siRNA Polyplexes Research Articles

Effective down-regulation of signal transducer and activator of transcription 3 (STAT3) by polyplexes of siRNA and lipid-substituted polyethyleneimine for sensitization of breast tumor cells to conventional chemotherapy

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that plays a major role in the development of resistance to conventional anti-cancer drugs in many types of cancer, when constitutively activated. Inhibition of STAT3 is considered as a promising strategy for inhibition of tumor growth and overcoming the drug resistance manifested. In this study, the capability of STAT3 knockdown by lipid substituted low molecular weight (2 kDa) polyethyleneimine (PEI2) complexes of STAT3–siRNA was assessed. The efficiency of PEI/STAT3-siRNA polyplexes in the induction of STAT3 associated cell death in wild type and drug-resistant MDA-MB-435 breast cancer cells as monotherapy and upon combination with chemotherapeutic agents, doxorubicin and paclitaxel, was also investigated. Our results identified linoleic acid-substituted (PEI-LA) polymer as the most efficient carrier among different lipid substituted PEI2 for siRNA delivery, leading to most STAT3 associated loss of cell viability in MDA-MB-435 cells. STAT3-siRNA delivery by the PEI-LA polymer resulted in efficient down-regulation of STAT3 at both mRNA and protein levels. Furthermore, pre-treatment of cancer cells with STAT3–siRNA formulation increased the cytotoxic effect of doxorubicin and paclitaxel in both wild type and drug resistant MDA-MB-435 cells. The results of this study point to the potential of PEI-LA polyplexes of STAT3–siRNA as inhibitors of STAT3 expression in breast tumor cells. The results also demonstrate an improved efficacy for chemotherapeutic drugs in combination with lipid-substituted low molecular weight PEI-LA/STAT3-siRNA complexes in comparison to drug therapy alone. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3216–3228, 2014.

Poly(trehalose): sugar-coated nanocomplexes promote stabilization and effective polyplex-mediated siRNA delivery.

When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of a common disaccharide, trehalose, which is well-known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly(methacrylamidotrehalose) or "poly(trehalose)" and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylamide (AEMA) at three degrees of polymerization (n = 34, 65, and 84). Two series of experiments were conducted to study these diblock copolymers in detail and to compare their properties to two control polymers [PEG-P(AEMA) and P(AEMA)]. First, we demonstrate that the poly(trehalose) coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying, allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC50 of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nanobased technologies and macromolecules, in particular, those related to nanomedicine applications.

Balancing Cationic and Hydrophobic Content of PEGylated siRNA Polyplexes Enhances Endosome Escape, Stability, Blood Circulation Time, and Bioactivity in Vivo

A family of pH-responsive diblock polymers composed of poly[(ethylene glycol)-b-[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)], PEG-(DMAEMA-co-BMA), was reversible addition-fragmentation chain transfer (RAFT) synthesized with 0-75 mol % BMA in the second polymer block. The relative mole % of DMAEMA and BMA was varied in order to identify a polymer that can be used to formulate PEGylated, siRNA-loaded polyplex nanoparticles (NPs) with an optimized balance of cationic and hydrophobic content in the NP core based on siRNA packaging, cytocompatibility, blood circulation half-life, endosomal escape, and in vivo bioactivity. The polymer with 50:50 mol % of DMAEMA:BMA (polymer "50 B") in the RAFT-polymerized block efficiently condensed siRNA into 100 nm NPs that displayed pH-dependent membrane disruptive behavior finely tuned for endosomal escape. In vitro delivery of siRNA with polymer 50 B produced up to 94% protein-level knockdown of the model gene luciferase. The PEG corona of the NPs blocked nonspecific interactions with constituents of human whole blood, and the relative hydrophobicity of polymer 50 B increased NP stability in the presence of human serum or the polyanion heparin. When injected intravenously, 50 B NPs enhanced blood circulation half-life 3-fold relative to more standard PEG-DMAEMA (0 B) NPs (p < 0.05), due to improved stability and a reduced rate of renal clearance. The 50 B NPs enhanced siRNA biodistribution to the liver and other organs and significantly increased gene silencing in the liver, kidneys, and spleen relative to the benchmark polymer 0 B (p < 0.05). These collective findings validate the functional significance of tuning the balance of cationic and hydrophobic content of polyplex NPs utilized for systemic siRNA delivery in vivo.

