Investigation and Comparison of Active and Passive Encapsulation Methods for Loading Proteins into Liposomes

Abstract

In this work, four different active encapsulation methods, microfluidic (MF), sonication (SC), freeze–thawing (FT), and electroporation (EP), were investigated to load a model protein (bovine serum albumin—BSA) into neutral liposomes made from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):cholesterol (Chol) and charged liposomes made from DSPC:Chol:Dioleoyl-3-trimethylammonium propane (DOTAP), DSPC:Chol:1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and DSPC:Chol:phosphatidylethanolamine (PE). The aim was to increase the protein encapsulation efficiency (EE%) by keeping the liposome size below 200 nm and the PDI value below 0.7, which warrants a nearly monodisperse preparation. Electroporation (100 V) yielded the best results in terms of EE%, with a dramatic increase in liposome size (>600 nm). The FT active-loading method, either applied to neutral or charged liposomes, allowed for obtaining suitable EE%, keeping the liposome size range below 200 nm with a suitable PDI index. Cationic liposomes (DSPC:Chol:DOTAP) loaded with the FT active method showed the best results in terms of EE% (7.2 ± 0.8%) and size (131.2 ± 11.4 nm, 0.140 PDI). In vitro release of BSA from AM neutral and charged liposomes resulted slower compared to PM liposomes and was affected by incubation temperature (37 °C, 4 °C). The empty charged liposomes tested for cell viability on Human Normal Dermal Fibroblast (HNDF) confirmed their cytocompatibility also at high concentrations (1010 particles/mL) and cellular uptake at 4 °C and 37 °C. It can be concluded that even if both microfluidic passive and active methods are more easily transferable to an industrial scale, the FT active-loading method turned out to be the best in terms of BSA encapsulation efficiencies, keeping liposome size below 200 nm.

Electroporation in Translational Medicine: From Veterinary Experience to Human Oncology

“…These new vesicles imitate the biophysical and biochemical features of EVs to bypass present limitations to the adoption of exosomes for therapeutic applications [ 92 ]. Again, several loading strategies have been proposed for these tools, and electroporation has been evaluated as being among the most promising techniques in terms of encapsulation efficiency [ 93 ].…”

Liposomes as Carriers of Bioactive Compounds in Human Nutrition

“…Liposomes improve the bioavailability of encapsulated compounds and can also encapsulate proteins. Loading proteins into liposomes enhances their stability and bioavailability, making them useful for therapeutic purposes [ 109 ]. Because of that, liposomes offer a versatile platform for encapsulating bioactive compounds, making them an attractive component in food.…”

Looking back, moving forward: protein corona of lipid nanoparticles

“…7,8 Moreover, conventional liposomes can be categorized by phospholipid type into cationic, anionic and neutral liposomes. 9,10 Among them, liposomes synthesized from neutral phospholipids such as phosphatidylcholine, 11 dipalmitoyl phosphatidylcholine (DPPC), 12,13 and 1,2-distearoyl- sn -glycero-3-phosphocholine (DSPC) 14,15 as lipid materials are neutral liposomes, while N -[1(2,3-dioloyloxy) propyl]- N , N , N -trimethylammonium chloride (DOTAP) 16 and oleic acid 17 are commonly used for the preparation of cationic and anionic liposomes, respectively. Ionic-rich liposome particles repel each other due to electrostatic interaction, reducing their aggregation ability, and thus enhancing physical stability.…”