In Vivo Bioimaging and Biodistribution of DiR-Labeled Solid SNEDDS of Black Sticky Rice Extract Using Neusilin® US2 as Solid Carrier

Abstract

Black Sticky Rice Extract (BSRE; Oryza sativa var. glutinosa Blanco) is rich in anthocyanins and flavonoids with well-documented antioxidant and hepatoprotective activities. Nevertheless, its clinical translation is hindered by poor aqueous solubility and limited stability under physiological conditions, resulting in low oral bioavailability. To overcome these limitations, a Self-Nanoemulsifying Drug Delivery System (SNEDDS) was developed to enhance BSRE solubilization, stability, and hepatic targeting efficiency. A 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR)-labeled SNEDDS was formulated using corn oil, Tween 80, and propylene glycol, followed by solidification onto Neusilin® US2 to obtain solid SNEDDS (S-SNEDDS). Physicochemical properties, including flowability, bulk and tapped density, and residual moisture content, were evaluated to assess processability and stability. In vivo biodistribution and hepatic targeting were investigated in mice using IVIS Spectrum fluorescence imaging (700–900 nm) after oral administration of three formulations: BSRE + corn oil + DiR, blank SNEDDS + DiR, and SNEDDS-BSRE + DiR. Quantitative region-of-interest (ROI) analysis was performed using Living Image® software, and statistical differences were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. The S-SNEDDS granules exhibited excellent flow properties (angle of repose = 27°, Carr’s index = 13.5%, Hausner’s ratio = 1.16) and optimal residual moisture (3.20%), confirming good solid-state stability and reconstitution performance. IVIS imaging revealed formulation-dependent biodistribution profiles: BSRE + corn oil produced diffuse systemic fluorescence, blank SNEDDS + DiR showed weak and transient signals, whereas SNEDDS-BSRE + DiR demonstrated moderate but sustained hepatic fluorescence. Quantitative ROI analysis confirmed significant intergroup differences (p < 0.001). Overall, incorporation of BSRE into the SNEDDS matrix enhanced nanocarrier stability and hepatic localization, highlighting the potential of SNEDDS-BSRE as a bioimaging-guided oral nutraceutical platform for liver-targeted delivery.

Introduction

Liver diseases constitute a major and growing global health burden arising from exposure to hepatotoxins, excessive alcohol consumption, viral infections, and metabolic disorders such as non-alcoholic fatty liver disease (NAFLD). Chronic liver disorders are responsible for more than two million deaths annually worldwide,  highlighting the urgent need for effective preventive and therapeutic interventions. As the primary organ responsible for metabolism and detoxification, the liver requires sufficient and sustained delivery of hepatoprotective agents to achieve therapeutic concentrations while minimizing premature degradation and systemic clearance. However, many natural hepatoprotective phytoconstituents, particularly flavonoids and anthocyanins, exhibit poor aqueous solubility, limited stability in the gastrointestinal tract, and extensive first-pass metabolism, which collectively restrict their oral bioavailability and clinical translation [1]. To address these challenges, lipid-based nanocarrier systems, especially Self-Emulsifying Drug Delivery Systems (SEDDS) and their nanoscale counterparts (SNEDDS), have gained increasing attention. Upon exposure to gastrointestinal fluids, SNEDDS spontaneously form fine oil-in-water nanoemulsions that markedly enhance solubilization and dissolution of poorly water-soluble compounds [2]. In addition, these systems promote intestinal permeability and lymphatic uptake, thereby partially bypassing presystemic hepatic metabolism and improving oral bioavailability [3]. Beyond pharmacokinetic enhancement, SNEDDS protect labile bioactives from enzymatic degradation, prolong the absorption window, and enable controlled biodistribution, making them particularly suitable for complex natural products with synergistic pharmacological profiles [4].

Black Sticky Rice Extract (BSRE; Oryza sativa var. glutinosa Blanco) represents a rich source of bioactive phytochemicals, dominated by anthocyanins, particularly cyanidin-3-O-glucoside (Cy3G), along with diverse phenolic and flavonoid compounds [5]. These constituents have been widely reported to exert potent antioxidant, anti-inflammatory, and hepatoprotective effects through modulation of oxidative stress and inflammatory pathways, including NF-κB and TNF-α signaling, in experimental models of liver injury [6]. Despite its promising biological activity, the therapeutic application of BSRE remains limited due to the inherent instability of anthocyanins. These compounds are highly susceptible to degradation under physiological conditions, including pH variation, enzymatic hydrolysis, and microbial metabolism in the gastrointestinal tract, resulting in poor intestinal absorption and rapid systemic elimination [7]. Lipid-based nanoformulation strategies, particularly SNEDDS, offer a rational approach to overcoming these limitations. Upon oral administration, SNEDDS generate nanosized emulsions that enhance the solubilization of lipophilic and semipolar constituents, protect unstable anthocyanins from degradation, and facilitate absorption via intestinal lymphatic pathways [8].

Importantly, SNEDDS have been shown to improve both the bioavailability and pharmacokinetic stability of polyphenols and flavonoids compared with conventional extract formulations [9]. However, despite extensive reports on bioavailability enhancement, systematic evaluation of the in vivo biodistribution of SNEDDS, especially with respect to hepatic localization, remains limited. Therefore, the present study aimed to investigate the in vivo biodistribution of DiR-labeled solid Self-Nanoemulsifying Drug Delivery Systems (S-SNEDDS) incorporating Black Sticky Rice Extract using IVIS Spectrum fluorescence imaging. The optimized formulation demonstrated favorable physicochemical characteristics, including droplet size, stability, and reconstitution behavior. Comparative bioimaging and quantitative region-of-interest (ROI) analyses were performed across three formulations: BSRE + corn oil + DiR, blank SNEDDS + DiR, and SNEDDS-BSRE + DiR, to elucidate formulation-dependent biodistribution patterns and hepatic targeting efficiency in live mice.

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Anggun Hari Kusumawati, Elfahmi Elfahmi, Afrillia Nuryanti Garmana, Rachmat Mauludin, In Vivo Bioimaging and Biodistribution of DiR-Labeled Solid SNEDDS of Black Sticky Rice Extract Using, Neusilin® US2 as Solid Carrier, Received: 2025.10.22; Accepted: 2026.01.20; Published: 2026.04.16, 2026; 10: 104-114. doi: 10.7150/ntno.127165, Nanotheranostics 2026, Vol. 10

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