Design and optimization of in situ self-functionalizing stress sensors

TL;DR

The paper tackles the challenge of in vivo mechanical stress measurement by introducing biocompatible inverted-emulsion oil droplets that act as self-functionalizing stress sensors. It presents a design that balances deformability (low interfacial tension) with controlled destabilization (self-functionalization) and demonstrates two scalable formation methods (SPG membranes and PVDF filters) to produce droplets of ~0.66–0.90 µm. The authors validate a mechanistic framework linking external tissue stress to droplet deformation within soft media using elastocapillary concepts, and they quantify local stresses with σ_loc = 2 γ (C_b − C_a) and a dimensionless collapse parameter ε* = (σ∞/E) / (6 + 15 γ/(E R_d)). They confirm functionalization and payload release in vitro and demonstrate in vivo deployment in brain organoids and zebrafish embryos, illustrating a practical approach for mapping stresses and delivering cargo in living tissues.

Abstract

Mechanical contributions are crucial regulators of diverse biological processes, yet their \textit{in vivo} measurement remains challenging due to limitations of current techniques, that can be destructive or require complex dedicated setups. This study introduces a novel method to synthesize biocompatible, self-functionalizing stress sensors based on inverted emulsions, that can be used to probe stresses inside tissues but can also locally perturb the biological environment through specific binder presentation or drug delivery. We engineered an optimal design for these inverted emulsions, focusing on finding the balance between the two contradictory constraints: achieving low surface tension for deformability while maintaining emulsion instability for efficient self-functionalization and drug release. Proof-of-concept experiments in both agarose gels and complex biological systems, including brain organoids and zebrafish embryos, confirm the droplets ability to deform in response to mechanical stress applied within the tissue, to self-functionalize and to release encapsulated molecules locally. These versatile sensors offer a method for non-invasive stress measurements and targeted chemical delivery within living biological tissues, giving the potential to overcome current technical barriers in biophysical studies.

Figures (10)

• Figure 1: Schematic illustration of an inverted emulsion oil droplet and its different functionalities. (A) Release of hydrophilic molecules from inner water droplets into the surrounding aqueous phase upon destabilization of the inverted emulsion. (B) Self-functionalization of an oil droplet. The surface of the inner water droplets is decorated with molecules through specific interactions with the lipids. Upon destabilization of the inverted emulsion, the inner water droplets fuse with the outer surface of the oil droplet, which results in a redistribution of surface molecules on surface of the oil droplet. (C) Schematic representation of surface functionalization with phospholipids. Here, the biotinylated phospholipids (16:0 PE biotin) are decorated with fluorescent streptavidin, onto which any biotinylated molecule can be grafted.
• Figure 2: Schematic illustration of the emulsification method through the Shirasu Porous Glass (SPG) membrane connector. (A) View of a connector filled with the mix of water and oil phases, with two syringes A and B, locked on both sides. Pushing the syringes back and forth successively results in the repeated extrusion of the aqueous phase through the porous membrane. (B) Scheme of the extrusion at the membrane pore scale. The water phase (light blue) is going through the 5µm pores of the membrane and the resulting shear creates water droplets in the oil phase (yellow) that are progressively stabilized with the amphiphilic molecules dispersed in the oil phase.
• Figure 3: Size distribution of the water-in-oil emulsion. (A) Typical field of view of an emulsion made through 5µm SPG membrane connector. The oil contains 0.5mgmL lipids and 0.25mgmL Tween 20. The water droplets are labelled with Streptavidin Alexa 546 and observed through spinning disk confocal microscopy (60X objective). (B) Surfacic Cumulative Distribution Function of water droplet diameters (each droplet has a ponderation in the distribution equal to its surface area) for emulsions prepared with the SPG connector at 0.5mgmL lipids + 0.25mgmL Tween 20 (dark pink circles, N=5228 droplets), emulsions using the same formulation but PVDF filters (light pink stars, N=617 droplets), and emulsions with only 0.5mgmL lipids prepared with PVDF filters (blue circles, N=1296 droplets).
• Figure 4: Scheme of an oil droplet deformed by a stress imposed on the surrounding medium. (A) Schematic representation of an oil droplet embedded in an elastic medium of Young modulus E. (B) When the medium is under compression, here with an imposed deformation $\epsilon$, it can deform of the oil droplet with a local extensile stress $\tau$, if capillary stresses $\gamma/R_d$ do not dominate.
• Figure 5: Interfacial tension measurements. (A) Schematic illustration of the rising drop tensiometry set-up. (B) Axisymmetric shape profile of the rising drop with the red arrow pointing the final converged guess for the solution of Young-Laplace equation, based on edge detection (see Methods). Illustration of pear-shape like droplet made of a continuous oil phase with surfactants (C) and made of an inverted emulsion (D). (E) Exponential decay of interfacial tension in time. The droplets made of oil (yellow circles) and emulsion (pink diamonds) contain 0.5mgmL 16:0 Biotinyl PE and 0.25mgmL Tween 20. Each time point represents a mean value of interfacial tension with the shaded area representing the 95% confidence intervals (CI).