) and quartz-crystal microbalance with dissipation (QCM-D). Additionally, the transfection efficiency of the polyplexes was tested in Neuro2A cells and tumor-bearing mice. AF4 and QCM results show a rapid degradation for HES with lower degrees of hydroxyethylation. Meanwhile, in vitro transfection experiments showed a better shielding for higher HES molar masses, as well as deshielding with a significant boost in transfection upon addition of AA. Finally, in vivo experiments showed that the biodegradable HES markedly reduced the non-specific lung transcription of the polyplexes, but maintained gene expression in the tumor, contrary to the non-degradable HES and PEG controls, which reduced both tumor and lung expression. This study shows that by controlling the molecular characteristics of HES it is possible to engineer the shielding and deshielding properties of the polyplexes for more efficient gene delivery. (C) 2013 Elsevier Ltd. All rights reserved.

The non-viral delivery of nucleic acids faces many extracellular and intracellular hurdles on the way from injection site to the site of action. Among these, aggregation in the blood stream and rapid elimination by the mononuclear phagocytic system (MPS) represent strong obstacles towards successful development of these promising therapeutic modalities. Even the state-of-the-art solutions using PEGylation show low transfection efficiency due to limited uptake and hindered endosomal escape. Engineering the carriers with sheddable coats reduces aggregation and phagocytosis due to the effective shielding, while the controlled deshielding at the desired site of action enhances the uptake and intracellular release. This work reports for the first time the use of hydroxyethyl starch (HES) for the controlled shielding/deshielding of polyplexes. HES, with different molar masses, was grafted to polyethylenimine (PEI) and characterized using 1H NMR, colorimetric copper-assay, and SEC. HES–PEI conjugates were used to generate polyplexes with the luciferase-expressing plasmid DNA pCMVluc, and were characterized by DLS and zeta potential measurements. Deshielding was tested in vitro by zeta potential measurements and, erythrocyte aggregation assay upon addition of α-amylase (AA) to the HES-decorated particles. The addition of AA led to gradual increase in the zeta potential of the nanoparticles over 0.5 to 1 h and to a higher aggregation tendency for erythrocytes due to the degradation of the HES-coat and exposure of the polyplexes' positive charge. In vitro transfection experiments were conducted in 2 cell-lines ± AA in the culture medium. The amylase-treated HES-decorated complexes showed up to 2 orders of magnitude higher transfection levels compared to the untreated HES-shielded particles, while AA had no effect on the transfection of PEG-coated or uncoated polyplexes. Finally, flow cytometry showed that the addition of AA increased the amount of delivered DNA per cell for the HES-shielded polyplexes. This study shows that decorating nanoparticles with HES can be a promising tool for the controlled shielding/deshielding of polyplexes.

Sequence defined oligo (ethane amino) amides produced by solid-phase supported synthesis using different building blocks and molecular shapes were tested for structure–activity relationships in siRNA delivery. Efficient reporter gene knockdown was obtained in a variety of cell lines using either branched three-armed structures, or lipid-modified structures with i-shape, T-shape, U-shape configuration. For the majority of structures (apart from U-shapes), the presence of 2 or 3 cysteines was strictly required for polyplex stabilization and silencing activity. Although all four building blocks contain the ethylenediamine proton sponge motif, only oligomers assembled with the tetraethylenepentamine based amino acids (Stp, Gtp, Ptp) but not with the triethylenetetramine based amino acid (Gtt) were able to mediate efficient gene silencing. For the lipopolymeric structures, out of the tested saturated (from C4 to C18) and unsaturated (C18) fatty acid moieties, two proximate oleic acids or linolic acids provided the oligomers with the best gene silencing activity and also pH specific lytic activity at pH 5.5, presumably facilitating endosomal escape of the polyplexes. Evaluation of oligomer chain length revealed a minimal number of at least two oligo (ethane amino) building blocks per oligomer arm as necessary for the vast majority of structures, but only marginal changes were found with higher numbers (structures with up to 60 ethane amino nitrogens were evaluated). Two promising carriers (T-shape 49, i-shape 229) were also evaluated for EG5 siRNA delivery. This resulted in tumor cell cycle arrest, and appearance of mitotic monoastral spindles both in vitro and in vivo upon systemic delivery. Repeated intratumoral treatment with EG5 siRNA polyplexes significantly reduced Neuro2A-eGFPLuc tumor growth in a siRNA-specific manner.

Although our understanding of RNAi and our knowledge on designing and synthesizing active and safe siRNAs significantly increased during the past decade, targeted delivery remains the major limitation in the development of siRNA therapeutics. On one hand, practical considerations dictate robust chemistry reproducibly providing precise carrier molecules. On the other hand, the multistep delivery process requires dynamic multifunctional carriers of substantial complexity. We present a monodisperse and multifunctional carrier system, synthesized by solid phase supported chemistry, for siRNA delivery in vitro and in vivo. The sequence-defined assembly includes a precise cationic (oligoethanamino)amide core, terminated at the ends by two cysteines for bioreversible polyplex stabilization, at a defined central position attached to a monodisperse polyethylene glycol chain coupled to a terminal folic acid as cell targeting ligand. Complexation with an endosomolytic influenza peptides-iRNA conjugate results in nanosized functional polyplexes of 6 nm hydrodynamic diameter. The necessity of each functional substructure of the carrier system for a specific and efficient gene silencing was confirmed. The nanosized polyplexes showed stability in vivo, receptor-specific cell targeting, and silencing of the EG5 gene in receptor-positive tumors. The nanosized appearance of these particles can be precisely controlled by the oligomer design (from 5.8 to 8.8 nm diameter). A complete surface charge shielding together with the high stability result in good tolerability in vivo and the absence of accumulation in nontargeted tissues such as liver, lung, or spleen. Due to their small size, siRNA polyplexes are efficiently cleared by the kidney.

