To date, the lipid liquid crystalline sponge phase (L3) has rarely been discussed in the context of encapsulation matrices for biofunctional molecules, such as enzymes. The sponge phase, however, has distinct advantages compared to other reversed lipid mesophases, both in terms of its increased flexibility and capacity to form large aqueous pores, which are able to encapsulate large active biomolecules. Additionally, the sponge phase can be dispersed into stable colloidal nanoparticles (NPs), in which entrapped active substances are protected from degradation, while simultaneously allowing control over their release and/or activity. We have investigated the encapsulation of two key industrial enzymes of different sizes, aspartic protease (34 kDa) and beta-galactosidase (476 kDa), in L3 phase dispersions, composed of diglycerol monooleate, glycerol monooleate, and the stabiliser polysorbate 80. Small angle neutron scattering (SANS) results revealed differences between the L3 NPs with and without enzyme, which can be interpreted as inclusion of the protein in the lipid liquid crystalline phase. Additionally, encapsulation in the L3 phase was shown to increase enzyme thermal stability, as, in both cases, the enzymes showed higher retained activity compared to the free enzyme solution after storage at room temperature. Lipid sponge phases have few distinct structural features in their diffraction patterns, compared to the related cubic phases. The dynamics are, therefore, very important. We performed neutron spin echo (NSE) measurements on these L3 NPs at NIST, USA, with the aim of revealing the dynamics of this system with and without the two enzymes, aspartic protease and b-galactosidase. To our knowledge, this was the first time that NSE measurements have been performed on sponge phase dispersions, in contrast to vesicular dispersions, which have been more widely studied. The bending moduli extracted from this data highlighted the specific effects of the encapsulated enzymes on the L3 phase. We observed that the sponge phase becomes stiffer when the enzymes are included, with a larger increase in the case of b-galactosidase than aspartic protease.

Lipid sponge phase as a matrix for enzyme encapsulation: Structure and dynamics

ACS Spring Meeting 2021

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