Implementation of Microfluidic FRAP for Characterization of Nanoparticle Transport in Porous Biopolymer Networks

Date

2025-08-01

Author

Dağistan, Ege
Esmaeilzadeh, Pouriya
Büküşoğlu, Emre
Özçelikkale, Altuğ

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Nanomedicine features nanoparticles (NPs) that can be designed in various sizes, shapes and surface functionalities for therapeutic or diagnostic applications. Despite the major promise of nanomedicine for targeted drug delivery and patient-specific treatments, its clinical translation remains limited due to challenges in its effective delivery to the target tissue [1]. Specifically, the extracellular matrix (ECM), a heterogeneous porous network of biopolymers such as collagen, poses a significant barrier against NP transport in the tissue interstitium. Transport of NPs in engineered tissue scaffolds also face similar challenges. Unfortunately, the mechanisms behind hinderance of NP transport within ECM are poorly understood, limiting the design of nanomedicine for delivery. The problem is further complicated by lack of specialized and accessible tools for characterization of diffusive and advective transport properties of NPs. This study addresses this gap by implementing Fluorescence Recovery after Photobleaching (FRAP) [2] for use in shallow (<10-50 µm high) microchannels where ECM mimetic hydrogels are injected in and polymerized. In what we refer to as the microfluidic FRAP technique, containment of specimens in shallow microchannels combined with relatively large length and time scales of interstitial transport enable use of wide-field fluorescence LED illumination rather than the laser illumination, confocal imaging and specialized modules typically required for FRAP. In addition, hydrogels in microchannels can be precisely perfused to characterize and delineate the diffusive and advective transport properties. In this study, we develop and validate this technique by measuring effective diffusivity of fluorescent silica and polymeric NPs up to 400 nm in diameter in nanofibrous collagen type I hydrogels with collagen concentrations varying between 1.5 mg/ml and 6.0 mg/ml. The microfluidic FRAP measurements are compared with those from mean-square displacement analysis of single particle tracking, stochastic simulations based on Brownian dynamics, and measurements from previous studies in the literature. This work provides microfluidic FRAP as an accessible wide-field quantitative microscopy tool for transport characterization and lays the groundwork for investigating the role of other NP design features and ECM components in transport. The insights from this study will ultimately help develop a mechanistic understanding of NP transport for rational design of nanomedicine for optimal delivery.

URI

https://drive.google.com/file/d/1OwEILS7TLr8A7pYpU6H78kAIUUV7L-LU/view
https://hdl.handle.net/11511/117621

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Department of Chemical Engineering, Article