Elucidating Structural Configuration of Lipid Assemblies for mRNA Delivery Systems.

The development of mRNA delivery systems utilizing lipid-based assemblies holds immense potential for precise control of gene expression and targeted therapeutic interventions. Despite advancements in lipid-based gene delivery systems, a critical knowledge gap remains in understanding how the biophysical characteristics of lipid assemblies and mRNA complexes influence these systems. Herein, we investigate the biophysical properties of cationic liposomes and their role in shaping mRNA lipoplexes by comparing various fabrication methods. Notably, an innovative fabrication technique called the liposome under cryo-assembly (LUCA) cycle, involving a precisely controlled freeze-thaw-vortex process, produces distinctive onion-like concentric multilamellar structures in cationic DOTAP/DOPE liposomes, in contrast to a conventional extrusion method that yields unilamellar liposomes. The inclusion of short-chain DHPC lipids further modulates the structure of cationic liposomes, transforming them from multilamellar to unilamellar structures during the LUCA cycle. Furthermore, the biophysical and biological evaluations of mRNA lipoplexes unveil that the optimal N/P charge ratio in the lipoplex can vary depending on the structure of initial cationic liposomes. Cryo-EM structural analysis demonstrates that multilamellar cationic liposomes induce two distinct interlamellar spacings in cationic lipoplexes, emphasizing the significant impact of the liposome structures on the final structure of mRNA lipoplexes. Taken together, our results provide an intriguing insight into the relationship between lipid assembly structures and the biophysical characteristics of the resulting lipoplexes. These relationships may open the door for advancing lipid-based mRNA delivery systems through more streamlined manufacturing processes.

Lipid Membrane Remodeling by the Micellar Aggregation of Long-Chain Unsaturated Fatty Acids for Sustainable Antimicrobial Strategies.

Antimicrobial fatty acids derived from natural sources and renewable feedstocks are promising surface-active substances with a wide range of applications. Their ability to target bacterial membrane in multiple mechanisms offers a promising antimicrobial approach for combating bacterial infections and preventing the development of drug-resistant strains, and it provides a sustainable strategy that aligns with growing environmental awareness compared to their synthetic counterparts. However, the interaction and destabilization of bacterial cell membranes by these amphiphilic compounds are not yet fully understood. Here, we investigated the concentration-dependent and time-dependent membrane interaction between long-chain unsaturated fatty acids-linolenic acid (LNA, C18:3), linoleic (LLA, C18:2), and oleic acid (OA, C18:1)-and the supported lipid bilayers (SLBs) using quartz crystal microbalance-dissipation (QCM-D) and fluorescence microscopy. We first determined the critical micelle concentration (CMC) of each compound using a fluorescence spectrophotometer and monitored the membrane interaction in real time following fatty acid treatment, whereby all micellar fatty acids elicited membrane-active behavior primarily above their respective CMC values. Specifically, LNA and LLA, which have higher degrees of unsaturation and CMC values of 160 µM and 60 µM, respectively, caused significant changes in the membrane with net |Δf| shifts of 23.2 ± 0.8 Hz and 21.4 ± 0.6 Hz and ΔD shifts of 5.2 ± 0.5 × 10-6 and 7.4 ± 0.5 × 10-6. On the other hand, OA, with the lowest unsaturation degree and CMC value of 20 µM, produced relatively less membrane change with a net |Δf| shift of 14.6 ± 2.2 Hz and ΔD shift of 8.8 ± 0.2 × 10-6. Both LNA and LLA required higher concentrations than OA to initiate membrane remodeling as their CMC values increased with the degree of unsaturation. Upon incubating with fluorescence-labeled model membranes, the fatty acids induced tubular morphological changes at concentrations above CMC. Taken together, our findings highlight the critical role of self-aggregation properties and the degree of unsaturated bonds in unsaturated long-chain fatty acids upon modulating membrane destabilization, suggesting potential applications in developing sustainable and effective antimicrobial strategies.

Recyclable and Reusable Natural Plant-Based Paper for Repeated Digital Printing and Unprinting.

