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RNA interference is an essential and powerful tool for targeting and verifying specific gene functions. Conditional control of small interfering RNA (siRNA) activity, especially using light activation, is a potential method for regulating target gene expression and functions. In this study, a series of photolabile siRNAs with amantadine modification have been rationally designed and developed through host-guest interactions between amantadine and β-cyclodextrin derivatives to enhance the blocking effect of siRNA binding and/or RNA-induced silencing complex processing. These caged siRNAs with amantadine modification at the 5' end of antisense-strand RNA were efficiently inactivated through the host-guest interactions between amantadine and β-cyclodextrin. Photomodulation of the gene silencing activity of these amantadine-modified caged siRNAs targeting both exogenous and endogenous genes was successfully achieved, which indicates that host-guest interactions could be a new strategy for developing new caged siRNAs for gene photoregulation with low leaking activity.

Hydrotalcites and amino acid functionalized hydrotalcites (aa-HTs) were synthesized using the co-precipitation method. These compounds were characterized using X-ray diffraction (XRD), inductively coupled plasma-optical emission spectroscopy (ICP-OES), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). The optimal siRNA binding capacity was determined using gel retardation assays. Cytotoxicity studies using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the siRNA:HT complexes were relatively non-cytotoxic to HeLa-tat-luc cells. Gene silencing studies showed that the aa-HTs elicited significantly higher levels of gene knockdown when compared to the non-functionalized HTs. Overall, these HTs have shown the potential to bind, protect and efficiently deliver siRNA, in vitro.

Transfecting nucleic acids into various cells is a key procedure in biological research also envisioned for therapeutic applications. In our effort to obtain simple reagents that would be readily accessible from commercial building blocks, we recently reported peptide dendrimers as single component siRNA transfection reagents accessible in pure form by solid-phase peptide synthesis. Here, we extend our studies of these dendrimers by identifying analogs bearing a coumarin or BODIPY fluorescent label in their core and displaying comparable siRNA transfection efficiencies, pH dependent aggregation, siRNA binding, and secondary structures. Fluorescence resonance energy transfer (FRET) studies show that the dendrimers are tightly associated with siRNA within the formed nanoparticles at pH 7.4 but are released into solution at pH 5.0 and can participate in endosome escape by destabilizing the membrane at this pH value. Colocalization studies furthermore suggest that peptide dendrimers and siRNA remain tightly associated throughout the transfection process.

Developing nucleic acid-based tools to control disease-relevant gene expression in human disorders, such as siRNAs, opens up potential opportunities for therapeutics. Because of their high molecular weight and polyanionic nature, synthetic siRNAs fail to cross biological membranes by passive diffusion and therefore, generally require transmembrane siRNA delivery technologies to access the cytoplasm of target cells. To create a biocompatible siRNA delivery agent, we chemically modified natural polysaccharide curdlan derivative 6AC-100 in a regioselective manner to introduce different ratios of imidazole rings in the amino units (denoted as Curimi) and evaluated their siRNA binding ability, cytotoxicity, endosome buffering capacity and siRNA transfection efficiency. The novel curdlan based Curimi polymers formed nanoparticles with siRNA at pH 7.4 in range of 85–105 nm and their size distribution increased along with decreasing pH condition. The zeta potential increased by lowering pH value as well. Curimi polymers showed lower toxicity and higher buffering capacity compared to 6AC-100, and efficiently delivered siRNA against to PLK1 into cancer cells, and subsequently, significantly inhibited target mRNA level. Our result suggested that novel curdlan based Curimi polymers may be used as efficient siRNA carrier for cancer therapy.

siRNA-mediated RNA interference (RNAi) against inflammation-related genes provides a promising modality for the treatment of myocardial ischemia reperfusion (IR) injury, and its success is critically dependent on the development of efficient yet safe siRNA delivery vehicles. Herein, we developed a bioreducible, branched poly(β-amino ester) with built-in redox-responsive domains (BPAE-SS) for the effective ICAM-1 siRNA delivery into injured rat cardiac microvascular endothelial cells (RCMECs). The branched BPAE-SS with a multivalent structure afforded potent siRNA binding affinity compared to its linear analogue, while upon internalization into RCMECs it was instantaneously degraded by intracellular glutathione (GSH) into small segments to mediate "on-demand" siRNA release and diminish the toxicity of post-transfection materials. By synchronizingly overcoming these critical barriers, BPAE-SS mediated remarkable ICAM-1 knockdown in IR-injured rats at 400 μg siRNA per kg via single i.v. injection, and subsequently suppressed myocardial inflammation, apoptosis, and fibrosis to recover the cardiac function. This study therefore provides a unique delivery system that can address the multiple critical challenges against non-viral siRNA delivery, and the potent therapeutic efficacy of BPAE-SS-mediated ICAM-1 silencing provides a promising strategy for the anti-inflammatory treatment of myocardial IR injury.

Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, is a major threat to economically important cucurbit crops worldwide. An attenuated strain (SH33b) derived from a severe strain (SH) of CGMMV caused a reduction in the viral RNA accumulation and the attenuation of symptoms, and it has been successfully used to protect muskmelon plants against severe strains in Japan. In this study, we compared GFP-induced silencing suppression by the 129K protein and the methyltransferase domain plus intervening region (MTIR) of the 129K protein between the SH and SH33b strains, respectively. As a result, silencing suppression activity (SSA) in the GFP-silenced plants was inhibited efficiently by the MTIR and 129K protein of SH strain, and it coincided with drastically reduced accumulation of GFP-specific small interfering RNAs (siRNAs) but not by that of SH33b strain. Furthermore, analyses of siRNA binding capability (SBC) by the MTIR of 129K protein and 129K protein using electrophoretic mobility shift assay revealed that SBC was found with the MTIR and 129K protein of SH but not with that of SH33b, suggesting that a single amino acid mutation (E to G) in the MTIR is responsible for impaired SSA and SBC of SH33b. These data suggest that a single amino acid substitution in the intervening region of 129K protein of CGMMV resulted in attenuated symptoms by affecting RNA silencing suppression.

Cationic liposomes have shown great potential in efficient siRNA delivery, and their positive charge is crucial for tight extracellular siRNA binding, effective intracellular siRNA disassembly and physiological toxicity. Thus, the development of novel cationic lipids with a suitable positive charge is desirable for safe and efficient siRNA delivery. Herein, we fabricated a library of 21 tertiary amine-derived cationic lipids (TA) to achieve a balance between effectiveness and safe siRNA delivery. The screened TA13 liposomes, which consisted of TA13 and helper lipid DOPE at a mole ratio of 1 : 1, readily condensed siRNA to form lipoplexes (TA13 LPs), achieving stronger gene silencing in diverse cells than the commercially available vector Lipo2000. Moreover, the TA13 LPs demonstrated effective in vivo gene silencing and good safety in normal mice. The improved gene silencing efficiency of the TA13 LPs is ascribed to their capability of sequentially conquering the barriers met by in vivo siRNA delivery. Notably, the TA13 LPs delivered ApoB-siRNA and obviously decreased ApoB mRNA expression in the liver and the total cholesterol and low-density lipoprotein in the serum of hypercholesterolemia mice, indicating a potential siRNA therapeutic for hypercholesterolemia treatment. It is anticipated that these novel tertiary amine-based liposomes can provide a simple and widely-used platform for the safe and effective delivery of siRNA, and their structure-activity relationships can aid in the further development of effective cationic lipids.

