Nanosuspensions, also known as crystalline or amorphous nanoparticles, are recognized as effective means to improve the oral bioavailability of poorly soluble drugs [1,2]. Preparing poorly soluble ingredients into nanoscale formulations can effectively improve their solubility and stability, thereby enhancing their oral bioavailability [3,4]. For orally administration, the transport and absorption of drugs through the gastrointestinal tract, are critical factors that affect their bioavailability [5]. In general, drugs are susceptible to the influence of the gastrointestinal environment and epithelial cells, making it difficult for their therapeutic effects to be fully realized. The intestinal mucus layer is the first major barrier for nanoparticles during their absorption in the intestine, while the intestinal epithelial cell barrier acts as the second major physiological barrier for nanoparticle absorption in the small intestine. Normally, the mucus layer hampered drug absorption, however, it was found that the ability of mucus layer to trap particles [6] and the high bioadhesion properties of the formulation [7] may contribute to the absorption of drugs. In addition, the absorption of drugs may depend on a variety of factors, such as particle size, shape, surface charge, hydrophilicity, composition, architecture and carrier material, as well as the molecular weight and solubility of drug [8].

Previous studies have shown that the absorption enhancement mechanisms of nanoparticle delivery systems mainly include the following aspects: increasing drug permeability across the normal intestinal epithelial cell membrane [9,10]; forming drug absorption pathways through M cells in the intestinal mucosa [11]; opening intercellular tight junctions to facilitate paracellular drug transport [12,13]; inhibiting the function of efflux proteins [[14], [15], [16]]. However, due to the lack of efficient particle qualitative and quantitative methods, the challenge of accurate online tracking of particles remains unresolved, which has led to an unclear understanding of the absorption and transport mechanisms of nanosuspensions.

In our previous study, an aggregation-caused quenching (ACQ) probe was used to successfully trace the process of nanoparticles in vivo [[17], [18], [19]], which proved that nanosuspensions is absorbed in the form of integral nanoparticles [20,21]. In this study, herpetrione, showed in Fig. 1A, a poorly soluble lignan ingredients extracted from the seeds of herpetospermum caudigerum Wall and being widely used in the treatment of liver diseases. As one of its main active ingredients, HPE belongs to BCS II class, with poor water solubility and low bioavailability. HPMC has a wide range of functions and is commonly used as a stabilizer, suspension aid and binder, and has been proven to be a good stabilizer for amorphous preparations and has good bioadhesion. We combined the safety and intestinal adhesion advantages of HPMC E15, herpetrione amorphous nanoparticles (HPE-ANPs) with hydroxypropylmethyl cellulose as stabilizer were prepared by physically embedding ACQ probes inside nanoparticles via anti-solvent method, aimed at improving the solubility and oral bioavailability. The ACQ probe possesses sensitive and accurate fluorescence signal conversion ability, which can accurately reflect the structural changes and fluorescence intensity of HPE-ANPs [22,23]. The concentration of nanoparticles was measured by fluorescence quantitative methods, the particles can be visualized and quantified in cells, which provides a new scientific and practical methods to reveal the uptake and transport process of HPE-ANP. At the same time, HPLC/LC-MS can quantitatively detect the total amount of drug absorbed by nanosuspensions. To evaluate and summarize the absorption and transport characteristics of different drug forms.