Development of rice bran-derived nanoparticles with excellent anti-cancer activity and their application for peritoneal dissemination

Abstract

Background: Rice bran a by-product of the rice milling process is currently underutilized. Recent studies have shown that plant-derived nanoparticles (pdNPs) can be mass-produced at a low cost and exhibit biological and therapeutic activities. Rice bran contains various anti-cancer compounds, including γ-oryzanol and γ-tocotrienol, and rice bran-derived nanoparticles (rbNPs) can be employed as novel therapeutic agents for cancer treatment.

Results: Koshihikari rice bran was suspended in water, and the suspension was centrifuged and filtered through a 0.45-µm-pore size syringe filter. The filtrate was ultracentrifuged, and the precipitates were suspended to obtain rbNPs. The rbNPs were negatively charged exosome-like nanoparticles with an average diameter of approximately 130 nm. The rbNPs exhibited cytotoxic activities against cancer cells but not against normal cells. The cytotoxic activity of rbNPs to murine colon adenocarcinoma colon26 cells was significantly greater than DOXIL^® or other pdNPs. The rbNPs induced cell cycle arrest and apoptosis, and reduced the expression of proliferative proteins, including β-catenin and cyclin D1. Intraperitoneal injections of rbNPs into mice bearing peritoneal dissemination of colon26 cells significantly suppressed tumor growth with no significant adverse effects.

Conclusion: These results indicated that rbNPs are promising nanoparticles, hold significant potential for anti-cancer applications, and are expected to play a vital role in cancer treatment.

Keywords: Apoptosis; Cancer therapy; Drug delivery system; Plant-derived nanoparticles; Rice bran.

Fig. 1

Preparation and characterization of rbNPs. (A) Schematic diagram of rbNP preparation. Rice bran suspension (rb-juice) was sequentially centrifuged, filtered, and ultracentrifuged to obtain rbNPs after filtration. (B) Size distribution of rbNPs determined by NanoSight NS300. The red area indicates the standard deviation of five measurements. (C) A TEM image of rbNPs. The image was obtained using H-7650 TEM. The scale bar indicates 100 nm. rbNPs, rice bran-derived nanoparticles; TEM, transmission electron microscopy

Fig. 2

Interaction of rbNPs with culture cells. (A) Cell number is measured by CCK-8 assay after 24 h incubation with rbNPs or PS-Lip at varying concentrations. Colon26, B16-BL6, HeLa, MDCK, HaCaT and RAW264.7 cells are incubated with 0.1−10 × 10⁹ rbNPs or PS-Lip/mL. Results are expressed as the mean ± SD of three samples. #p < 0.01 vs. no treatment (NT) group. (B) Confocal microscopic images of colon26 cells after the addition of DiI-labeled rbNPs (DiI-rbNPs). Colon26 cells are incubated with 0.1−10 × 10⁹ DiI-rbNPs/mL for 1, 3, and 12 h. Scale bars indicate 50 μm. White arrows indicate DiI-rbNPs. (C) Cellular uptake of DiO-labeled rbNPs (DiO-rbNPs) in colon26 cells. Colon26 cells are incubated with DiO-rbNPs for 3, 6, 12, and 24 h at 37 °C, then fixed with paraformaldehyde. The fluorescence intensity of colon26 cells is quantified by flow cytometry, and the mean fluorescence intensity (MFI) is calculated. Results are expressed as the mean ± SD of three samples. #p < 0.01 vs. NT group. Colon26, murine colon adenocarcinoma cell line; B16-BL6, murine melanoma cell line; HeLa, human cervix adenocarcinoma cell line; MDCK, canine kidney cell line; HaCaT, human keratinocyte cell line; and RAW264.7, murine macrophage cell line