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 6months. 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.

Salt-Induced Stability and Serum-Resistance of Polyglutamate Polyelectrolyte Brushes/Nuclear Factor-κB p65 siRNA Polyplex Enhance the Apoptosis and Efficacy of Doxorubicin

Short interfering RNAs (siRNAs) as chemotherapeutic RNAi agents hold great promise for a significant improvement in cancer therapy. Despite the promise, effective transport of siRNA with minimal side effects remains a challenge. The common problem associated with the low delivery efficiencies of current polycation-based gene delivery systems is their low stability in the presence of salt and serum. In the present study we developed the polyglutamate derivatives (PGS) polyelectrolyte brushes for NF-κB p65 siRNA delivery. The PGS polyelectrolyte brushes/siRNA polyplex was colloidally stable (150 nm diameter) in physiological saline (150 mM NaCl), likely due to the osmotic brushes of PGS. The size-controlled siRNA/PGS polyplex also showed the serum resistance resulting in their efficient cellular uptake was not negatively influenced by the presence of serum. The endothermic profile of ITC, their low values of Gibbs free energy and binding constants Kb under salt conditions provided the direct evidence that PGS polyelectrolyte brushes had a much lower binding affinity for serum proteins, compared with PEI 25KDa. PGS polyelectrolyte brushes delivering NF-κB p65 siRNA achieved efficient down-regulation of NF-κB p65 protein in HeLa cells. The NF-κB p65 down-regulation mediated by PGS polyelectrolyte brushes was more significant than PEI 25KDa and comparable to Lipofectamine 2000. Furthermore, the combination treatment with PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex and doxorubicin demonstrated synergistic apoptotic and cytotoxic effects on HeLa cancer cells. The high stability in physiological saline and salt-induced serum resistance of PGS polyelectrolyte brushes/siRNA polyplex has potential applications together with standard chemotherapies such as doxorubicin to be a viable method to improve the clinical outcomes in cancer therapies.

Anti-apoptotic cardioprotective effects of SHP-1 gene silencing against ischemia–reperfusion injury: Use of deoxycholic acid-modified low molecular weight polyethyleneimine as a cardiac siRNA-carrier

The cardiomyocyte apoptosis plays a critical role in the development of myocardial injury after ischemia and reperfusion. Thus, alteration of the major apoptosis-regulatory factors during myocardial ischemia–reperfusion is expected to have favorable cardioprotective effects. Herein, we report ischemic–reperfused myocardial infarction (MI) repair with siRNA against Src homology region 2 domain-containing tyrosine phosphatase-1 (SHP-1), which is known as a key factor involved in regulating the progress of apoptosis in many cell types. A low molecular weight polyethyleneimine modified with deoxycholic acid (PEI1.8–DA)-based delivery strategy was suggested for the cardiac application of SHP-1 siRNA to overcome the poor gene delivery efficiency to myocardium due to the highly charged structures of the compact cardiac muscles. The PEI1.8–DA conjugates formed stable nanocomplexes with SHP-1 siRNA via electrostatic and hydrophobic interactions. The PEI1.8–DA/SHP-1 siRNA polyplexes effectively silenced SHP-1 gene expression in cardiomyocytes, leading to a significant inhibition of cardiomyocyte apoptosis under hypoxia. In comparison to conventional gene carriers, relatively large amounts of siRNA molecules remained after treatment with the PEI1.8–DA/SHP-1 siRNA polyplexes. Cardiac administration of the PEI1.8–DA/SHP-1 siRNA polyplexes resulted in substantial improvement in SHP-1 gene silencing, which can be explained by the enhancement of cardiac delivery efficiency of the PEI1.8–DA conjugates. In addition, in vivo treatment with the PEI1.8–DA/SHP-1 siRNA polyplexes induced a highly significant reduction in myocardial apoptosis and infarct size in rat MI models. These results demonstrate that the PEI1.8–DA/SHP-1 siRNA polyplex formulation is a useful system for efficient gene delivery into the compact myocardium that provides a fundamental advantage in treating ischemic–reperfused MI.

