TABLE 3.

Overview of the use of liposomes for bacteriophage encapsulation.

Envisioned application	Material and construct	Embedded phage	Relevant results				Reference
			Study type	Manufacturing and encapsulation process	Phage release / Protective properties	Antimicrobial effects
Food processing industry	Niosomes, liposomes and transfersomes from commercially available phospholipid formulations (Pronanosome Lipo-NTM and Pronanosome Nio-NTM)	Staphylococcal phage phiIPLA-RODI	In vitro	For liposomes and Niosomes. the commercial formulations were dissolved in 0.05M HEPES buffer. The solutions were agitated by manual shaking and homogenization. Transferosomes were fabricated with thin film hydration.	Niosomes and liposome showed a 2-log reduction in PFU after acid exposure. Niosomes protected the encapsulated phage against elevated temperatures of 60°C, again showing a 2-log reduction where other formulations expressed no active phage.	Not Assessed	Gonzalez-Menendez et al., 2018
Wound dressing	Uni-lamellar liposomes (diameter ± 200 nm) containing: ∙ Phosphatidylcholine ∙ Cholesterol ∙ Tween 80 ∙ Stearylamine (weight ratio: 7/3/1/0.5)	S. aureus phage MR-5 and MR-10	In vivo	Encapsulation efficiency of 87% for both phages.	Phage was quantifiable for 10 days at the site of the wound. Liposome entrapped phages initially showed higher local PFU count as free phage.	Encapsulated phage in liposomes performed similarly as the antibiotic control group, where free phage was unable to clear infection. Wound healing was more rapid for liposome treated group	Wroe et al., 2020
Uptake of encapsulated phage by intestinal cells	Liposomes containing: ∙ phospholipids ∙ cholesteryl ∙ polyethylene glycol 600 ∙ cholesterol ∙ cholesteryl 3β-N-(dimethylaminoethyl) carbamate hydrochloride (weight ratio: 1/0.1/0.2/0.7)	S. enterica phage UAB_Phi20	In vivo	1011 PFU/mL dispersed phage could be encapsulated though lipid layer hydration methods with an efficacy of 46%.	Upon oral administration in mice, phage labeled with infrared marker was detected in several organs, including the stomach, intestine, spleen, liver and muscle.	Not assessed	Otero et al., 2019
			Study type	Manufacturing and encapsulation process	Phage release / Protective properties	Antimicrobial effects
Phage delivery for internal medicine	Liposomes containing: ∙ DSCP phospholipids ∙ Cholesterol	S. aureus phage K ATCC 1985-B1 and E. coli phage ATCC 11303-B3	In vitro	Liposomes were fabricated using microfluidic hydrodynamic flow focusing, with a phage load of ∼108 PFU/mL	Encapsulation in liposomes offered limited protection against acidic environments, with a 3log10 reduction of phage.	Not assessed	Cinquerrui et al., 2018
Intestinal phage delivery	Liposomes (diameter ± 300 nm) containing: ∙ 1,2-dilauroyl-rac-glycero-3-phosphocholine (DOPC) ∙ Cholesteryl ∙ polyethylene glycol 600 sebacate ∙ cholesterol ∙ cholesteryl 3β-N-(dimethylaminoethyl) carbamate hydrochloride (molar ratio: 1/0.1/0.2/0.7)	Salmonella phages UAB_Phi20/Phi78/Phi87	In vivo	Liposomes formed by thin-film hydration method. Encapsulation efficiency between 47 and 49% for all three phages.	Liposomes provided limited protection against acidic environments (4–5log10 PFU titer reduction).	Presence of phage in the excrement of chickens is 90% for chickens where encapsulated phage was administered. For free phage, this was only 35%. After 15 days, a 2log10 reduction in Salmonella CFU was observed in the excrement of chickens treated with phage loaded liposomes.	Chhibber et al., 2018
Intracellular phage delivery	Liposomes (diameter ± 100 nm) ∙ Phosphatidyl choline ∙ Cholesterol ∙ Tween 80 ∙ Stearylamine (weight ratio: 9/1/2/0.5)	K. pneumoniae phage KPO1K2 (MTCC 5831)	In vivo	Thin film hydration at 40°C.	Liposomes provided full protection against phage neutralizing antibodies and serum. Liposome encapsulated phage was observed in the spleen of BALB/c mice for 14 days, while free phage was not quantified after 2 days.	95% intracellular bacteria were killed by liposome encapsulated phages, while only a 21% reduction was seen for free phage.	Singla et al., 2016
Intracellular phage delivery	Giant unilamellar Liposomes (diameter in μm range) ∙ DOPC ∙ 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (weight ratio: 50/50)	Mycobacterium smegmatis phage TM4 and E. coli phage λeyfp	In vitro	Gel-assisted thin film hydration and inverse emulsion	Only intracellular phage presence was measured. Approximately 4-fold increase of intracellular phage was observed by fluorescent confocal microscopy.	Not assessed	Nieth et al., 2015a

Abbreviations: CFU: Colony-forming units; HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); PFU: Plaque-forming units.