The discovery of RNA interference (RNAi) more than two decades ago opened avenues for avant-garde cancer treatments that possess the ability to evade issues hampering current chemotherapeutic strategies, owing to its specific gene sequence-driven mechanism of action. A potent short interfering RNA (siRNA) delivery vehicle designed to overcome physiological barriers is imperative for successful RNAi therapy. For this purpose, this study explored the characteristics and therapeutic efficacy of low-molecular weight (MW) polyethylenimine (PEI) with high cholesterol substitution, yielding water-insoluble polymers, in chronic myeloid leukemia (CML) K562 cells. A strong impact of cholesterol grafting on the physicochemical attributes of the resultant polymers and their corresponding complexes with siRNA was observed, with the siRNA binding capacity of polymers increasing and complex dissociation sensitivity decreasing with increase in cholesterol content of the polymers. The modified polymer complexes were significantly smaller in size and possessed higher cationic charge compared to the parent polymer. The interaction with anionic heparan sulfate preoteoglycans present on the cell surface was significant in cellular uptake of the complexes. The therapeutic efficacy of siRNA/polymer complexes was reflected in their ability to effectively silence the reporter green fluorescent protein gene and endogenous CML oncogene BCR-ABL as well as significantly inhibit colony formation by K562 cells post BCR-ABL silencing. The results of this study demonstrated beneficial effects of high levels of hydrophobic substitution on low MW PEI on their functional performance bestowing them the potential to be potent RNAi agents for CML therapy.

We propose to combine two therapeutic anti-inflammatory approaches with different mechanisms of action in a single drug delivery system consisting of cationic dexamethasone palmitate nanoparticles (CDXP-NP) associated with TNF-alpha siRNA. The CDXP-NPs are obtained by the solvent emulsion evaporation technique using dexamethasone palmitate, a prodrug of dexamethasone, associated with a cationic lipid, DOTAP. Their physicochemical properties as well as their ability to bind siRNA were evaluated through gel electrophoresis and siRNA binding quantification. SiRNA cellular uptake was assessed by flow cytometry and confocal microscopy on RAW264.7 macrophages. TNF-alpha inhibition was determined on LPS-activated RAW264.7 macrophages. Stable and monodisperse nanoparticles around 100 nm with a positive zeta potential (+59 mV) were obtained with an encapsulation efficiency of the prodrug of 95%. A nitrogen/phosphate (N/P) ratio of 10 was selected that conferred the total binding of siRNA to the nanoparticles. Using these CDXP-siRNA-NPs, the siRNA was strongly internalized by RAW264.7 macrophage cells and localized within the cytoplasm. on the LPS-induced RAW264.7 macrophages, a larger inhibition of TNF-alpha was observed with CDXP-siRNA-NPs compared to CDXP-NPs alone. in conclusion, from these data, it is clear that a combination of DXP and TNF-alpha siRNA therapy could be a novel strategy and optimized alternative approach to cure inflammatory diseases., TUB.ITAK 2214/A International Research Fellowship Program for PhD students; ANRFrench National Research Agency (ANR) [ANR-10-LABX-33]; CNRSCentre National de la Recherche Scientifique (CNRS)European Commission; l'Agence Nationale de la RechercheFrench National Research Agency (ANR) [ANR-11-EQPX-0029, ANR-10-INBS-04, ANR-11-IDEX-0003-02], S. Gurcan was supported by TUB.ITAK 2214/A International Research Fellowship Program for PhD students. This research received support from Laboratory of Excellence LERMIT thanks to an ANR grant (ANR-10-LABX-33) and from CNRS. The present work has benefited from Imagerie-Gif core facility supported by l'Agence Nationale de la Recherche (ANR-11-EQPX-0029/Morphoscope, ANR-10-INBS-04/FranceBioImaging; ANR-11-IDEX-0003-02/Saclay Plant Sciences) with the precious help of C. Boulogne and C. Gillet. The authors would like to thank V. Nicolas (MIPSIT, Universite Paris-Saclay) for help with confocal microscopy.

Owing to the unique physical and chemical properties of carbon nanotubes, they have been widely explored as delivery vectors for proteins, and nucleic acid etc. after functionalization. Particularly, the modification of carbon nanotubes suited for the delivery of siRNA has been intensely studied over the past decade. The assay described in this chapter allows for realizable quantification of siRNA binding on carbon nanotube-based materials using gel electrophoresis and silencing by flow cytometry when the siRNA complexes are delivered in vitro.

RNA interference (RNAi) has emerged as a promising therapeutic approach for the treatment of a wide range of disorders. Small interfering RNAs (siRNAs), i.e., non-coding double-stranded RNA molecules, have been mainly used for RNAi. Because siRNA is susceptible to enzymatic degradation and is rapidly cleared from the bloodstream, the success of RNAi is strongly related to the design of efficient delivery technologies. Among auspicious carriers for siRNA, polymeric micelles self-assembled by polyion complexation between block ionomers and siRNA have attracted much attention due to their well-defined size, efficient complexation and potential for delivery in vivo. In this regard, we have recently demonstrated that the polycation flexibility influences the complexation with single stranded RNA molecules, affecting the delivery capability of the resulting micelles. On the other hand, the effects of the catiomer flexibility on micelles loading double stranded siRNA remains unknown. Thus, herein, we studied the effects of the polycation backbone flexibility on siRNA-loaded polyion complex (PIC) micelles by using complementary block copolymers, i.e., the relatively flexible poly(ethylene glycol)-poly(glycidylbutylamine) (PEG-PGBA) and the more rigid PEG-poly(L-lysine) (PEG-PLL). By mixing these polymers with siRNA at different N/P ratios, we found that PEG-PGBA effectively promoted self-assembly of PIC micelles at lower N/P ratios and lower siRNA concentrations than PEG-PLL. Computational studies of siRNA binding with polycations and PEG-polycations further supported the favorable binding process of flexible polycations with siRNA. The micelles based on PEG-PGBA were stable in physiological conditions and promoted effective intracellular delivery of siRNA for efficient gene knockdown. Our results indicate the importance of polycation flexibility for the assembly of PIC micelles with siRNA, and its potential for developing innovative carrier systems.