Cellular internalization and gene silencing of siRNA polyplexes by cytocleavable cationic polyrotaxanes with tailored rigid backbones

To achieve successful delivery of siRNA therapeutics, cytocleavable cationic polyrotaxanes (PRXs) composed of N,N-dimethylaminoethyl (DMAE) group-modified α-cyclodextrins (CDs) that were threaded onto a poly(ethylene glycol) (PEG) axis and capped with a bulky stopper using cytocleavable disulfide linkages (DMAE-PRX) were utilized as an siRNA carrier. DMAE-PRXs with various numbers of threading CDs and modified DMAE groups were synthesized, and the physicochemical properties, cellular internalization, and gene silencing activity of DMAE-PRX/siRNA were investigated to elucidate the relationship between its supramolecular structure and its function. When the numbers of modified DMAE groups were increased, the DMAE-PRXs formed closely associated polyplexes with siRNA and increased their polyanion exchange resistance. Additionally, the DMAE-PRXs with 52 threading CDs (52CD-PRXs) showed greater binding capabilities with siRNA and greater resistance to polyanion competition than 31CD-PRXs, indicating that the highly CD-threaded PRX structure in the 52CD-PRXs is superior in forming stable polyplexes with siRNA. Indeed, 52CD-PRX/siRNA showed greater intracellular uptake of siRNA than 31CD-PRX/siRNA with comparable numbers of DMAE groups. 52CD-PRX/siRNA successfully induced gene silencing of a targeted luciferase expressed in human cervical carcinoma without marked cytotoxicity and non-specific gene silencing. Although the gene silencing activities of DMAE-PRX/siRNA were comparable to those of linear poly(ethylenimine) (L-PEI), L-PEI showed cytotoxicity and non-specific gene silencing. Additionally, DMAE-PRXs with cytocleavable capabilities were found to enhance gene silencing, in comparison with non-cleavable DMAE-PRX. Thus, the cytocleavable cationic PRXs are suggested to be attractive supermolecules for the delivery of therapeutic siRNAs.

Molecular weight and architectural dependence of well-defined star-shaped poly(lysine) as a gene delivery vector.

A series of well-defined star-shaped polypeptides were successfully synthesised by the ring opening polymerisation (ROP) of the N-carboxyanhydride (NCA) of ε-carbobenzyloxy-l-lysine (ZLL) using a range of generations of polypropylene imine (PPI) dendrimers as multifunctional initiators. The monomer feed ratio and dendrimer generation were varied to afford a series of polypeptide dendrimer hybrids with superior structural versatility and functionality. Subsequent protecting group removal yielded star-shaped poly(lysine) of controlled variation in polypeptide chain length and arm multiplicity. Star-shaped PLL polymers were used to prepare pDNA and siRNA to form "polyplexes" to determine their ability to complex different nucleic acid cargoes and were compared with linear PLL polyplex controls. Significant differences in size and surface charge were seen between star-shaped PLL polyplexes and linear PLL polyplexes for both cargoes. The star-shaped polypeptides were capable of more effective complexation of both nucleic acids at low N/P ratios compared to linear PLL as evidenced by zeta potential and electrophoretic data. This was particularly evident in siRNA polyplexes as linear PLL failed to completely complex siRNA into nanocomplexes of appropriate size for cell transfection i.e. <200 nm in size, while star poly(lysine) formed siRNA polyplexes <100 nm at certain N/P ratios, albeit strongly dependent on the particular molecular weight and architecture, as analysed by dynamic light scattering (DLS). Atomic force microscopy (AFM) identified discrete spherically shaped polyplexes for all star-shaped polypeptide-based polyplexes while linear PLL formed elongated irregular shaped complexes. This difference in morphology may go some way towards explaining the 300-fold increase in luciferase expression seen for star-shaped PLL polyplexes G5(64)-PLL40 compared to linear PLL pGLuc polyplexes in epithelial cells. Each of the PPI-PLL polymers appeared to be capable of protecting the nucleic acid cargoes from degradation by the relevant nuclease enzyme as effectively as the positive control polyethyleneimine (PEI) polyplexes. Overall the promising nucleic acid complexation, sizing, morphology and protection capacity of two different genetic "cargoes" highlight the potential of polypeptide dendrimer hybrids as gene delivery vectors.