Although paperless technologies are becoming ubiquitous, paper and paper-based materials remain one of the most widely used resources, predicted to exceed an annual total of 460 million metric tons by 2030. Given the environmental challenges, deleterious impact on natural resources, and waste associated with conventional wood-based paper manufacturing, developing more sustainable strategies to source, produce, and recycle paper from natural materials is essential. Here, the development and production of reusable and recyclable paper are reported. This approach offers a pathway for easily producing natural pollen grains via ecofriendly, economical, scalable, and low-energy fabrication routes. It is demonstrated that the pollen-based paper exhibits high-quality printability, readability, and erasability, enabling its reuse. Based on the pH-responsive morphological responses of engineered pollen materials, a method for hygro stable printing and on-demand unprinting is presented. The reusability of the pollen paper renders it more advantageous than conventional single-print wood-based paper. This study thus provides possible pathways to utilize non-allergenic pollen, which is renewable and naturally abundant, as a sustainable source of reusable paper. While this work primarily deals with paper, the methods described here can be extended to produce other products such as cartons and containers for the storage and transport of liquid and solid materials.

Selective Recognition of Phosphatidylinositol Phosphate Receptors by C-Terminal Tail of Mitotic Kinesin-like Protein 2 (MKlp2).

The mitotic kinesin-like protein 2 (MKlp2) plays a key role in the proper completion of cytokinetic abscission. Specifically, the C-terminal tail of MKlp2 (CTM peptides) offers a stable tethering on the plasma membrane and microtubule cytoskeleton in the midbody during abscission. However, little is known about the underlying mechanism of how the CTM peptides bind to the plasma membrane of the intercellular bridge. Herein, we identify the specific molecular interaction between the CTM peptides and phosphatidylinositol phosphate (PIP) receptors using quartz crystal microbalance-dissipation and atomic force microscopy force spectroscopic measurements. To systematically examine the effects of amino acids, we designed a series of synthetic 33-mer peptides derived from the wild-type (CTM1). First, we evaluated the peptide binding amount caused by electrostatic interactions based on 100% zwitterionic and 30% negatively charged model membranes, whereby the nonspecific attractions were nearly proportional to the net charge of peptides. Upon incubating with PIP-containing model membranes, the wild-type CTM1 and its truncated mutation showed significant PI(3)P-specific binding, which was evidenced by a 15-fold higher binding mass and 6-fold stronger adhesion force compared to other negatively charged membranes. The extent of the specific binding was predominantly dependent on the existence of S21, whereby substitution or deletion of S21 significantly hindered the binding affinity. Taken together, our findings based on a correlative measurement platform enabled the quantification of the nonelectrostatic, selective binding interactions of the C-terminal of MKlp2 to certain PIP receptors and contributed to understanding the molecular mechanisms on complete cytokinetic abscission in cells.

Thermodynamic Modeling of Solvent-Assisted Lipid Bilayer Formation Process.

The solvent-assisted lipid bilayer (SALB) formation method provides a simple and efficient, microfluidic-based strategy to fabricate supported lipid bilayers (SLBs) with rich compositional diversity on a wide range of solid supports. While various studies have been performed to characterize SLBs formed using the SALB method, relatively limited work has been carried out to understand the underlying mechanisms of SALB formation under various experimental conditions. Through thermodynamic modeling, we studied the experimental parameters that affect the SALB formation process, including substrate surface properties, initial lipid concentration, and temperature. It was found that all the parameters are critically important to successfully form high-quality SLBs. The model also helps to identify the range of parameter space within which conformal, homogeneous SLBs can be fabricated, and provides mechanistic guidance to optimize experimental conditions for lipid membrane-related applications.

Nanoarchitectured air-stable supported lipid bilayer incorporating sucrose-bicelle complex system.

Cell-membrane-mimicking supported lipid bilayers (SLBs) provide an ultrathin, self-assembled layer that forms on solid supports and can exhibit antifouling, signaling, and transport properties among various possible functions. While recent material innovations have increased the number of practically useful SLB fabrication methods, typical SLB platforms only work in aqueous environments and are prone to fluidity loss and lipid-bilayer collapse upon air exposure, which limits industrial applicability. To address this issue, herein, we developed sucrose-bicelle complex system to fabricate air-stable SLBs that were laterally mobile upon rehydration. SLBs were fabricated from bicelles in the presence of up to 40 wt% sucrose, which was verified by quartz crystal microbalance-dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP) experiments. The sucrose fraction in the system was an important factor; while 40 wt% sucrose induced lipid aggregation and defects on SLBs after the dehydration-rehydration process, 20 wt% sucrose yielded SLBs that exhibited fully recovered lateral mobility after these processes. Taken together, these findings demonstrate that sucrose-bicelle complex system can facilitate one-step fabrication of air-stable SLBs that can be useful for a wide range of biointerfacial science applications.