Grapevine leafroll-associated virus 1 (GLRaV-1) is a major pathogen associated with grapevine leafroll disease. However, the molecular mechanisms underlying GLRaV-1 interactions with plant cells are unclear. Using Agrobacterium infiltration-mediated RNA-silencing assays, we demonstrated that GLRaV-1 p24 protein (p24G1) acts as an RNA-silencing suppressor (RSS), inhibiting local and systemic RNA silencing. Electrophoretic mobility shift assays showed that p24G1 binds double-stranded 21-nucleotide small interfering RNA (siRNA), and that siRNA binding is required but not sufficient for its RSS activity. p24G1 localizes in the nucleus and can self-interact through its amino acid 10 to 210 region. Dimerization is needed for p24G1 interaction with importin &alpha, 1 before moving to the nucleus, but is not required for its siRNA binding and RSS activity. Expression of p24G1 from a binary pGD vector or potato virus X-based vector elicited a strong hypersensitive response in Nicotiana species, indicating that p24G1 may be a factor in pathogenesis. Furthermore, p24G1 function in pathogenesis required its RSS activity, dimerization and nuclear localization. In addition, the region of amino acids 122&ndash, 139 played a crucial role in the nuclear import, siRNA binding, silencing suppression and pathogenic activity of p24G1. These results contribute to our understanding of the molecular mechanisms underlying GLRaV-1 infection.

Grapevine leafroll-associated virus 1 (GLRaV-1) is a major pathogen associated with grapevine leafroll disease. However, the molecular mechanisms underlying GLRaV-1 interactions with plant cells are unclear. Using Agrobacterium infiltration-mediated RNA-silencing assays, we demonstrated that GLRaV-1 p24 protein (p24G1) acts as an RNA-silencing suppressor (RSS), inhibiting local and systemic RNA silencing. Electrophoretic mobility shift assays showed that p24G1 binds double-stranded 21-nucleotide small interfering RNA (siRNA), and that siRNA binding is required but not sufficient for its RSS activity. p24G1 localizes in the nucleus and can self-interact through its amino acid 10 to 210 region. Dimerization is needed for p24G1 interaction with importin α1 before moving to the nucleus, but is not required for its siRNA binding and RSS activity. Expression of p24G1 from a binary pGD vector or potato virus X-based vector elicited a strong hypersensitive response in Nicotiana species, indicating that p24G1 may be a factor in pathogenesis. Furthermore, p24G1 function in pathogenesis required its RSS activity, dimerization and nuclear localization. In addition, the region of amino acids 122–139 played a crucial role in the nuclear import, siRNA binding, silencing suppression and pathogenic activity of p24G1. These results contribute to our understanding of the molecular mechanisms underlying GLRaV-1 infection.

The development of delivery vehicles for small interfering RNAs (siRNAs) remains a bottleneck to widespread clinical use. Cationic polymers represent an important class of potential delivery vehicles. In this study, we used alkyne-azide click chemistry to synthesize a variety of cationic poly(propargyl glycolide) backbone polymers to bind and deliver siRNAs. We demonstrated control over the binding interactions of these polymers and siRNAs by varying binding strength by more than three orders of magnitude. Binding strength was found to meet or exceed that of commercially available transfection agents. Our polymers effectively delivered siRNAs with no detectable cytotoxicity. Despite accumulation of siRNAs at levels comparable with commercial reagents, we did not observe silencing of the targeted protein. The implications of our results for future siRNA delivery vehicle design are discussed.

Surface modification of titanium implants by siRNA is quite efficient for improving implant osseointegration. Loading siRNA onto their surface is a crucial factor for siRNA-functionalized implants to realize their biological function. Direct binding of siRNA to implants has low siRNA binding and releasing rate, so usually it needs to be mediated by vectors. Polymeric, nonmaterial-mediated and lipid-based vectors are types of non-viral vectors which are commonly used for delivering siRNA. Three major methods of loading process, namely simple physical adsorption, layer-by-layer assembly and electrodeposition, are also summarized. A brief introduction, the basic principle and the general procedure of each method are included. The loading efficiency, which can be measured both qualitatively and quantitatively, together with gene knockdown efficiency, cytotoxicity assay and osteogenesis of the three methods are compared. A good many applications in osteogenesis have also been described in this review.

CSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNAs targeting most germline-expressed genes. Here we show that the two isoforms of CSR-1 have distinct expression patterns; CSR-1B is ubiquitously expressed throughout the germline and during all stages of development while CSR-1A expression is restricted to germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine modifications, which are necessary for the preferential binding of CSR-1A to spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory mechanism for C. elegans Argonaute proteins that allows for specificity of small RNA binding between similar Argonaute proteins with overlapping temporal and spatial localization., The Argonaute protein CSR-1 is essential for fertility and viability in C. elegans. Here the authors show that CSR-1A isoform associates preferentially with small RNAs mapping to spermatogenesis-specific genes while CSR-1B isoform binds small RNAs mapping to oogenesis-specific genes. Arginine methylation of CSR-1A promotes small RNA-binding specificity.

To use siRNA molecule as a therapeutic agent in gene silencing, an efficient delivery system is necessary. Stability and clearance by reticuloendothelial of siRNA still remains the major challenges for clinical application. Herein, we could develop new lipid-polymer hybrid nanoparticles (LPHNP) as a siRNA carrier to silence insulin-like growth factor type I (IGF-1R) gene overexpression in MCF-7 human breast cancer cell line.Dimethyldioctadecylammonium bromide-methoxy poly(ethylene glycol)-poly (ε-caprolactone) (DDAB-mPEG-PCL) LPHNPs were synthesized using a single step nanoprecipitation method and characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM) microscope. Cytotoxicity of the nanoparticles was assessed in the MCF7 cell line using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Desired LPHNP-siRNA complex was determined using different Nitrogen:Phosphate ratio (N/P) ratios and gel retardation. To determine the encapsulation efficiency of siRNA (%) in LPHNP, its absorbance was measured. The effect of the siRNA-LPHNP complex on IGF-1R silencing was assessed by reverse transcription-polymerase chain reaction (RT-PCR) RESULTS: LPHNP was synthesized using a single-step sonication method with a size below 100 nM. The viability of cells treated with hybrid nanoparticles was significantly greater than the corresponding cationic lipid (P 0.01). As demonstrated by gel retardation assay, efficient siRNA binding to LPHNP occurred at N/P equal to 40 and siRNA encapsulation efficiency was found to be 95% ± 4 at this ratio. LPHNP-IGF-1R siRNA complex could be able to down-regulate the target more efficiently when it compared with the corresponded controls (P 0.001).In conclusion, our results suggest that DDAB cationic lipid and mPEG-PCL copolymer hybrid nanoparticle may be a good candidate for efficient siRNA delivery.

A safe and effective delivery system is considered a key to the success of nucleic acid therapeutics. It has been reported that pulmonary surfactants or their components could facilitate the uptake of small interfering RNA (siRNA) into the lung epithelial cells. Previously, our group investigated the use of KL4 peptide, a synthetic cationic peptide that simulates the structural properties of surfactant protein B (SP-B), as siRNA delivery vector. Although KL4 peptide exhibits good in vitro siRNA transfection efficiency on lung epithelial cells, its therapeutic potential is limited by its poor aqueous solubility due to the presence of a high proportion of hydrophobic leucine residues. In this study, we aim to address the solubility issue, designing five different modified peptides by replacing the hydrophobic leucine with alanine or valine, and assess their potential as siRNA delivery vectors. While the modified peptides retain the overall cationic property, their siRNA binding is also affected and their transfection efficiency is inferior to the parent KL4 peptide. A closer examination of the conformation of these peptides by circular dichroism shows that substitution of leucine residues leads to the change of the secondary structure from α-helical content to either β-sheet or more disordered, β-turn conformations. Relatively conservative amino acid substitutions, in terms of hydrophobicity bulk, lead to substantial conformational alteration, heavily impacting siRNA binding and release, cellular uptake, and transfection efficiency. Although the peptide modification strategy employed in this study was unsuccessful in developing an improved version of KL4 peptide for siRNA delivery, it highlights the importance of the α-helical conformation for efficient siRNA transfection, providing useful insights for future development of peptide-based RNA delivery system.

