Efficacy of neutral and negatively charged liposome-loaded gentamicin on planktonic bacteria and biofilm communities

Figures & data

Table 1 Characterization of liposomal formulations

Liposomal formulations	Molar ratio	EE, %	Size, nm	Charge, mV
DPPC:Chol (NLG)	2:1	1.8±0.15	625.0±5.5	−0.22±0.17
DPPC:DMPG:Chol (NELG-1)	2:3:1	37.2±0.46	691.3±3.3	−25.3±2.5
DPPC:DMPG:Chol (NELG-2)	2:4:1	43.6±0.65	806.6±3.4	−31.7±1.7

Notes: The EE, size, and charge of liposomal formulations are shown. Each value is represented as the mean ± SEM.

Abbreviations: Chol, cholesterol; DMPG, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol); DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; EE, encapsulation efficiency; NLG, dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol; NELG-1, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1); NELG-2, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1); SEM, standard error of the mean.

Figure 1 Stability of NLG formulation in PBS at 4°C (circle), in PBS (square) at 37°C, sputum (diamond) at 37°C, in BAL (triangle up) at 37°C and plasma (triangle down) 37°C.

Abbreviations: BAL, bronchoalveolar lavage; NLG, dipalmitoyl-sn-glycero- 3-phosphocholine and cholesterol.

Figure 2 Stability of NELG-1 formulation in PBS at 4°C (circle), in PBS (square) at 37°C, sputum (diamond) at 37°C, in BAL (triangle up) at 37°C and plasma (triangle down) 37°C.

Abbreviations: BAL, bronchoalveolar lavage; NELG-1, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1).

Figure 3 Stability of NELG-2 formulation in PBS at 4°C (circle), in PBS (square) at 37°C, sputum (diamond) at 37°C, in BAL (triangle up) at 37°C and plasma (triangle down) 37°C.

Abbreviations: BAL, bronchoalveolar lavage; NELG-2, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1).

Table 2 Antibacterial activities of NLG, NELG-1, NELG-2, and free gentamicin against selected bacterial strains (in mg/L)

Bacterial strains	NLG		NELG-1		NELG-2		Free gentamicin
	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
Pseudomonas aeruginosa ATCC 27853	0.5	1	1	2	1	2	1	2
Acinetobacter baumannii BAA747	0.5	1	2	2	1	4	2	4
Klebsiella oxytoca 700324	0.5	2	1	4	1	4	1	8
P. aeruginosa BAA1744	0.25	2	1	4	1	4	1	4
P. aeruginosa 9721	0.5	1	2	4	2	2	2	4
Proteus vulgaris 6380	0.5	1	2	4	2	4	2	4
Staphylococcus aureus BAA976	0.25	0.25	1	2	1	1	1	1
S. aureus BAA977	0.06	0.06	0.25	0.25	0.25	0.5	0.5	0.5
Listeria monocytogenes BAA751	0.25	0.5	1	1	1	1	1	1

Abbreviations: MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; NLG, dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol; NELG-1, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1); NELG-2, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1).

Figure 4 Killing curve of bacterial strain Pseudomonas aeruginosa BAA1744 exposed to 0.25, 0.5, and 1 mg/L of NLG and 1, 2, and 4 mg/L of free gentamicin.

Abbreviations: CFU, colony forming unit; MIC, minimum inhibitory concentration; NLG, dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol.

Figure 5 Killing curve of bacterial strain Pseudomonas aeruginosa BAA1744 exposed to 1, 2, and 4 mg/L of NELG-1 and free gentamicin.

Abbreviations: CFU, colony forming unit; MIC, minimum inhibitory concentration; NELG-1, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1).

Figure 6 Killing curve of bacterial strain Pseudomonas aeruginosa BAA1744 exposed to 1, 2, and 4 mg/L of NELG-2 and free gentamicin.

Abbreviations: CFU, colony forming unit; MIC, minimum inhibitory concentration; NELG-2, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1).

Figure 7 Killing curve of bacterial strain Klebsiella oxytoca 700324 exposed to 0.5, 1, and 2 mg/L of NLG and 1, 2, and 4 mg/L of free gentamicin.

Abbreviations: CFU, colony forming unit; MIC, minimum inhibitory concentration; NLG, dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol.

Figure 8 Killing curve of bacterial strain Klebsiella oxytoca 700324 exposed to 1, 2, and 4 mg/L of NELG-1 and free gentamicin.

Abbreviations: CFU, colony forming unit; MIC, minimum inhibitory concentration; NELG-1, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1).

Figure 9 Killing curve of bacterial strain Klebsiella oxytoca 700324 exposed to 1, 2, and 4 mg/L of NELG-2 and free gentamicin.

Abbreviations: CFU, colony forming unit; MIC, minimum inhibitory concentration; NELG-2, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1).

Figure 10 Prevention effect of 1/2× MIC of free gentamicin, NLG, NELG-1, and NELG-2 on Pseudomonas aeruginosa biofilm. *P<0.05, **P<0.01, and ***P<0.001.

Abbreviations: MIC, minimum inhibitory concentration; NLG, dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol; NELG-1, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1); NELG-2, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimy-ristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1).

Figure 11 Prevention effect of 1/2× MIC of free gentamicin, NLG, NELG-1, and NELG-2 on Klebsiella oxytoca biofilm. ***P<0.001.

Abbreviations: MIC, minimum inhibitory concentration; NLG, dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol; NELG-1, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1); NELG-2, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimy-ristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1).

Figure 12 Reducing effect of 16× MIC of free gentamicin, NLG, NELG-1, and NELG-2 on Pseudomonas aeruginosa structured biofilm. *P<0.05 and **P<0.01.

Abbreviations: MIC, minimum inhibitory concentration; NLG, dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol; NELG-1, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1); NELG-2, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimy-ristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1).

Figure 13 Reducing effect of 16× MIC of free gentamicin, NLG, NELG-1, and NELG-2 on Klebsiella oxytoca structured biofilm. *P<0.05, **P<0.01, and ***P<0.001.

Abbreviations: MIC, minimum inhibitory concentration; NLG, dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol; NELG-1, dipalmitoyl-sn-glycero-3-phos-phocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1); NELG-2, dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimy ristoyl-sn-gly-cero-3-phospho-(1′-rac-glycerol), and cholesterol (2:3:1).