Controlling the Kinetics of an Enzymatic Reaction through Enzyme or Substrate Confinement into Lipid Mesophases with Tunable Structural Parameters.

Marco Mendozza,Debora Berti,Costanza Montis,Arianna Balestri

doi:10.3390/ijms21145116

Abstract

Lipid liquid crystalline mesophases, resulting from the self-assembly of polymorphic lipids in water, have been widely explored as biocompatible drug delivery systems. In this respect, non-lamellar structures are particularly attractive: they are characterized by complex 3D architectures, with the coexistence of hydrophobic and hydrophilic regions that can conveniently host drugs of different polarities. The fine tunability of the structural parameters is nontrivial, but of paramount relevance, in order to control the diffusive properties of encapsulated active principles and, ultimately, their pharmacokinetics and release. In this work, we investigate the reaction kinetics of p-nitrophenyl phosphate conversion into p-nitrophenol, catalysed by the enzyme Alkaline Phosphatase, upon alternative confinement of the substrate and of the enzyme into liquid crystalline mesophases of phytantriol/H2O containing variable amounts of an additive, sucrose stearate, able to swell the mesophase. A structural investigation through Small-Angle X-ray Scattering, revealed the possibility to finely control the structure/size of the mesophases with the amount of the included additive. A UV–vis spectroscopy study highlighted that the enzymatic reaction kinetics could be controlled by tuning the structural parameters of the mesophase, opening new perspectives for the exploitation of non-lamellar mesophases for confinement and controlled release of therapeutics.

Highlights

Polymorphic lipids have been widely studied over the years, for their unique self-assembly properties: in water, they can form lyotropic liquid crystals with long-range order promoted by hydrophobic forces and H-bonds [1], with different arrangements and a complex phase diagram highly dependent on temperature, pressure [2], water content and ionic strength [3,4,5,6].Glycerol monooleate (GMO, a glycerol monoester) and phytantriol (Phyt, a terpenoid polyalcohol) are among the most studied polymorphic amphiphiles and are characterized by a rich phase diagram
Depending on the experimental conditions, they assemble into hexagonal mesophases, lamellar mesophases, or inverse bicontinuous cubic mesophases
In order to homogenize the reaction solution, the buffer was stirred with cuvette stirrer bars during all the time of the experiments. This contribution reports on the enzymatic kinetics of the conversion of p-nitrophenyl phosphate in p-nitrophenol, catalysed by alkaline phosphatase, upon confinement either of the enzyme or of the substrate in phytantriol liquid crystalline mesophases, as the structural parameters are varied

Summary

Introduction

Polymorphic lipids have been widely studied over the years, for their unique self-assembly properties: in water, they can form lyotropic liquid crystals with long-range order promoted by hydrophobic forces and H-bonds [1], with different arrangements and a complex phase diagram highly dependent on temperature, pressure [2], water content and ionic strength [3,4,5,6].Glycerol monooleate (GMO, a glycerol monoester) and phytantriol (Phyt, a terpenoid polyalcohol) are among the most studied polymorphic amphiphiles and are characterized by a rich phase diagram. While gyroid and diamond phase are the thermodynamically stable phases with excess water, in different experimental conditions, the primitive cubic phase arrangement is generally observed in the presence of additives [8] and polymers [9] swelling the water channels Due to their unique structure, characterized by the coexistence of extended hydrophobic and hydrophilic regions arranged in a continuous 3D complex architecture, cubic mesophases, in their disperse form, i.e., cubosomes, have been widely studied as possible alternatives to the most common lamellar phases (in particular in their dispersed form, as liposomes) for the development of vectors for drugs of different size and polarity for biomedical applications [6,10,11,12]. It has been shown that cubic mesophases have a strong ability to interact with phospholipid cell membranes, making them interesting for the intracellular delivery of drugs

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