Purpose Paclitaxel is a first-line drug for the therapy of lung cancer, however, drug resistance is a serious limiting factor, related to overexpression of anti-apoptotic proteins like survivin. To overcome this phenomenon, developing novel ultrasound responsive nanobubbles - nanosized drug delivery system- for the delivery of paclitaxel and siRNA in order to silence survivin expression in the presence of ultrasound was aimed. Methods Paclitaxel-carrying nanobubble formulation was obtained by modifying the multistep method. Then, the complex formation of the nanobubbles - paclitaxel formulation with survivin-siRNA, was examined in terms of particle size, polydispersity index, zeta potential, and morphology. Furthermore, siRNA binding and protecting ability, cytotoxicity, cellular uptake, gene silencing, and induction of apoptosis studies were investigated in terms of lung cancer cells. Results Developed nanobubbles have particle sizes of 218.9-369.6 nm, zeta potentials of 27-34 mV, were able to protect siRNA from degradation and delivered siRNA into the lung cancer cells. Survivin expression was significantly lower compared with the control groups and enhanced apoptosis was induced by the co-delivery of survivin-siRNA and paclitaxel. Furthermore, significantly higher effects were obtained in the presence of ultrasound induction. Conclusion the ultrasound responsive nanobubble system carrying paclitaxel and survivin-siRNA is a promising and effective approach against lung cancer cells., Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [116S213], This study has been financially supported by the Scientific and Technological Research Council of Turkey (TUBITAK) under grant code 116S213. the authors would like to thank Ege University, Faculty of Pharmacy, Department of Pharmaceutical Technology (for DLS measurement), Ege University, Pharmaceutical Sciences Research Center (for Varioskan Multiplate Reader), Izmir Katip Celebi University, Central Research Labs (for SEM imaging) and Middle East Technical University, Central Laboratory (for TEM imaging).

Hydrophobic modification on polycations were commonly used to improve the stability and transfection efficiency of polyplexes. However, the improved stability often means undesired release of the encapsulated siRNA, limiting the application of cationic micelles for siRNA delivery. The current strategy of preparing bioresponsive micelles based on the cleavage of sensitive linkages between polycation and hydrophobic part was far from sufficient, owing to the siRNA binding of the separated polycations from micelles leading to the incomplete release of siRNA. In this study, we propose a new strategy by the combination of micelles disassembly and separated polycations charge reversal. FPBA (3-fluoro-4-carboxyphenylboronic acid) grafted PEI 1.8 k (polyethylenimine) as the polycations of PEI-FPBA and dopamine (with diol-containing moiety) conjugated with cholesterol as the hydrophobic part (Chol-Dopa). The PFCDM micelles was assembled by PEI-FPBA and Chol-Dopa, based on the FPBA-Dopa conjugation. The prepared PFCDM showed strong siRNA loading ability and superior stability in the presence of PBS or serum. Besides, the PFCDM exhibited excellent ATP sensibility. The intracellular ATP could effectively trigger the disassembly of micelles and charge reversal of PEI-FPBA, resulting in the burst release of siRNA in the cytosol. With the property of extracellular stability and intracellular instability, PFCDM displayed good performance on in vitro and in vivo luciferase silencing on 4T1 cells. It should also be noted that the assembly of low molecular weight PEI was relatively safe to cells compared with 25 k PEI. To sum up, the ATP-fueled assembly and charge reversible micelles gave examples for polyplexes to achieve better stability and on demand cargo release at the same time and shows potential to be used for in vitro and in vivo siRNA transfection.

The development of non-viral vectors for gene delivery has gained attention over the past decades. Specifically, poly(β-amino ester) (PBAE) has shown great potential for improving the delivery of gene therapeutics. It has been observed that low-molecular-weight PBAE displayed low transfection activities, while quaternization could enhance the transgene expression efficacy of PBAE. Herein, PBAE quaternary ammonium salt (PBAEQAS) was synthesized to increase the positive charge of the polymers, which resulted in an increase in siRNA binding efficiency based on self-assembly electrostatic interaction. Specifically, the nanoparticle surface was positively charged, which increased the uptake ability of siRNA. Compared with acrylate-PBAEQAS/siNC nanoparticles and amine-PBAEQAS/siNC nanoparticles, acrylate-PBAEQAS/siSurvivin nanoparticles and amine-PBAEQAS/siSurvivin nanoparticles induced more-efficient cell apoptosis and gene silencing. All these results suggest that PBAEQAS would be a promising gene delivery vector for cancer treatment.

Small interfering RNAs (siRNAs) are invaluable research tools for studying gene functions in mammalian cells. siRNAs are mainly produced by chemical synthesis or by enzymatic digestion of double-stranded RNA (dsRNA) produced in vitro. Recently, bacterial cells, engineered with ectopic plant viral siRNA binding protein p19, have enabled the production of "recombinant" siRNAs (pro-siRNAs). Here, we describe an optimized methodology for the production of milligram amount of highly potent recombinant pro-siRNAs from Escherichia coli cells. We first optimized bacterial culture medium and tested new designs of pro-siRNA production plasmid. Through the exploration of multiple pro-siRNA related factors, including the expression of p19 protein, (dsRNA) generation method, and the level of RNase III, we developed an optimal pro-siRNA production plasmid. Together with a high-cell density fed-batch fermentation method in a bioreactor, we have achieved a yield of ~10 mg purified pro-siRNA per liter of bacterial culture. The pro-siRNAs produced by the optimized method can achieve high efficiency of gene silencing when used at low nanomolar concentrations. This new method enables fast, economical, and renewable production of pure and highly potent bioengineered pro-siRNAs at the milligram level. Our study also provides important insights into the strategies for optimizing the production of RNA products in bacteria, which is an under-explored field.

In this studies, three fatty acyl derivatives of CGKRK homing peptides were coupled successfully to chitosan oligosaccharides (COS) using sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate sodium salt (sulfo-SMCC). The COS-SMCC was prepared by direct coupling between COS and sulfo-SMCC in PBS (pH 7.5) at RT for 48 h. The structure of COS-SMCC and the three fatty acyl-CGKRK-SMCC-COS conjugates were characterized by FT-IR, 13C NMR, and SEM. The ability of three conjugates to condense siRNA into nanosized polyplexes and their efficacy in protecting siRNA from serum nucleases degradation were investigated. Among the investigated derivatives, S-CGKRK-COS showed higher siRNA binding affinity as compared to the P-CGKRK-COS and O-CGKRK-COS, respectively. At a ratio of 10:1, complete protection for siRNA from early enzymatic degradation was achieved. The polymers and the polymer/siRNA polyplexes showed negligible cytotoxicity on human breast cancer cell line MDA-MB-231 at all investigated ratios. However, the polyplexes prepared with palmitoyl and oleoyl derivatives at polymer concentration 10 μg/mL reduced the cell viability by 21.5% and 35%, respectively. The results of this study revealed the potential use of fatty acyl-CGKRK-COS as a siRNA carrier and confirmed the importance of incorporating a hydrophobic moiety into chitosan to improve its capacity in complexing with siRNA and protection from degradation.

