Alginate nanoparticles as non-toxic delivery system for miltefosine in the treatment of candidiasis and cryptococcosis

Figures & data

Table 1 Physicochemical characteristics of unloaded (Nano.Alg) and miltefosine-loaded (MFS.Alg) alginate nanoparticles before and after the freeze-drying process

	Size (nm)	Pdi	Zeta potential (mV)
Nano.Alg before freeze-drying	335.7±10.9	0.35±0.06	−21.1±1.3
Nano.Alg after freeze-drying	346.5±30.6	0.40±0.07	−36.2±6.8*
MFS.Alg before freeze-drying	320.2±37.9	0.43±0.14	−19.0±3.1
MFS.Alg after freeze-drying	279.1±56.7	0.42±0.15	−39.7±5.2*

Notes: Data represent mean ± standard deviation. *P<0.05 compared with nanoparticles before freeze-drying (Student’s t-test).

Figure 1 Transmission electron microscopy (A, B) and scanning electron microscopy (C, D) of alginate nanoparticles. Unloaded (Nano.Alg) (A, C) and miltefosine-loaded (MFS.Alg) (B, D) nanoparticles. Bars in (A) and (B): 2 μm; bars in (C) and (D): 0.5 μm.

Figure 2 Release of miltefosine (MFS) from alginate nanoparticles. In vitro release at 37 °C, with constant agitation (200 rpm).

Figure 3 Toxicity of free miltefosine (MFS) and miltefosine-loaded alginate nanoparticles (MFS.Alg) in Galleria mellonella larvae. *P<0.05 when compared with untreated larvae (PBS group). Unloaded alginate nanoparticles (Nano.Alg) were also tested and no toxicity was observed.

Table 2 Susceptibility of Candida spp. and Cryptococcus spp. strains to fluconazole (FLC), amphotericin B (AMB), free miltefosine (MFS) and miltefosine-loaded alginate nanoparticles (MFS.Alg)

Strains	Concentrations (µg/mL)
	FLC^a		AMB^a		MFS		MFS.Alg
MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC
C. albicans SC5314	2	4	0.12	0.12	1	1	300	600
C. albicans IAL-40	64^R	>64	0.12	0.12	1	1	600	>600
C. glabrata ATCC 2001	16^R	>64	0.12	0.25	0.5	0.5	150	>600
C. glabrata IAL-23	8^SDD	>64	0.12	0.25	0.5	1	37.5	>600
C. krusei ATCC 6258	64^R	>64	0.5	0.5	2	4	600	>600
C. krusei IAL-30	64^R	>64	0.25	0.5	1	1	600	>600
C. parapsilosis ATCC 22019	2	4	0.12	0.25	1	1	600	>600
C. parapsilosis IAL-17	>64^R	>64	0.25	0.25	2	2	600	>600
C. tropicalis ATCC 200956	>64^R	>64	2^R	2	1	2	600	>600
C. tropicalis IAL-01	2	16	0.25	0.25	2	2	300	>600
C. neoformans CAP59	1	32	0.03	0.03	1	1	1.56	1.56
C. neoformans H99	4	4	0.25	0.25	2	2	25	400
C. gattii ATCC 56990	2	32	0.25	0.25	0.5	0.5	12.5	25

Note: ^aThe assay was repeated and the results obtained here corroborated with previously published data.⁶⁶

Abbreviations: MIC, minimum inhibitory concentration; MFC, minimum fungicidal concentration; R, resistant; SDD, susceptibility dose-dependent.

Figure 4 Survival curves of Galleria mellonella larvae infected with yeasts and treated 30 mins post-infection with free miltefosine (MFS) and miltefosine-loaded alginate nanoparticles (MFS.Alg). (A) Candida albicans SC5314, (B) Candida albicans IAL-40, (C) Cryptococcus neoformans H99, (D) Cryptococcus gattii ATCC 56990. P-values when compared with the untreated group.

Figure 5 Fungal burden and histopathological analysis of Galleria mellonella larval tissue infected with yeasts and treated or not with free miltefosine (MFS) and miltefosine-loaded alginate nanoparticles (MFS.Alg). Fungal burden data is represented by the mean ± standard deviation. *P<0.05 and  **P<0.01 when compared with the untreated group (one-way ANOVA with Dunnet’s post-test). Fungal burden, filamentation and dissemination of yeasts in Galleria mellonella larvae were analyzed by visualization of larval tissue histological sections of larvae untreated, treated with MFS (40 mg/kg), or treated with MFS.Alg (200 mg/kg). Black arrows indicate yeasts and white arrows indicate C. albicans filamentation. Bars: 50 μm.

Muñoz JE, Rossi DCP, Ishida K, et al. Antifungal activity of the biphosphinic cyclopalladate C7a against Candida albicans yeast forms In vitro and In vivo. Front Microbiol. 2017;8:771. doi:10.3389/fmicb.2017.0077128515716

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