Synergistic interactions between antimicrobial peptides derived from plectasin and lipid nanocapsules containing monolaurin as a cosurfactant against Staphylococcus aureus

Figures & data

Figure 1 Schematic representation of preparation of ML-LNCs and peptide-loaded ML-LNCs.

Abbreviation: ML-LNCs, monolaurin-lipid nanocapsules.

Figure 2 Flow curve of monolaurin-lipid nanocapsules.

Table 1 Structure and properties of antimicrobial peptides

Peptide	AP114 = NZ2114	AP138
Sequence	GFGCNGPWNEDDLRCHNHCKSIKGYKGGYCAKGGFVCKCY	GFGCNGPWSEDDLRCHRHCKSIKGYRGGYCAKGGFVCKCY
	Cyclic Cys4–Cys30, Cys15–Cys37, Cys19–Cys39	Cyclic Cys4–Cys30, Cys15–Cys37, Cys19–Cys39
Molecular weight	4,417 Da	4,460.3 Da
Theoretical pI	8.62	8.87
Number of amino acids	40	40
Number of cationic amino acids	6	7
Number of anionic amino acids	3	3
Net charge at pH 7	+3	+4

Note: The information was obtained from the manufacturer (AP114: Adenium Biotech, Denmark; AP138: PolyPeptide Laboratories, Sweden).

Table 2 Composition and properties of ML-LNCs and peptide-loaded ML-LNCs

Peptide	LNC/peptide MMR	Adsorption efficiency (%)	Peptide loading (%)	Particle diameter (nm)	Polydispersity index
–	N/A	N/A	N/A	36.0±0.30	0.026±0.020
AP138	100	61.96±0.94	0.6196±0.0094	36.2±0.18	0.024±0.009
AP138	50	46.15±0.62	0.9230±0.0125	36.4±0.13	0.037±0.017
AP138	25	37.39±0.81	1.4955±0.0322	36.8±0.17*	0.051±0.011
AP114	50	47.80±0.47	0.9560±0.0094	36.3±0.13	0.021±0.009
AP114	25	42.71±1.58	1.7802±0.0631	36.6±0.25	0.023±0.010
AP114	12.5	33.91±2.88	2.7130±0.2305	36.8±0.25*	0.036±0.011

Notes: Peptide concentration was 1,000 µg/mL.

* P<0.05 versus blank ML-LNCs.

Abbreviations: MMR, mass mixing ratio; ML-LNCs, monolaurin-lipid nanocapsules; N/A, not applicable.

Table 3 MICs of ML-LNCs and plectasin derivatives against different Staphylococcus aureus isolates

Strain	Strain ID	MIC AP114	MIC AP138	MIC ML-LNCs
MRSA	0706C0025	4	2	200
MRSA	11004533801	4	2	200
MRSA	11004691801	4	2	200
MRSA	11004787401	8	2	100
MRSA	11006153901	8	4	200
MRSA	070170095	4	2	200
MRSA	0702E0196	4	2	200
MSSA	11004327701	1	0.125	100
MSSA	11004480701	8	2	400
MSSA	11004697301	4	1	100
MSSA	11004010401	4	2	100
MSSA	0703H0036	4	1	100
MSSA	0701A0095	4	2	200
MSSA	ATCC 25923	8	4	100

Notes: MICs are expressed in µg/mL. ML-LNCs contain 15% of monolaurin.

Abbreviations: MICs, minimum inhibitory concentrations; ML-LNC, monolaurin-lipid nanocapsules; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible Staphylococcus aureus.

Figure 3 Time-kill curves of ML-LNCs against MSSA ATCC strain.

Abbreviations: ML-LNCs, monolaurin-lipid nanocapsules; MSSA, methicillin-susceptible Staphylococcus aureus.

Figure 4 Transmission electron microscope images of MSSA ATCC strain untreated control (A) and treated with 3.2 mg/mL (32× MIC) of ML-LNCs (B) for 1 hour. Scale bars: (A) left: 0.2 µm, right: 0.2 µm; (B) left: 0.5 µm, right: 100 nm.

Abbreviations: MIC, minimum inhibitory concentration; ML-LNCs, monolaurin-lipid nanocapsules; MSSA, methicillin-susceptible Staphylococcus aureus.

Figure 5 Time-kill curves of plectasin derivatives AP114 (A) and AP138 (B) against MSSA ATCC 25923 strain. The MIC values were 8 and 4 µg/mL for AP114 and AP138, respectively.

Abbreviations: MIC, minimum inhibitory concentration; MSSA, methicillin-susceptible Staphylococcus aureus.

Figure 6 Transmission electron microscope images of MSSA ATCC 25923 strain treated with 128 µg/mL (32× MIC) of AP114 (A) and 64 µg/mL (32× MIC) of AP138 (B) for 1 hour. Scale bars: (A) left: 0.2 µm, right: 100 nm; (B) left: 0.2 µm, right: 200 nm.

Abbreviations: MIC, minimum inhibitory concentration; MSSA, methicillin-susceptible Staphylococcus aureus.

Table 4 MICs of ML-LNCs and plectasin derivatives used alone and in combination against MSSA and MRSA

Strain	Peptide	MIC of peptide alone	MIC of peptide in mixture	MIN of ML-LNCs alone	MIC of ML-LNCs in mixture	FIC index
MRSA 0702E0196	AP138	2	0.5	200	25	0.375
MRSA 0702E0196	AP114	4	1	200	50	0.5
MSSA ATCC 25923	AP138	4	1	100	25	0.5
MSSA ATCC 25923	AP114	8	2	100	25	0.5

Note: MICs are expressed in µg/mL.

Abbreviations: FIC, fractional inhibitory concentration; MICs, minimum inhibitory concentrations; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible Staphylococcus aureus.

Figure 7 Time-kill curves of ML-LNCs and plectasin derivatives AP114 (A) and AP138 (B) used alone and in combination against MSSA ATCC strain.

Abbreviations: ML-LNCs, monolaurin-lipid nanocapsules; MSSA, methicillin-susceptible Staphylococcus aureus.

Figure 8 Transmission electron microscope images of MSSA ATCC strain treated with containing 1.6 mg/mL of ML-LNCs and plectasin derivative: 64 µg/mL of AP114 (A) or 32 µg/mL of AP138 for 1 hour. Scale bars: (A) left: 0.2 µm, right: 0.2 µm; (B) left: 0.5 µm, right: 0.2 µm.

Abbreviations: ML-LNCs, monolaurin-lipid nanocapsules; MSSA, methicillin-susceptible Staphylococcus aureus.