Photochemical Regulation of Gene Expression Using Caged siRNAs with Single Terminal Vitamin E Modification

Caged siRNAs with a single photolabile linker and/or vitamin E (vitE) modification at the 5′ terminal were rationally designed and synthesized. These virtually inactive caged siRNAs were successfully used to photoregulate both firefly luciferase and GFP gene expression in cells with up to an 18.6‐fold enhancement of gene silencing activity, which represents one of the best reported photomodulation of gene silencing efficiencies to date. siRNA tracking and vitE competition experiments indicated that the inactivity of vitE‐modified siRNAs was not due to the bulky moiety of vitE; rather, the involvement of vitE‐binding proteins has a large contribution to caged siRNA inactivation by preventing the dissociation of siRNA/lipo complexes and/or siRNA release. Further patterning experiments revealed the ability to spatially regulate gene expression through simple light irradiation.     

Tetrazine-Induced Bioorthogonal Activation of Vitamin E-Modified siRNA for Gene Silencing

The temporal activation of siRNA provides a valuable strategy for the regulation of siRNA activity and conditional gene silencing. The bioorthogonal bond-cleavage reaction of benzonorbonadiene and tetrazine is a promising trigger in siRNA temporal activation. Here, we developed a new method for the bio-orthogonal chemical activation of siRNA based on the tetrazine-induced bond-cleavage reaction. Small-molecule activatable caged siRNAs were developed with the 5′-vitamin E-benzonobonadiene-modified antisense strand targeting the green fluorescent protein (GFP) gene and the mitotic kinesin-5 (Eg5) gene. The addition of tetrazine triggered the reaction with benzonobonadiene linker and induced the linker cleavage to release the active siRNA. Additionally, the conditional gene silencing of both exogenous GFP and endogenous Eg5 genes was successfully achieved with 5′-vitamin E-benzonobonadiene-caged siRNAs, which provides a new uncaging strategy with small molecules.     

Surmounting tumor resistance to metallodrugs by co-loading a metal complex and siRNA in nanoparticles

Copper complexes are promising anticancer agents widely studied to overcome tumor resistance to metal-based anticancer drugs. Nevertheless, copper complexes per se encounter drug resistance from time to time. Adenosine-5′-triphosphate (ATP)-responsive nanoparticles containing a copper complex CTND and B-cell lymphoma 2 (Bcl-2) small interfering RNA (siRNA) were constructed to cope with the resistance of cancer cells to the complex. CTND and siRNA can be released from the nanoparticles in cancer cells upon reacting with intracellular ATP. The resistance of B16F10 melanoma cells to CTND was terminated by silencing the cellular Bcl-2 gene via RNA interference, and the therapeutic efficacy was significantly enhanced. The nanoparticles triggered a cellular autophagy that amplified the apoptotic signals, thus revealing a novel mechanism for antagonizing the resistance of copper complexes. In view of the extensive association of Bcl-2 protein with cancer resistance to chemotherapeutics, this strategy may be universally applicable for overcoming the ubiquitous drug resistance to metallodrugs.

Bcl-2-related tumor resistance to anticancer drugs can be overcome by silencing the cellular Bcl-2 gene via RNA interference. The realization of the goal is exemplified by delivering Bcl-2 siRNA and a tumor-resistant Cu complex to cancer cells with an ATP-responsive nanocarrier.     

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.     

A photoresponsive antibody–siRNA conjugate for activatable immunogene therapy of cancer

Tumor-targeted delivery of small-interfering RNAs (siRNAs) for cancer therapy still remains a challenging task. While antibody–siRNA conjugates (ARCs) provide an alternative way to address this challenge, the uncontrollable siRNA release potentially leads to undesirable off-tumor side effects, limiting their in vivo therapeutic efficacy. Here, we report a photoresponsive ARC (PARC) for tumor-specific and photoinducible siRNA delivery as well as photoactivable immunogene therapy. PARC is composed of an anti-programmed death-ligand 1 antibody (αPD-L1) conjugated with a siRNA against intracellular PD-L1 mRNA through a photocleavable linker. After targeting cancer cells through the interaction between αPD-L1 and membrane PD-L1, PARC is internalized and it liberates siPD-L1 upon light irradiation to break the photocleavable linker. The released siPD-L1 then escapes from the lysosome into the cytoplasm to degrade intracellular PD-L1 mRNA, which combines the blockade of membrane PD-L1 by αPD-L1 to boost immune cell activity. Owing to these features, PARC causes effective cancer suppression both in vitro and in vivo. This study thus provides a useful conditional delivery platform for siRNAs and a novel means for activatable cancer immunogene therapy.

A photoresponsive antibody–siRNA conjugate (PARC) enables tumor-targeted siRNA delivery and photoactivatable gene silencing for cancer immunotherapy.     

