Dimaki, M. | Search bioRxiv

bioRxiv2026

3D Printed X-ray Compatible Microfluidics for Online Characterization of Hexosomes: A Synchrotron SAXS-on-Chip Study with Molecular Dynamics Insights

Online structural characterization during microfluidic lipid self-assembly is important for understanding and controlling the formation of nonlamellar liquid crystalline nanodispersions. Here, we report a 3D-printed, X-ray-compatible hydrodynamic flow-focusing microfluidic chip with variable channel dimensions, integrated with synchrotron small-angle X-ray scattering (SAXS), for position-resolved SAXS-on-chip monitoring of Ca2+-triggered hexosome formation. Hexosomes were produced under continuous flow by mixing ethanolic solutions of docosahexaenoic acid monoglyceride (MAG-DHA), the negatively charged phosphatidylglycerol DOPG, and -tocopherol with Ca2+-containing PIPES buffer. Online SAXS-on-chip measurements detected three Bragg reflections characteristic of the internal inverse hexagonal (H2) phase on a tens-of-milliseconds residence-time scale, revealing rapid structural evolution during microfluidic mixing. Complementary ex situ SAXS identified the DOPG/Ca2+ molar ratio as a key parameter modulating the direct vesicle-to-hexosome transformation and the compactness of the internal H2 nanostructures. Dynamic light scattering showed that the flow-rate ratio modulated nanoparticle size, yielding hexosomes with mean hydrodynamic diameters in the range of approximately 120-175 nm and polydispersity index values down to 0.14 at a total flow rate of 200 {micro}L min-1. Cryo-TEM revealed coexistence of hexosomes and vesicular nanostructures, highlighting morphological heterogeneity, while Coarse-Grained Molecular Dynamics simulations supported a central role of Ca2+-DOPG association in promoting a direct lamellar-H2 phase transition. Overall, this work shows that 3D-printed SAXS-compatible microfluidics can integrate continuous production with online structural characterization, providing a basis for future formulation and process optimization of drug-loaded cubosomes, hexosomes, and related nonlamellar liquid crystalline nanodispersions.

biophysics↗

bioRxiv2025

Butterflies Are Shrinking: Evidence from the Past Century

Climate change has had strong impacts on biodiversity, including well-documented shifts in the distributions and phenology of species. Reductions in body size represent a third pervasive biological response; it has garnered significantly less attention despite great ecological relevance. Theoretical frameworks-the temperature-size rule, Bergmanns rule, and Jamess rule-predict that warmer temperatures are associated with smaller body sizes in ectotherms. In contrast, empirical evidence concerning this pattern is mixed across both taxa and environments. In the present study, we applied computer vision techniques to historical data from two large butterfly museum collections, totaling 593 species across 10 terrestrial biomes over more than a century, in order to investigate long-term trends in butterfly body size. We measured forewing length through both manual image analyses and automated computer vision algorithms proxying body size and analyzed trends by using generalized additive models in order to consider a temporal, biome-specific pattern assessment. We have tested two hypotheses: that butterfly body size (1) declines over time, in conjunction with increasing ambient temperatures, in agreement with the temperature-size rule, and (2) its variation is more marked in warm, dry biomes. Results indicate a significant overall reduction in the body size of butterflies during the last century and that this reduction is indeed more pronounced in those biomes facing higher rises in temperature. These findings constitute large-scale evidence in support of the temperature-size rule and indicate a potential ecological impact of climate change on butterfly populations.

ecology↗