Influence of surface charge on the potential toxicity of PLGA nanoparticles towards Calu-3 cells

Figures & data

Table 1 Physicochemical properties of unlabeled and rhodamine-labeled nanoparticles

Nanoparticle type	Mean diameter (nm)	PDI	Zeta potential (mV)	Associated stabilizers (mg/100 mg PLGA)
PLGA/CS	230 ± 6	0.20	+32 ± 3	15.3 ± 3.3 (CS)
				30.4 ±1.8 (PVA)
PLGA/PVA	230 ± 2	0.12	−5 ± 1	11.5 ± 1.1 (PVA)
PLGA/PF68	105 ± 2	0.13	−24 ± 1	10.7 ± 3.7 (PF68)
Rhod-PLGA/CS	174 ± 3	0.20	+13 ± 1	20.5 ± 4.8 (CS)
				31.8 ± 2.6 (PVA)
Rhod-PLGA/PVA	226 ± 4	0.09	−5 ± 1	12.4 ± 1.0 (PVA)
Rhod-PLGA/PF68	100 ± 2	0.13	−20 ± 2	10.6 ± 2.1 (PF68)

Abbreviations: PLGA, poly (lactide-co-glycolide); PVA, poly (vinyl alcohol); CS, chitosan; Rhod, rhodamine B; PF68, Pluronic^® F68; PDI, polydispersity index.

Figure 1 Transmission electron microscopic images of (A) PLGA/CS nanoparticles, (B) PLGA/PVA nanoparticles, and (C) PLGA/PF68 nanoparticles.

Abbreviations: PLGA, poly (lactide-co-glycolide); PVA, poly (vinyl alcohol); PF68, Pluronic^® F68; CS chitosan.

Table 2 Evolution of mean diameter of PLGA/CS, PLGA/PVA, and PLGA/PF68 nanoparticles at 37°C as a function of time (size is expressed in nm unless otherwise stated). Nanoparticles were incubated and measured in water, cell culture medium containing 10% fetal bovine serum, and Ca²⁺ and Mg²⁺ free phosphate buffer

Experimental medium	Time (h)	Nanoparticle type
		PLGA/CS	PLGA/PVA	PLGA/PF68
	0	230 ± 8	210 ± 6	103 ± 2
	4	218 ± 6	205 ± 5	102 ± 5
H20	24	227 ± 7	203 ± 5	101 ± 2
	48	212 ± 3	201 ± 6	98 ± 4
	72	225 ± 8	201 ± 4	103 ± 2
	96	215 ± 3	204 ± 3	102 ± 3
	4	217 ± 36	159 ± 12	113 ± 4
	24	166 ± 13	141 ± 6	123 ± 8
DMEM + 10% FBS	48	173 ± 10	157 ± 5	131 ± 9
	72	216 ± 26	156 ± 5	128 ± 5
	96	188 ± 12	156 ± 6	134 ± 9
	4	688 ± 86	198 ± 9	114 ± 4
	24	851 ± 113	197 ± 5	102 ± 3
PBS	48	>1 μm	200 ± 4	115 ± 5
	72	>1 μm	202 ± 4	112 ± 2
	96	>1 μm	201 ± 3	111 ± 3

Note: Data represent the mean ± standard deviation (n = 3).

Abbreviations: FBS, fetal bovine serum; PLGA, poly (lactide-co-glycolide); PVA, poly (vinyl alcohol); PF68, Pluronic^® F68; CS, chitosan; PBS, phosphate-buffered solution; DMEM, Dulbecco’s modified Eagle’s medium.

Figure 2 Calu-3 cell viability (MTT assay) after 72 hours of exposure to PLGA/CS, PLGA/PVA, and PLGA/PF68 nanoparticles (0.030–5 mg/mL). Each experiment was repeated eight times from three independent incubation preparations. Results are expressed as percentages of absorption for treated cells (± standard deviation) in comparison with untreated control cells.

Notes: Statistical significance was indicated as P < 0.05. *PLGA/CS nanoparticles versus PLGA/PVA nanoparticles (0.030–5 mg/mL); **PLGA/CS versus PLGA/PF68 nanoparticles (3–5 mg/mL); ^§PLGA/PVA nanoparticles versus PLGA/PF68 nanoparticles (0.030–1 mg/mL).

Abbreviations: PLGA, poly (lactide-co-glycolide); PVA, poly (vinyl alcohol); CS, chitosan; NPs, nanoparticles; PF68, Pluronic^® F68.

Figure 3 Calu-3 cell viability (MTT assay) after 72 hours of exposure to (A) PLGA/CS, (B) PLGA/PVA, and (C) PLGA/PF68 nanoparticles or (A) CS, (B) PVA, and (C) PF68 solutions as a function of stabilizer concentration. Each experiment was repeated eight times from three independent incubation preparations. Results are expressed as percentages of absorption for treated cells (± standard deviation) in comparison with untreated control cells.

Note: Statistical significance was indicated as *P < 0.05 (stabilizer solution versus PLGA nanoparticles).

Abbreviations: PLGA, poly (lactide-co-glycolide); PVA, poly (vinyl alcohol); PF68, Pluronic^® F68; CS, chitosan; NPs, nanoparticles.

Figure 4 Evolution of Calu-3 cell viability (MTT assay) after exposure to (A) PLGA/CS, (B) PLGA/PVA, and (C) PLGA/PF68 nanoparticles as a function of time. Each experiment was repeated eight times from three independent incubation preparations. Results are expressed as percentages of absorption for treated cells (± standard deviation) in comparison with untreated control cells.