Design and enhanced gene silencing activity of spherical 2′-fluoroarabinose nucleic acids (FANA-SNAs)

Drug delivery vectors for nucleic acid therapeutics (NATs) face significant barriers for translation into the clinic. Spherical nucleic acids (SNAs) – nanoparticles with an exterior shell made up of DNA strands and a hydrophobic interior – have recently shown great potential as vehicles to improve the biodistribution and efficacy of NATs. To date, SNA design has not taken advantage of the powerful chemical modifications available to NATs. Here, we modify SNAs with 2′-deoxy-2′-fluoro-d-arabinonucleic acid (FANA-SNA), and show increased stability, enhanced gene silencing potency and unaided uptake (gymnosis) as compared to free FANA. By varying the spacer region between the nucleic acid strand and the attached hydrophobic polymer, we show that a cleavable DNA based spacer is essential for maximum activity. This design feature will be important when implementing functionalized nucleic acids into nanostructures for gene silencing. The modularity of the FANA-SNA was demonstrated by silencing two different targets. Transfection-free delivery was superior for the modified SNA compared to the free FANA oligonucleotide.

Optimizing FANA modified spherical nucleic acids (FANA-SNAs) for highly efficient delivery of nucleic acid therapeutics.     

Free-standing metal–organic framework (MOF) monolayers by self-assembly of polymer-grafted nanoparticles

We report a general method for the synthesis of free-standing, self-assembled MOF monolayers (SAMMs) at an air–water interface using polymer-brush coated MOF nanoparticles. UiO-66, UiO-66-NH₂, and MIL-88B-NH₂ were functionalized with a catechol-bound chain-transfer agent (CTA) to graft poly(methyl methacrylate) (PMMA) from the surface of the MOF using reversible addition-fragmentation chain transfer polymerization (RAFT). The polymer-coated MOFs were self-assembled at the air–water interface into monolayer films ∼250 nm thick and capable of self-supporting at a total area of 40 mm². Mixed-particle films were prepared through the assembly of MOF mixtures, while multilayer films were achieved through sequential transfer of the monolayers to a glass slide substrate. This method offers a modular and generalizable route to fabricate thin-films with inherent porosity and sub-micron thickness composed of a variety of MOF particles and functionalities.

We report a general method for the synthesis of free-standing, self-assembled MOF monolayers (SAMMs) at an air–water interface using polymer-brush coated MOF nanoparticles.     

Photoregulation of Gene Expression with Amantadine-Modified Caged siRNAs through Host–Guest Interactions

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.     

Gene silencing by siRNA microhydrogels via polymeric nanoscale condensation

The first attempt to prepare biologically active siRNA-based microhydrogels is reported. The self-assembled microhydrogels were fabricated using sense/antisense complementary hybridization between single-stranded linear and Y-shaped trimeric siRNAs. The siRNA microhydrogels were condensed using a popular cationic polymer such as LPEI to form compact, stable siRNA/polymeric nanoparticles that exhibited superb cellular uptake efficiency and gene silencing activity.     

Confined space design by nanoparticle self-assembly

Nanoparticle (NP) self-assembly has led to the fabrication of an array of functional nanoscale systems, having diverse architectures and functionalities. In this perspective, we discuss the design and application of NP suprastructures (SPs) characterized by nanoconfined compartments in their self-assembled framework, providing an overview about SP synthetic strategies reported to date and the role of their confined nanocavities in applications in several high-end fields. We also set to give our contribution towards the formation of more advanced nanocompartmentalized SPs able to work in dynamic manners, discussing the opportunities of further advances in NP self-assembly and SP research.

This perspective gives an outlook on the design of interparticle confined nanocavities in self-assembled NP systems and their functional relevance.     

Regiodivergent construction of medium-sized heterocycles from vinylethylene carbonates and allylidenemalononitriles

Medium-sized heterocycles exist in a broad spectrum of biologically active natural products and medicinally important synthetic compounds. The construction of medium-sized rings remains challenging, particularly the assembly of different ring sizes from the same type of substrate. Here we report palladium-catalyzed, regiodivergent [5 + 4] and [5 + 2] annulations of vinylethylene carbonates and allylidenemalononitriles. We describe the production of over 50 examples of nine- and seven-membered heterocycles in high isolated yields and excellent regioselectivities. We demonstrate the synthetic utility of this approach by converting a nine-membered ring product to an interesting polycyclic caged molecule via a [2 + 2] transannulation. Mechanistic studies suggest that the [5 + 2] annulation proceeds through palladium-catalyzed ring-opening/re-cyclization from the [5 + 4] adducts.

Here we report palladium-catalyzed, regiodivergent [5 + 4] and [5 + 2] annulations of vinylethylene carbonates and allylidenemalononitriles affording over 50 medium-sized heterocycles in high isolated yields with excellent regioselectivities.     

