Harnessing tomato-derived small extracellular vesicles as drug delivery system for cancer therapy

Abstract

Aim: This study aims to explore a sustainable and scalable approach using tomato fruit-derived sEVs (TsEVs) to deliver calcitriol for enhanced anticancer effects, addressing challenges of low yield and high costs associated with mammalian cell-derived sEVs.

Methods: TsEVs were isolated by centrifugation and ultrafiltration and characterized using DLS, TEM, and biochemical assays. Calcitriol was loaded into TsEVs via loading methods, with efficiency measured by spectrophotometry and HPLC. HCT116 and HT29 colon cancer cells were treated with TsEV-calcitriol and assessed for viability, colony formation, migration, ROS levels, and apoptosis gene expression.

Results: Isolated TsEVs ranged from 30-200 nm with a protein-to-lipid ratio of ∼1. Calcitriol encapsulation efficiencies were 15.4% (passive), 34.8% (freeze-thaw), and 47.3% (sonication). TsEV-calcitriol reduced HCT116 cell viability with IC50 values of 4.05 µg/ml (24 h) and 2.07 µg/ml (48 h). Clonogenic assays showed reduced colony formation and migration. Elevated ROS levels and increased Bax/Bcl-2 ratio were observed in treated HCT116 and HT29 colon cancer cells.

Conclusion: These findings highlight TsEVs as a promising alternative drug delivery platform to mammalian cell-derived sEV for enhancing the therapeutic efficiency of calcitriol and other anticancer agents.

Keywords: Plant exosomes; calcitriol; cancer therapy; cell death; drug delivery; vitamin D.

Graphical abstract

Figure 1.

Isolation and characterization of TsEVs. (A) TsEVs isolation workflow: A combination of differential centrifugation, ultrafiltration, and PEG precipitation techniques was employed to efficiently purify sEVs from ripen tomatoes, ensuring high-quality extraction. (B) size distribution of TsEVs determined by DLS: The size distribution profile indicating a range of 30–200 nm in diameter. (C) Morphology of TsEVs under TEM: Representative TEM image of TsEVs showing the vesicle shaped morphology and confirming their size to be less than 200 nm. (D) Protein and Lipid Estimation: Quantitative assessment of protein content was performed using the BCA assay, and lipid content was measured using the sulfo-phospho-vanillin assay. (E) Protein to Lipid Ratio: The protein to lipid ratio was calculated based on the concentrations obtained from the BCA and phosphovanillin assays, showing a compositional balance of approximately 1, indicating the purity of TsEVs. (F) Particle Concentration: NTA quantified the concentration of TsEVs, indicating approximately 9 × 10¹³ particles per kg. (G) RNA profile: An electropherogram generated using the Agilent 2100 Bioanalyzer, representing the TsEVs RNA length ranging between 20- 200 nucleotides.

Figure 2.

Evaluation of drug loading and release characteristics of TsEVs. (A) Loading efficiency estimation: The loading efficiency of calcitriol into TsEVs was determined using UV–visible spectrophotometry and HPLC. The table shows that the sonication method achieved the highest loading efficiency, with 47.30% ± 1.6% by spectrophotometry and 45.3% ± 1.3% by HPLC. (B) TEM Analysis of drug-loaded TsEVs: TEM image of TsEVs after drug loading via sonication confirm that the structural integrity of the nanoparticles is maintained, with the particles displaying a typical round vesicle morphology. (C) Size and Zeta Potential Changes After Drug Loading: The size of TsEVs increased slightly from 55.7 nm to 88.09 nm, and the zeta potential changed from -12.3 mV to -15.4 mV after loading with calcitriol. (D) In vitro calcitriol release from TsEVs: The release profile of calcitriol from TsEVs was evaluated under acidic (pH 4.5) and neutral (pH 7.4) conditions using a dialysis method. The graph indicates a sustained and significantly higher release of calcitriol at pH 4.5 compared to pH 7.4, demonstrating the acid-stimulated drug release properties of the TsEVs formulation.

Figure 3.

