ROS-responsive microspheres for on demand antioxidant therapy in a model of diabetic peripheral arterial disease

Abstract

A new microparticle-based delivery system was synthesized from reactive oxygen species (ROS)-responsive poly(propylene sulfide) (PPS) and tested for "on demand" antioxidant therapy. PPS is hydrophobic but undergoes a phase change to become hydrophilic upon oxidation and thus provides a useful platform for ROS-demanded drug release. This platform was tested for delivery of the promising anti-inflammatory and antioxidant therapeutic molecule curcumin, which is currently limited in use in its free form due to poor pharmacokinetic properties. PPS microspheres efficiently encapsulated curcumin through oil-in-water emulsion and provided sustained, on demand release that was modulated in vitro by hydrogen peroxide concentration. The cytocompatible, curcumin-loaded microspheres preferentially targeted and scavenged intracellular ROS in activated macrophages, reduced in vitro cell death in the presence of cytotoxic levels of ROS, and decreased tissue-level ROS in vivo in the diabetic mouse hind limb ischemia model of peripheral arterial disease. Interestingly, due to the ROS scavenging behavior of PPS, the blank microparticles also showed inherent therapeutic properties that were synergistic with the effects of curcumin in these assays. Functionally, local delivery of curcumin-PPS microspheres accelerated recovery from hind limb ischemia in diabetic mice, as demonstrated using non-invasive imaging techniques. This work demonstrates the potential for PPS microspheres as a generalizable vehicle for ROS-demanded drug release and establishes the utility of this platform for improving local curcumin bioavailability for treatment of chronic inflammatory diseases.

Keywords: Antioxidant; Diabetes; Hind limb ischemia; Inflammation; ROS; Smart polymer.

Fig. 1

The size distribution of curcumin-PPS microspheres, as analyzed by SEM, indicated that average microparticle diameter was 1.33 μm with a standard deviation of 0.55 μm. Scale bar is 10 μm.

Fig. 2

Curcumin release from PPS microparticles was ROS dose-dependent and on demand. A) In vitro release of curcumin from PPS microspheres exposed to temporally-constant H₂O₂ concentrations ranging from 0 mM to 3 wt% (882 mM) showed H₂O₂ dose-dependent release. B) In vitro release of curcumin from PPS microspheres with intermittent exposure to 0.1 mM, 1 mM, and 2 mM SIN-1 showed SIN-1 dose-dependent, on demand release during temporal phases when SIN-1 was present. n=3 for all samples.

Fig. 3

Curcumin loaded PPS microparticles reduce the cytotoxicity of H₂O₂. Cell survival was measured for luciferase-expressing 3T3 fibroblasts incubated for 24 hours with blank PPS microspheres, curcumin-PPS microspheres, or vehicle in media containing varied doses of H₂O₂. Curcumin dose is 3.4 μM in (A) and 27.1 μM in (B). Blank PPS microparticles represent the equivalent polymer dose (2.5 μg/mL PPS in (A) and 20.4 μg/mL PPS in (B)) used to deliver the corresponding curcumin dose. *p<0.05 relative to other treatment groups within each H₂O₂ dose. n=3 per group.

Fig. 4

Curcumin-PPS microspheres are preferentially internalized by activated macrophages and exert functional effects on ROS generation and MCP-1 secretion. A) Confocal microscopy revealed that curcumin-PPS microspheres were internalized to a greater degree by LPS/IFN-γ stimulated RAW cells relative to non-activated macrophages and fibroblasts, suggesting size dependent targeting of the microparticles to pro-inflammatory macrophages. Scale bar is 20 μm. Inset in stimulated RAW macrophages is the center slice of a z-stack, confirming cell internalization of microspheres. Inset scale bar is 5 μm. B) Quantitative analysis of microsphere uptake was performed using flow cytometry to measure intracellular curcumin fluorescence, and significant differences were observed between all groups (p<0.05). C) Intracellular ROS levels are reduced in LPS/IFN-γ-stimulated RAW macrophages by treatment with PPS microspheres and Cur-PPS microspheres (p<0.05). Intracellular ROS levels in activated macrophages treated with CUR-PPS microspheres were statistically equivalent to the non-activated RAW cells. D) Secretion of MCP-1 is reduced in LPS/IFN-γ-stimulated RAW macrophages by treatment with Cur-PPS microspheres relative to blank PPS microspheres (p<0.05). Microsphere doses contain 3.4 μM curcumin or the equivalent polymer dose. *p<0.05 for differences between indicated groups. #p<0.05 relative to unstimulated macrophages (LPS/IFN-γ(−)/NT group).

Fig. 5

PPS microspheres provide sustained, on demand local curcumin release and reduce tissue ROS levels in the ischemic limb in vivo. A) Curcumin-PPS microspheres release curcumin more rapidly in the ischemic limb in comparison to the control limb. B) ROS levels in the gastrocnemius muscle are increased at day 1 post-surgery (level of ROS is 2.3-fold greater in ischemic versus control gastrocnemius). C) Blank PPS microspheres and curcumin-loaded PPS microspheres significantly reduce ROS in gastrocnemius muscles extracted from ischemic limbs. Data presented as mean ± SEM. Saline group n=8, blank PPS group n=11, curcumin-PPS group n=10. *p<0.05 relative to saline treatment.

Fig. 6

Curcumin-loaded PPS microspheres improved ischemic limb recovery in the setting of diabetes in vivo. A) Representative images from the time course of hemoglobin oxygen saturation recovery from each treatment group delivered to the ischemic limb of diabetic mice. B) Hemoglobin saturation is significantly increased in the curcumin-PPS treated group (n=10) relative to the blank PPS (n=11) and saline-treated (n=8) groups over the time course of ischemic recovery. At day 2, the curcumin PPS group has a significantly higher hemoglobin saturation ratio compared to the saline group. Data presented as mean ± SEM. *Cur-PPS group is significantly different from PPS and Saline groups over the time course from day 2 to 6 (p<0.05). PPS and Saline groups are not significantly different (p>0.9). #Cur-PPS and Saline differ significantly (p<0.05).

Fig. 7

Vessel morphology was imaged non-invasively on day 7 with speckle variance OCT (n=4–5/group). A) Representative images of vessel morphology from each treatment group. Scale bar is 1 mm. B) Curcumin-PPS treated mice had a significant increase in length of vasculature with diameters between 25 μm and 125 μm relative to the blank PPS microsphere group (p<0.05).