7-Dehydrocholesterol Encapsulated Polymeric Nanoparticles As a Radiation-Responsive Sensitizer for Enhancing Radiation Therapy

Abstract

Therapeutics that can be activated by radiation in situ to enhance the efficacy of radiotherapy are highly desirable. Herein, 7-Dehydrocholesterol (7-DHC), a biosynthetic precursor of cholesterol, as a radiosensitizer, exploiting its ability to propagate the free radical chain reaction is explored. The studies show that 7-DHC can react with radiation-induced reactive oxygen species and in turn promote lipid peroxidation, double-strand breaks, and mitochondrial damage in cancer cells. For efficient delivery, 7-DHC is encapsulated into poly(lactic-co-glycolic acid) nanoparticles, forming 7-DHC@PLGA NPs. When tested in CT26 tumor bearing mice, 7-DHC@PLGA NPs significantly enhanced the efficacy of radiotherapy, causing complete tumor eradication in 30% of the treated animals. After treatment, 7-DHC is converted to cholesterol, causing no detectable side effects or hypercalcemia. 7-DHC@PLGA NPs represent a radiation-responsive sensitizer with great potential in clinical translation.

Keywords: 7-dehydrocholesterol; colon carcinoma; nanoparticles; radiation therapy; radiosensitizers.

Figure 1.

Physicochemical characterizations of 7-DHC@PLGA NPs. (a) Photograph of 7-DHC@PLGA NPs in water. (b) Negative staining TEM image of 7-DHC@PLGA NPs. Scale bar, 100 nm. (c) Zeta potential of 7-DHC@PLGA NPs in PBS and water. (d) DLS analysis of 7-DHC@PLGA NPs in water. (e) Size change of 7-DHC@PLGA NPs, when incubated in PBS at 37 °C incubation for 48 h. (f) 7-DHC release from 7-DHC@PLGA NPs, tested in solutions with pH 7.4, 6.5, and 5.5, respectively.

Figure 2.

Evaluate the impact of 7-DHC@PLGA NPs on cell lipid peroxidation and viability in CT26 cells. (a) Cell viability in the absence of radiation, measured by MTT assays at 24 h. Both 7-DHC@PLGA NPs and free 7-DHC showed minimal toxicity when 7-DHC concentration was below 12.5 μg/mL. (b) Cell viability in the absence of radiation, measured by ATP bioluminescence at 24 h. c, d) Impact on cell lipid peroxidation, tested with 7-DHC@PLGA NPs (equivalent to 5 μg 7-DHC per mL) in the presence of radiation (5 Gy). PBS, 7-DHC@PLGA NPs, and ionizing radiation alone (IR) were tested as controls. (c) Caspase-3 activity, measured by FAM-FLICA Caspase 3/7 assay. (d) Cell viability, evaluated by MTT assays at 24 h. For comparison, carnosine, which inhibits the formation of alpha-beta unsaturated aldehydes, was added during the treatment for comparison. (e) Clonogenic assay, tested with PBS, IR, and 7-DHC@PLGA NPs plus IR (7-DHC@PLGA NPs+IR). Survival fractions relative to the unirradiated control were fit into the linear quadratic (LQ) model. *, p < 0.05; ns, no significant difference.

Figure 3.

Evaluate the impact of 7-DHC@PLGA NPs on the mitochondria and DNA. 7-DHC@PLGA NPs (equivalent to 5 μg 7-DHC per mL) were incubated with CT26 cells, followed by 5 Gy radiation at 24 h (7-DHC@PLGA NPs+IR). PBS, irradiation (IR), and 7-DHC@PLGA NPs only were tested as a comparison unless specified otherwise. (a) Proposed mechanisms for 7-DHC-enhanced lipid peroxidation under radiation. 7-DHC easily loses one H‧ to radiation-induced ROS. The resulting radical quickly reacts with O₂ to form a peroxide, which then undergoes autoxidation to form oxysterols such as DHCEO. Alternatively, the radical reacts with PUFAs in cell membranes, triggering lipid peroxidation. The products of lipid oxidation include reactive aldehydes and ketones such as MDA. (b) BODIPY C11 lipid peroxidation assay results. A decrease in the 590/510 nm fluoresce intensity ratio indicates an increased level of lipid peroxidation. (c) MDA levels, measured by TBARS assay. Cells treated with 7-DHC@PLGA NPs+IR showed a dramatic increase of MDA level. (d) MnSOD activity results. (e) DHE assay results. An increase in fluorescence indicates an elevated superoxide level in cells. (f) DNA damage. Double-strand breaks were measured by anti-γH2AX staining (left). Blue, DAPI; Red, γH2AX. Positively stained foci per cell was quantified by ImageJ using ITCN (right). (g) Cytochrome c release. Cells received anti-cytochrome c and mitochondrial complex Vα double staining (left). Green, cytochrome C; Red, complex Vα. Signal colocalization was computed by ImageJ (right). A decrease in colocalization rate indicates increased cytochrome c release. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, no significant difference.

Figure 4.

Evaluate radiosensitizing effects in vivo in CT26 tumor bearing nude mice. (a) Whole body fluorescence imaging. Images were taken 0.5 min, 4 h and 24 h post injection of DiR-labeled 7-DHC@PLGA NPs. (b) Tumor-to-muscle ratios, based on ROI analyses of results from (a). (c) Biodistribution of nanoparticles, based on ex vivo imaging performed at 24 h. (d-g) Therapy studies. 7-DHC@PLGA NPs (10 mg/kg) were intravenously (i.v.) injected, followed by beam radiation (3 Gy) applied to tumors at 4 h (7-DHC@PLGA NPs+IR; n=10). A total of three doses of treatment was given two days apart. Irradiation only (IR), 7-DHC@PLGA NPs only (7-DHC@PLGA NPs), and carrier only (PBS) were tested (n=5). (d) Schematic illustration of the treatment schedule. (e) Tumor growth curves. 7-DHC@PLGA NPs+IR caused the most effective tumor suppression, with 30% of the animals being tumor-free on Day 43. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, no significant difference. (f) Animal survival. Sixty percent of the animals in the 7-DHC@PLGA NPs+IR group remained alive on Day 43, while all animals in control groups had either died or reached a humane endpoint by the time. (g) TUNEL, Ki67, and H&E staining of tumor samples from different treatment groups. Scale bars, 200 nm.

Figure 5.

Evaluate toxicity of 7-DHC@PLGA NPs. (a) Body weight changes. No significant body weight drop was observed throughout the therapy study. (b) Post-mortem H&E staining of normal tissues. No sign of toxicity was observed. Scale bars, 200 μm. (c) Serum calcium level change. 7-DHC@PLGA NPs (1 mg/kg), calcitriol (200 μg/kg), 7-DHC (200 μg/kg), and PBS were intraperitoneally injected into healthy balb/c mice (n=3). Blood samples were collected at 0, 2, 6, and 24 h for analysis. No calcium level increase was observed in animals injected with 7-DHC@PLGA NPs.

Scheme 1.

Schematic illustration of radiosensitization with 7-DHC@PLGA-NPs. 7-DHC@PLGA NPs accumulate in tumors through the EPR effect. 7-DHC is released from the nanoparticles and enriched in cancer cells’ plasma and mitochondrial membrane. Under radiation, 7-DHC triggers and propagates radical chain reactions that cause lipid peroxidation, exacerbating oxidative stress in cells. Meanwhile, lipid peroxidation also produces toxic oxysterols and aldehydes that may react with DNA and other biomolecules. All these events culminate at inducing cancer cell death.