Background

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is characterized by chronic inflammation of the gastrointestinal (GI) tract and poses significant therapeutic challenges [1,2]. Conventional treatments often suffer from limited efficacy and significant systemic side effects due to nonspecific drug biodistribution. Lipid nanoparticles (LNPs) have emerged as a promising drug delivery platform, offering targeted delivery capability and enhanced biocompatibility [3,4]. However, assessing their cellular targets and biodistribution in the GI tract, particularly under inflammatory conditions, remains a challenge.

Existing methods for studying in vivo nanoparticle uptake, such as basic flow cytometry, provide useful insights but lack the dimensionality needed to resolve complex cellular heterogeneity. Recent advances in data analysis techniques, particularly t-distributed stochastic neighbor embedding (t-SNE), enable high-dimensional visualization and clustering, improving the identification of intricate cellular subpopulations [5,6].

This protocol builds upon previously established methods for studying nanoparticle–cell interactions by integrating t-SNE analysis into flow cytometric workflows [7]. This approach enables precise identification of colonic epithelial and macrophage populations in complex inflamed colon tissues and enhances the detection of subtle cellular patterns that traditional gating strategies may overlook. Unlike previous studies relying on single-dimensional gating, this protocol offers a robust multidimensional assessment of nanoparticle targeting and uptake.

Beyond its application to LNPs, this protocol can be adapted for other drug delivery systems, such as polymeric nanoparticles or exosomes, and in various disease models. Additionally, the integration of t-SNE into flow cytometry establishes a framework for studying cellular heterogeneity in other inflamed or complex tissues, such as respiratory epithelia. By addressing key limitations of traditional methodologies, this protocol advances nanoparticle drug delivery research and provides a powerful tool for exploring in vivo targeting mechanisms of nanoparticles.