(B-60) Characterization of plant-derived nanoparticles for drug loading application

Introduction:: Animal-derived extracellular vesicles (EVs) hold promises as advanced platforms for targeted drug/gene delivery but their practical applications can be limited due to the high cost of production, poor yield and the time-consuming and low reproducibility of the isolation process. Interestingly, plant kingdom can also be a source of EV-like particles. These plant-derived vesicles in form of lipid nanoparticles (pNPs) have shown to exhibit low immunological risk and good bioavailability but as opposed to EVs, also offer many more resources of raw materials, cost efficient production and do not harbor zoonotic or human pathogens. To use these pNPs in clinical setting, as either new therapeutic compound through the natural cargo they can carry, or as an efficient drug delivery system, there is a need to better characterizing them to understand their nature and the potential mechanisms of their biological effects.


Materials and Methods:: More than 16 plants, including fruits, leaves and roots, have been first blended, and debris removed by serial low-speed centrifugations. Total pNPs have been then pelleted by ultracentrifugation and purified later through sucrose gradient (UC-SUC). Physical characterization and stability, as well as storage condition, of pNPs, involved nano-tracking analysis (NTA), electron microscopy, and membrane fluidity assessment. pNPs intake by human cells have been assessed by lipid membrane labelling and immunofluorescence (IF). Finally, anti-inflammatory and viability assays were performed to assess biological activity and drug loading efficiency.


Results, Conclusions, and Discussions:: Sixteen plants have been successfully used for the pNPs isolation using our serial protocol. For olives-derived NPs (ODNPs), UC-SUC produced 5 distinct fractions that show different size distribution. By following this approach, we were able to efficiently isolate a fraction with mono dispersion and an average size of 103 nm in diameter (fitting drug delivery target range, under 200 nm) with a production yield up to 10¹² obtained in only 2 days. TEM showed that the ODNPs have a round shape and formed by a lipid bilayer. Membrane thermal resistance was proved by the preservation of membrane integrity at high temperature (50 °C) for 1 h. The ODNPs maintain their diameter and concentration stable at refrigerated temperature for up to a month, suggesting the robustness of such material compared to EVs. Additional studies are currently investigating several protocols to increase storage condition and to assess stability of biochemical activity. IF showed that PKH67-labeled pNPs are present in cells after 24 h incubation, suggesting cytoplasmic internalization. On-going studies are evaluating the internalization mechanisms by blocking different endocytosis pathways. Interestingly, ODNPs did not show any cytotoxic effect on human cells and have promising anti-inflammatory effects. As such, ODNPs have been selected as potential drug delivery system and fluorescent doxorubicin (Dox) has been loaded by passive incubation. Results showed that Dox concentration within ODNPs is about 1.3 µM and was enough to decrease proliferation of cancer cells in a greater extent that the same concentration of free Dox, suggesting a potential synergic effect of ODNPs for Dox distribution. Further improvements involve optimization of loading protocol for ODNPs. Together these data showed that pNPs can be easily isolated, undergo endocytosis by human cells, are not cytotoxic and can be loaded with external compounds to modify cellular biological response. For better understanding, it is now required to perform deep omics analysis to inform about the membrane composition and the different cargo that these pNPs can carry, as well as to compare biological and physical pNPs properties with synthetic reference (e.g., liposome), or EVs, to pave the way towards translational clinical application.

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