The Assessment of Lipid Liquid Crystalline Nanoparticles for Agricultural Applications

Non-lamellar liquid lipid crystal nanoparticles (LCNPs), produced using biocompatible,
commercially available, inverse type lipids, have complex internal nanostructures that possess
2 or 3-dimensional periodicity. These internal nanostructures are based on the inverse bi
continuous cubic and 2D-hexagonal (HII) phases which, when dispersed as colloidally stable
nanoparticles (NPs), are termed cubosomes and hexosomes, respectively. LCNPs, like all liquid
crystals, exhibit the typical long-range order of solids whilst maintaining certain characteristic
of liquids. LCNPs have advantages over traditional liposomal adjuvants such as higher surface
areas, the ability to contain both hydrophobic and hydrophilic active ingredients (AIs),
mechanical robustness and the opportunity to convert between different liquid crystalline
phases, each with specific properties. These properties mean that LCNPs have shown promise
as adjuvants, particularly in biomedical applications. Further research, however, has indicated
that these same advantageous properties mean LCNPs may also possess utility in agriculture.
The twin pressures of a growing population and an environment less amenable to prodigious
growth of crops challenges humanity’s ability to meet the calorific needs of our contemporary,
and near future, societies. The critical nature of this challenge compels us to develop more
efficient, sustainable, and effective agricultural praxis in which NPs have demonstrable
application as AI adjuvants, nano-seed primers, pesticides, fungicides and more. Phytantriol
(PYT) cubosomes are known to assist the delivery of the pesticide 2,4- dichlorophenoxyacetic
acid to plants whilst PYT and glycerol monooleate (GMO) cubosomes do not show visible
phytotoxic damage to target and non-target plants. Some evidence suggests however, that
cubosomes can stimulate callose production in leaves and solubilise cuticular waxes.
Current research into the potential application of LCNPs for agricultural applications is largely
limited to the application of PYT and GMO cubosomes, produced using low-output methods, to
the leaves of plants. To advance the use of LCNPs in agriculture more research is required to
develop a more comprehensive understanding of the interaction of numerous LCNP platforms
with diverse plant organs alongside the development of new LCNP formation methods that
overcome the productive limitations of current LCNP production methods. In the thesis
outlined below LCNPs of diverse liquid crystalline phase, and stabilised with three non-ionic
surfactants, were produced from the lipids PYT, GMO and Selachyl alcohol (SA). The produced
LCNP platforms were evaluated with regard to the loading of the model AI 2-Methyl-4
Chlorophenoxyacetic acid (MCPA) and spraying. Results demonstrated for the first known time
that SA hexosomes possess potential utility for agricultural applications, showing impressive
structural robustness to both spraying and MCPA loading. In contrast, PYT based cubosomes
were structurally labile to spraying. Importantly, the results also present for the first known time
the successful generation of SA and PYT based LCNPs via mixing within a high-volume output
and scalable microfluidic mixer based on a multi-inlet vortex mixer (MIVM). The physical
characteristics of the LCNPs formed compared well with LCNPs generated from more
traditional LCNP synthetic pathways such as sonication. Furthermore, it was shown that with
variations in both the total flow rate (TFR) and input solution concentrations a degree of Z
average diameter (DH) and polydispersity index (PDI) control could be achieved.

As a result of mammalian cell studies which have identified different cytotoxicity patterns
depending on LCNP composition and formation procedure, the potential phyto- and cyto-
damaging properties of LCNPs and their bulk stabilisers were investigated with respect to
variation of the lipid, stabiliser, and method of production. This involved the first known
investigation of the interaction of LCNPs with seeds, roots, and plant cells as well as the first
evaluation of the implications of SA hexosome exposure to different plant organs. Results
showed some general trends such as the stimulation of Allium cepa root length when rooted in
suspensions of F-127 stabilised LCNPs and a general increase in the observed number of
clastogenic aberrations in A. cepa cells with LCNP exposure. In general, however, LCNP at low
intermediate concentrations were safe to apply to a range of plant targets and no single variable
of LCNP composition could be identified as a determinant of altered phyto-damage.
Furthermore, LCNP suspensions compared favourably with commercial non-ionic agricultural
surfactants.

Finally, the response of plants, particularly Triticum aestivum, to SA hexosome and PYT
cubosome application under more realistic glasshouse conditions was evaluated. Results
demonstrated that regardless of the growth stage of T. aestivum at the time of LCNP application
selected growth parameters were unaffected and plants remained phenotypically normal.
Photosynthetic parameters such as PSII quantum efficiency and chloroplast structure were
unaffected by LCNP application. Interestingly, under more realistic growth conditions both SA
hexosomes and PYT cubosomes enhanced both seedling emergence (%) and seedling
emergence rate suggesting that LCNPs may show utility as novel nano-seed primers.