Oleic acid-loaded nanostructured lipid carrier inhibit neutrophil activities in the presence of albumin and alleviates skin inflammation

Figures & data

Table 1 The physicochemical characteristics of OA-NLC in the absence or presence of albumin

	Size (nm)	PI	ZP (mV)	EE (%)
Without albumin
OA-NLC	208.26 ± 0.93	0.19 ± 0.93	−40.59 ± 1.14	100.00 ± 0.00
With albumin
OA-NLC	421.72 ± 0.83	0.29 ± 0.02	−12.61 ± 0.06	N/A

Notes: The particle size, polydispersity index, zeta potential, and drug EE values nanoparticles are presented.

Abbreviations: OA-NLC, oleic acid within nanostructured lipid carrier; PI, polydispersity index; ZP, zeta potential; EE, entrapment efficiency; N/A, not applicable.

Figure 1 Transmission electron micrograph images of OA-NLC.

Notes: OA–NLC was applied to a copper grid coated with formvar/carbon film, and then stained with 1% phosphotungstic acid. The sample was subsequently dried in the air prior to observation under TEM. Scale bar, 200 nm. Representative data from four independent experiments are shown.

Abbreviations: OA-NLC, oleic acid within nanostructured lipid carriers; TEM, transmission electron microscopy.

Figure 2 OA and OA-NLC do not have cytotoxicity effect.

Notes: Neutrophils (6×10⁵ cells/mL) were treated with various concentrations of OA (A, C) or OA-NLC (B, D) for 17 min in the absence (A, B) or presence (C, D) of 0.1% BSA. LDH activity was assessed with a commercial LDH assay kit. Measured colorimetric signal was presented as a percentage of total LDH activity, as determined by measuring the fluorescence of lysed neutrophils treated with 0.1% Triton X-100. Data are expressed as mean $\pm$ standard error of the mean, n=4.

Abbreviations: OA, oleic acid; OA-NLC, oleic acid within nanostructured lipid carriers; BSA, bovine serum albumin; LDH, lactate dehydrogenase.

Figure 3 Effects of OA and OA-NLC on superoxide generation in activated neutrophils.

Notes: Human neutrophils (6×10⁵ cells/mL) were pre-incubated with OA or OA-NLC and then activated with cytochalasin B (1 μg/mL) and fMLF (0.1 μM) in the absence or presence of 0.1% BSA. (A, B) In the absence of BSA, superoxide release was reduced by both OA and OA-NLC. (C, D) In the presence of BSA, only OA-NLC inhibited superoxide release. Superoxide generation was detected spectrophotometrically using ferricytochrome c. Data are expressed as the mean ± standard error of the mean, n=6, **P<0.01, ***P<0.001, as compared to the control assay.

Abbreviations: OA, oleic acid; OA-NLC, oleic acid within nanostructured lipid carriers; BSA, bovine serum albumin.

Figure 4 Effects of OA and OA-NLC on elastase release in fMLF-activated neutrophils.

Notes: Human neutrophils (6×10⁵ cells/mL) were pre-incubated with OA or OA-NLC and then activated with cytochalasin B (0.5 μg/mL) and fMLF (0.1 μM) in the absence or presence of 0.1% BSA. (A, B) In the absence of BSA, elastase release was reduced by both OA and OA-NLC. (C, D) In the presence of BSA, only OA-NLC inhibited elastase release. Elastase release was detected spectrophotometrically using elastase substrate. Data are expressed as the mean ± standard error of the mean, n=6, **P<0.01, ***P<0.001, as compared to the control assay.

Abbreviations: OA, oleic acid; OA-NLC, oleic acid within nanostructured lipid carriers; BSA, bovine serum albumin.

Figure 5 Effects of OA and OA-NLC on elastase release in LTB4-activated neutrophils.

Notes: Human neutrophils (6×10⁵ cells/mL) were pre-incubated with OA or OA-NLC and then activated with cytochalasin B (0.5 μg/mL) and LTB4 (0.1 μM) in the absence or presence of 0.1% BSA. (A, B) In the absence of BSA, elastase release was reduced by both OA and OA-NLC. (C, D) In the presence of BSA, only OA-NLC inhibited elastase release. Elastase release was detected spectrophotometrically using elastase substrate. Data are expressed as the mean ± standard error of the mean, n=6, **P<0.01, ***P<0.001, as compared to the control assay.

Abbreviations: OA, oleic acid; OA-NLC, oleic acid within nanostructured lipid carriers; BSA, bovine serum albumin.

