Mitochondrion-Directed Nanoparticles Loaded with a Natural Compound and a microRNA for Promoting Cancer Cell Death via the Modulation of Tumor Metabolism and Mitochondrial Dynamics

Figures

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  Figure 1. Schematic, morphological characteristics, particle size, and zeta potential of E/LPN-KL and miR-125/SLN-KL formulations. (a) A schematic of E/LPN-KL and miR-125/SLN-KL, (b,c) TEM images of (b) E/LPN-KL and (c) miR-125/SLN-KL, (d) size distribution and (e) zeta potential of E/LPN-KL, and (f) size distribution and (g) zeta potential of miR-125/SLN-KL. The representative plots are shown (n = 3). E, ellagic acid; LPN, lipid-polymer nanoparticle; SLN, solid lipid nanoparticles.

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  Figure 2. Cellular internalization and uptake of DiI or FAM-miR125/SLN-KL into human tongue squamous carcinoma SAS cells. (a,b) SAS cells were treated with (a–c) DiI in LPN-KL or (b–d) FAM-miR125/SLN-KL for the indicated time intervals and observed with CLSM. (a) Green, MitoGreen; red, DiI; blue, DAPI; gray, EGFR. (b) Green, FAM-miR125; red, MitoRed; blue, DAPI; gray, EGFR. (c) Green, LysoGreen; red, DiI; blue, DAPI; gray, EEA1. (d) Green, FAM-miR125; red, LysoRed; blue, DAPI; gray, EEA1. Magnification: 1500×. The representative images are shown (n = 3). DiI, 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; FAM, carboxyl fluorescein; EEA1, early endosome antigen 1.

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  Figure 3. Effect of different treatments on mitochondrial reactive oxygen species (ROS), bioenergenesis, and dynamics in SAS cells. (a) Relative ROS percentage. (b) Relative ATP percentage. (c) Relative mitochondrial membrane potential (MMP) percentage. (a–c) Control (CTR) was normalized as 100%. The mean fluorescence intensity of the other treatments was normalized relative to CTR. Data are presented as means ± SD from n = 3. * p < 0.05 compared with CTR, † p < 0.05 compared with E, ‡ p < 0.05 compared with E/LPN, ¶ p < 0.05 compared with E/LPN-KL, and § p < 0.05 compared with miR-125/SLN-KL. (d) Oxygen consumption rate (OCR) and (e) extracellular acidification rate (ECAR) related to mitochondrial dynamics in SAS cells.

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  Figure 4. Effect of different formulations on the relative levels of (a) glucose uptake and (b) lipid accumulation. For (a,b): * p < 0.05: compared with CTR, † p < 0.05 compared with E/LPN, ‡ p < 0.05 compared with E/LPN, ¶ p < 0.05 compared with E/LPN-KL, and § p < 0.05 compared with miR125/LPN-KL via Student’s t-test analysis, respectively. (c,d) The expression of proteins associated with (c) lipid or glucose metabolism and (d) mitochondrial dynamics, fusion/fission, mitophagy, and necroptosis in SAS cells. After various treatments, the cells were evaluated with Western blot, and the representative blots were shown (n = 3).

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  Figure 5. Effect of various treatments on migration and multidrug resistance (MDR) pathway in SAS cells. (a) The migration assay was conducted to evaluate the SAS cells treated with different formulations of E and miR-125 for 15 h. Cellular images were then observed under a microscope. (b) Relative percentages of the cell migration area. * p < 0.05: compared with CTR, † p < 0.05 compared with E/LPN, ‡ p < 0.05 compared with E/LPN, ¶ p < 0.05 compared with E/LPN-KL, and § p < 0.05 compared with miR125/LPN-KL via Student’s t-test analysis. (c) The protein expression levels of epithelial–mesenchymal transition (EMT) and MDR were evaluated via Western blot after the SAS cells were given various treatments for 48 h. The experiments were performed in triplicate with similar results. (d) The cellular uptake of coumarin-6 (C6; a probe of E) was monitored by detecting the relative green fluorescence intensity of C6 by using a flow cytometer. * p < 0.05 compared with C6, † p < 0.05 compared with C6/LPN, ‡ p < 0.05 compared with C6/LPN-KL, and § p < 0.05 compared with miR125+C6/LPN-KL via Student’s t-test analysis, respectively.

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  Figure 6. (a,b) Cytotoxicity of E and miR-125 in various formulations on normal and cancer cells. (a) NOK (normal oral keratinocyte) and (b) SAS cells were treated with E and/or miR-125 in various formulations for 48 h. Cell viability was measured with sulforhodamine B (SRB) assay. Values are the mean ± SD (n = 3). For (a): * p < 0.05 compared with CTR; † p < 0.05 compared with E. For (b): * p < 0.05 compared with CTR; † p < 0.05 compared with E; ‡ p < 0.05 compared with E/LPN-KL; ¶ p < 0.05 compared with miR-125; § p < 0.05 compared with miR-125/SLN-KL. (c) The relative percentage of the apoptotic, necrotic, and dead cell population by Annexin V and propidium iodide (PI) assay. * p < 0.05 compared with CTR; † p < 0.05 compared with E; ‡ p < 0.05 compared with E/LPN; ¶ p < 0.05 compared with E/LPN-KL. (d) Effect of different treatments on the expression of proteins associated with the apoptosis of SAS cells. After various treatments, the cells were evaluated with Western blot, and their representative images were shown (n = 3).

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  Figure 7. In vivo quantitation of blood (a) cholesterol, (b) glucose, (c) creatine kinase-myocardial band (CKMB), (d) glutamate pyruvate transaminase (GPT), and (e) creatinine (CRE) levels in SAS-bearing mice in different treatment groups. * p < 0.05 compared with CTR, † p < 0.05 compared with E, ‡ p < 0.05 compared with E/LPN, ¶ p < 0.05 compared with E/LPN-KL, and § p < 0.05 compared with miR-125/SLN-KL.

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  Figure 8. Antitumor efficacy and body weight studies on SAS-bearing mice treated with different formulations. (a) Antitumor efficacy of various formulations intravenously injected into SAS-bearing mice. Tumor growth was measured with digital calipers every 2 days (statistical significance: ** p < 0.01; *** p < 0.001.); (b) body weight as a function of time in CT-26-bearing mice.