Micellar curcumin improves the antibacterial activity of the alkylphosphocholines erufosine and miltefosine against pathogenic Staphyloccocus aureus strains

Figures & data

Figure 1. Loading of curcumin (µg/mL micellar dispersion) depending on the loading method and Pluronic^® type.

Table 1. Size and zeta-potential of the developed micelles loaded with CRM.

CRM-loaded micelles (NCRM)	Size (nm)	Zeta-potential (mV)
P123	20.2 ± 1.5	−5.4
F127	47.0 ± 3.0	−6.3
P123/F127	55 ± 5.5	−7.1

Figure 2. In vitro release of curcumin in a phosphate buffer.

Figure 3. Effect of single drugs on colony formation.

Note: CRM-Et, ethanol solution of curcumin; MLT, miltefosine; ERF, erufosine.

Table 2. Antibacterial activity of examined drugs against MSSA and MRSA.

Drugs	Parameters of antimicrobial susceptibility	Staphylococcus aureus strains
		ATCC6538 (MSSA)	MRSA
CRM, ethanol solution	MIC [µmol/L]/[mg/L]	125/46	125/46
	CFU/mL at MIC	7.2 × 10^3	∼10^5
	Redox activity at MIC [%]	9.2 ± 0.7	13.8 ± 0.9
	MBC [µmol/L]/[mg/L]	500/184	>1000*
	MBIC50 [µmol/L]/[mg/L]	–	24.84/8.83
	BIC80 [µmol/L]/[mg/L]	–	100/36.8
CRM-loaded P123 micelles (NCRM1)	MIC [µmol/L]/[mg/L]	500/184	500/184
	CFU/mL at MIC	≥10^5	≥10^5
	Redox activity at MIC [%]	51.2 ± 3.1	63.5 ± 5.8
	MBC [µmol/L]/[mg/L]	bacteriostatic	bacteriostatic
	MBIC50 [µmol/L]/[mg/L]	–	88.2/32.46
	BIC90 [µmol/L]/[mg/L]	–	200/73.6
CRM-loaded P123/F127 micelles (NCRM2)	MIC [µmol/L]/[mg/L]	500/184	500/184
	CFU/mL at MIC	≥10^5	≥10^5
	Redox activity at MIC [%]	56 ± 2.4	61 ± 4.7
	MBC [µmol/L]/[mg/L]	bacteriostatic	bacteriostatic
	MBIC50[µmol/L]/[mg/L]	–	62/22.82
	BIC90[µmol/L]/[mg/L]	–	200/73.6
Miltefosine (MLT)	MIC [µmol/L]/[mg/L]	62.5/25.47	80/32.61
	CFU/mL at MIC	1.35 × 10^2	4 × 10^2
	Redox activity at MIC [%]	8.8 ± 0.3	9 ± 5.8
	MBC [µmol/L]/[mg/L]	500/203.8	500/203.8
	MBIC50[µmol/L]/[mg/L]	–	6.0/2.45
	BIC90[µmol/L]/[mg/L]	–	7.8/3.18
	BIC100[µmol/L]/[mg/L]	–	20/8.15
Erufosine (ERF)	MIC [µmol/L]/[mg/L]	50/25.29	60/30.22
	CFU/mL at MIC	1.7×10^3	1×10^1
	Redox activity at MIC [%]	10.5 ± 0.1	8.4 ± 0.08
	MBC [µmol/L]/[mg/L]	1000/503.8	500/251.9
	MBIC50[µmol/L]/[mg/L]	–	1.87/0.94
	BIC90[µmol/L]/[mg/L]	–	7.1/3.58
	BIC99[µmol/L]/[mg/L]	–	20/10.08
Penicillin (reference drug)	MIC [µmol/L]/[mg/L]	0.024/0.008	>5.98*/2**
	MBC [µmol/L]/[mg/L]	0.1/0.032	–
	Redox activity at MIC [%]	33.5 ± 5.5	–
Gentamycin (reference drug)	MIC [µmol/L]/[mg/L]	–	0.52/0.25
	MBC [µmol/L]/[mg/L]	–	2.08/1
	Redox activity at MIC [%]	–	11.1 ± 1.7

Note: MBIC₅₀, minimal biofilm inhibitory concentration (inhibits up to 50% the biofilm formation); BIC₈₀, BIC₉₀, BIC₉₉ and BIC₁₀₀, biofilm inhibitory concentrations that inhibit the biofilm formation by 80, 90, 99 and 100%.

