Poster X13 - Comprehensive characterization of olive-derived nanoparticles (ODNPs) as a new drug delivery system.

Introduction: Extracellular vesicles (EV) have shown great promise as drug delivery system (DDS). However, their complex and costly production limit their development for clinical use. Interestingly, like animal, plants can produce nanovesicles. These plant-derived nanoparticles (PDNPs) can be easily isolated and purified from a large quantity of raw material at high yield. Moreover, PDNPs’ chemical and structural properties are close to animal EVs. A lot of research has proved PDNPs are a source of many therapeutic compounds, and their capacity to load and transport drugs, but there is still limited knowledge in this relatively new field. Here, we deeply characterized olive-derived nanoparticles (ODNPs) physical and biological features to demonstrate their promising potential to be used as a DDS.

Materials and

Methods: ODNPs were isolated from raw fruits through sequential steps including blending, serial centrifugations, and ultimately sucrose gradient ultracentrifugation. Their size, surface charge, and morphology were characterized using nano-tracking analysis (NTA), dynamic light scattering (DLS), and cryogenic electron microscopy (CryoEM), respectively. The expression of plant EVs surface biomarkers, TET8 and PEN1, was evaluated by ELISA, while lipidomic analysis was performed to inform on membrane composition. The impact of various physical stressors on ODNPs, such as pH, salt concentration, temperature, and extrusion, was assessed using NTA. Additionally, the effects of different storage conditions over a 14-day period, including refrigeration (4°C), room temperature, deep freezing (-80°C), and lyophilization in various solvents (H2O and PBS with and without 25 mM trehalose), were investigated using NTA. Cell viability assay and immunofluorescence imaging were performed on both 2D and 3D cell culture of A549 and SKOV3 cells lines to explore the cytotoxicity and internalization of ODNPs. Finally, the anticancer drug doxorubicin (Dox) was loaded into ODNPs, and the physical stability of Dox-loaded ODNPs as well as their biological effects were assessed by NTA and viability assays.

Results, Conclusions, and Discussions: NTA indicated an average size of 108 nm and yield of 1012 ODNPs extracted from 100 g of raw fruits. Zeta potential charge of ODNPs, at 25 °C, is -23.94 ± 0.51 mV. CryoEM showed the presence of a lipid-bilayer membrane, multivesicular vesicles and transmembrane proteins, similar to animal EVs. ELISA results demonstrated a fourfold increase of PEN1 and TET8 proteins levels in ODNPs compared to raw fruits, confirming the enrichment of the ODNPs fraction in plant EVs. However, ODNPs showed a 60% less enrichment in TET8 and PEN1 compared to the positive control (nanoparticles derived from Arabidopsis leaves), indicating that ODNPs might have different surface biomarkers not identified so far. Lipidomic analysis revealed glycerolipids, sterol lipids, and fatty acyls as major lipid components of ODNPs. Interestingly, ODNPs size and yield stay unchanged after exposure to 70°C for 1 h, pH 5-10 for 1 day, 0-10 mM salt for 1 day and 50/100 nm extrusion for at least 20 cycles and 10 cycles respectively, demonstrating a strong robustness of ODNPs to multiple stress factors. ODNPs size and yield also stayed stable in water at 4°C and room temperature up to a month. For longer storage, ODNPs could also be frozen in water but the addition of 25 mM trehalose was required to avoid degradation. Biological characterization showed that ODNPs were internalized in the cytoplasm of A549 and SKOV3 cells when cultured either in 2D or 3D, without triggering cytotoxicity even at high concentration (1011 ODNPs/mL). Dox was then loaded into ODNPs by passive incubation, and cells exposed to Dox-loaded ODNPs exhibited reduced cell viability by 90% compared to unexposed cells. Interestingly, viability of cells exposed to free unencapsulated Dox at equivalent concentrations only decreased by 70%, suggesting that encapsulation in ODNPs improved Dox cytotoxicity effect. Importantly the size, concentration, and efficiency of Dox-loaded ODNPs remained unchanged after 2 weeks of storage in water at 4°C. In summary, ODNPs are promising DDS candidates as demonstrated by their biocompatibility, high resistance to stress, good stability in minimal environment, and improvement of anticancer drug efficacy.

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