The Droplet-Size Effect Of Squalene@cetylpyridinium Chloride Nanoemulsions On Antimicrobial Potency Against Planktonic And Biofilm MRSA

Figures & data

Table 1 The Characterization Of Micelles And Cationic Nanoemulsions By Droplet Size, Polydispersity Index (PDI), And Zeta Potential

Formulation	Size (nm)	PDI	Zeta Potential (mV)
Micelles	1434.67±102.47	1.0±0	9.27±2.23
CN-S	54.54±1.93	0.40±0.12	46.53±1.86
CN-M	164.73±1.44	0.19±0.05	46.37±2.35
CN-L	244.70±2.36	0.26±0.01	51.10±0.56

Note: Each value represents the mean±SD (n=3).

Figure 1 The physicochemical characteristics of micelles and squalene@CPC nanoemulsions: (A) the size change of micelles and nanoemulsions at 50 °C and 75% relative humidity for 14 days; and (B) fluorescence emission of Nile red incorporation in micelles and nanoemulsions for determining molecular environment polarity. The stability data are presented as the mean of four experiments±S.D.

Table 2 The Minimum Inhibitory Concentration (MIC, µg/mL) And Minimum Bactericidal Concentration (MBC, µg/mL) Of Cetylpyridinium Chloride (CPC) In Micelles And Cationic Nanoemulsions Against Different Strains Of Bacteria

	Strain	Micelles	CN-S	CN-M	CN-L
MIC	MRSA	0.98	0.98	0.98	0.98
	S. aureus	0.49~0.98	0.49	0.49	0.49
	KM-1	0.49~0.98	0.49	0.49	0.49
	KV-2	0.49~0.98	0.49	0.49	0.49
	E. coli	3.91	3.91~7.81	3.91~7.81	7.81
	C. albicans	1.95~3.91	3.91	1.95~3.91	1.95~3.91
MBC	MRSA	1.95	1.95	1.95	0.98~1.95
	S. aureus	0.49~0.98	0.98~1.95	0.98	0.49~0.98
	KM-1	0.98	0.98	0.98	0.98
	KV-2	0.98~1.95	1.95	0.98~1.95	0.98
	E. coli	3.91	7.81	7.81	7.81~15.63
	C. albicans	3.91~7.81	7.81~15.63	7.81~15.63	7.81~15.63

Note: KM-1 and KV-2 are the clinical isolates of MRSA and VISA, respectively. Each value represents the mean±S.D. (n=3).

Figure 2 Determination of the antibacterial activity of micelles and squalene@CPC nanoemulsions against planktonic MRSA: (A) zone of inhibition measured from agar diffusion assay; (B) morphological changes of MRSA viewed under SEM; and (C) the enlarged images of B. The agar diffusion data are presented as the mean of three experiments±S.D.

Figure 3 The live/dead imaging by treatment of micelles and squalene@CPC nanoemulsions against planktonic MRSA: (A) treatment with CPC at 10 μg/mL; and (B) treatment with CPC at 100 μg/mL.

Figure 4 Determination of the antibacterial activity of micelles and squalene@CPC nanoemulsions against biofilm MRSA: (A) MRSA CFU inside the biofilm; (B) MRSA CFU outside the biofilm; (C) the three-dimensional images of biofilm visualized by confocal microscopy; (D) the corresponding biofilm green intensity; and (E) the corresponding biofilm thickness. All data are presented as the mean of three experiments±S.D. ***p < 0.001; **p < 0.01; *p < 0.05.

Table 3 Differentially Expressed Proteins In MRSA Following The Treatments Of Micelles And Cationic Nanoemulsions