La presente invention concerne un procede d'administration controlee de molecules d'acide nucleique dans une ou plusieurs cellules cibles, en particulier des cellules tumorales, ledit procede comprenant : l'elaboration d'un complexe polymere forme entre une ou plusieurs molecules d'acide nucleique a administrer et une ou plusieurs molecules de support cationique, au moins une partie de la molecule ou des molecules de support dans le complexe polymere etant fixee de maniere covalente a de l'hydroxyalkyl-amidon, et l'hydroxyalkyl-amidon protegeant le complexe polymere; l'etape consistant a laisser le complexe polymere protege entrer en contact avec la cellule ou les cellules cibles; la deprotection du complexe polymere en eliminant l'hydroxyalkyl-amidon; et l'etape consistant a laisser le complexe polymere deprotege s'internaliser dans la cellule ou les cellules cibles. L'elimination de l'hydroxyalkyl-amidon peut s'accomplir par voie enzymatique en exposant le complexe polymere a une amylase. L'invention a egalement pour objet l'utilisation d'un tel procede pour la prevention et/ou le traitement d'une affection choisie dans le groupe constitue d'un cancer, de maladies auto-immunes, de maladies cardiovasculaires, de maladies neuronales, d'infections, et de maladies inflammatoires.

The pseudodendritic, biodegradable polymer HD-O, consisting of an OEI800 core with several OEI800 molecules attached to it via 1.6-hexanediol diacrylate linkers, has potent pDNA but poor siRNA delivery ability, due to instability of the resulting siRNA polyplexes. Stabilization of such nanoparticles by crosslinking surface amines of HD-O in the polyplexes with dithiobis-(succinimidylpropionate) (DSP) greatly enhanced gene silencing efficiency. Successful crosslinking on the polyplex surface was indicated by a decrease of the positive Zeta potential of the polyplexes. Tuning the polymer/siRNA ratio in combination with adjustment of the linker to a molar ratio of 0.05/1 between linker and polymer amines proved essential for transfection efficiency and prevention of particle aggregation. Gene silencing ability of the crosslinked particles was demonstrated in murine neuroblastoma N2A and human hepatoma HUH-7 cells. Flow cytometry showed efficient cellular uptake already after 1 h incubation with the crosslinked but not with unstabilized particles. Downregulation of endogenous AHA1 mRNA (85% knockdown compared to control) by crosslinked HD-O/AHA1-siRNA particles was detected by quantitative real-time PCR.

Artificial oligo(ethylene amino) acids, together with selected natural amino acids and fatty acid modifications, have been used for the solid-phase-assisted synthesis of polymers with precise sequence, topology, and modifications (see picture). First proof-of-concept studies demonstrate the high potential of such polymers in pDNA and siRNA delivery. K=lysine, A=alanine. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Künstliche Oligoethylenaminosäuren wurden zusammen mit natürlichen Aminosäuren und optional Fettsäuren zur Festphasen-basierten Synthese von Polymeren mit präziser Sequenz, Topologie und Modifikation eingesetzt. Erste Konzeptstudien demonstrieren das große Potenzial solcher Polymere in der Transfektion von pDNA und siRNA.

Polymers present an interesting option for the delivery of genes and other therapeutic nucleic acids. In the delivery process, the polymeric carriers facemany different delivery tasks and different physiological microenvironments. Polymers can be designed to respond to microenvironmental differences with changes in their physio-chemical properties, enabling them to perform individual delivery tasks. Cleavage of covalent bonds, disassembly of noncovalent interactions, changes of protonation, conformation, or hydrophilicity/lipophilicity, can trigger such dynamic physicochemical adjustments. The polymeric carrier has to stably bind the therapeutic nucleic acid during the extracellular delivery phase and protect it against degradation in the bloodstream. At the intracellular site of action, the polyplex has to disassemble to an extent that the nucleic acid is functionally accessible. Polyplexes need to be shielded in the circulation and be inert against numerous possible biological interactions, but should actively interact with the target cell surface by electrostatic or ligand receptor interactions. Lipid-membrane destabilization at the cell membrane or nontarget sites is usually associated with undesired cytotoxicity, the analogous biophysical event, however, is required within an endocytic vesicle for polyplex transfer into the cytosol. Strategies will be presented how bioresponsive polymers can be designed and incorporated into polyplexes. Examples include dynamic stabilization of the polymer/nucleic acid core and transient activation of properties required for crossing lipid-membrane barriers. Bioresponsive delivery domains at the polyplex surface required for shielding, deshielding, and cell targeting also contribute to better performance. (C) 2010 John Wiley & Sons, Inc. WIREs Nanomed Nanobiotechnol 2011 3 33-46 DOI: 10.1002/wnan.97