Development, characterization, and in vivo assessment of mucoadhesive nanoparticles containing fluconazole for the local treatment of oral candidiasis

Figures & data

Table 1 The composition of NPs for preliminary studies

Formulation code	EUD RS 100 (%)	EUD RL 100 (%)
N1	2.5	–
N2	5	–
N3	7.5	–
N4	10	–
N5	–	2.5
N6	–	5
N7	–	7.5
N8	–	10
N9	1.25	1.25
N10	2.5	2.5
N11	3.75	3.75
N12	5	5

Abbreviations: NPs, nanoparticles; EUD, EUDRAGIT^®.

Table 2 The composition of main NP formulations

Formulation code	EUD RS 100 (%)	EUD RL 100 (%)	CSH (%)	FLZ (%)
N13	2.5	–	–	0.5
N14	–	2.5	–	0.5
N15	1.25	1.25	–	0.5
N16	2.5	–	0.025	–
N17	–	2.5	0.025	–
N18	1.25	1.25	0.025	–
N19	2.5	–	0.025	0.5
N20	–	2.5	0.025	0.5
N21	1.25	1.25	0.025	0.5

Abbreviations: NPs, nanoparticles; EUD, EUDRAGIT^®; CSH, chitosan; FLZ, fluconazole.

Table 3 PS, PI, and ZP of NPs

Formulation code	PS (nm) ± SD	PI ± SD	ZP (mV) ± SD
N1	173.4±6.4	0.183±0.016	30.0±0.9
N2	223.1±0.9	0.192±0.009	30.8±1.9
N3	348.3±3.8	0.175±0.002	25.9±0.3
N4	447.9±3.9	0.161±0.022	23.5±0.3
N5	134.2±1.4	0.228±0.018	21.5±1.5
N6	200.5±1.5	0.164±0.021	20.2±0.7
N7	292.3±3.9	0.161±0.002	19.3±1.0
N8	372.7±3.7	0.191±0.010	17.9±0.7
N9	147.2±0.6	0.200±0.001	25.1±0.7
N10	227.8±0.7	0.166±0.016	24.3±1.4
N11	306.8±2.1	0.226±0.031	23.9±0.6
N12	388.7±1.9	0.182±0.031	23.5±1.4
N13	181.1±1.3	0.189±0.040	28.3±1.3
N14	141.0±1.3	0.207±0.019	18.7±0.8
N15	173.7±2.2	0.240±0.014	21.9±0.5
N16	194.5±1.5	0.251±0.007	33.0±0.4
N17	165.0±3.7	0.262±0.010	22.9±0.9
N18	213.1±3.8	0.270±0.025	26.2±0.3
N19	210.2±3.0	0.255±0.012	29.7±0.5
N20	197.7±2.8	0.294±0.019	14.8±1.2
N21	216.2±1.4	0.275±0.020	20.8±0.4

Note: Data is presented as mean ± SD.

Abbreviations: PS, particle size; PI, polydispersity index; ZP, zeta potential; NPs, nanoparticles; SD, standard deviation.

Figure 1 TEM photographs of the NP formulations.

Notes: (A) N1 (the scale bar for image represents 500 nm), (B) N13 (the scale bar for image represents 5,000 nm), (C) N16 (the scale bar for image represents 20,000 nm), and (D) N19 (the scale bar for image represents 100 nm).

Abbreviations: TEM, transmission electron microscopy; NP, nanoparticle.

Figure 2 (A) Estimation of the ZP of the CSH-coated NPs during incubation in 0.1% aqueous mucin dispersion (B) estimation of the interaction between CSH-coated NPs and mucin dispersion by turbidimetric assay.

Abbreviations: ZP, zeta potential; NPs, nanoparticles; CSH, chitosan.

Table 4 EE and DL capacity of the NPs

Formulation code	EE (%)	DL (%)
N13	87.39±1.59	99.22±0.67
N14	87.39±1.44	98.68±1.62
N15	87.07±0.50	99.86±0.51
N19	87.47±1.30	99.50±1.52
N20	85.79±1.62	99.93±0.21
N21	86.08±1.6	99.55±1.80

Note: Data is presented as mean ± SD.

Abbreviations: EE, drug entrapment efficiency; DL, drug loading; NPs, nanoparticles; SD, standard deviation.

Figure 3 (A) in vitro release of noncoated NPs and (B) in vitro release of CSH-coated NPs.

Abbreviations: FLZ, fluconazole; CSH, chitosan; NPs, nanoparticles.

Table 5 Release parameters of FLZ from NPs

Formulation code	n	Log k	r^2
N13	1.220	−0.150	0.977
N14	0.507	0.794	0.954
N15	0.767	0.530	0.999
N19	0.796	0.324	0.964
N20	0.559	0.808	0.973
N21	0.866	0.337	0.982

Abbreviations: FLZ, fluconazole; NPs, nanoparticles.

Figure 4 Cytotoxic effect of selected NPs evaluated by MTT assay.

Note: Bars represent “mean ± standard deviation” values from five individual experiments. *P<0.0005, group compared to control group (taken as 100%). Triton X-100 used as control for cytotoxic activity.

Abbreviations: NPs, nanoparticles; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 5 Zones caused by the formulations on G-MB-MHA medium (A) N16, (B) N19.

Abbreviation: G-MB-MHA, glucose- and methylene blue-added Mueller-Hinton Agar.

Figure 6 (A) Fungicidal effect of N19-related in vitro release study; (B) Time-dependent fungicidal effects of N19.

Figure 7 Images of histological examination.

Notes: (A) Palatal mucosa of a cured animal (1: epithelium, 2: lamina propria) (HE ×10). (B) Palatal mucosa of an animal with ongoing infection (1: epithelium, 2: lamina propria) (HE ×10). (C) Tongue mucosa of a cured animal (HE ×10) (1: epithelium, 2: lamina propria, 3: muscle, [→] papillae of the tongue). (D) Tongue mucosa of an animal with ongoing infection (HE ×10) (1: epithelium, 2: lamina propria, 3: muscle, [→] papillae of the tongue). (E) Buccal mucosa of a cured animal (HE ×10) (1: epithelium, 2: lamina propria, 3: muscle). (F) Buccal mucosa of an animal with ongoing infection (HE ×10) (1: epithelium, 2: lamina propria, 3: muscle, [→] degradation in the stratified epithelium).

Abbreviation: HE, hematoxylin–eosin.