Optimising microfluidic preparation of lipid sponge phase nanoparticles for biomolecule encapsulation

Abstract: Drug delivery with lipid nanoparticles, especially sponge phase lipid nanoparticles (LNP), has been well studied because of its ability for drug encapsulation and transferring the drug through the cell membrane. The sponge phase LNP formulation could be affected by the molecular structure of the lipid, the temperature, pH, dilution, the buffer, and the addition of a stabiliser. This project aimed to replace the sonication method with microfluidics because the sonication damages the sample by using ultrasound waves. However, in microfluidics more parameters were involved in the formulation, the total flow rate, and the lipid/buffer flow ratio. In this study, a mixture of mono-, di- and triglycerides and polysorbate 80 were used to investigate the formulation of sponge phase LNP by a microfluidic device. The effect of flow rate, flow ratio, stabilizer concentration, buffer and pH were studied to optimise the sponge phase LNP formulation method. Protein encapsulation was also tested to see how it would affect the size and structure of the LNPs. The formulation method used a T-crossed channel chip where the channel has a rectangular shape, 140x200 µm, with three inputs and one output. A statistical experimental design by the software Design Expert was done to plan the project and decide the different sample preparation conditions. The effect of the buffer was studied by comparing the formulation in milli-Q water, tris(hydroxymethyl)aminomethane (TRIS) buffer at pH=7.2 and pH=8.9, and in phosphate buffer (PB) at pH=7.0. The protein encapsulation was tested with different protein concentrations. The size and the zeta potential of the LNP were measured by dynamic light scattering (DLS), and the structure of the LNP was determined by small-angle X-ray scattering (SAXS). In addition, some samples have been imaged in transmission electron microscope (cryo-TEM). The size measurements of the LNP were fitted into a model that describes the effect of the parameters, total flow rate, and final lipid concentration that depended on the flow ratio and the concentration of the P80 as a stabiliser. According to the model, a high flow rate decreased the particle size, a high P80 concentration helped form smaller particles and a larger flow ratio led to lower lipid concentration and smaller particles. However, the pH also influenced the size where pH=7 helped form smaller particles than pH=8.9. Furthermore, TRIS and phosphate buffer formed smaller particles than milli-Q water and had clearer signals for the sponge phase. Even the encapsulation showed to influence the size and the phase of the LNP. High protein concentration disturbed the formulation of the sponge phase and led to bigger particles. Despite all, the microfluidic method showed its ability to form sponge phase LNP with different big sizes.