Development of New Targeted Inulin Complex Nanoaggregates for siRNA Delivery in Antitumor Therapy

Here, a novel strategy of formulating efficient polymeric carriers based on the already described INU-IMI-DETA for gene material whose structural, functional, and biological properties can be modulated and improved was successfully investigated. In particular, two novel derivatives of INU-IMI-DETA graft copolymer were synthesized by chemical functionalisation with epidermal growth factor (EGF) or polyethylenglycol (PEG), named INU-IMI-DETA-EGF and INU-IMI-DETA-PEG, respectively, in order to improve the performance of already described “inulin complex nanoaggregates” (ICONs). The latter were thus prepared by appropriately mixing the two copolymers, by varying each component from 0 to 100 wt% on the total mixture, named EP-ICONs. It was seen that the ability of the INU-IMI-DETA-EGF/INU-IMI-DETA-PEG polymeric mixture to complex siGL3 increases with the increase in the EGF-based component in the EP-ICONs and, for each sample, with the increase in the copolymer:siRNA weight ratio (R). On the other hand, the susceptibility of loaded siRNA towards RNase decreases with the increase in the pegylated component in the polymeric mixture. At all R values, the average size and the zeta potential values are suitable for escaping from the RES system and suitable for prolonged intravenous circulation. By means of biological characterisation, it was shown that MCF-7 cells are able to internalize mainly the siRNA-loaded into EGF-decorated complexes, with a significant difference from ICONs, confirming its targeting function. The targeting effect of EGF on EP-ICONs was further demonstrated by a competitive cell uptake study, i.e., after cell pre-treatment with EGF. Finally, it was shown that the complexes containing both EGF and PEG are capable of promoting the internalisation and therefore the transfection of siSUR, a siRNA acting against surviving mRNA, and to increase the sensitivity to an anticancer agent, such as doxorubicin.     

A Versatile Solid-Phase Approach to the Synthesis of Oligonucleotide Conjugates with Biodegradable Hydrazone Linker

One of the ways to efficiently deliver various drugs, including therapeutic nucleic acids, into the cells is conjugating them with different transport ligands via labile or stable bonds. A convenient solid-phase approach for the synthesis of 5′-conjugates of oligonucleotides with biodegradable pH-sensitive hydrazone covalent bonds is proposed in this article. The approach relies on introducing a hydrazide of the ligand under aqueous/organic media to a fully protected support-bound oligonucleotide containing aldehyde function at the 5′-end. We demonstrated the proof-of-principle of this approach by synthesizing 5′-lipophilic (e.g., cholesterol and α-tocopherol) conjugates of modified siRNA and non-coding RNAs imported into mitochondria (antireplicative RNAs and guide RNAs for Mito-CRISPR/system). The developed method has the potential to be extended for the synthesis of pH-sensitive conjugates of oligonucleotides of different types (ribo-, deoxyribo-, 2′-O-methylribo-, and others) with ligands of different nature.     

Synthesis of Site‐Specifically Phosphate‐Caged siRNAs and Evaluation of Their RNAi Activity and Stability

A complete set of new photolabile nucleoside phosphoramidites were synthesized, then site‐specifically incorporated into sense or antisense strands of siRNA for phosphate caging. Single caging modification was made along siRNA strands and their photomodulation of gene silencing were examined by using the firefly luciferase reporter gene. Several key phosphate positions were then identified. Furthermore, multiple caging modifications at these key positions led to significantly enhanced photomodulation of gene silencing activity, suggesting a synergistic effect. The caging group on both the terminally phosphate‐caged siRNA and the single‐stranded caged RNA has comparatively high stability, whereas hydrolysis of the caged group from the internally caged siRNA was observed, irrespective of the presence of Mg²⁺. Molecular dynamic simulations demonstrated that enhanced hydrolysis of the caging group on internally phosphate‐caged siRNAs was due to easy fragmentation of the caging group upon formation of the pentavalent intermediate of the phosphotriester with attack by water. The caging group in the terminally phosphate‐caged siRNA or single‐stranded caged RNA prefers to form π–π stacks with nearby nucleobases. In addition to providing explanations for previous observations, this study sheds further light on the design of caged oligonucleotides and indicates the direction of future development of nucleic acid drugs with phosphate modifications.     

