3D Printed X-ray Compatible Microfluidics for Online Characterization of Hexosomes: A Synchrotron SAXS-on-Chip Study with Molecular Dynamics Insights
- 1University of Copenhagen
Abstract
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.
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BibTeX
@misc{babaie2026printed,
title = {{3D Printed X-ray Compatible Microfluidics for Online Characterization of Hexosomes: A Synchrotron SAXS-on-Chip Study with Molecular Dynamics Insights}},
author = {Z. Babaie and M. Valerio and F. Schuhmann and M. Dimaki and B. Rezaei and W. Pezeshkian and S. S. Keller and W. E. Svendsen and P. C. T. d. Souza and A. Yaghmur},
howpublished = {bioRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.31.748233},
url = {https://www.biorxiv.org/content/10.64898/2026.08.31.748233v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - 3D Printed X-ray Compatible Microfluidics for Online Characterization of Hexosomes: A Synchrotron SAXS-on-Chip Study with Molecular Dynamics Insights
AU - Z. Babaie
AU - M. Valerio
AU - F. Schuhmann
AU - M. Dimaki
AU - B. Rezaei
AU - W. Pezeshkian
AU - S. S. Keller
AU - W. E. Svendsen
AU - P. C. T. d. Souza
AU - A. Yaghmur
PY - 2026
DA - 2026/08/31
PB - bioRxiv
DO - 10.64898/2026.08.31.748233
UR - https://www.biorxiv.org/content/10.64898/2026.08.31.748233v1
AB - 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.
ER -