*This value is the highest tested concentration.

**MIC of penicillin higher than 2 mg/L is usually typical for methicillin resistant strains according to EUCAST.

Figure 4. Redox activity of S. aureus strains after single drug treatment – MM modified model.Note: NEA, normalized enzyme activity, [%]; Vm, overall normalized maximum enzyme activity, [%]; Ki, inhibition constant, [µmol/L]; R, correlation coefficient, [-].

Figure 5. Redox activity of S. aureus strains after single drug treatment – LP model.

Note: NEA, normalized enzyme activity; [%]; Dose, concentration of drug, [µmol/L]; P1, inhibitory constant, which may be interpreted IC₅₀ in medical studies or as the dose achieving 50% inhibition, [µmol/L]; P2, slope, [-]; R, correlation coefficient, [-].

Figure 6. Quantitative biofilm evaluation after treatment with ethanol solution of curcumin, curcumin-loaded micelles, miltefosine and erufosine.

Figure 7. Biofilm formation of MRSA after treatment with curcumin (in the form of ethanol solution or nano-micelles) and alkylphosphocholines: microscopic documentation of the biofilm inhibition.

Table 3. Antibacterial activity of drug combinations.

Bacterial strain	Drug A			Drug B			∑FIC	Combination ratio	Effect
	Name	MICC [µmol/L]	FIC	Name	MICC [µmol/L]	FIC
MRSA		62.5	0.78	CRM	3.9	0.03	0.81	16:1 [A:B]	Additive
	MLT	31.25	0.39	NCRM1	31.25	0.06	0.45	1:1 [A:B]	Synergy
		31.25	0.39	NCRM2	31.25	0.06	0.45	1:1 [A:B]	Synergy
		62.5	1.04	CRM	3.9	0.03	1.07	16:1 [A:B]	Indifference (nearly additive)
	ERF	62.5	1.04	NCRM1	62.5	0.125	1.129	1:1 [A:B]	Indifference
		31.25	0.5	NCRM2	31.25	0.06	0.56	1:1 [A:B]	Additive
ATCC 6538		62.5	1	CRM	3.9	0.03	1.03	16:1 [A:B]	Indifference (nearly additive)
	MLT	31.25	0.5	NCRM1	7.813	0.02	0.52	4:1 [A:B]	Additive (nearly synergy)
		62.5	1	NCRM2	15.625	0.03	1.03	4:1 [A:B]	Indifference (nearly additive)
		62.5	1.25	CRM	3.9	0.03	1.28	16:1 [A:B]	Indifference
	ERF	31.25	0.625	NCRM1	7.813	0.02	0.645	4:1 [A:B]	Additive
		31.25	0.625	NCRM2	7.8125	0.02	0.645	4:1 [A:B]	Additive
		0.05	2	CRM	62.5	0.5	2.5	1:1250[A:B]	Indifference
	Pen	0.01	0.5	NCRM1	15.625	0.03	0.53	1:1562.5[A:B]	Additive (nearly synergy)
		0.005	0.25	NCRM2	15.625	0.03	0.28	1:3125 [A:B]	Synergy

Note: Drug A, MLT (miltefosine) or ERF (erufosine); Drug B, CRM-Et (ethanol solution of surcumin), NCRM1 (P123 based micelles) or NCRM2 (P123/F127 based micelles); MIC_C, minimal inhibitory concentration in combination; FIC, fractional inhibitory concentration; [A:B], ratio between drug A and drug B.