Therapeutic Delivery of Phloretin by Mixed Emulsifier-Stabilized Nanoemulsion Alleviated Cerebral Ischemia/Reperfusion Injury

Abstract

Background: Cerebral ischemia/reperfusion injury (CIRI) is a major challenge in ischemic stroke treatment. Phloretin (PHL), despite its potent antioxidant and anti-inflammatory properties, has limited clinical application due to poor oral bioavailability. This study aimed to develop an orally administered phloretin-loaded nanoemulsion (NE-PHL) to enhance brain delivery and neuroprotective efficacy against CIRI. Methods: NE-PHL was optimized via an orthogonal experimental design combined with ultrasonication. The optimized formulation was characterized for physicochemical properties and evaluated for pharmacokinetics and brain bioavailability. Its therapeutic efficacy was assessed in middle cerebral artery occlusion (MCAO) rats by measuring infarct volume, neurological scores, oxidative stress markers, and inflammatory cytokines. RNA sequencing analysis was performed to elucidate the underlying mechanisms. Results: The optimized NE-PHL exhibited a small droplet size (96.26 ± 0.86 nm), high encapsulation efficiency (84.58 ± 3.03%), and good storage stability over a period of 120 days. Pharmacokinetic studies showed a 2.72-fold increase in AUC _0-12h for NE-PHL compared to free PHL. In MCAO rats, NE-PHL treatment significantly improved neurological function, reduced cerebral infarct volume, attenuated oxidative stress, and modulated inflammatory responses by suppressing pro-inflammatory cytokines and enhancing anti-inflammatory activity. RNA sequencing analysis further confirmed coordinated downregulation of key pathways related to oxidative stress and inflammation. Conclusions: NE-PHL represents a promising oral nanotherapeutic strategy for the effective management of CIRI, offering enhanced bioavailability and significant neuroprotection.

Keywords: cerebral ischemia/reperfusion injury; mixed emulsifiers; nanoemulsion; oxidative stress; phloretin.

Figure 1

Characterization of NE-PHL. Optimization on the ultrasonic times (A) and ultrasonic powers (B) of NE-PHL. (C) Macroscopic appearance of the optimized NE-PHL. The size (D) and zeta potential (E) of NE-PHL. (F) TEM image of NE-PHL. (G) Changes in the average size and PDI of NE-PHL during 30-day storage at 4 °C. Data are presented as mean ± SD (n = 3).

Figure 2

Results of the in vitro drug release and in vivo pharmacokinetics of NE-PHL. The in vitro release profile of NE-PHL in PBS (A), in SGF (B), and in SIF (C) (n = 3). (D) The concentration–time curves of free PHL and NE-PHL after oral administration in rats. (E–F) Qualitative and quantitative analysis of the fluorescence signals of NE-PHL in brain tissue at 3 h and 6 h following oral administration. (G) Bio-distribution of the fluorescence signal of NE-PHL 6 h after oral administration in rats at 6 h. Data are expressed as mean ± SD (n = 6). * p < 0.05, ** p < 0.01 compared to the control group.

Figure 3

Protective effects of NE-PHL on CIRI following oral administration. (A) Schematic representation of the experimental design for NE-PHL treatment in MCAO Rats; (B) Laser scatter imaging for monitoring alterations in CBF; (C) Semi-quantitative analysis of CBF. Data are presented as mean ± SD (n = 5). ** p < 0.01, **** p < 0.0001; (D) Neurological deficit scores of all experimental groups; (E) Representative images of TTC-stained brain slices; (F) Analysis of cerebral infarct volume. Data are presented as mean ± SD (n = 5). *** p < 0.001, **** p < 0.0001 compared to the sham group; ^# p < 0.05, ^### p < 0.001 compared to the MCAO group. The dash lines from up to down indicate 75th percentile, median line and 25th percentile.

Figure 4

NE-PHL mitigated oxidative stress in the cerebral tissue of rats with CIRI. The activities of oxidative stress markers were quantified, including (A) ROS, (B) MDA, (C) H₂O₂, (D) SOD, (E) GSH, and (F) GSH-Px (n = 5). (G) Radar chart analysis of the effects of NE-PHL on oxidative stress markers. Data are presented as mean ± SD ** p < 0.01, **** p < 0.0001 compared to the sham group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 compared to the NE-PHL group.

Figure 5

Post-stroke administration of NE-PHL notably reduced neuroinflammation in the cerebral tissue of MCAO rats. Levels of (A) IL-4, (B) IL-10, (C) IL-1β, (D) IL-6, and (E) TNF-α were quantified using ELISA (n = 5). (F) Radar chart analysis of the effects of NE-PHL on inflammatory cytokines. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001 compared to the sham group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 compared to the NE-PHL group.

Figure 6

RNA sequencing analysis of differential gene expression. (A) Heatmap showing correlated mRNA expression across MCAO, PHL, and NE-PHL groups. (B–D) Volcano plots of DEGs for NE-PHL vs. MCAO (B), PHL vs. MCAO (C), and NE-PHL vs. PHL (D) comparisons. The dashed line separates upregulated genes from downregulated genes. (E) Venn diagram. (F) KEGG pathway analysis of DEGs between MCAO and NE-PHL groups. (G) KEGG pathway enrichment network visualized with Cytoscape 3.7.1. (n = 3).

Figure 7

In vivo biosafety of NE-PHL. (A) H&E staining of the major organs of rat. Scale bar: 50 μm. Frequency of lymphocytes (B), monocytes (C), neutrophils (D), red blood cells (E), white blood cells (F), and platelets (G) determined in peripheral blood using a veterinary autohematology analyzer. Data are presented as mean ± SD (n = 7). Dashed lines stand for the normal level range of lymphocytes, monocytes, neutrophils, red blood cells, white blood cells and platelet in blood.