Electroporation microarray for parallel transfer of small interfering RNA into mammalian cells

This paper describes the fabrication of microarrays that enable the parallel electroporation of small interfering RNAs (siRNAs) into mammalian cells. To optimize the conditions of microarray preparation and electric pulsing, a self-assembled monolayer was formed on a gold electrode, and a cationic polymer was adsorbed by the entire surface of the monolayer. siRNA was then adsorbed by the cationically modified electrode through electrostatic interactions. Human embryonic kidney cells stably transformed with the expression construct of green fluorescent protein (GFP) were used to examine the electric pulse-triggered transfer of GFP-specific siRNA. A single electric pulse was applied to the cells cultured on the electrode at a field strength of 240 V cm(-1). The expression of GFP was significantly suppressed in a sequence-specific manner two days after pulsing. Microscopic observation and flow-cytometric analysis revealed that the expression of GFP was attenuated in the majority of cells in a loading-dependent manner. Moreover, the effect of siRNA could be temporally controlled by changing the culture periods before pulsing. When a micropatterned self-assembled monolayer was used as a platform for loading siRNA in an array format, gene silencing was spatially restricted to the regions where specific siRNA was loaded. From these results, we conclude that array-based electroporation provides an excellent means of individual transfer of siRNAs into mammalian cells for high-throughput gene function studies.     

Hierarchical two-dimensional molecular assembly through dynamic combination of conformational states at the liquid/solid interface

Self-sorting of multiple building blocks for correctly positioning molecules through orthogonal recognition is a promising strategy for construction of a hierarchical self-assembled molecular network (SAMN) on a surface. Herein we report that a trigonal molecule, dehydrobenzo[12]annulene (DBA) derivative having three tetradecyloxy chains and three hydroxy groups in an alternating manner, forms hierarchical triangular clusters of different sizes ranging from 2.4 to 16.4 nm, consisting of 3 to 78 molecules, respectively, at the liquid/graphite interface. The key is the dynamic combination of three different conformational states, which is solvent and concentration dependent. The present knowledge extends design strategies for production of sophisticated hierarchical SAMNs using a single component at the liquid/solid interface.

Self-sorting of multiple building blocks for correctly positioning molecules through orthogonal recognition is a promising strategy for construction of a hierarchical self-assembled molecular network (SAMN) on a surface.     

Sustainable existence of solid mercury (Hg) nanoparticles at room temperature and their applications

Although liquid mercury (Hg) has been known since antiquity, the formation of stable solid nano forms of Hg at room temperature has not been reported so far. Here, for the first time, we report a simple sonochemical route to obtain solid mercury nanoparticles, stabilized by reduced graphene oxide at ambient conditions. The as-formed solid Hg nanoparticles were found to exhibit remarkable rhombohedral morphology and crystallinity at room temperature. Extensive characterization using various physicochemical techniques revealed the unique properties of the solid nanoparticles of Hg compared to its bulk liquid metal phase. Furthermore, the solid nature of the Hg nanoparticles was studied electrochemically, revealing distinctive properties. We believe that solid Hg nanoparticles have the potential for important applications in the fields of electroanalytical chemistry and electrocatalysis.

Formation of the stable solid mercury nanoparticles by acoustic cavitation and their electrochemical activity compared to its bulk liquid metal.     

Light-intensity switch enabled nonsynchronous growth of fluorinated raspberry-like nanoparticles

Raspberry-like (RB) nanoparticles hold potential for diverse applications due to their hierarchical morphology. Here we developed a novel tandem synthetic approach of nonsynchronous growth based on photo-mediated reversible-deactivation radical polymerization, enabling simple, efficient and bottom-up synthesis of RB nanoparticles of uniform sizes at quantitative conversions of fluorinated monomers. Chain transfer agents of different chain lengths, concentrations and chemical compositions were varied to tune the diameter of RB particles. Importantly, fluorinated RB nanoparticles obtained with this method allow facile post modifications via both covalent bond formation and intermolecular physical interactions without disrupting the RB morphology. The facile nature of this method and versatility of the obtained fluorinated RB materials open new opportunities for the development of functional materials using nanoparticles.

Nonsynchronous growth of raspberry-like (RB) nanoparticles in a one-pot and bottom-up fashion, enabling simple post-modification of RB colloids through both covalent bond formation and supramolecular interaction.     

A chemical circular communication network at the nanoscale

In nature, coordinated communication between different entities enables a group to accomplish sophisticated functionalities that go beyond those carried out by individual agents. The possibility of programming and developing coordinated communication networks at the nanoscale—based on the exchange of chemical messengers—may open new approaches in biomedical and communication areas. Here, a stimulus-responsive circular model of communication between three nanodevices based on enzyme-functionalized Janus Au–mesoporous silica capped nanoparticles is presented. The output in the community of nanoparticles is only observed after a hierarchically programmed flow of chemical information between the members.

