Increased photodynamic therapy sensitization in tumors using a nitric oxide-based nanoplatform with ATP-production blocking capability

Abstract

Photodynamic therapy (PDT) efficacy in cancer cells is affected by sub-physiological hypoxia caused by dysregulated and "chaotic" tumor microvasculature. However, current traditional O₂-replenishing strategies are undergoing their own intrinsic deficiencies. In addition, resistance mechanisms activated during PDT also lead the present situation far from satisfactory. Methods: We propose a nitric oxide (NO)-based theranostic nanoplatform by using biocompatible poly-lactic-co-glycolic acid nanoparticles (PLGA NPs) as carriers, in which the outer polymeric layer embeds chlorin e6 (Ce6) and incorporates L-Arginine (L-Arg). This nanoplatform (L-Arg@Ce6@P NPs) can reduce hyperactive O₂ metabolism of tumor cells by NO-mediated mitochondrial respiration inhibition, which should raise endogenous O₂ tension to counteract hypoxia. Furthermore, NO can also hinder oxidative phosphorylation (OXPHOS) which should cause intracellular adenosine triphosphate (ATP) depletion, inhibiting tumor cells proliferation and turning cells more sensitive to PDT. Results: When the L-Arg@Ce6@P NPs accumulate in solid tumors by the enhanced permeability and retention (EPR) effect, locally released L-Arg is oxidized by the abundant H₂O₂ to produce NO. In vitro experiments suggest that NO can retard hypoactive O₂ metabolism and save intracellular O₂ for enhancing PDT efficacy under NIR light irradiation. Also, lower intracellular ATP hinders proliferation of DNA, improving PDT sensitization. PDT phototherapeutic efficacy increased by combining these two complementary strategies in vitro/in vivo. Conclusion: We show that this NO-based nanoplatform can be potentially used to alleviate hypoxia and sensitize tumor cells to amplify the efficacy of phototherapy guided by photoacoustic (PA) imaging.

Keywords: nitric oxide; photodynamic therapy, hypoxia relief, mitochondrial respiration, adenosine triphosphate.

Scheme 1

Schematic illustration of the L-Arg@Ce6@P NPs, the NO-based theranostic nanoplatform which amplifies PDT efficacy by hypoxia relief and ATP depletion.

Figure 1

Morphology and characterization. (A) Schematic illustration of the synthesis process of L-Arg@Ce6@P NPs. (B) TEM image of L-Arg@Ce6@P NPs (scale bar: 0.2 µm). (C) SEM image of L-Arg@Ce6@P NPs (scale bar: 1.0 µm). (D) Size distribution of L-Arg@Ce6@P NPs as measured by DLS. (E) Zeta potential of different NPs (PLGA, L-Arg@P, Ce6@P, L-Arg@ Ce6@P). (n = 3). (F) UV-vis-NIR absorbance spectra of free Ce6 at elevated concentrations and the standard curve of free Ce6. (G) Absorbance spectra of different NPs (PLGA, Free Ce6, Ce6@P, L-Arg@Ce6@P) as recorded by UV-vis-NIR spectrophotometer.

Figure 2

Cells uptake of NPs and NO-mediated hypoxia relief. (A-B) Intracellular uptake of L-Arg@Ce6@P NPs as observed by CLSM and quantified by flow-cytometry analysis. (Scale bar: 50 µm). (C) Cumulative NO release from NPs. (n = 3, ***p < 0.001). (D-E) Cells stained with DAF-FM DA observed by fluorescence microscope and quantified by flow-cytometry analysis. (Scale bar: 50 µm). (F) Activity of cytochrome c oxidase (CcO) after different treatments. (n = 3, ***p < 0.001). (G) Schematic illustration about measuring the O₂ consumption of different groups. (H) Relative dissolved oxygen (DO) content changes in the cell medium of different groups. (I-J) Cells stained with ROS-ID in different groups observed by CLSM and quantified by flow-cytometry. (Scale bar: 20 µm).

Figure 3

ATP-depletion and effects on cells. (A) Mitochondria membrane potential changes of cells after different treatments observed by CLSM. Cells treated with CCCP (ETC inhibitor) as the positive control group. (scale bar: 50 µm). (B) Flow-cytometry-based JC-1 assay as a measure of mitochondrial depolarization. (C) Schematic illustration of hypoxia relief and ATP-depletion for amplifying PDT. (D) Content of ATP after incubating with different concentrations of free L-Arg. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001). (E) Content of ATP after different treatments. (n = 3, **p < 0.01, ***p < 0.001). (F) Cell cycle of cells after different treatments. (G) The percentage of each phases on cell cycle in different groups.

Figure 4

In vitro PDT of cancer cells. (A) Concentration-dependent ¹O₂ generation of L-Arg@Ce6@P NPs irradiated by 660 nm laser (5 mW cm^-2) for 4 min. (B-C) Intracellular ROS level observed by CLSM and quantified by flow-cytometry analysis after different treatments (5 mW cm^-2 ×3 min). (scale bar: 50 µm). (D-F) Relative cell viability of cells after different treatments (5 mW cm^-2 ×4 min or 8 min). (n = 3, *p < 0.05, **p < 0.01). (G-H) Cells stained with Calcein-AM/PI staining after different treatments (5 mW cm^-2 ×4 min) observed by CLSM and apoptosis quantified by flow-cytometry analysis. (-) means without laser irradiation, (+) means with laser irradiation. (Scale bar: 50 µm).

Figure 5

PA imaging of L-Arg@Ce6@P NPs in vitro and in vivo. (A) In vitro PA contrast images and PA values of L-Arg@Ce6@P NPs at different concentrations. (B) In vivo PA images of tumors in tumor-bearing mice after i.v. injection of L-Arg@Ce6@P NPs at different time points. (C) Changes of PA-signal intensities within tumor regions at corresponding time points. (n = 3).

Figure 6

Tumor-hypoxia status in vivo. (A) Schematic illustration of measuring oxyhemoglobin saturation by PA imaging. (B) PA images of tumor sites in oxy-hemoglobin mode at different time points. (C) Quantification of oxyhemoglobin saturation at tumor sites by measuring the ratios of oxygenated hemoglobin (λ = 850 nm) and deoxygenated hemoglobin (λ = 750 nm). (n = 3, ***p < 0.001). (D) Immunofluorescent images of tumor slices stained by the hypoxia probe and blood vessel probe. (scale bar: 50 µm). (E) Mean Fluorescence intensity quantitative analysis of HIF-α and anti-CD₃₁. (n = 3, **p < 0.01).

Figure 7

In vivo PDT of L-Arg@Ce6@P NPs. (A) Schematic illustration of the L-Arg@ Ce6@P NPs based PDT process. (B) Tumor growth curves of five groups after various treatments. (n = 5, ***p < 0.001). (C) Photographs of tumors dissected from mice of five groups after various treatments. (D)Weight of tumors 18 days post various treatments. (n = 5, **p < 0.01, ***p < 0.001). (E) Body-weight curves of five groups after various treatments. (F) H&E staining, immunochemical staining of TUNEL and PCNA on tumor sections from MDA-MB-231 tumor-bearing mice after various treatments. (scale bar: 100 µm). (G) Mean fluorescence intensity quantitative analysis of PCNA and TUNEL.