Advanced Microfluidic Technologies for Lipid Nano-Microsystems from Synthesis to Biological Application.

Microfluidics is an emerging technology that can be employed as a powerful tool for designing lipid nano-microsized structures for biological applications. Those lipid structures can be used as carrying vehicles for a wide range of drugs and genetic materials. Microfluidic technology also allows the design of sustainable processes with less financial demand, while it can be scaled up using parallelization to increase production. From this perspective, this article reviews the recent advances in the synthesis of lipid-based nanostructures through microfluidics (liposomes, lipoplexes, lipid nanoparticles, core-shell nanoparticles, and biomimetic nanovesicles). Besides that, this review describes the recent microfluidic approaches to produce lipid micro-sized structures as giant unilamellar vesicles. New strategies are also described for the controlled release of the lipid payloads using microgels and droplet-based microfluidics. To address the importance of microfluidics for lipid-nanoparticle screening, an overview of how microfluidic systems can be used to mimic the cellular environment is also presented. Future trends and perspectives in designing novel nano and micro scales are also discussed herein.

Hybrid polymer/lipid vesicle synthesis: Association between cationic liposomes and lipoplexes with chondroitin sulfate.

The association of cationic carriers with different anionic mucoadhesive biopolymers has been widely explored as an alternative to improve their delivery routes and specific targeting. This work presents a complete analysis of the association between chondroitin sulfate (CS) and cationic liposomes (CLs)/lipoplex (CL-pDNA). In this study, plasmid DNA (pDNA) was used as a genetic cargo for association with carriers. Firstly, we measured the stoichiometry of pseudo complexes and evaluated their colloidal properties, structural and morphological characteristics. Optimized CL-pDNA lipoplexes (positive z-potential) and CL-CS / CL-pDNA-CS (negative z-potential with CS mass ratio of 9% (w/w)) were further studied in detail. Small-angle X-ray scattering analysis and cryo-transmission electron microscopy micrographs revealed that the electrostatic interaction between CS and CL / CL-pDNA easily reorganized the lipid bilayers resulting in nanoscale uni/multilamellar vesicles. A high CS mass ratio (9% (w/w)) led to the reassembly of liposomal structure, wherein the pDNA was easily exchanged for CS chains, forming more than 50% of dense multilamellar vesicles. This data evidenced that the association between CS and CLs is not a conventional coating process since it generates complex and hybrid structures. We believe that these obtained colloidal data may be used in the future to investigate polymer-tailored nanocarriers and their production process. In brief, the colloidal study of hybrid structures may open interesting perspectives for developing novel carriers for drug and gene delivery applications.

Layer-by-Layer Biomimetic Microgels for 3D Cell Culture and Nonviral Gene Delivery.

Localized release of nucleic acid therapeutics is essential for many biomedical applications, including gene therapy, tissue engineering, and medical implant coatings. We applied the substrate-mediated transfection and layer-by-layer (LbL) technique to achieve an efficient local gene delivery. In the experiments presented herein, we embeded lipoplexes containing plasmid DNA encoding for enhanced green fluorescent protein (pEGFP) within polyelectrolyte alginate-based microgels composed of poly(allylamine hydrochloride) (PAH), chondroitin sulfate (CS), and poly-l-lysine (PLL) with diameters between 70 and 90 µm. Droplet-based microfluidics was used as the main process to produce the alginate (ALG)-based microgels with discrete size, shape, and low coefficient of variation. The physicochemical and morphological properties of the polyelectrolyte microgels were characterized via optical microscopy, scanning electron microscopy (SEM), and zeta potential analysis. We found that polyelectrolyte microgels provide low cytotoxicity and cell-material interactions (adhesion, spreading, and proliferation). In addition, the microsystem showed the ability to load lipoplexes and a loading efficiency equal to 83%, and it enabled in vitro surface-based transfection of MCF-7 cells. This approach provides a new suitable route for cell adhesion and local gene delivery.

Recent advances in co-delivery nanosystems for synergistic action in cancer treatment.

Nanocarrier delivery systems have been widely studied to carry unique or dual chemical drugs. The major challenge of chemotherapies is to overcome the multidrug-resistance (MDR) of cells to antineoplastic medicines. In this context, nano-scale technology has allowed researchers to develop biocompatible nano-delivery systems to overcome the limitation of chemical agents. The development of nano-vehicles may also be directed to co-deliver different agents such as drugs and genetic materials. The delivery of nucleic acids targeting specific cells is based on gene therapy principles to replace the defective gene, correct genome errors or knock-down a particular gene. Co-delivery systems are attractive strategies due to the possibility of achieving synergistic therapeutic effects, which are more effective in overcoming the MDR of cancer cells. These combined therapies can provide better outcomes than separate delivery approaches carrying either siRNA, miRNA, pDNA, or drugs. This article reviews the main design features that need to be associated with nano-vehicles to co-deliver drugs, genes, and gene-drug combinations with efficacy. The advantages and disadvantages of co-administration approaches are also overviewed and compared with individual nanocarrier systems. Herein, future trends and perspectives in designing novel nano-scale platforms to co-deliver therapeutic agents are also discussed.

Mechanisms of Nanonewton Mechanostability in a Protein Complex Revealed by Molecular Dynamics Simulations and Single-Molecule Force Spectroscopy.

Can molecular dynamics simulations predict the mechanical behavior of protein complexes? Can simulations decipher the role of protein domains of unknown function in large macromolecular complexes? Here, we employ a wide-sampling computational approach to demonstrate that molecular dynamics simulations, when carefully performed and combined with single-molecule atomic force spectroscopy experiments, can predict and explain the behavior of highly mechanostable protein complexes. As a test case, we studied a previously unreported homologue from Ruminococcus flavefaciens called X-module-Dockerin (XDoc) bound to its partner Cohesin (Coh). By performing dozens of short simulation replicas near the rupture event, and analyzing dynamic network fluctuations, we were able to generate large simulation statistics and directly compare them with experiments to uncover the mechanisms involved in mechanical stabilization. Our single-molecule force spectroscopy experiments show that the XDoc-Coh homologue complex withstands forces up to 1 nN at loading rates of 105 pN/s. Our simulation results reveal that this remarkable mechanical stability is achieved by a protein architecture that directs molecular deformation along paths that run perpendicular to the pulling axis. The X-module was found to play a crucial role in shielding the adjacent protein complex from mechanical rupture. These mechanisms of protein mechanical stabilization have potential applications in biotechnology for the development of systems exhibiting shear enhanced adhesion or tunable mechanics.