A community of three nanodevices communicates through a hierarchically programmed circular flow of chemical information between members.     

Optimised power harvesting by controlling the pressure applied to molecular junctions

A major potential advantage of creating thermoelectric devices using self-assembled molecular layers is their mechanical flexibility. Previous reports have discussed the advantage of this flexibility from the perspective of facile skin attachment and the ability to avoid mechanical deformation. In this work, we demonstrate that the thermoelectric properties of such molecular devices can be controlled by taking advantage of their mechanical flexibility. The thermoelectric properties of self-assembled monolayers (SAMs) fabricated from thiol terminated molecules were measured with a modified AFM system, and the conformation of the SAMs was controlled by regulating the loading force between the organic thin film and the probe, which changes the tilt angle at the metal-molecule interface. We tracked the thermopower shift vs. the tilt angle of the SAM and showed that changes in both the electrical conductivity and Seebeck coefficient combine to optimize the power factor at a specific angle. This optimization of thermoelectric performance via applied pressure is confirmed through the use of theoretical calculations and is expected to be a general method for optimising the power factor of SAMs.

A major potential advantage of creating thermoelectric devices using self-assembled molecular layers is their mechanical flexibility.     

Aggregation-free and high stability core–shell polymer nanoparticles with high fullerene loading capacity,variable fullerene type,and compatibility towards biological conditions

Fullerenes have unique structural and electronic properties that make them attractive candidates for diagnostic, therapeutic, and theranostic applications. However, their poor water solubility remains a limiting factor in realizing their full biomedical potential. Here, we present an approach based on a combination of supramolecular and covalent chemistry to access well-defined fullerene-containing polymer nanoparticles with a core–shell structure. In this approach, solvophobic forces and aromatic interactions first come into play to afford a micellar structure with a poly(ethylene glycol) shell and a corannulene-based fullerene-rich core. Covalent stabilization of the supramolecular assembly then affords core-crosslinked polymer nanoparticles. The shell makes these nanoparticles biocompatible and allows them to be dried to a solid and redispersed in water without inducing interparticle aggregation. The core allows a high content of different fullerene types to be encapsulated. Finally, covalent stabilization endows nanostructures with stability against changing environmental conditions.

A polymer nanoparticle approach to biorelevant and robust fullerene nanoparticles is presented.     

Ambient microdroplet annealing of nanoparticles

Conversion of polydisperse nanoparticles to their monodisperse analogues and formation of organized superstructures using them involve post synthetic modifications, and the process is generally slow. We show that ambient electrospray of preformed polydisperse nanoparticles makes them monodisperse and the product nanoparticles self-assemble spontaneously to form organized films, all within seconds. This phenomenon has been demonstrated with thiol-protected polydisperse silver nanoparticles of 15 ± 10 nm diameter. Uniform silver nanoparticles of 4.0 ± 0.5 nm diameter were formed after microdroplet spray, and this occurred without added chemicals, templates, and temperature, and within the time needed for electrospray, which was of the order of seconds. Well organized nanoparticle assemblies were obtained from such uniform particles. A home-made and simple nanoelectrospray set-up produced charged microdroplets for the generation of such nanostructures, forming cm² areas of uniform nanoparticles. A free-standing thin film of monodisperse silver nanoparticles was also made on a liquid surface by controlling the electrospray conditions. This unique method may be extended for the creation of advanced materials of many kinds.

Polydisperse silver nanoparticles were converted to a highly ordered assembly of nanoparticles by microdroplet-induced chemistry, under ambient conditions, within seconds.     

Heterobimetallic Eu(iii)/Pt(ii) single-chain nanoparticles: a path to enlighten catalytic reactions

We introduce the formation and characterization of heterometallic single-chain nanoparticles entailing both catalytic and luminescent properties. A terpolymer containing two divergent ligand moieties, phosphines and phosphine oxides, is synthesized and intramolecularly folded into nanoparticles via a selective metal complexation of Pt(ii) and Eu(iii). The formation of heterometallic Eu(iii)/Pt(ii) nanoparticles is evidenced by size exclusion chromatography, multinuclear NMR (¹H, ³¹P{¹H}, ¹⁹F, ¹⁹⁵Pt) as well as diffusion-ordered NMR and IR spectroscopy. Critically, we demonstrate the activity of the SCNPs as a homogeneous and luminescent catalytic system in the amination reaction of allyl alcohol.

A bifunctional terpolymer containing two orthogonal ligand moieties was synthesized, giving way to the facile formation of heterometallic Eu(iii)/Pt(ii) single-chain nanoparticles, which display both catalytic and luminescent properties.