Small interfering RNA (siRNA) is a promising therapeutic modality, however, its successful clinical application is still challenging due to the lack of safe and efficient carrier systems. Superparamagnetic iron oxide nanoparticles (SPIONs)-based drug or gene carrier systems have displayed tremendous promise in nanomedicine. They possess intrinsic unique superparamagnetism that provides them to be concentrated in the targeted therapeutic site of action where an external magnetic field is applied. SPIONs can be also designed as theranostic agents to achieve simultaneous therapeutic and diagnostic purposes. Despite these favorable features, SPIONs are colloidally unstable and can be easily eliminated in the circulation. More importantly, the toxicological concerns associated with SPIONs, which often lead to the generation of reactive oxygen species (ROS), need to be thoroughly considered. Various types of polymers have been proposed so far to cover the surface of SPION to overcome these disadvantages. Silk protein, sericin can be ideal as a coating material due to its high biocompatibility, good biodegradability, and in vivo stability. In terms of the development of SPIONs as siRNA carriers, to the best of our knowledge, no protein was used as the coating material, and SPIONs coated with sericin have not been reported in the literature as a drug or gene carrier system. The present study aimed to design and develop a novel siRNA carrier system based on SPIONs. We prepared and characterized a nanomaterial comprised of SPIONs covalently coated with a biocompatible protein, sericin (Ser), and modified with a cationic polymer, poly- l -lysine (PLL) to incorporate the negatively charged siRNA. Transmission electron microscopy (TEM) images revealed that obtained nanoparticles had narrow size distribution with spherical shape and coated nanoparticles presented black spheres in the SPION core surrounded with a gray layer, which indicated sericin coating. Adding PLL to Ser-SPIONs did not cause any dramatic change in nanoparticle size whereas it provided a net positive charge to the particles. Additionally, electron spin resonance (ESR) analysis showed that sericin coating and PLL modification did not efface the superparamagnetic properties of the particles. We successfully attached the control-siRNAs to the surface of PLL/Ser-SPIONs, forming siRNA-PLL/Ser-SPION nanoplatforms and achieving high siRNA binding efficiencies ranged between 81.90% and 93.50%. The ability of SPIONs-based nanocarriers to fulfill their promises in clinical use depends on keeping their nanotoxicity properties to a minimum. All the nanoparticle formulations tested herein were found to be biocompatible against non-cancerous and cancer cells by cytotoxicity assays. We also investigated the effect of resulting bare particles, PLL/Ser-SPIONs, on the clonogenicity of cancer cells. According to the results, the cells exhibited colony formation ability depending on the concentration of the nanoparticles and PLL/Ser-SPIONs did not block clonogenicity, except at very high doses. Taken together, our findings imply that these new PLL/Ser-SPIONs can be considered as a safe and promising carrier candidate to be used and developed for further siRNA-based applications.

Triple negative breast cancer (TNBC) is one of the deadliest types of cancer for women of different age groups. Frequently this cancer does not respond to conservative treatment. Combinatorial RNAi can be suggested as an advanced approach to TNBC therapy. Due to the fact that TNBC cells overexpress chemokine receptor 4 we used modular L1 peptide-based nanoparticles modified with CXCR4 ligand for combinatorial delivery of siRNAs suppressing major transduction pathways. TNBC cell line MDA-MB-231 was used as a cellular model. Genes encoding the AQP3, CDC20, and COL4A2 proteins responsible for proliferative activity in TNBC cells were selected as RNAi targets. The siRNA binding ability of the carrier was studied at different charge ratios. The silencing specificity was demonstrated for all siRNAs studied. Alamar Blue proliferation assay has shown significant reduction in the anti-proliferative activity after combinatorial siRNA transfection compared to single siRNA delivery. The most significant synergistic effect has been demonstrated for combinatorial transfection of anti-COL4A2 and anti-CDC20 siRNAs what resulted in 1.5–2 fold inhibition of proliferation and migration of TNBC cells. Based on our findings, we have concluded that combinatorial treatment by CXCR4-ligand modified L1-polyplexes formed with AQP3, CDC20, and COL4A2 siRNAs effectively inhibits proliferation of TNBC cells and can be suggested as useful tool for RNAi-mediated cancer therapy.

The development of potent yet nontoxic membrane-penetrating materials is in high demand for effective intracellular gene delivery. We have recently developed α-helical polypeptides which afford potent membrane activities to facilitate intracellular DNA delivery via both endocytosis and the nonendocytic "pore formation" mechanism. Endocytosis will cause endosomal entrapment of the DNA cargo, while excessive "pore formation" would cause appreciable cytotoxicity. Additionally, helical polypeptides with stiff, rodlike structure suffer from low siRNA binding affinity. To address such critical issues, we herein incorporated various aromatic domains (benzyl, naphthyl, biphenyl, anthryl, and pyrenyl) into the side-chain terminals of guanidine-rich, helical polypeptides, wherein the flat-rigid shape, π-electronic structures of aromatic motifs "self-activated" the membrane-penetrating capabilities of polypeptides to promote intracellular gene delivery. Benzyl (Bn)- and naphthyl (Naph)-modified polypeptides demonstrated the highest DNA uptake level that outperformed the unmodified polypeptide, P2, by ∼4 fold. More importantly, compared with P2, Bn- and Naph-modified polypeptides allowed more DNA cargos to be internalized via the nonendocytic pathway, which significantly bypassed the endosomal entrapment and accordingly enhanced the transfection efficiency by up to 42 fold, outperforming PEI 25k as the commercial reagent by 3-4 orders of magnitude. The aromatic modification also improved the siRNA condensation capability of polypeptides, achieving notably enhanced gene-silencing efficiency against tumor necrosis factor-α to treat acute hepatic inflammation. Furthermore, we revealed that aromaticity-augmented membrane activity was accompanied by comparable or even significantly reduced "pore formation" capability, thus leading to diminished cytotoxicity at high concentrations. This study therefore provides a promising approach to manipulate the membrane activities and penetration mechanisms of polycations, which overcomes the multiple critical barriers preventing effective and safe gene delivery.

Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.

