Development of Focused Ultrasound-Assisted Nanoplexes for RNA Delivery.

RNA-based therapeutics, including siRNA, have obtained recognition in recent years due to their potential to treat various chronic and rare diseases. However, there are still limitations to lipid-based drug delivery systems in the clinical use of RNA therapeutics due to the need for optimization in the design and the preparation process. In this study, we propose adaptive focused ultrasound (AFU) as a drug loading technique to protect RNA from degradation by encapsulating small RNA in nanoliposomes, which we term nanoplexes. The AFU method is non-invasive and isothermal, as nanoplexes are produced without direct contact with any external materials while maintaining precise temperature control according to the desired settings. The controllability of sample treatments can be effectively modulated, allowing for a wide range of ultrasound intensities to be applied. Importantly, the absence of co-solvents in the process eliminates the need for additional substances, thereby minimizing the potential for cross-contaminations. Since AFU is a non-invasive method, the entire process can be conducted under sterile conditions. A minimal volume (300 µL) is required for this process, and the treatment is speedy (10 min in this study). Our in vitro experiments with silencer CD44 siRNA, which performs as a model therapeutic drug in different mammalian cell lines, showed encouraging results (knockdown > 80%). To quantify gene silencing efficacy, we employed quantitative polymerase chain reaction (qPCR). Additionally, cryo-electron microscopy (cryo-EM) and atomic force microscopy (AFM) techniques were employed to capture images of nanoplexes. These images revealed the presence of individual nanoparticles measuring approximately 100-200 nm in contrast with the random distribution of clustered complexes observed in ultrasound-untreated samples of liposome nanoparticles and siRNA. AFU holds great potential as a standardized liposome processing and loading method because its process is fast, sterile, and does not require additional solvents.

Probing the Gelation Synergies and Anti-Escherichia coli Activity of Fmoc-Phenylalanine/Graphene Oxide Hybrid Hydrogel.

The N-fluorenyl-9-methyloxycarbonyl (Fmoc)-protected amino acids have shown high antimicrobial application potential, among which the phenylalanine derivative (Fmoc-F) is the most well-known representative. However, the activity spectrum of Fmoc-F is restricted to Gram-positive bacteria only. The demand for efficient antimicrobial materials expanded research into graphene and its derivatives, although the reported results are somewhat controversial. Herein, we combined graphene oxide (GO) flakes with Fmoc-F amino acid to form Fmoc-F/GO hybrid hydrogel for the first time. We studied the synergistic effect of each component on gelation and assessed the material's bactericidal activity on Gram-negative Escherichia coli (E. coli). GO flakes do not affect Fmoc-F self-assembly per se but modulate the elasticity of the gel and speed up its formation. The hybrid hydrogel affects E. coli survival, initially causing abrupt bacterial death followed by the recovery of the surviving ones due to the inoculum effect (IE). The combination of graphene with amino acids is a step forward in developing antimicrobial gels due to their easy preparation, chemical modification, graphene functionalization, cost-effectiveness, and physicochemical/biological synergy of each component.

Diversity at the nanoscale: laser-oxidation of single-layer graphene affects Fmoc-phenylalanine surface-mediated self-assembly.

We report the effects of a laser-oxidized single layer graphene (SLG) surface on the self-assembly of amphiphilic gelator N-fluorenylmethoxycarbonyl-L-phenylalanine (Fmoc-Phe) towards an gel-SLG interface. Laser oxidation modulates the levels of hydrophobicity/hydrophilicity on the SLG surface. Atomic force, scanning electron, helium ion and scattering scanning nearfield optical microscopies (AFM, SEM, HIM, s-SNOM) were employed to assess the effects of surface properties on the secondary and tertiary organization of the formed Fmoc-Phe fibres at the SLG-gel interface. S-SNOM shows sheet-like secondary structures on both hydrophobic/hydrophilic areas of SLG and helical or disordered structures mainly on the hydrophilic oxidized surface. The gel network heterogeneity on pristine graphene was observed at the scale of single fibres by s-SNOM, demonstrating its power as a unique tool to study supramolecular assemblies and interfaces at nanoscale. Our findings underline the sensitivity of assembled structures to surface properties, while our characterization approach is a step forward in assessing surface-gel interfaces for the development of bionic devices.

Nanoscale Probing of the Supramolecular Assembly in a Two-Component Gel by Near-Field Infrared Spectroscopy.

The design of soft biomaterials requires a deep understanding of molecular self-assembly. Here a nanoscale infrared (IR) spectroscopy study of a two-component supramolecular gel is introduced to assess the system's heterogeneity and supramolecular assembly. In contrast to far-field IR spectroscopy, near-field IR spectroscopy revealed differences in the secondary structures of the gelator molecules and non-covalent interactions at three distinct nano-locations of the gel network. A ß-sheet arrangement is dominant in single and parallel fibres with a small proportion of an α-helix present, while the molecular assembly derives from strong hydrogen bonding. However, at the crossing point of two fibres, only the ß-sheet motif is observed, with an intense π-π stacking contribution. Near-field nanospectroscopy can become a powerful tool for the nanoscale distinction of non-covalent interactions, while it is expected to advance the existing spectroscopic assessments of supramolecular gels.

Triggering a transient organo-gelation system in a chemically active solvent.

A transient organo-gelation system with spatiotemporal dynamic properties is described. Here, the solvent actively controls a complex set of equilibria that underpin the dynamic assembly event. The observed metastability is due to the in situ formation of a secondary solvent, acting as an antagonist against the primary solvent of the organogel.

Tuning protein adsorption on graphene surfaces via laser-induced oxidation.

An approach for controlled protein immobilization on laser-induced two-photon (2P) oxidation patterned graphene oxide (GO) surfaces is described. Selected proteins, horseradish peroxidase (HRP) and biotinylated bovine serum albumin (b-BSA) were successfully immobilized on oxidized graphene surfaces, via non-covalent interactions, by immersion of graphene-coated microchips in the protein solution. The effects of laser pulse energy, irradiation time, protein concentration and duration of incubation on the topography of immobilized proteins and consequent defects upon the lattice of graphene were systemically studied by atomic force microscopy (AFM) and Raman spectroscopy. AFM and fluorescence microscopy confirmed the selective aggregation of protein molecules towards the irradiated areas. In addition, the attachment of b-BSA was detected by a reaction with fluorescently labelled avidin-fluorescein isothiocyanate (Av-FITC). In contrast to chemically oxidized graphene, laser-induced oxidation introduces the capability for localization on oxidized areas and tunability of the levels of oxidation, resulting in controlled guidance of proteins by light over graphene surfaces and progressing towards graphene microchips suitable for biomedical applications.