Endotoxin Nanovesicles: Hydrophilic Gold Nanodots Control Supramolecular Lipopolysaccharide Assembly for Modulating Immunological Responses.

In this study, we sought to control the assembly of an endotoxin known as the biologically supramolecular lipopolysaccharide (LPS, which consists of three portions: an O antigen, a core carbohydrate, and a lipid A molecule) in order to modulate immunological responses in a manner that has the potential for utilization in vaccine development. Changing the structures of LPS aggregates from lamellas to specific nonlamellas (i.e., cubosomes and hexosomes) can dramatically enhance the strength of LPS in causing inflammatory responses, leading to highly active responses. In order to control the formation of cubosome-free and hexosome-free nonlamellas, we designed a simple strategy based on the use of hydrophilic gold nanodots (AuNDs) to control LPS assembly to facilitate the formation of stable endotoxin nanovesicles, which are stable precursors of cubosomes and hexosomes with specific immunological effects. Structurally, the wall thicknesses of these nanovesicles are exactly twice the lengths of a single LPS molecule, indicating that the LPS molecules adopt a tail-to-tail arrangement (with the lipid A portions acting as the tail domain). The involvement of the hydrophilic AuNDs to laterally link polar domains of LPS can result in the progressive extension of an endotoxically active zone of lipid A assembly, leading to the eventual formation of large-size nanovesicles. Our results showed that endotoxin nanovesicles with such dense lipid A units can elicit the stronger inflammatory gene expressions, including interleukin 6 (IL-6), IL-1A, TNF-α, C-X-C chemokine ligand (CXCL) 1, 2, and 11, which have characteristics of T-helper 1 adjuvants. These findings provide evidence that the concept of manipulating the surface hydrophilicity of AuNDs to control LPS assembly in order to avoid the formation of highly active cubosomes and hexosomes, and thereby modulate immunological responses appropriately, could prove useful in vaccine development.

5,5'-Bis[(2,2,2-trifluoro-eth-oxy)meth-yl]-2,2'-bipyridine.

The complete molecule of the title compound, C(16)H(14)F(6)N(2)O(2), is generated by crystallographic inversion symmetry, which results in two short intramolecular C-H⋯N hydrogen-bond contacts per molecule. In the crystal, aromatic π-π stacking [centroid-centroid distance = 3.457 (2) Å] and weak C-H⋯π inter-actions occur. A short H⋯H [2.32 (3) Å] contact is present.

4,4'-Bis(2,2,2-trifluoroethoxymethyl)-2,2'-bipyridine.

As part of a homologous series of novel polyfluorinated bipyridyl (bpy) ligands, the title compound, C(16)H(14)F(6)N(2)O(2), contains the smallest fluorinated group, viz. CF(3). The molecule resides on a crystallographic inversion centre at the mid-point of the pyridine C(ipso)-C(ipso) bond. Therefore, the bpy skeleton lies in an anti conformation to avoid repulsion between the two pyridyl N atoms. Weak intramolecular C-H...N and C-H...O interactions are observed, similar to those in related polyfluorinated bpy-metal complexes. A pi-pi interaction is observed between the bpy rings of adjacent molecules and this is probably a primary driving force in crystallization. Weak intermolecular C-H...N hydrogen bonding is present between one of the CF(3)CH(2)- methylene H atoms and a pyridyl N atom related by translation along the [010] direction, in addition to weak benzyl-type C-H...F interactions to atoms of the terminal CF(3) group. It is of note that the O-CH(2)CF(3) bond is almost perpendicular to the bpy plane.

Designed nucleus penetrating thymine-capped dendrimers: a potential vehicle for intramuscular gene transfection.

A nucleus penetrating vehicle is indispensible when seeking to deliver plasmid DNA for gene transfection. In this study, dendrimers with terminal thymine groups were synthesized to meet this objective. Through modifications of the hydrophilic and neutral thymine moieties on hyperbranched peripheries, these dendrimers can achieve biosafety, efficient endosomal escape ability, cytosolic accessibility, and eventually, nuclear entry for the purposes of gene transfection. After optimization of the thymine coverages, better gene expression can only be achieved while replacing ∼50% of the amine groups of a dendrimer with thymine moieties. Presumably, a specific dendrimer comprising thymine and primary amines might possess a synergistic effect to promote pDNA condensation via the cooperation of electrostatic interaction and hydrogen bonding. In comparison, a dendrimer entirely capped by thymine can lose external amines, decreasing pDNA complexity and stability, which would cause poor gene transfection. The utility of specific thymine-capped dendrimers in vivo level was demonstrated to successfully and efficiently deliver plasmid DNA at a low complex ratio into mouse muscle by intramuscular injection. Upon the easy accessibility of intramuscular administration, the capability of thymine-capped dendrimers might be potentially used in immunotherapeutic gene transfection in the future.

Co-caged gold nanoclusters and methyl motifs lead to detoxification of dendrimers and allow cytosolic access for siRNA transfection.

Nonviral vectors used in gene delivery, such as cationic polymers and dendrimers, exhibit problems of inherent toxicity and inefficient cytosolic access that must be overcome. In this work, a simple co-caging strategy focused on overcoming the two limitations of dendrimers for siRNA transfection is reported. By embedding gold nanoclusters within a dendrimer, the structure of the dendrimer becomes compact and allows an irreversible backfolding of exterior primary amines from the branch to the core, which dramatically eliminates dendrimer toxicity and enhances safety. Gold nanoclusters with strong emissions can confer a trackable function to dendrimers acting as a transfection vector (TV) for siRNA transfection. In order to maximize efficiency of complexing with siRNA, the TV further incorporated caged methyl motifs, transforming the partially tertiary amines into quaternary ammonium ions to form a methylated TV (MTV). The cellular responses to the MTV were similar to those of the TV, but the responses to the MTV can also enhance cytosolic access to better deliver siRNA for mRNA knockdown. This finding offers a novel perspective to facilitate the use of various cationic polymers for detoxification in biological applications through a co-caging strategy without further chemical modifications.

Recent progress in copolymer-mediated siRNA delivery.

RNAi-mediated gene silencing has great potential for treating various diseases, including cancer, by delivering a specific short interfering RNA (siRNA) to knock down pathogenic mRNAs and suppress protein translation. Although many researchers are dedicated to devising polymer-based vehicles for exogenous in vitro siRNA transfection, few synthetic vehicles are feasible in vivo. Recent studies have presented copolymer-based vectors that are minimally immunogenic and facilitate highly efficient internalizing of exogenous siRNA, compared with homopolymer-based vectors. Cationic segments, organelle-escape units, and degradable fragments are essential to a copolymer-based vehicle for siRNA delivery. The majority of these cationic segments are derived from polyamines, including polylysine, polyarginine, chitosan, polyethylenimines and polyamidoamine dendrimers. Not only do these cationic polyamines protect siRNA, they can also promote disruption of endosomal membranes. Degradable fragments of copolymers must be derived from various polyelectrolytes to release the siRNA once the complexes enter the cytoplasm. This review describes recent progress in copolymer-mediated siRNA delivery, including various building blocks for biocompatible copolymers for efficient in vitro siRNA delivery, and a useful basis for addressing the challenges of in vivo siRNA delivery.