Fig. 3

Comparison of rbNPs with other pdNPs, DOXIL^® or rb-sup. (A) The number of colon26 cells 24 h after the addition of grape, ginger, and lemon NPs, and rbNPs at varying concentrations. Colon26 cells are incubated with 0.1−10 × 10⁹ NPs/mL, and the cell number is measured at 24 h using CCK8 assay. Results are expressed as the mean ± SD of four samples. #p < 0.01 vs. NT group. (B) The number of colon26 cells 24 h after the addition of rbNPs or DOXIL^®. Colon26 cells are incubated with 0.1−10 × 10⁹ NPs/mL of rbNP or DOXIL^®, and the cell number is measured as described earlier. Results are expressed as the mean ± SD of three samples. #p < 0.01 vs. DOXIL^®. (C) The number of colon26 cells 24 h after addition of rbNPs and rb-sup. Colon26 cells are incubated with 1,000 µg/mL of rbNP or rb-sup, and the cell number is measured at 24 h using CCK8 assay. Results are expressed as the mean ± SD of three samples. *p < 0.05. #p < 0.01. (D) Western blot analysis of β-catenin, cyclin D1, and β-actin in colon26 cells. Colon26 cells are treated with 1,000 µg/mL rbNP or rb-sup for 24 h, and the cellular proteins are extracted for the analysis The bands of each protein are visualized using Invitrogen iBright Imaging Systems. (E) DNA fragmentation of colon26 cells after addition of rbNPs, rb-sup, or actinomycin D. Colon26 cells are incubated with 1 µM actinomycin D or 1,000 µg/mL rbNP or rb-sup. The DNA of the cells is extracted and subjected to 3% agarose gel electrophoresis, followed by visualization using Invitrogen iBright Imaging Systems. (F) Confocal images of colon26 cells stained with DAPI. Colon26 cells are incubated with 1,000 µg/mL rb-sup or rbNP for 24 h at 37 °C, fixed with paraformaldehyde, and the nuclei of the cells are stained with DAPI. Scale bars indicate 50 μm (low magnification) and 10 μm (high magnification). White arrows indicate chromatin condensation

Fig. 4

Anti-cancer effect of rbNPs in peritoneal dissemination model mice. (A) Flow diagram for the evaluation of the anti-cancer effect of rbNPs in peritoneal dissemination model mice. Colon26/fluc cells are transplanted to the peritoneal cavity of mice, and rbNPs are injected with three cycles of 3 daily injections and 1 injection-free day between cycles. At day 12, mice are subjected to in vivo imaging. (B) In vivo imaging of colon26/fluc cells in mice. Mice are anesthetized, and injected with VivoGlo™ Luciferin, and the luminescence derived from colon26/fluc cells in mice is detected. (C) The sum intensity of luciferase activity is calculated based on the images of Fig. 4B. Results are expressed as the mean ± SD of three or six. #p < 0.01. ns, not significant. (D) Body weight changes of mice. The body weight of mice is measured daily. Results are expressed as the mean ± SD of three or six mice. *p < 0.05 vs. NT group; ns, not significant. (E) Fluorescence images of mouse organs harvested 15 min, 1, 3, 6, and 24 h after injection of DiR-rbNPs or DiR. BALB/c mice are intraperitoneally injected with DiR-rbNPs or DiR. At 15 min, and one, three, six, and 24 h after injection, the fluorescence intensity of organs is visualized using an in vivo imaging system

Fig. 5

Adverse effects of rbNPs in mice. Mice are injected with rbNPs according to the same cycle described in the anti-tumor experiment. (A-E) The serum levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (Cre). Phosphate buffered saline (PBS, the vehicle) or rbNPs are injected into mice, and the blood is collected at day zero, four, eight, and 12 after the first injection. Subsequently, the serum is obtained and the levels of (A) TNF-α and (B) IL-6 are determined by ELISA. The serum levels of (C) Cre, (D) ALT, and (E) AST are also measured. Results are expressed as the mean ± SD of three samples. ns, not significantly different from one another; N.D., not detected. Cre, creatinine; ALT, alanine aminotransferase; AST, aspartate aminotransferase