The incorporation of anionic excipients into polyplexes is a promising strategy for modulating siRNA binding versus release and integrating diagnostic capabilities; however, specific design criteria and structure-function relationships are needed to facilitate the development of nanocarrier-based theranostics. Herein, we incorporated poly(acrylic acid) (PAA) and quantum dot (QD) excipients into photolabile siRNA polyplexes to increase gene silencing efficiencies by up to 100% and enable self-reporting of nanocarrier disassembly. Our systematic approach identified the functional relationships between gene silencing and key parameters such as excipient loading fractions and molecular weights that facilitated the establishment of design rules for optimization of nanocarrier efficacy. For example, we found that PAA molecular weights ∼10-20× greater than that of the coencapsulated siRNA exhibited the most efficient release and silencing. Furthermore, siRNA release assays and RNAi modeling allowed us to generate a PAA "heat map" that predicted gene silencing a priori as a function of PAA molecular weight and loading fraction. QDs further promoted selective siRNA release and provided visual as well as Förster resonance energy transfer (FRET)-based monitoring of the dynamic changes in nanostructure in situ. Moreover, even with the addition of anionic components, our formulations exhibited substantially improved stability and shelf life relative to typical formulations, with complete stability after a week of storage and full activity in the presence of serum. Taken together, this study enabled synergistic improvements in siRNA release and diagnostic capabilities, along with the development of mechanistic insights that are critical for advancing the translation of nucleic acid theranostics into the clinic.

The targeted delivery of polymers to neurons is a challenging yet important goal for polymer based drug delivery. We prepared a fentanyl based atom transfer radical polymerization (ATRP) initiator to target the Mu opioid receptor (MOR) for neuronal targeting. We incorporated our recently discovered rigid acrylate linking group into the initiator to retain a high degree of binding to the MOR and grafted random or block copolymers of poly(oligo(ethylene oxide) methacrylate)-block-(glycidyl methacrylate). Trifluoroethanol promoted amine ring opening of the glycidyl methacrylate was used for post-polymerization modification of the fentanyl initiated polymers to attach a near-infrared fluorescent dye (ADS790WS) or to build a targeted siRNA delivery system via modification with secondary amines. We examined the biocompatibility, cellular internalization, and siRNA binding properties of our polymer library in a green fluorescent protein expressing SY SH5Y neuroblastoma cell-line.

The recent discovery of small interfering RNAs (siRNAs) has opened new avenues for designing personalized treatment options for various diseases. However, the therapeutic application of siRNAs has been confronted with many challenges because of short half-life in circulation, poor membrane penetration, difficulty in escaping from endosomes, and insufficient release into the cytosol. To overcome these challenges, we designed a diethylenetriamine (DETA)- and tetraethylenepentamine (TEPA)-modified polyamidoamine dendrimer generation 4.5 (PDG4.5), and characterized it using 1H nuclear magnetic resonance (NMR), 13C NMR, correlation spectroscopy (COSY), heteronuclear single-quantum correlation spectroscopy (HSQC), and Fourier transform infrared (FTIR) spectroscopy followed by conjugation with siRNA. The PDG4.5-DETA and PDG4.5-TEPA polyplexes exhibited spherical nanosize, ideal zeta potential, and effective siRNA binding ability, protected the siRNA from nuclease attack, and revealed less cytotoxicity of PDG4.5-DETA and PDG4.5-TEPA in HeLa cells. More importantly, the polyplexes also revealed good cellular internalization and facilitated translocation of the siRNA into the cytosol. Thus, PDG4.5-DETA and PDG4.5-TEPA can act as potential siRNA carriers in future medical and pharmaceutical applications.

Synthetic siRNA technology has emerged as a promising approach for molecular therapy of cancer but, despite its potential for post-transcriptional gene silencing, there is an urgent need to develop efficient delivery systems particularly for difficult-to-transfect, anchorage-independent cells. In this study, we designed highly hydrophobic cationic lipopolymers by grafting cholesterol (Chol) onto low-molecular weight (0.6, 1.2, and 2.0 kDa) polyethylenimines (PEIs) to enable specific siRNA therapy to chronic myeloid leukemia (CML) cells. The siRNA binding by PEI-Chol led to nano-sized (100-200 nm diameter) polyplexes with enhanced ζ-potential (+20 to +35 mV) and ability to protect the loaded siRNA completely in fresh serum. The siRNA delivery to CML (K562) cells was proportional to degree of substitution and, unexpectedly, inversely proportional to molecular size of the polymeric backbone. Chol grafting with as little as ~1.0 Chol/PEI on 0.6 and 1.2 kDa PEIs enabled silencing of the reporter Green Fluorescent Protein gene as well as the endogenous BCR-Abl oncogene in K562 cells. The PEI-Chol mediated delivery of siRNAs specific for BCR-Abl and KSP genes significantly arrested the growth the cells which was significantly reflected in colony formation potency of K562 cells. BCR-Able siRNA mediated therapeutic efficacy was also observed in significantly increased caspase activity and apoptosis of K562 cells. Thus, Chol-grafted low-molecular weight PEIs appear to be unique siRNA carriers to realize the molecular therapy in CML cells.

Polydiacetylene micelles were assembled from four different cationic amphiphiles and photopolymerized to reinforce their architecture. The produced micelles were systematically investigated, in interaction with siRNAs, for intracellular delivery of the silencing nucleic acids. The performances of the carrier systems were rationalized based on the cell penetrating properties of the micelles and the nature of their cationic complexing group, responsible for efficient siRNA binding and further endosomal escape.

Small interfering RNA (siRNA)-based gene silencing strategy has high potential on suppressing specific molecular targets, involved in cancer progression. However, the lack of an effective nanocarrier system that safely delivers siRNA to its target still limits the clinical applications of siRNA. This study aimed to develop albumin-sericin nanoparticles (Alb-Ser NPs) as a novel siRNA delivery system for laryngeal cancer treatment. Nanoparticle formulations composed of albumin and sericin at different ratios (1:1, 2:1, 1:2 w/w) were synthesized by desolvation method. The nanoparticles were modified with poly-L-lysine (PLL) for siRNA binding and decorated with hyaluronic acid (HA) to target laryngeal cancer cell line, Hep-2. HA/PLL/Alb-Ser NPs were individually loaded with siRNAs for casein kinase 2 (CK2), Absent, Small, or Homeotic-Like (ASH2L), and Cyclin D1 genes, which are overexpressed in Hep-2 cells. Downregulation of genes was confirmed by real-time PCR (RT-PCR). Size, morphological, and thermogravimetric characterizations revealed that Alb-Ser NPs having 2:1 (w/w) ratio are the most optimized formulation. Between 36.8 and 61.3% of siRNA entrapment efficiencies were achieved. HA/PLL-siRNA/Alb-Ser (2:1) NPs-mediated gene silencing resulted in a significant inhibition of cell growth and induction of apoptosis in cells. Our findings showed that HA/PLL/Alb-Ser (2:1) NPs were promising as a siRNA carrier.

Recent advances in agarose gel electrophoresis protocols established conditions for the high-resolution separation of DNA and RNA using higher voltages combined with short run times. We subsequently developed a protocol for using these conditions to measure the binding affinity of a protein for an RNA ligand on an agarose gel. This native gel mobility shift assay is highly accessible, using common molecular biology reagents found in most laboratories. Here, we describe the protocol for carrying out native agarose gel electrophoresis to characterize the binding affinity of a protein for an RNA ligand. The electrophoresis time is less than 10 min, which minimizes the dissociation of protein and ligand. We have used the p19 siRNA binding protein and its cognate dsRNA ligand to demonstrate strategies for identifying optimal conditions to measure apparent binding constants using this agarose gel shift system.