Intracellular uptake and inhibition of colon cancer cell growth by TsEVs-Calcitriol in HCT116 and HT29 cells. (A, B) Representative Fluorescence Microscopy Images: Fluorescence microscopy images of HCT116 (A) and HT29 (B) cells incubated with TsEVs or TsEVs-calcitriol labeled with BODIPY TR ceramide for 6 h. The images show the cellular uptake of labeled TsEVs, with DAPI staining the nuclei (blue) and BODIPY TR ceramide, indicating the presence of TsEVs (red). The merged images confirm the intracellular localization of TsEVs. (C, D) Quantitative analysis of BODIPY TR fluorescence intensity in HCT116 (C) and HT29 (D) cells using ImageJ software. The bar graphs represent the relative fluorescence intensity, indicating the uptake of TsEVs and TsEVs-calcitriol (***p < 0.001 vs. control). (E, F) Viability of HCT116 Cells: MTT assay results showing the viability of HCT116 cells exposed to calcitriol and TsEVs-calcitriol for 24 h (E) and 48 h (F). The data indicate that TsEVs-calcitriol treatment results in higher cell death than calcitriol alone. (G, H) Viability of HT29 Cells: MTT assay results demonstrating the viability of HT29 cells exposed to calcitriol and TsEVs-calcitriol for 24 h (G) and 48 h (H). The results show that TsEVs-calcitriol leads to significantly higher cell death than calcitriol alone. (I, J) Comparative IC50 Values for calcitriol and TsEVs-calcitriol: The IC50 data calculated from the MTT assay show that TsEVs-calcitriol has a lower IC50 value in both HCT116 (I) and HT29 (J) cell lines, indicating greater efficacy in reducing cell viability.

Figure 4.

TsEVs-calcitriol inhibits clonogenicity and cell migration in colon cancer cells: (A, B) Representative colony formation assay images: Colony formation assay images of HCT116 (A) and HT29 (B) cells after seven days of treatment with TsEVs-calcitriol. The images show a significant reduction in the number of colonies in cells treated with TsEVs-calcitriol compared to those treated with calcitriol alone. (C, D) Quantification analysis of clonogenic efficiency: The bar graphs represent the quantification of clonogenic efficiency in HCT116 (C) and HT29 (D) cells. The data demonstrate that TsEVs-calcitriol treatment significantly inhibited the clonogenic potential of both cell lines compared to the calcitriol and control groups. Quantification data are presented as the mean ± SD (n = 3). Statistical differences are indicated: ***P < 0.001 vs. control, ^##P < 0.01 vs. calcitriol. (E, F) Representative images of HCT116 (E) and HT29 (F) monolayer wounded areas at 0 and 24 h. Wound closure was significantly reduced in TsEVs-calcitriol-treated cells compared to cells treated with calcitriol alone. (G, H) Quantitative estimation of wound closure after 24 h in HCT116 (G) and HT29 (H) cells. The data indicated significantly lower wound closure in TsEVs-calcitriol-treated cells compared to calcitriol alone (***P < 0.001 compared to control, ^#P < 0.05 vs. calcitriol), highlighting the enhanced inhibitory effect on cell migration.

Figure 5.

Enhanced ROS generation and modulation of apoptosis-related gene expression in colon cancer cells treated with TsEVs-calcitriol. (A, B) Fluorescence microscopy images of DCF staining: Fluorescence microscopy images of HCT116 (A) and HT29 (B) cells stained with DCFDA, indicating increased DCF fluorescence in TsEVs-calcitriol-treated cells. (C, D) Total ROS production measured by DCFDA fluorometric assay: The bar graphs show significantly higher levels of DCF fluorescence in TsEVs-calcitriol-treated HCT116 (C) and HT29 (D) cells after 12 h compared to calcitriol alone. All data are expressed as mean ± SD. Statistical differences are indicated as follows: ***P < 0.001 vs. control, ^##P < 0.01 vs. calcitriol. The data corroborate the microscopy results, demonstrating that TsEVs-calcitriol-treated cells exhibit higher amounts of DCF fluorescence, reflecting increased ROS generation. (E, F) The mRNA expression levels: The mRNA expression levels of survivin, Bax and Bcl-2 showing a significant difference between treatment with calcitriol and TsEVs-calcitriol in HCT116 (E) and HT29 (F) cells. (G, H) Bax/Bcl-2 ratio: Treatment with TsEVs-calcitriol for 12 h significantly decreased Bcl-2 expression while increasing Bax expression, leading to a significantly higher Bax/Bcl-2 ratio, indicative of apoptosis induction. Statistical differences: **P < 0.01 vs. control, ***P < 0.001 vs. control, ^##P < 0.01 vs. calcitriol, ^###P < 0.001 vs. calcitriol. These findings suggest that TsEVs-calcitriol treatment effectively modulated apoptosis-related genes and promoted apoptosis in both HCT116 and HT29 cells, with enhanced efficacy compared to calcitriol treatment alone.