Table 2 Effects of OA and OA-NLC on Ca²⁺ mobilization in the absence or presence of albumin

	Without albumin		With albumin
	Peak [Ca^2+]i (nM)	t1/2 (sec)	Peak [Ca^2+]i (nM)	t1/2 (sec)
Control	297.34 ± 12.86	25.85 ± 2.92	346.40 ± 11.43	22.84 ± 0.94
OA	298.17 ± 17.62	6.90 ± 0.09***	334.62 ± 5.12	23.57 ± 0.87
OA-NLC	325.38 ± 10.05	9.40 ± 0.25***	371.04 ± 10.68	6.53 ± 0.41***

Notes: In the absence of albumin, neither OA nor OA-NLC affected peak [Ca²⁺]_i values in activated neutrophils; however, they significantly decreased t_1/2. In the presence of albumin, OA-NLC, but not OA, affected t_1/2 values. ***P<0.001, as compared to the control assay.

Abbreviations: t_1/2, the time required for [Ca₂₊]i to return to half of the peak value; OA, oleic acid; OA-NLC, oleic acid within nanostructured lipid carriers.

Figure 6 Effects of OA and OA-NLC on Ca²⁺ mobilization.

Notes: Fluo 3-loaded neutrophils were incubated with OA or OA-NLC for 5 min. Cells were then activated by fMLF (0.1 μM). Mobilization of Ca²⁺ was determined in real time in a spectrofluorometer. (A, B) In the absence of BSA, neither OA nor OA-NLC affected peak [Ca²⁺]i values in fMLF-activated neutrophils; however, they significantly decreased the time required for [Ca²⁺]i to return to half of the peak value (t_1/2). (C, D) In the presence of BSA, OA-NLC, but not OA, affected t_1/2 values. Representative traces from four independent experiments are shown.

Abbreviations: OA, oleic acid; OA-NLC, oleic acid within nanostructured lipid carriers; BSA, bovine serum albumin.

Figure 7 NLC uptakes by neutrophils.

Notes: Neutrophils (1.8×10⁷ cells/mL were co-incubated with (A) rhodamine 800 labeled-OA-NLC, and (B) H₂0 layer of rhodamine 800-loaded OA-NLC after configuration, at 37 °C for 5 min. The intracellular fluorescence signal was then observed by CLSM. (A) Fluorescence signals were observed throughout the neutrophil cytoplasm. Scale bar, 20 μm.

Abbreviations: CLSM, confocal laser scanning microscopy; NLC, nanostructured lipid carriers; OA-NLC, oleic acid within nanostructured lipid carriers.

Figure 8 TEM images of NLC interaction with albumin.

Notes: OA-NLC was co-incubated with or without BSA (5%), subsequently applied to a 200-mesh copper grid coated with formvar/carbon film. The sample was then stained with 1% phosphotungstic acid and observed under TEM. The diameter of the NLC-albumin complex with a protein corona (arrow) is larger than that of the NLC without a protein corona (Figure 1).

Abbreviations: NLC, nanostructured lipid carriers; OA-NLC, oleic acid within nanostructured lipid carriers; BSA, bovine serum albumin; TEM, transmission electron microscopy.

Figure 9 Effects of OA and OA-NLC on LTB4-induced skin inflammation.

Notes: OA or OA-NLC were applied topically to ear of the mice, followed by the topical application of LTB4 (0.5 μg/mL, in ethanol). Skin inflammation was characterized by epidermal neutrophil infiltration, which was alleviated by OA and OA-NLC. Representative histology images from six independent experiments are shown. Magnification, 400×; inset, 800×, Scale bar represent 50 μm; 25 μm (A) sham-operated mice. (B) Mice with LTB4-induced skin inflammation. (C) Mice with LTB4-induced skin inflammation pretreated with OA. (D) Mice with LTB4-induced skin inflammation pretreated with OA-NLC. (E) Administration of OA and OA-NLC suppress MPO activity, which had a significantly increased in inflamed skin induced with LTB4. Data are expressed as the mean ± standard error of the mean, n=6, **P<0.01, ***P<0.001, as compared to the control assay.

Table S1 Effects of unsaturated fatty acids on superoxide generation and elastase release

	Superoxide generation	Elastase release
	IC50 (μg/ml)
OA	0.72±0.07	0.32±0.04
LA	0.84±0.06	1.04±0.39
αLA	1.63±0.22	1.61±0.09

Notes: OA, C18:1, n-9; LA, C18:2, n-6; αLA, C18:3, n-3. Data are expressed as mean ± standard error of the mean.

Abbreviations: IC₅₀, half maximal inhibitory concentration; OA, oleic acid; LA, linoleic acid; αLA, α-linolenic acid.