Polyethylenimine (PEI) is widely used as delivery vector for nucleic acids. However, the clinical application is limited by relatively low transfection efficacy and high toxicity. To address this problem, here fluorinated polymer technology have been proposed to modify PEI. We synthesized fluorinated PEI and prepared nanomicelles with a fluorinated core for siRNA delivery. Nanomicelles were formed by fluorous interactions. The nanomicelles possessed good buffering capacity and siRNA binding ability. Nanomicelles could protect siRNA from the RNase A degradation and show excellent colloidal stability. More importantly, the fluorinated PEI and nanomicelles achieved efficient gene transfection capacity at relatively low w/w ratios, which ensured low cytotoxicity of the formulations. In addition, the nanomicelles exhibited high levels of cellular uptake and endosomal escape, which was consistent with high transfection activity. The results suggest that fluorination of PEI and preparation of nanomicelles are a promising strategy in the design of highly efficient siRNA delivery vectors.

Histone Deacetylase(HDAC) is an enzyme that eliminates acetyl group from the histone octamer complex. The acetylation state of histone proteins is a major interest of epigenetic gene expression. HDAC1 inhibitors are used for anticancer therapeutics by controlling multiple signaling protein kinases like SAPK, ERK and TNF-alpha. Here, we used single strand DNA 18-oligomer to mimic RNA interference technology. We modeled the HDAC1 mRNA secondary structure and identified the possible four siRNA binding sites by higher possibility than miR-449 targeting site. Also, its possible configuration was modeled according to binding energies. Three places, where the possibility of siRNA binding is low, were randomly identified as positive controls. As a Result, the 18-omer single strand DNA was generated according to the identified sequences. This preliminary data can be further warranted to generate HDAC1 knockdown activity and its comparison to the current HDAC1 inhibitors. Furthermore, generation of single strand DNA as a antisense sequence to a specific mRNA can be utilized for therapeutics along with RNAi. With the thermodynamically stable structure of DNA compared to RNA, it can be applied for long term usage.

Herein, chiral selenium nanoparticles (L-SeNPs/D-SeNPs) modified with a dinuclear Ruthenium (II) complex were used to effectively deliver siRNA targeting the MDR1 gene. In this co-delivery system, the luminescent dinuclear Ruthenium (II) complex was developed to act as a gene carrier and anti-tumor drug, while offering luminescent imaging to follow the intracellular trafficking. Interestingly, Ru@L-SeNPs exhibited a stronger protein and pDNA affinity than Ru@D-SeNPs, indicating that chirality may have an effect on pDNA/siRNA binding and biocompatibility. Cisplatin-resistant A549R cells treated with Ru@L-SeNPs-siRNA demonstrated significant downregulation of P-glycoprotein (P-gp) expression, resulting in unprecedented enhanced cytotoxicity through the induction of apoptosis with the involvement of phosphorylation of p53, MAPK and PI3K/Akt signaling pathways. In vivo investigation confirmed that Ru@L-SeNPs-siRNA nanoparticles exhibited high tumor-targeted fluorescence, enhanced anti-tumor efficacy, and decreased systemic toxicity. These results suggest that Ru@L-SeNPs are promising vectors for the delivery of siRNA and for real-time tracking of treatment. From the Clinical Editor In this study, the authors designed bi-functional selenium nanoparticles with specific chirality to deliver siRNA, for targeting tumor MDR1 gene. The underlying ruthenium (II) complex could also offer fluorescence for real-time imaging. This new system has been shown to have enhanced efficacy against drug resistant tumor cells in both in-vitro and in-vivo experiments.

Background Silencing of excessive secreted tumour necrosis factor (TNF)-α from macrophages might be an effective therapy of ulcerative colitis (UC), which acquires improvements on small interfering RNA (siRNA) delivery vectors. Thus, in the present study, the effects of particle size and binding affinity of four polymeric nanoparticles on siRNA delivery for the treatment of UC were evaluated. Methods Galactosylated trimethyl chitosan-cysteine (GTC) nanoparticles of varying particle size and binding affinity for siRNA were prepared and TNF-α siRNA was encapsulated. Their cellular transport was investigated in murine macrophages and Caco-2 cell monolayers were utilized to analysis the intestinal permeation. Finally, in vivo anti-inflammatory efficacy was assessed in a mouse model of UC. Results Although marginal effects of particle size on the in vitro gene silencing efficiency were detected, GTC nanoparticles with a particle size of 450 nm and stronger binding affinity for siRNA showed reduced intestinal epithelial permeability and enhanced in vivo anti-inflammatory efficacy compared to those with a particle size of 200 nm. By contrast, the delivery processes were significantly affected by the binding affinity for siRNA, where smaller GTC nanoparticles (200 nm) with moderate siRNA binding strength exhibited remarkable cytoplasmic distribution and sufficient intracellular release of siRNA, as well as a sustained in vitro and in vivo gene silencing effect. Conclusions Nanoparticles with a particle size of 450 nm or balanced binding affinity for siRNA might be preferable for the treatment of ulcerative colitis. Copyright © 2015 John Wiley & Sons, Ltd.

Assembly of the RNA-induced silencing complex (RISC) requires formation of the RISC loading complex (RLC), which contains the Dicer-2 (Dcr-2)-R2D2 complex and recruits duplex siRNA to Ago2 in Drosophila melanogaster. However, the precise composition and action mechanism of Drosophila RLC remain unclear. Here we identified the missing factor of RLC as TATA-binding protein-associated factor 11 (TAF11) by genetic screen. Although it is an annotated nuclear transcription factor, we found that TAF11 also associated with Dcr-2/R2D2 and localized to cytoplasmic D2 bodies. Consistent with defective RLC assembly in taf11(-/-) ovary extract, we reconstituted the RLC in vitro using the recombinant Dcr-2-R2D2 complex, TAF11, and duplex siRNA. Furthermore, we showed that TAF11 tetramer facilitates Dcr-2-R2D2 tetramerization to enhance siRNA binding and RISC loading activities. Together, our genetic and biochemical studies define the molecular nature of the Drosophila RLC and elucidate a cytoplasmic function of TAF11 in organizing RLC assembly to enhance RNAi efficiency.

Delivery of small interfering RNA (siRNA) by nanocarriers has shown promising therapeutic potential in cancer therapy. However, poor understanding of the correlation between the physicochemical properties of nanocarriers and their interactions with biological systems has significantly hindered its anticancer efficacy. Herein, in order to identify the optimal size of nanocarriers for siRNA delivery, different sized cationic micellar nanoparticles (MNPs) (40, 90, 130, and 180 nm) are developed that exhibit similar siRNA binding efficacies, shapes, surface charges, and surface chemistries (PEGylation) to ensure size is the only variable. Size-dependent biological effects are carefully and comprehensively evaluated through both in vitro and in vivo experiments. Among these nanocarriers, the 90 nm MNPs show the optimal balance of prolonged circulation and cellular uptake by tumor cells, which result in the highest retention in tumor cells. In contrast, larger MNPs are rapidly cleared from the circulation and smaller MNPs are inefficiently taken up by tumor cells. Accordingly, 90 nm MNPs carrying polo-like kinase 1 (Plk1)-specific siRNA (siPlk1) show superior antitumor efficacy, indicating that 90 nm could either be the optimal size for systemic delivery of siRNA or close to it. Our findings provide valuable information for rationally designing nanocarriers for siRNA-based cancer therapy in the future.

