Targeting Recycling Endosomes to Potentiate mRNA Lipid Nanoparticles.

mRNA lipid nanoparticles (LNPs) have emerged as powerful modalities for gene therapies to control cancer and infectious and immune diseases. Despite the escalating interest in mRNA-LNPs over the past few decades, endosomal entrapment of delivered mRNAs vastly impedes therapeutic developments. In addition, the molecular mechanism of LNP-mediated mRNA delivery is poorly understood to guide further improvement through rational design. To tackle these challenges, we characterized LNP-mediated mRNA delivery using a library of small molecules targeting endosomal trafficking. We found that the expression of delivered mRNAs is greatly enhanced via inhibition of endocytic recycling in cells and in live mice. One of the most potent small molecules, endosidine 5 (ES5), interferes with recycling endosomes through Annexin A6, thereby promoting the release and expression of mRNA into the cytoplasm. Together, these findings suggest that targeting endosomal trafficking with small molecules is a viable strategy to potentiate the efficacy of mRNA-LNPs.

Designer Nanodiscs to Probe and Reprogram Membrane Biology in Synapses.

Signal transduction at the synapse is mediated by a variety of protein-lipid interactions, which are vital for the spatial and temporal regulation of synaptic vesicle biogenesis, neurotransmitter release, and postsynaptic receptor activation. Therefore, our understanding of synaptic transmission cannot be completed until the elucidation of these critical protein-lipid interactions. On this front, recent advances in nanodiscs have vastly expanded our ability to probe and reprogram membrane biology in synapses. Here, we summarize the progress of the nanodisc toolbox and discuss future directions in this exciting field.

Regional Control of Multistimuli-Responsive Structural Color-Switching Surfaces by a Micropatterned DNA-Hydrogel Assembly.

Structural colors have advantages compared with chemical pigments or dyes, such as iridescence, tunability, and unfading. Many studies have focused on developing the ability to switch ON/OFF the structural color; however, they often suffer from a simple and single stimulus, remaining structural colors, and target selectivity. Herein, we present regionally controlled multistimuli-responsive structural color switching surfaces. The key part is the utilization of a micropatterned DNA-hydrogel assembly on a single substrate. Each hydrogel network contains a unique type of stimuli-responsive DNA motifs as an additional cross-linker to exhibit swelling/deswelling via stimuli-responsive DNA interactions. The approach enables overcoming the existing limitations and selectively programming the DNA-hydrogel to a decrypted state (ON) and an encrypted state (OFF) in response to multiple stimuli. Furthermore, the transitions are reversible, providing cyclability. We envision the potential of our method for diverse applications, such as sensors or anticounterfeiting, requiring multistimuli-responsive structural color switching surfaces.

A 4D Printable Shape Memory Vitrimer with Repairability and Recyclability through Network Architecture Tailoring from Commercial Poly(ε-caprolactone).

Vitrimers have shown advantages over conventional thermosets via capabilities of dynamic network rearrangement to endow repairability as well as recyclability. Based on such characteristics, vitrimers have been studied and have shown promises as a 3D printing ink material that can be recycled with the purpose of waste reduction. However, despite the brilliant approaches, there still remain limitations regarding requirement of new reagents for recycling the materials or reprintability issues. Here, a new class of a 4D printable vitrimer that is translated from a commercial poly(ε-caprolactone) (PCL) resin is reported to exhibit self-healability, weldability, reprocessability, as well as reprintability. Thus, formed 3D-printed vitrimer products show superior heat resistance in comparison to commercial PCL prints, and can be repeatedly reprocessed or reprinted via filament extrusion and a handheld fused deposition modeling (FDM)-based 3D printing method. Furthermore, incorporation of semicrystalline PCL renders capabilities of shape memory for 4D printing applications, and as far as it is known, such demonstration of FDM 3D-printed shape memory vitrimers has not been realized yet. It is envisioned that this work can fuel advancement in 4D printing industries by suggesting a new material candidate with all-rounded capabilities with minimized environmental challenges.

Tuning lipid layer formation on particle surfaces by using DNA-containing recruiter molecules.

Biofunctional interfaces containing DNA-conjugated molecules have been explored for various bioengineering applications. However, there is still a lack of understanding of the interaction between DNA conjugates and surrounding biomolecules. In this study, we prepare DNA-containing recruiter molecules and incorporate them onto DNA immobilized gold nanoparticles through DNA hybridization. Liposomes composed of different phospholipids are then applied to investigate supported lipid layer formation on these recruiter-containing surfaces. We find that the morphology and the amount of lipid layers formed are determined by both the liposome concentration and the type of recruiter molecule. When liposomes are applied in excess above a critical concentration, surface chemistry determines the lipid layers formed, leading to lipid multilayers on hydrophilic DNA recruiter containing surfaces and lipid monolayers on hydrophobic DNA-lipid recruiter containing surfaces. When the liposome concentration is below the critical value, the surface molecules take on a more direct role and recruit lipids through hydrophobic interaction. The total amount of the lipid layers formed is further modulated by the overall charge and the fluidity of the liposomes applied. These results provide quantitative analysis on the interaction of DNA conjugates with lipid molecules and introduce a new approach to fine-tune lipid layer formation behavior.