Biophysical Measurement Strategies for Antiviral Drug Development: Recent Progress in Virus-Mimetic Platforms Down to the Single Particle Level.

The rapid growth in the global human population has increased the prevalence of emerging infectious diseases, which poses a major risk to public health. In search of effective clinical solutions, the acquisition of knowledge and understanding of biomolecular processes associated with viral pathogens represents a prerequisite. In this context, biophysical engineering approaches are particularly promising since they can resolve biomolecular interactions systematically by circumventing the complexities associated with experiments involving natural biological systems. The engineering approaches encompass the design and construction of biomimetic platforms that simulate the physiological system. This approach enables us to characterize, measure, and quantitatively analyze biomolecular interactions.In this Account, we summarize biophysical measurements that our group has successfully adopted to develop broad-spectrum antiviral drugs based on the lipid envelope antiviral disruption (LEAD) strategy, targeting the structural integrity of the outer viral membrane to abrogate viral infectivity. We particularly focus on the engineering aspects related to the design and construction of the tethered lipid vesicle platform, which closely mimics the viral membrane. We first outline the development of the LEAD agents screening platform that integrates soft matter design components with biomaterials and surface functionalization strategies to facilitate parallel measurements tracking peptide-induced destabilization of nanoscale, virus-mimicking vesicles with tunable size and composition. Then, we describe how this platform can be effectively employed to gain insights into the membrane curvature dependency of certain peptides. The fundamental knowledge acquired through this systematic process is crucial in the identification and subsequent development of antiviral drug candidates. In particular, we highlight the development of curvature-sensitive α-helical (AH) peptides as a broad-spectrum antiviral agent that has been demonstrated as an effective therapeutic treatment against multiple enveloped viruses. Also, we introduce a tethered cluster of vesicles to mimic clusters of enveloped viruses, exhibiting higher infectivity levels in the biological system. Then, we discuss key considerations, including experimental artifacts, namely dye leakage and imaging-related photobleaching, and corresponding corrective measures to improve the accuracy of quantitative interpretation. With the ongoing development and application of the tethered lipid vesicle platform, there is a compelling opportunity to explore fundamental biointerfacial science and develop a new class of broad-spectrum antiviral agents to prepare for the future membrane-enveloped viral pandemics.

Entrepreneurial Talent Building for 21st Century Agricultural Innovation.

Agricultural innovation is a key component of the global economy and enhances food security, health, and nutrition. Current innovation efforts focus mainly on supporting the transition to sustainable food systems, which is expected to harness technological advances across a range of fields. In this Nano Focus, we discuss how such efforts would benefit from not only supporting farmer participation in deciding transition pathways but also in fostering the interdisciplinary training and development of entrepreneurial-minded farmers, whom we term "AgTech Pioneers", to participate in cross-sector agricultural innovation ecosystems as cocreators and informed users of developing and future technologies. Toward this goal, we discuss possible strategies based on talent development, cross-disciplinary educational and training programs, and innovation clusters to build an AgTech Pioneer ecosystem, which can help to reinvigorate interest in farming careers and to identify and address challenges and opportunities in agriculture by accelerating and applying advances in nanoscience, nanotechnology, and related fields.

Fabrication of schizophyllan hydrogel via orthogonal thiol-ene photopolymerization.

Schizophyllan (SPG) is a water soluble ß-glucan, which is obtained from Schizophyllum commune. SPG has been widely explored with its unique properties such as triple helical structure, immune-modulation, and anti-tumoral activity. In this study, we tried to fabricate SPG hydrogel via thiol-ene photopolymerization. SPG-norbornene and SPG-thiol were synthesized from ultrasonicated SPG. Two types of SPG hydrogels (SPG and PEG/SPG hybrid) were formed via thiol-ene photo-click reaction. By controlling the SPG content and stoichiometric balance of norbornene and thiol groups, swelling ratio and shear elastic modulus of SPG hydrogel could be controlled in the range of 20-60 and 0.5-10kPa, respectively. For PEG/SPG hybrid hydrogel, we found that the triple-helical structure of SPG played a role in physical network formation with thiol-ene cross-linking. The degradability of SPG hydrogel could be also modulated by varying formulation. Therefore, such highly tunable SPG hydrogels would be utilized for various applications.