Caenorhabditis elegans as an alternative in vivo model to determine oral uptake, nanotoxicity, and efficacy of melatonin-loaded lipid-core nanocapsules on paraquat damage

Figures & data

Table 1 Concentration of PCL and LNC used in Caenorhabditis elegans during acute treatment

	Concentration of PCL* (mg/mL)	Concentration of LNC (LNC/mL)	Surface area of LNC (m^2/mL)
Control	–	–	–
Group I	0.05	2.37×10^12	0.57×10^2
Group II	0.1	4.74×10^12	1.14×10^2
Group III	0.25	11.85×10^12	2.85×10^2
Group IV	0.5	23.70×10^12	5.70×10^2
Group V	1	47.40×10^12	11.40×10^2
Group VI	2.5	118.50×10^12	28.50×10^2

Note:

* PCL was the polymer used in LNC formulation.

Abbreviations: LNC, lipid-core nanocapsules; PCL, poly(ε-caprolactone).

Table 2 Physicochemical characterization of LNC and Mel-LNC (batch of three samples)

Formulation	D[4,3] (nm)	Span	Z-average (nm)	PDI	Zeta potential (mV)	pH
LNC	243±2	1.75±0.02	205±4	0.10±0.02	−6.4±0.6	5.45±0.4
Mel-LNC	203±3	1.75±0.03	203±3	0.11±0.01	−5.2±0.2	5.58±0.3
Mel-LNC-RhodB	305±15	1.44±0.11	215±3	0.12±0.01	−9.5±0.9	5.24±0.2

Note: Results expressed as mean ± standard deviation.

Abbreviations: PDI, polydispersity index; LNC, unloaded nanocapsules; Mel-LNC, melatonin-loaded lipid-core nanocapsules; RhoB, rhodamine B.

Figure 1 Granulometric profile (laser diffraction).

Notes: (A) Unloaded lipid-core nanocapsules. (B) Melatonin-loaded lipid-core nanocapsules.

Figure 2 Microscope images (400× magnification) of worms exposed to RhoB-labeled Mel-LNC.

Notes: (A) Control worm (treated with saline solution). (B) Worm fluorescence image after 30 minutes of treatment, indicating an oral uptake. (C) Fluorescence image of worm 3 hours posttreatment, showing the distribution in the worm body. (D–F) Phase contrast microscopy images of worms; images were acquired at 400× magnification.

Abbreviations: Mel-LNC, melatonin-loaded lipid-core nanocapsules; RhoB, Rhodamine B.

Figure 3 Log dose–response curve for lethal dose 50% determination of lipid-core nanocapsules after acute treatment.

Note: Data are expressed as mean ± standard deviation.

Figure 4 Body areas of Caenorhabditis elegans after acute treatment with LNC in different concentrations.

Notes: (A) Control group. (B) Group III (11.85×10¹² LNC/mL). (C) Group VI 118.5×10¹² LNC/mL). (D) Graphical representation of concentration (LNC/mL) × body area (µm²). *P<0.01 compared with control group.

Abbreviation: LNC, unloaded lipid-core nanocapsules.

Figure 5 Reactive oxygen species production assessment after exposure with lipid-core nanocapsules in acute treatment.

Note: *P<0.01 compared to the control group.

Abbreviation: DCF, dichlorofluorescein.

Figure 6 Survival rate of worms pretreated with Mel-LNC and Mel after PQ exposure.

Notes: Control group was exposed to M9 buffer. *P<0.01 compared to the control group; ^#P<0.01 compared to the PQ group.

Abbreviations: Mel, free melatonin aqueous solution; Mel-LNC, melatonin-loaded lipid-core nanocapsules; PQ, paraquat; LNC, unloaded lipid-core nanocapsules.

Figure 7 Mel-LNC pretreatment protected Caenorhabditis elegans from abnormal development caused by PQ-poisoning.

Notes: (A) Control group. (B) Paraquat group. (C) LNC + PQ group. (D) Mel + PQ group. (E) Mel-LNC + PQ group. (F) Graphical representation of statistic difference among the groups. *P<0.01compared to the control group; ^#P<0.01 compared to the PQ group.

Abbreviations: LNC, unloaded lipid-core nanocapsules; Mel, free melatonin aqueous solution; Mel-LNC, melatonin-loaded lipid-core nanocapsules; PQ, paraquat.

Figure 8 Pretreatment with Mel-LNC protected against PQ-induced ROS production in Caenorhabditis elegans.

Notes: ROS were measured by DCF-DA assay. *P<0.01 compared to control group (treated with saline solution). ^#P<0.01 compared to the Mel + PQ group and PQ group.

Abbreviations: DCF, dichlorofluorescein; DCF-DA, 2′,7′-dichlorofluorescein diacetate; LNC, unloaded lipid-core nanocapsules; Mel, free melatonin aqueous solution; Mel-LNC, melatonin-loaded lipid-core nanocapsules; PQ, paraquat; ROS, reactive oxygen species.