Preparation and evaluation of antimicrobial activity of nanosystems for the control of oral pathogens Streptococcus mutans and Candida albicans

Figures & data

Figure 1 X-ray powder diffraction patterns of (A) CHX and βCD, (B–D) physical mixture and inclusion complexes obtained by solubilization-freeze-drying and kneading at 1:1 (B), 1:2 (C), and 1:3 (D) molar ratios.

Abbreviations: βCD, β-cyclodextrin; CHX, chlorhexidine; K, kneading; PM, physical mixture; S, solubilization-freeze-drying.

Figure 2 Differential scanning calorimetry curves of CHX, βCD, MβCD, HPβCD, and inclusion complexes obtained by kneading at a 1:1 molar ratio.

Abbreviations: βCD, β-cyclodextrin; CHX, chlorhexidine; HPβCD, hydroxypropyl- β-cyclodextrin; K, kneading; MβCD, methyl-β-cyclodextrin; Temp, temperature.

Table 1 Inclusion process results obtained with MMTNa and CHX

Intercalation conditions			2θ (°)	Basal spacing (Å)	Indirect yield (%)
MMTNa
MMTNa			6.55	13.81	–
CHX:MMTNa
CHX:MMTNa 60%	1 h	C1	5.25	16.83	71
	18 h	C2	5.40	16.37	71
	24 h	C3	5.05	17.50	70
	48 h	C4	5.20	16.99	79
CHX:MMTNa 80%	1 h	C5	5.00	17.67	50
	18 h	C6	5.05	17.50	58
	24 h	C7	5.10	17.33	61
	48 h	C8	5.20	16.99	76
CHX:MMTNa 100%	1 h	C9	5.00	17.67	36
	18 h	C10	5.00	17.67	45
	24 h	C11	5.10	17.33	46
	48 h	C12	4.95	17.85	50

Abbreviations: CHX, chlorhexidine; h, hour; MMTNa, sodium montmorillonite.

Figure 3 DSC curves of MMTNa, CHX, and nanosystems with 60% CEC – 24 hours (C3).

Abbreviations: CEC, cation exchange capacity; CHX, chlorhexidine; DSC, differential scanning calorimetry; MMTNa, sodium

Table 2 Inclusion process results obtained with MMTNa and NYS

Intercalation conditions		2θ (°)		Basal spacing (Å)	Indirect yield (%)
NYS:MMTNa 60%	1 h	N1	5.90	14.98	51
	18 h	N2	5.65	15.64	52
	24 h	N3	5.20	16.99	54
	48 h	N4	5.95	14.85	46
NYS:MMTNa 80%	1 h	N5	5.45	16.22	53
	18 h	N6	5.60	15.78	49
	24 h	N7	5.20	16.99	55
	48 h	N8	5.75	15.37	43
NYS:MMTNa 100%	1 h	N9	5.40	16.37	47
	18 h	N10	5.60	15.78	46
	24 h	N11	5.55	15.92	51
	48 h	N12	6.00	14.73	44

Abbreviations: h, hour; MMTNa, sodium montmorillonite; NYS, nystatin.

Figure 4 X-ray powder diffraction patterns of MMTNa and NYS-and-MMTNa nanosystems (NYS:MMTNa) with 60% CEC – 24 hours (N3).

Abbreviations: CEC, cation exchange capacity; MMTNa, sodium montmorillonite; NYS, nystatin.

Figure 5 DSC curves of MMTNa, NYS, physical mixture of 60% CEC (NYS:MMTNa PM), and NYS:MMTNa 60% CEC – 24-hour nanosystem (N3).

Abbreviations: CEC, cation exchange capacity; DSC, differential scanning calorimetry; MMTNa, sodium montmorillonite; NYS, nystatin; PM, physical mixture; Temp, temperature.

Table 3 Inhibition halo diameters of CHX (control) and inclusion complexes against Streptococcus mutans and Candida albicans

	S. mutans			C. albicans
Inhibition halo diameters (mm)
CHX:βCD	17	17	17	15	15	15
CHX:MβCD	18	18	18	16	16	16
CHX:HPβCD	17	17	17	16	15	15
CHX	17	17	17	16	16	15

Abbreviations: βCD, β-cyclodextrin; CHX, chlorhexidine; HPβCD, hydroxypropyl-β-cyclodextrin; MβCD, methyl-β-cyclodextrin.

Table 4 Inhibition halo diameters of pure substances and developed derivatives against Streptococcus mutans and Candida albicans strains using three different plating techniques

	S. mutans			C. albicans
	Plate 1	Swab 1a	Swab 2b	Plate 1	Swab 1a	Swab 2b
Inhibition halo diameters (mm)
MMT	0	0	0	0	0	0
CHX	27	26	27	18	19	26
C3c	22	21	21	11	12	20
NYS	0	0	0	16	19	27
N3d	0	0	0	0	0	0

a Notes: Swab 1 samples applied in drops;

b Swab 2 samples applied to wells;

c C3 = CHX:MMTNa 60% CEC – 24 hours;

d N3 = NYS:MMTNa 60% CEC – 24 hours.

Abbreviations: CEC, cation exchange capacity; CHX, chlorhexidine; MMT, montmorillonite; MMTNa, sodium montmorillonite; NYS, nystatin.

Figure 6 Inhibition halo analysis using (A) Streptococcus mutans (n = 3) and (B) Candida albicans strains (n = 3) of CHX aqueous solution 0.12%, CHX derivative 60% CEC – 24 hours (C3), NYS, NYS derivative 60% CEC – 24 hours (N3), and MMTNa.

Note: The inoculum in all Petri dishes followed the order displayed on the first dish.

Abbreviations: CHX, chlorhexidine; MMTNa, sodium montmorillonite; NYS, nystatin.

Figure 7 Stability profile of mouth wash formulation containing CHX not included, and inclusion complexes of βCD, MβCD, and HPβCD stored at 40°C ± 2°C and 75% ± 5% relative humidity for 6 months.

Abbreviations: βCD, β-cyclodextrin; CHX, chlorhexidine; HPβCD, hydroxypropyl-β-cyclodextrin; MβCD, methyl-β-cyclodextrin.