Buffer and pH effects on encapsulation of myoglobin in sponge phase nanoparticles via microfluidics

Abstract

The thesis focuses on optimizing microfluidic methodology to create lipid sponge phase nanoparticles (L3NPs) for myoglobin encapsulation, while investigating buffer and pH effect on particle characteristics. Traditional LNP production methods like ultrasonication are time consuming and requires large material quantities. Microfluidic techniques offer a more precise and cost effective alternative but requires optimization due to equipment and cost constrains. The study aims to optimize microfluidic techniques for L3NP production, investigating influence of microfluidic parameters, buffer composition, and pH on particle size and phase using dioleoyl phosphatidylcholine, glycerol monooleate (Capmul GMO-50) and Polysorbate 80 in buffer.
The microfluidic technique for lipid nanoparticle formation uses three pumps and a T.cross channel chip (dimensions 200μm x 200μm x 87mm) with three inlets and one outlet, monitored by an optical microscope. Flow rates and lipid+surfactant (L&S) to solvent ratio were varied, and dispersion are prepared using different DOPC/GMO-50/P80 amounts. Hydrodynamic size and polydispersity index are examined using Dynamic Light Scattering, structure and particle morphology via Small Angle X-ray Scattering. Cryo-TEM was performed to visualize the particles and their structures.
This study demonstrates that microfluidic and formulation parameters significantly affected L3NP size and phase. Optimal conditions include a 250 μl/min flow rate, 1:4 flow ratio L&S:solvent, increasing lipid and surfactant concentration and a DOPC/GMO-50/P80 ratio of 28/42/30 in 20 mM TRIS buffer at pH 7.4 and 5 mM CaCl buffer at pH 8.5. This study proved that nanoparticle size increased with a majority of the buffers, while TRIS and CaCl were chosen due to the low PDI, observed in DLS. Myoglobin encapsulation minimally impacts size and structure, but may affect the intra-particle interactions.
The results demonstrate that microfluidic techniques effectively create L3NPs for myoglobin encapsulation under specific conditions, confirmed by using Cryo-EM. Conditions include the importance to alter the DOPC/GMO-50 ratio to prevent phase separation, the necessity for P80 for nanoparticle colloidal stability and using a flow ratio of higher solvent content. These results confirm that by adjusting microfluidic and formulation parameters, buffer and pH, it is possible to optimize particle size and phase for myoglobin encapsulation, with L3NPs bilayer form factor and internal structure unaffected implying no change in intra-particle interactions.

Popular Abstract

Lipids, known as fats and oils, play essential roles in the body. Lipid nanoparticles (LNPs) are small lipid in the nanometric scale and can be made in the lab and designed to deliver drugs or genetic material in the body, protecting their cargo until it reaches the target. Ingredients like DOPC, GMO-50, and P80 are used to form LNPs in this study: both DOPC and GMO-50 served as main building block, while GMO-50 also aids stabilization and delivery, and P80 maintains the integrity of LNPs by colloidal stability. LNPs are created by first dissolving the ingredients in a organic solvent, such as ethanol. This solution is mixed with water or buffer under controlled conditions. In this method the particles are build up during mixing in contrast to stirring or the usage of high-frequency sound waves (ultrasonication) which is based on the principle to divide larger clusters (aggregates) into smaller once. These methods, known as conventional, are straightforward but limited in controlling particle size and distribution, often resulting in variability between batches. In this study we have used microfluidic techniques, often used since it entails with better control over particle properties and composition suitable for reproduction but are also known for its rapid and efficient nanoparticle production. Microfluidics, which is a small plumbing system with tiny channels, allows improved precise control over LNP properties by adjusting the rate and ratio of fluid mixing with help of pumps that controls the flow of liquids from syringes. By altering the composition of lipid, surfactants and stabilizers (DOPC, GMO-50 and P80, also referred to as lipid-mixture) and introducing different charges from the buffer, LNP properties and pH stability can be optimized. These adjustments are made to find the optimal method to prepare the LNPs for the intended cargo to enclose, the protein myoglobin. Myoglobin is a protein that stores and releases oxygen in muscles. Once energy is requires, during training for example, myoglobin releases oxygen to produce energy required for exercise.
In this study we’re using two different scattering techniques: Dynamic Light Scattering (DLS) which gives information about size and size distribution while Small Angle X-ray Scattering (SAXS) is used to examine the structure of the LNPs.
This study proved that altering DOPC and GMO-50 content play a crucial role in LNP formation and maintenance. An increase of DOPC and decrease of GMO-50 were suitable to prevent phase separation between water and lipid-mixture. P80 seemed to have an effect on size and size distribution while the structure seemed unaffected. The fluid mixing were seen optimal when all three pumps used were pumping fluid in a rate of 250 μl/min in total, in a ratio of 1:4 of Lipid-mixture:water. A higher water content were seen to lower size distribution. Size generally increased with most buffers, with TRIS and CaCl chosen for their low size distribution. Myoglobin introduction, enclosed by the LNPs, didn’t alter LNPs structure or size but could affect how particles interact with each other.

• author: Nildén, Alice
• supervisor: Tommy Nylander
• organization: Department of Chemistry; Computational Chemistry
• course: KEMR20 20241
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Lipid nanoparticles, Lipid sponge phase, Microfluidics, Myoglobin, Protein encapsulation, Physical chemistry
• language: English
• id: 9158039
• date added to LUP: 2024-06-11 10:58:42
• date last changed: 2024-06-11 10:58:42