Opposites attract: influence of the molar mass of branched poly(ethylene imine) on biophysical characteristics of siRNA-based polyplexese

Polymer-based carriers, in particular polycations, represent an interesting alternative to viral vectors, as they form so-called polyplexes with nucleic acids by entropic driven, electrostatic interactions. In this study, we investigate in detail polyplexes based on small interfering RNA (siRNA), the delivery of which into eukaryotic cells represents an attractive route for treating genetic diseases by inhibition of harmful gene expression. Although plasmid DNA (pDNA) based polyplexes are well characterized, we show that not all knowledge can be adopted from pDNA, as siRNA is around 250 times smaller and shows a higher rigidity. The used polymer component is the polycation branched poly(ethylene imine) (B-PEI) of a high range of molar masses (0.6, 1.8, 10, 25 kDa), which are further analyzed by potentiometric titration and cytotoxicity tests. The formation, size, and net-charge of the polyplexes are examined at different ratios of nitrogen of the different polymers and phosphates of the RNA (N/P). Moreover, the stability of siRNA polyplexes against heparin and time was investigated. The obtained physicochemical parameters were then correlated to the cellular internalization of polyplexes. A strong dependency of the molar mass on the polyplex characteristics of the used B-PEI was found. Thereby, high molar mass B-PEI ≥ 10 kDa forms smaller polyplexes of around 50 nm radius with zeta potentials > 25 mV, increased long-term stability, and enhanced cellular uptake compared to low molar mass ones. To gain deeper insight into the differences and characteristics of siRNA based polyplexes, the characterization by analytical and preparative ultracentrifugation (AUC, PUC) is applied on siRNA polyplexes for the first time and referred to conventional characterization methods such as DLS. AUC was also used to identify non-complexed PEI in the polyplex solutions. A virtual N/P of 1.3 for siRNA was measured, independent of the used molar masses of B-PEI. Additionally, differences in cellular uptakes of siRNA and pDNA based polyplexes were found. The results of this study will help to understand the properties of siRNA-based polyplexes and could lead to more efficient polymer design.

In vivo disassembly of IV administered siRNA matrix nanoparticles at the renal filtration barrier

Intravenous administration of siRNA nanocarriers may provide unique therapeutic opportunities for tissue-specific gene silencing. Although often engineered to overcome the numerous barriers that exist in the systemic circulation, many nanocarriers fail in extending the circulation time of the siRNA. A more detailed assessment of the different clearance mechanisms that are in play after intravenous injection could therefore be of value to improve siRNA nanocarrier design. In this report, the biodistribution in mice of siRNA loaded dextran nanogels was investigated in detail. Both single photon emission computed tomography (SPECT) imaging and fluorescence single particle tracking (fSPT) indicate that the particles are rapidly cleared from the circulation. PEGylation of the nanogels was not able to increase the half-life in the bloodstream. Carrier disassembly in the systemic circulation and phagocytic clearance are known to facilitate the elimination of siRNA nanoparticles. Additionally, it is demonstrated for dextran nanogels that also the kidneys play an important role in their elimination from the bloodstream. SPECT imaging revealed an accumulation of the siRNA loaded dextran nanogels in the kidneys shortly after intravenous injection and a significantly delayed transition of siRNA from kidney to bladder, as opposed to the injection of free siRNA. These data indicate that components of the glomerular filtration barrier may contribute to the dissociation of siRNA from its carrier, as was recently suggested for cationic cyclodextrin siRNA polyplexes. This clearance mechanism should therefore be taken into account when designing siRNA nanocarriers for intravenous administration.