Cationic Vesicles Based on Amphiphilic Pillar[5]arene Capped with Ferrocenium: A Redox‐Responsive System for Drug/siRNA Co‐Delivery

A novel ferrocenium capped amphiphilic pillar[5]arene (FCAP) was synthesized and self‐assembled to cationic vesicles in aqueous solution. The cationic vesicles, displaying low cytotoxicity and significant redox‐responsive behavior due to the redox equilibrium between ferrocenium cations and ferrocenyl groups, allow building an ideal glutathione (GSH)‐responsive drug/siRNA co‐delivery system for rapid drug release and gene transfection in cancer cells in which higher GSH concentration exists. This is the first report of redox‐responsive vesicles assembled from pillararenes for drug/siRNA co‐delivery; besides enhancing the bioavailability of drugs for cancer cells and reducing the adverse side effects for normal cells, these systems can also overcome the drug resistance of cancer cells. This work presents a good example of rational design for an effective stimuli‐responsive drug/siRNA co‐delivery system.     

Effect of Cationic Lipid Nanoparticle Loaded siRNA with Stearylamine against Chikungunya Virus

Chikungunya is an infectious disease caused by mosquito-transmitted chikungunya virus (CHIKV). It was reported that NS1 and E2 siRNAs administration demonstrated CHIKV inhibition in in vitro as well as in vivo systems. Cationic lipids are promising for designing safe non-viral vectors and are beneficial in treating chikungunya. In this study, nanodelivery systems (hybrid polymeric/solid lipid nanoparticles) using cationic lipids (stearylamine, C9 lipid, and dioctadecylamine) and polymers (branched PEI-g-PEG -PEG) were prepared, characterized, and complexed with siRNA. The four developed delivery systems (F1, F2, F3, and F4) were assessed for stability and potential toxicities against CHIKV. In comparison to the other nanodelivery systems, F4 containing stearylamine (Octadecylamine; ODA), with an induced optimum cationic charge of 45.7 mV in the range of 152.1 nm, allowed maximum siRNA complexation, better stability, and higher transfection, with strong inhibition against the E2 and NS1 genes of CHIKV. The study concludes that cationic lipid-like ODA with ease of synthesis and characterization showed maximum complexation by structural condensation of siRNA owing to high transfection alone. Synergistic inhibition of CHIKV along with siRNA was demonstrated in both in vitro and in vivo models. Therefore, ODA-based cationic lipid nanoparticles can be explored as safe, potent, and efficient nonviral vectors overcoming siRNA in vivo complexities against chikungunya.     

Size- and Surface- Dual Engineered Small Polyplexes for Efficiently Targeting Delivery of siRNA

Though siRNA-based therapy has achieved great progress, efficient siRNA delivery remains a challenge. Here, we synthesized a copolymer PAsp(-N=C-PEG)-PCys-PAsp(DETA) consisting of a poly(aspartate) block grafted with comb-like PEG side chains via a pH-sensitive imine bond (PAsp(-N=C-PEG) block), a poly(l-cysteine) block with a thiol group (PCys block), and a cationic poly(aspartate) block grafted with diethylenetriamine (PAsp(DETA) block). The cationic polymers efficiently complexed siRNA into polyplexes, showing a sandwich-like structure with a PAsp(-N=C-PEG) out-layer, a crosslinked PCys interlayer, and a complexing core of siRNA and PAsp(DETA). Low pH-triggered breakage of pH-sensitive imine bonds caused PEG shedding. The disulfide bond-crosslinking and pH-triggered PEG shedding synergistically decreased the polyplexes’ size from 75 nm to 26 nm. To neutralize excessive positive charges and introduce the targeting ligand, the polyplexes without a PEG layer were coated with an anionic copolymer modified with the targeting ligand lauric acid. The resulting polyplexes exhibited high transfection efficiency and lysosomal escape capacity. This study provides a promising strategy to engineer the size and surface of polyplexes, allowing long blood circulation and targeted delivery of siRNA.     

Flexible Nanosomes (SECosomes) Enable Efficient siRNA Delivery in Cultured Primary Skin Cells and in the Viable Epidermis of Ex Vivo Human Skin

The extent to which nanoscale‐engineered systems cross intact human skin and can exert pharmacological effects in viable epidermis is controversial. This research seeks to develop a new lipid‐based nanosome that enables the effective delivery of siRNA into human skin. The major finding is that an ultraflexible siRNA‐containing nanosome—prepared using DOTAP, cholesterol, sodium cholate, and 30% ethanol—penetrates into the epidermis of freshly excised intact human skin and is able to enter into the keratinocytes. The nanosomes, called surfactant‐ethanol‐cholesterol‐osomes (SECosomes), show excellent size, surface charge, morphology, deformability, transfection efficiency, stability, and skin penetration capacity after complexation with siRNA. Importantly, these nanosomes have ideal characteristics for siRNA encapsulation, in that the siRNA is stable for at least 4 weeks, they enable highly efficient transfection of in vitro cultured cells, and are shown to transport siRNA delivery through intact human skin where changes in the keratinocyte cell state are demonstrated. It is concluded that increasing flexibility in nanosomes greatly enhances their ability to cross the intact human epidermal membrane and to unload their payload into targeted epidermal cells.