Band No.	Protein	Accession No.	Mw (Da)	Matched Peptides	Coverage % (SCORE)	Ratios To MRSA^a,b				Biological Function
Micelles	CN-S	CN-M	CM-L
1	DNA-directed RNA polymerase subunit β	P60279	133,409	19	16% (100)	−2.33	−3.23	−8.34	−3.45	DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates.
2	DNA-directed RNA polymerase subunit β’	P60286	134,327	24	24% (115)	−2.78	−0.71	−3.57	−0.64	DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates.
3	Transketolase	Q6GH64	72,235	15	28% (136)	0.64	2.07	1.16	2.44	Catalyzes the transfer of a two-carbon ketol group from a ketose donor to an aldose acceptor, via a covalent intermediate with the cofactor thiamine pyrophosphate.
4	Phosphoenolpyruvate carboxykinase (ATP)	Q8NVZ8	59,599	26	56% (168)	−0.59	−0.21	−0.96	−0.54	Involved in the gluconeogenesis. Catalyzes the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) through direct phosphoryl transfer between the nucleoside triphosphate and OAA.
5	Catalase	Q8NWV5	51,221	14	47% (141)	−0.28	0.95	0.97	2.20	Decomposes hydrogen peroxide into water and oxygen; serves to protect cells from the toxic effects of hydrogen peroxide.
6	Enolase	P64079	46,036	14	47% (90)	0.91	−1.59	1.82	1.18	Catalyzes the reversible conversion of 2-phosphoglycerate into phosphoenolpyruvate. It is essential for the degradation of carbohydrates via glycolysis.
7	Pyruvate dehydrogenase E1 component subunit alpha	P60090	36,459	7	34% (84)	−3.03	−2.50	−3.57	−1.94	The pyruvate dehydrogenase complex catalyzes the overall conversion of pyruvate to acetyl-CoA and CO2.
8	L-lactate dehydrogenase	P65257	34,650	15	58% (96)	−2.33	−1.87	−2.22	−2.21	Catalyzes the conversion of lactate to pyruvate. Appears to be the primary factor that allows S.aureus growth during nitrosative stress in both aerobically and anaerobically cultured cells.
9	Cysteine synthase	P63872	33,013	9	45% (84)	−2.86	−0.27	−0.31	−1.85	O-acetyl-L-serine + hydrogen sulfide = L-cysteine + acetate.
10	Pyridoxal 5ʹ-phosphate synthase subunit PdxS	P60799	32,114	8	42% (95)	−0.07	1.13	0.99	2.34	Catalyzes the formation of pyridoxal 5ʹ-phosphate from ribose 5-phosphate (RBP), glyceraldehyde 3-phosphate (G3P) and ammonia.
11	2,3-bisphosphoglycerate-dependent phosphoglycerate mutase	P65709	26,697	24	86% (204)	1.08	1.19	−0.12	−0.21	Catalyzes the interconversion of 2-phosphoglycerate and 3-phosphoglycerate.
12	DUF1642 domain-containing protein	PF07852.6	22,691	9	40% (82)	−1.79	1.22	−0.95	1.25	Protein of unknown function (DUF1642)
13	Superoxide dismutase [Mn/Fe] 2	P66832	23,098	10	72% (92)	1.06	1.09	1.99	1.82	Destroys superoxide anion radicals which are normally produced within the cells and which are toxic to biological systems.
14	Ferritin, partial	Q7A0H5	16,627	7	52% (112)	1.04	1.78	1.55	2.17	Iron-storage protein.
15	DNA protection during starvation protein 2	Q8RPQ2	16,749	9	76% (88)	1.23	1.16	0.93	1.58	Protects DNA from oxidative damage by sequestering intracellular Fe^2+ ion and storing it in the form of Fe^3+ oxyhydroxide mineral.
16	DNA-binding protein HU	Q7A0U9	9,606	7	72% (96)	1.14	1.25	1.28	1.61	Histone-like DNA-binding protein which is capable of wrapping DNA to stabilize it, and thus to prevent its denaturation under extreme environmental conditions.

Notes: ^aRatios to MRSA indicated the fold changes in protein volume among micelles, CN-S, CN-M, and CN-L versus MRSA samples, respectively. The ratios > 1.0 mean the proteins whose expression levels were increased upon treatments of compounds, while ratios < −1.0 indicate the proteins were downregulated under the exposure to compounds. ^bAnalyzing the gel images using GeneTools software.

Figure 5 The genomic and proteomic profiles of MRSA treated by micelles and squalene@CPC nanoemulsions: (A) analysis of the quality of MRSA genomic DNA by agarose gel electrophoresis; (B) total protein concentration in MRSA; and (C) the protein change of MRSA analyzed by SDS-PAGE and MALDI-TOF/TOF mass. All data are presented as the mean of three experiments±S.D. ***p < 0.001; **p < 0.01; *p < 0.05.