Buffering Capacity and Size of siRNA Polyplexes Influence Cytokine Levels

Induction of cytokines by small interfering RNA (siRNA) polyplexes has been a significant concern of researchers attempting to minimize the toxicity of this promising therapy. Although cationic carriers of siRNA are known to increase cytokine levels, few systematic studies have been done to determine what properties of the carrier are important to modulate cytokines. Because branched histidine-lysine (HK) peptides are effective carriers of siRNA and their sequence can be readily modified, we selected this class of carrier to determine which sequences of the peptide were important for cytokine induction. With the use of peripheral blood mononuclear cells (PBMCs), the HK peptide with a higher number of histidines (H3K(+H)4b) in complex with siRNA induced lower levels of cytokines compared with other HK (e.g., H2K4b, H3K4b, H3K(+N)4b) siRNA nanoplexes. Notably, these peptides' siRNA polyplexes showed a similar pattern of cytokine induction when injected intravenously in a mouse model, i.e., the HK with higher content of histidines induced cytokines the least. As indicated by the pH-sensitive dye within acidic endosomes, the greater pH-buffering capacity of H3K(+H)4b compared with other HK peptides may explain why cytokine levels were reduced. In addition to buffering capacity, the size of HK polyplexes markedly influenced cytokine production.

Stabilizing effect of tyrosine trimers on pDNA and siRNA 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.

Silica nanogelling of environment-responsive PEGylated polyplexes for enhanced stability and intracellular delivery of siRNA

In this study, poly(ethylene glycol) (PEG)-block-polycation/siRNA complexes (PEGylated polyplexes) were wrapped with a hydrated silica, termed "silica nanogelling", in order to enhance their stability and functionality. Silica nanogelling was achieved by polycondensation of soluble silicates onto the surface of PEGylated polyplexes comprising a disulfide cross-linked core. Formation of silica nanogel layer on the PEGylated cross-linked polyplexes was confirmed by particle size increase, surface charge reduction, and elemental analysis of transmission electron micrographs. Silica nanogelling substantially improved polyplex stability against counter polyanion-induced dissociation under non-reductive condition, without compromising the reductive environment-responsive siRNA release triggered by disulfide cleavage. Silica nanogelling significantly enhanced the sequence-specific gene silencing activity of the polyplexes in HeLa cells without associated cytotoxicity, probably due lower endosomal entrapment (or lysosomal degradation) of delivered siRNA. The lower endosomal entrapment of the silica nanogel system could be explained by an accelerated endosomal escape triggered by deprotonated silanol groups in the silica (the proton sponge hypothesis) and/or a modulated intracellular trafficking, possibly via macropinocytosis, as evidenced by the cellular uptake inhibition assay. Henceforth, silica nanogelling of PEGylated siRNA polyplexes is a promising strategy for preparation of stable and functional siRNA delivery vehicles.

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 (∼120nm). Small particles (<40nm) 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.

Stabilization of polyplexes via polymer crosslinking for efficient siRNA delivery

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 1h 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.

Nanosized Multifunctional Polyplexes for Receptor-Mediated SiRNA Delivery

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 peptide-siRNA 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.

Cell Targeting Peptide Conjugation to siRNA Polyplexes for Effective Gene Silencing in Cardiomyocytes

To deliver siRNA specifically to cardiomyocytes with a high transfection efficiency, primary cardiomyocyte-targeting (PCM) and/or cell-penetrating (Tat) peptides were incorporated into the siRNA. With the addition of plasmid DNA, these peptide-conjugated siRNAs were able to form compact and stable nanosized polyplex particles with bioreducible poly(CBA-DAH). The peptide-modified siRNA polyplexes enhanced the cellular uptake and the gene-silencing capacity of the siRNA in cardiomyocytes without significant immunogenicity or cytotoxicity. These findings demonstrate that the cell-targeting peptide and/or cell-penetrating peptide conjugation of siRNA may be a potentially important strategy for cell-specific gene therapy in gene-mediated disease states.