Utilization of a Multiple Cloning Site as a Versatile Platform for DNA Triblock Copolymer Synthesis.

DNA-containing block copolymers have utility in a wide range of biomedical applications. However, synthesis of these hybrid materials, especially ones with complex chain structures, remains to be a major challenge. Here, we report the use of a combination of restriction enzyme sites and ligation enzymes to synthesize DNA triblock copolymers. In contrast to triblock structures held together by DNA hybridization, the newly synthesized DNA triblocks have all blocks connected by covalent bonds. The improved stability of the triblocks against environmental factors such as urea denaturing is confirmed. Furthermore, we incorporate a multiple cloning site (MCS) into the DNA block copolymers and show that the restriction sites can be cut by their corresponding restriction enzymes, generating diblocks with different sticky ends. By utilizing these sticky ends of specific sequences, the cut diblocks are further ligated to create a variety of triblock copolymers with different DNA center blocks and synthetic polymer end blocks. This study presents a versatile platform based on MCS for the synthesis and regeneration of a range of DNA-containing block copolymers.

Protective effect of recombinant soluble neprilysin against ß-amyloid induced neurotoxicity.

A few decades ago, researchers found emerging evidence showing that a number of sequential events lead to the pathological cascade of Alzheimer's disease (AD) which is caused by the accumulation of amyloid beta (Aß), a physiological peptide, in the brain. Therefore, regulation of Aß represents a crucial treatment approach for AD. Neprilysin (NEP), a membrane metallo-endopeptidase, is a rate-limiting peptidase which is known to degrade the amyloid beta peptide. This study investigated soluble NEP (sNEP) produced by recombinant mammalian cells stably transfected with a non-viral NEP expression vector to demonstrate its protective effect against Aß peptides in neuronal cells in vitro. Stably transfected HEK 293 cells were used to purify the soluble protein. sNEP and Aß peptide co-treated hippocampal cells had a decreased level of Aß peptides shown by an increase in cell viability and decrease in apoptosis measured by the CCK-8 and relative caspase-3 activity ratio assays, respectively. This study shows that stably transfected mammalian cells can produce soluble NEP proteins which could be used to protect against Aß accumulation in AD and subsequently neuronal toxicity. Additionally, approaches using protein therapy for potential targets could change the pathological cascade of Alzheimer's disease.

Protective Effect of Tat PTD-Hsp27 Fusion Protein on Tau Hyperphosphorylation Induced by Okadaic Acid in the Human Neuroblastoma Cell Line SH-SY5Y.

Alzheimer's disease (AD) is an age-related disorder that causes a loss of brain function. Hyperphosphorylation of tau and the subsequent formation of intracellular neurofibrillary tangles (NFTs) are implicated in the pathogenesis of AD. Hyperphosphorylated tau accumulates into insoluble paired helical filaments that aggregate into NFTs; therefore, regulation of tau phosphorylation represents an important treatment approach for AD. Heat shock protein 27 (Hsp27) plays a specific role in human neurodegenerative diseases; however, few studies have examined its therapeutic effect. In this study, we induced tau hyperphosphorylation using okadaic acid, which is a protein phosphatase inhibitor, and generated a fusion protein of Hsp27 and the protein transduction domain of the HIV Tat protein (Tat-Hsp27) to enhance the delivery of Hsp27. We treated Tat-Hsp27 to SH-SY5Y neuroblastoma cells for 2 h; the transduction level was proportional to the Tat-hsp27 concentration. Additionally, Tat-Hsp27 reduced the level of hyperphosphorylated tau and protected cells from apoptotic cell death caused by abnormal tau aggregates. These results reveal that Hsp27 represents a valuable protein therapeutic for AD.

DNA duplex stabilization in crowded polyanion solutions.

The melting temperature of duplex DNA is much higher in polyanions than in non-ionic polymers with similar ionic strength, suggesting an additional electrostatic contribution on top of the excluded volume effect.

DNA-functionalized gold nanoparticles in macromolecularly crowded polymer solutions.

DNA-functionalized gold nanoparticles (AuNPs) are one of the most commonly used reagents in nanobiotechnology. They are important not only for practical applications in analytical chemistry and drug delivery, but also for fundamental understanding of nanoscience. For biological samples such as blood serum or for intracellular applications, the effects of crowded cellular proteins and nucleic acids need to be considered. The thermodynamic effect of crowding is to induce nanoparticle aggregation. But before such aggregation can take place, there might also be a depletion repulsive barrier. Polyethylene glycol (PEG) is one of the most frequently used polymers to mimic the crowded cellular environment. We show herein that while DNA-functionalized AuNPs are very stable in buffer (e.g., no PEG) and citrate-capped AuNPs are very stable in PEG, DNA-functionalized AuNPs are unstable in PEG and are easily aggregated. Although such aggregation in PEG is mediated by DNA, no sharp melting transition typical for DNA-linked AuNPs is observed. We attribute this broad melting to depletion force instead of DNA base pairing. The effects of PEG molecular weight, concentration and temperature have been studied in detail and we also find an interesting PEG phase separation and AuNP partition into the water-rich phase at high temperature.