Controllable release is particularly important for the delivery of small interfering RNA (siRNA), as siRNAs have a high susceptibility to enzymatic degradation if release is premature, yet lack silencing activity if they remain inaccessible within the cytoplasm. To overcome these hurdles, novel and tailorable mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)n) diblock copolymers containing light-sensitive o-nitrobenzyl moieties and pendant amines are employed to provide both efficient siRNA binding, via electrostatic and hydrophobic interactions, as well as triggered charge reversal and nucleic acid release. In particular, siRNA/mPEG-b-P(APNBMA)23.6 polyplexes show minimal aggregation in physiological salt and serum, and enhanced resistance to polyanion-induced unpackaging compared to polyethylenimine preparations. Cellular delivery of siRNA/mPEG-b-P(APNBMA)23.6 polyplexes reveal greater than 80% cellular transfection, as well as rapid and widespread cytoplasmic distribution. Additionally, UV irradiation indicates ~70% reduction in targeted gene expression following siRNA/mPEG-b-P(APNBMA)23.6 polyplex treatment, as compared to 0% reduction in polyplex treated cells without UV irradiation, and only ~30% reduction for Lipofectamine treated cells. The results herein highlight the potential of these light-sensitive copolymers with a well-defined on/off switch for applications including cellular patterning for guided cell growth and extension, and cellular microarrays for exploring protein and drug interactions that require enhanced spatiotemporal control of gene activation.

We present an approach to tuning the multifunctionality of iron oxide nanoparticles (IONs) using mixed self-assembled monolayers of cationic lipid and anionic polyethylene glycol (PEG) lipid. By forming stable, monodispersed lipid-coated IONs (L-IONs) through a solvent-exchange technique, we were able to demonstrate the relationship between surface charge, the magnetic transverse relaxivity (r2 from T2-weighted images), and the binding capacity of small interfering ribonucleic acids (siRNAs) as a function of the cationic-to-anionic (PEG) lipid ratio. These properties were controlled by the cationic charge and the PEG conformation; relaxivity and siRNA binding could be varied in the mushroom and brush regimes but not at high brush densities. In vitro results combining cell viability, uptake, and transfection efficiency using HeLa cells suggest that the functional physicochemical and biological properties of L-IONs may be best achieved using catanionic lipid coatings near equimolar ratios of cationic to ani...

Small Interfering RNAs (siRNAs) are one of the valuable tools to investigate the functions of genes, and are also used for gene silencing. It has a wide scope in drug discovery through in vivo target validation. siRNA therapeutics are not optimal drug-like molecules due to poor bioavailability, immunogenic and off-target effects. In order to overcome the challenges associated with siRNA therapeutics, identification of appropriate chemical modifications that improves the stability, specificity and potency of siRNA is essential. This review focuses on the various chemical modifications and their implications in siRNA therapy.

(1) Background: The great potential of RNA interference (RNAi)-based gene therapy is premised on the effective delivery of small interfering RNAs (siRNAs) to target tissues and cells. Hence, we aimed at developing and examining a novel integrin αvβ3-specific delivery carrier for targeted transfection of siRNA to malignant tumor cells; (2) Methods: Arginine-glycine-aspartate motif (RGD) was adopted as a tissue target for specific recognition of integrin αvβ3. To enable siRNA binding, a chimeric peptide was synthesized by adding nonamer arginine residues (9R) at the carboxy terminus of cyclic-RGD dimer, designated as c(RGD)2-9R. The efficiency of 9R peptide transferring siRNA was biologically evaluated in vitro by flow cytometry, confocal microscopy, and Western blot; (3) Results: An optimal 10:1 molar ratio of c(RGD)2-9R to siRNA was confirmed by the electrophoresis on agarose gels. Both the flow cytometry and confocal microscopy results testified that transfection of c(RGD)2-9R as an siRNA delivery carrier was obviously higher than the naked-siRNA group. The results of Western blot demonstrated that these 9R peptides were able to transduce siRNA to HepG2 cells in vitro, resulting in efficient gene silencing; and (4) Conclusion: The chimeric peptide of c(RGD)2-9R can be developed as an effective siRNA delivery carrier and shows potential as a new strategy for RNAi-based gene therapy.

The sequence-defined polycationic polymers with or without Cys-Arg-Cys motifs conjugated with targeting and shielding segments were synthesized as siRNA carriers via native chemical ligation (NCL) reaction. After purification, the structures and physicochemical characteristics were determined by a variety of experimental techniques. The particle size of siRNA/CRC-polymer polyplex was much smaller than that of polyplex without CRC motifs. The buffer capacity and siRNA binding ability of CRC motifs modified polymers were significantly improved, resulting from the twin disulfides and hexatomic ring formulation. The critical micelle concentrations of the polymers were10mg/L, indicating formation of polymeric micelles and sufficient stability of the system. The CRC motifs modified polymers with folate targeted ligands exhibited a strongly enhanced cellular uptake than the negative control and the unmodified analogues. The results of gene transfection showed that the folate-PEG-ligated polymer modified with CRC motifs had much better gene transfection compared to the alanine-ended control and other analogues. Furthermore, they showed barely cytotoxicity. By the way, there was no distinctly improvement for pDNA transfection. All above results suggested that folate-PEG-ligated polymers modified with CRC motifs and their self-assembly polymeric micelles could be promising non-viral siRNA carriers.

[Aim] To study the structural requirements that a cyclooligosaccharide-based nanoparticle must fulfill to be an efficient siRNA transfection vector.[Materials & methods] siRNA protection from degradation by RNAses, transfection efficiency and the thermodynamic parameters of the nanoparticle/siRNA interactions were studied on pairs of amphiphilic molecules using biochemical techniques and molecular dynamics., [Results] The lower the siRNA solvent accessible surface area in the presence of the nanoparticle, higher the protection from RNAse-mediated degradation in the corresponding nanocomplex; a moderate nanoparticle/siRNA binding energy value further facilitates reversible complexation and binding to the target cellular mRNA., [Conclusion] The use, in advance, of these parameters will provide a useful indication of the potential of a molecular nanoparticle as siRNA transfecting vector.

A novel magnetobiosensing approach for the rapid, sensitive and selective miR-21 detection is reported involving the use of a specific RNA probe (antimiR-21), streptavidin-magnetic beads (Strep-MBs), the siRNA Binding Protein p19 as detector bioreceptor, and amperometric detection with the H2O2/hydroquinone (HQ) system at disposable screen-printed carbon electrodes. The magnetosensor exhibited a dynamic range from 1.4 to 10 nM and a detection limit of 4.2 fmol of synthetic target miR-21 without any amplification step in 75 min. The usefulness of the approach was evaluated by analyzing total RNA (RNAt) extracted from metastatic breast cancer cell lines, human tissues and breast cytologies.