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. 2017 Sep 18:12:6949-6961.
doi: 10.2147/IJN.S141709. eCollection 2017.

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

Moayad Alhariri  1 , Majed A Majrashi  2 , Ali H Bahkali  3 , Faisal S Almajed  4 , Ali O Azghani  5 , Mohammad A Khiyami  2 , Essam J Alyamani  2 , Sameera M Aljohani  6 , Majed A Halwani  1
Affiliations

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

Moayad Alhariri et al. Int J Nanomedicine. .

Abstract

We investigated the efficacy of liposomal gentamicin formulations of different surface charges against Pseudomonas aeruginosa and Klebsiella oxytoca. The liposomal gentamicin formulations were prepared by the dehydration-rehydration method, and their sizes and zeta potential were measured. Gentamicin encapsulation efficiency inside the liposomal formulations was determined by microbiologic assay, and stability of the formulations in biologic fluid was evaluated for a period of 48 h. The minimum inhibitory concentration and the minimum bactericidal concentration were determined, and the in vitro time kill studies of the free form of gentamicin and liposomal gentamicin formulations were performed. The activities of liposomal gentamicin in preventing and reducing biofilm-forming P. aeruginosa and K. oxytoca were compared to those of free antibiotic. The sizes of the liposomal formulations ranged from 625 to 806.6 nm in diameter, with the zeta potential ranging from -0.22 to -31.7 mV. Gentamicin encapsulation efficiency inside the liposomal formulation ranged from 1.8% to 43.6%. The liposomes retained >60% of their gentamicin content during the 48 h time period. The minimum inhibitory concentration of neutral formulation was lower than that of free gentamicin (0.25 versus 1 mg/L for P. aeruginosa and 0.5 versus 1 mg/L for K. oxytoca). The negatively charged formulation exhibited the same bacteriostatic concentration as that of free gentamicin. The minimum bactericidal concentration of neutral liposomes on planktonic bacterial culture was twofold lower than that of free gentamicin, whereas the negatively charged formulations were comparable to free gentamicin. The killing time curve values for the neutral negatively charged formulation against planktonic P. aeruginosa and K. oxytoca were better than those of free gentamicin. Furthermore, liposomal formulations prevent the biofilm-formation ability of these strains better than free gentamicin. In summary, liposomal formulations could be an effective lipid nanoparticle to combat acute infections where planktonic bacteria are predominant.

Keywords: antibacterial activity; biofilm; drug delivery; stability.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
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
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
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).
Figure 4
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
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
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
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
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
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
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
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
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
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).
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