Optimising microfluidic preparation of lipid sponge phase nanoparticles for biomolecule encapsulation
(2023) KETM05 20231Chemical Engineering (M.Sc.Eng.)
- 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... (More)
- 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. (Less) - Popular Abstract (Swedish)
- Läkemedelstransportering med lipidnanopartiklar (LNP), särskilt svampfas lipidnanopartiklar,
har varit intressanta på grund av dess möjlighet att inkapsla läkemedel och överföra
läkemedlet genom cellmembranet. Svampfas LNP formuleringen kan påverkas av lipidens
molekylära struktur, temperaturen, pH-värdet, utspädningen, bufferten och existerandet av
en stabilisator. Projektets syfte var att ersätta ultraljudsformuleringsmetoden med
mikrofluidik, eftersom ultraljud skulle skada provet genom de inskickade energifulla
ultraljudsvågor. I mikrofluidik system var fler parametrar involverade i formuleringen, den
totala flödeshastigheten och lipid/buffertflödesförhållandet. I denna studie undersöktes
blandningar av olika monooleat lipider... (More) - Läkemedelstransportering med lipidnanopartiklar (LNP), särskilt svampfas lipidnanopartiklar,
har varit intressanta på grund av dess möjlighet att inkapsla läkemedel och överföra
läkemedlet genom cellmembranet. Svampfas LNP formuleringen kan påverkas av lipidens
molekylära struktur, temperaturen, pH-värdet, utspädningen, bufferten och existerandet av
en stabilisator. Projektets syfte var att ersätta ultraljudsformuleringsmetoden med
mikrofluidik, eftersom ultraljud skulle skada provet genom de inskickade energifulla
ultraljudsvågor. I mikrofluidik system var fler parametrar involverade i formuleringen, den
totala flödeshastigheten och lipid/buffertflödesförhållandet. I denna studie undersöktes
blandningar av olika monooleat lipider och polysorbat 80 för att formulera svampfas LNP med
hjälp av en mikrofluidik system. Effekten av flödeshastighet, flödesförhållande,
stabilisatorkoncentration, buffert och pH studerades för att optimera LNP-
formuleringsmetoden med svampfas. Proteininkapsling testades också för att se hur det
skulle påverka storleken och strukturen på LNP.
Formuleringsmetoden använde ett T-korsat kanalchip där kanalen har en rektangulär form,
140x200 μm, med tre ingångar och en utgång. En statistisk experimentell design av
programvaran Design Expert gjordes för att planera projektet och bestämma de olika
provberedningsvillkoren. Genom resultatet studerades effekten av bufferten genom att
jämföra formuleringen i milli-Q vatten, tris(hydroximetyl)aminometan (TRIS) buffert vid
pH=7,2 och pH=8,9, och i fosfatbuffert (PB) vid pH=7,0. Proteininkapslingen testades med
olika proteinkoncentrationer. Storleken och zetapotentialen för LNP mättes med dynamisk
ljusspridning (DLS), och fasen för LNP bestämdes med liten vinkelröntgenspridning (SAXS).
Dessutom har några prover avbildats i transmissionselektronmikroskop (cryo-TEM).
Storleksmätningarna av LNP monterades in i en modell som beskrev effekten av
parametrarna, total flödeshastighet, slutlig lipidkoncentration som berodde på
flödesförhållandet och koncentrationen av P80 som stabilisator. Enligt modellen minskade en
hög flödeshastighet partikelstorleken, en hög P80-koncentration hjälpte till att bilda mindre
partiklar, och ett mer signifikant flödesförhållande led till lägre lipidkoncentration och mindre
partiklar. pH påverkade också storleken där pH=7 hjälpte till att bilda mindre LNP jämfört med
pH=8,9. Dessutom bildade TRIS och fosfatbuffert mindre LNP än milli-Q-vatten och de hade
tydligare signaler för svampfasen i SAXS mättningarna. Även proteininkapslingen visade sig
påverka storleken och fasen av LNP. Hög proteinkoncentration störde formuleringen av
svampfasen och ledde till större partiklar. Trots allt visade mikrofluidik metoden sin förmåga
att bilda svampfas LNP med olika stora storlekar. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9126273
- author
- Khalil, Owais LU
- supervisor
- organization
- course
- KETM05 20231
- year
- 2023
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Sponge phase, lipid nanoparticles, microfluidics, SAXS, cryo-TEM, DLS, chemical engineering
- language
- English
- id
- 9126273
- date added to LUP
- 2023-06-22 11:10:39
- date last changed
- 2023-06-22 11:10:39
@misc{9126273,
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.}},
author = {{Khalil, Owais}},
language = {{eng}},
note = {{Student Paper}},
title = {{Optimising microfluidic preparation of lipid sponge phase nanoparticles for biomolecule encapsulation}},
year = {{2023}},
}