Structure–activity relationships of siRNA carriers based on sequence-defined oligo (ethane amino) amides

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.

Facial amphipathic deoxycholic acid-modified polyethyleneimine for efficient MMP-2 siRNA delivery in vascular smooth muscle cells

Clinical applications of RNA interference-based therapeutics such as small interfering RNAs (siRNAs) have been limited mainly due to low intracellular delivery efficiency in vitro and in vivo. In this study, facially amphipathic deoxycholic acid (DA)-modified polyethyleneimine (PEI1.8) (DA-PEI1.8) was synthesized and used as a potent carrier system for siRNA targeted against matrix metalloproteinase-2 (MMP-2) to inhibit the migration of vascular smooth muscle cells (SMCs), which is the major pathomechanism in the development of atherosclerosis and restenosis after arterial injury. A representative facial amphipathic bile acid DA having a high membrane permeability was conjugated to the terminal amine groups of the low molecular weight PEI1.8 via amide bonds. The DA-PEI1.8 conjugates formed self-assembled nanoparticles with siRNA molecules in an aqueous phase and the DA-PEI1.8/siRNA polyplexes became stabilized and condensed as particle incubation time increased from 0 to 4h. Both cellular internalization and target gene silencing were enhanced as the DA-PEI1.8/siRNA polyplexes stabilized. When vascular SMCs were transfected with MMP-2 siRNA, the DA-PEI1.8/siRNA polyplex formulation led to a significant decrease in MMP-2 gene expression, resulting in the suppression of cell migration. These results suggest that the DA-PEI1.8/MMP-2 siRNA delivery system may be useful in anti-restenotic treatment for various vasculoproliferative disorders such as atherosclerosis, in-stent restenosis, and vein graft failure.

Comparison of Cationic and Amphipathic Cell Penetrating Peptides for siRNA Delivery and Efficacy

Cell penetrating peptides (CPPs) are short strands of arginine- and/or lysine-rich peptides (<30 amino acids) that use their cationic nature for efficient intracellular accumulation. CPPs have been used for small interfering RNA (siRNA) delivery by direct complexation with the siRNA anionic phosphate backbone. During this process, however, part of the CPP cationic charges are neutralized, and the resultant loss of free positive charges may substantially compromise CPP's internalization capabilities and eventually reduce siRNA delivery efficiency. The purpose of this study was to design a novel type of polyplex for siRNA delivery to overcome the CPP neutralization issue. This novel polyplex consists of three components: siRNA, 21mer oligolysine (K21) chemically modified to incorporate CPP conjugation sites (K21-PDP), and CPP delivery moiety. The siRNA was first neutralized by cationic charges of K21-PDP to form a polyplex. Then a cationic (hexaarginine, R6) or an amphipathic (model amphipathic peptide, MAP) CPP was conjugated to the polyplex. Agarose gel shift assays indicated that the siRNA could be released from the polyplex after K21-PDP degradation or polyplex dilution. Furthermore, the total intracellular internalization of these two CPP-polyplexes was studied. Compared with R6-polyplex, MAP-polyplex exhibited 170- and 600-fold greater uptake of fluorescently labeled siRNA at 1 and 6 h post-transfection, respectively. MAP-polyplex also exhibited comparable GFP silencing effects as Lipofectamine 2000 complex in Huh7.5 cells stably transfected to express GFP-light chain 3 protein, whereas R6-polyplex did not demonstrate significant silencing activity. Further studies indicated that the K21-PDP-siRNA polyplex formation and conjugation of MAP to the polyplex were essential for siRNA polyplex uptake and gene silencing. MAP-polyplex was also shown to be unaffected by the presence of 10% FBS during transfection. In addition, MAP-polyplex uptake was dependent on vesicle formation and fusion due to 70 and 54% loss of uptake at 4 and 16 °C, respectively, compared to incubation at 37 °C. Therefore, the amphipathic CPP is a more suitable carrier moiety for delivery of siRNA polyplex.