Notes: The significance was indicated as *P < 0.05 (24 hours versus 4 hours) and ^#P < 0.05 (72 hours versus 24 hours).

Abbreviations: PLGA, poly (lactide-co-glycolide); PVA, poly (vinyl alcohol); PF68, Pluronic^® F68; CS, chitosan; NPs, nanoparticles.

Figure 5 Real time confocal laser scanner microscopy images of Calu-3 cells exposed to Rhod-PLGA/PVA nanoparticles for 24 hours and subsequent washing of the medium. (A) Nomarski image, (B) fluorescent image, and (C) superimposition of Nomarski and fluorescence images, and (D) enlarged region of (C).

Note: Scale bars = 20 μm.

Abbreviations: PLGA, poly (lactide-co-glycolide); PVA, poly (vinyl alcohol); Rhod, rhodamine B alcohol.

Figure 6 (A) IL-6 and (B) IL-8 secretion by Calu-3 cells exposed for 24 hours and 48 hours to lipopolysaccharide (gray bars), PLGA/CS (red bars), PLGA/PVA (green bars), and PLGA/PF68 nanoparticles (blue bars). White bars represent cytokine secretion from untreated control cells.

Notes: Data represent the mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01 (lipopolysaccharide-exposed versus control and nanoparticle-treated cells).

Abbreviations: PLGA, poly (lactide-co-glycolide); PVA, poly (vinyl alcohol); PF68, Pluronic^® F68; CS, chitosan; IL, interleukin.

Table S1 X-ray photoelectron spectroscopic analysis of the different C1s and O1s peak fitting intensities of the polymers before and after particle formulation

Sample	XPS C1s envelope ratio (%)			XPS O1s envelope ratio (%)
	CC-C	CC-O	CC=O	OC=O	OO-C=O	OC-OH	OC-O-C
PLGA	24.8	35.0	40.2	62.4	37.6	–	–
CS	14.0	69.0(2)	17.0	–	–	39.7	60.3
PVA	47.9	45.1	7.0	36.4	–	63.6	–
PF68	100.0(1)	–	–	–	–	24.1	75.9
Rhod-OH	48.4	45.2	6.4	8.9	–	75	16.1
Rhod-PLGA	49.9	43.4	6.7	10.1	–	71.8	18.1
PLGA/CS nanoparticles	25.6	54.0(2)	20.4	–	–	76.1	23.9
PLGA/PVA nanoparticles	37.7	35.7	26.6	42.3	14.0	43.7	–
PLGA/PF68 nanoparticles	35.1	41.9	23.0	30.2	19.3	–	50.5
Rhod-PLGA/CS nanoparticles	28.0	45.0(2)	27.0	41.2	–	42.3	16.5
Rhod-PLGA/PVA nanoparticles	35.0	36.4	28.6	41.3	12.8	45.9	–
Rhod-PLGA/PF68 nanoparticles	31.0	51.0	18.0	83.0(3)	17.0	–	–

(1) Notes: Peak corresponds to C_C-O and C_C-N environments;

(2) Peak corresponds to C_C-O and C_C-N environments;

(3) Peak corresponds to O_C=O and C_C-O-C environments.

Abbreviations: Rhod, rhodamine B; PLGA, polylactide-co-glycolide; PVA, polyvinyl alcohol; PF68, Pluronic^® F68; CS, chitosan; XPS, x-ray photoelectron spectroscopy.

Table S2 Fluorescence variation with Calu-3 cells after exposure to Rhod-PLGA/CS, Rhod-PLGA/PVA, and Rhod-PLGA/PF68 nanoparticles. Red fluorescence was measured at 4, 12, and 24 hours

	Percentage of cells above fluorescence threshold
	4 hours	12 hours	24 hours
Rhod-PLGA/CS	9.1 ± 1.1	13.4 ± 1.8	21.5 ± 4.4
Rhod-PLGA/PVA	7.6 ± 2.3	14.4 ± 4.7	20.3 ± 3.8
Rhod-PLGA/PF68	9.0 ± 1.0	15.8 ± 1.6	28.4 ± 4.3

Notes: The results were obtained after gating and selection of a fluorescence threshold. 10,000 cells were counted per sample. Data are expressed as the mean ± standard deviation (n = 3).

Abbreviations: Rhod, rhodamine B; PLGA, poly (lactide-co-glycolide); PVA, poly (vinyl alcohol); PF68, Pluronic^® F68; CS, chitosan.

Figure S1 Oxygen O1 s envelopes of x-ray photon spectroscopic analysis from (A) PLGA/CS nanoparticles and (B) Rhod-PLGA/CS nanoparticles.

Abbreviations: Rhod, rhodamine B; PLGA, poly (lactide-co-glycolide); CS, chitosan.

Figure S2 Real time confocal laser scanner microscopy images of Calu-3 cells exposed to Rhod-PLGA/CS nanoparticles for 24 hours and subsequent washing of the medium. (A) Nomarski image, (B) fluorescent image, and (C) superimposition of Nomarski and fluorescence images. (D, E, and F) show enlarged pictures.

Note: Scale bars = 20 μm.

Abbreviations: Rhod, rhodamine B; PLGA, poly (lactide-co-glycolide); CS, chitosan.

Figure S3 Real time confocal laser scanner microscopy images of Calu-3 cells exposed to Rhod-PLGA/PF68 nanoparticles for 24 hours and subsequent washing of the medium. (A) Nomarski image, (B) fluorescent image, and (C) superimposition of Normaski and fluorescence images. (D, E, and F) show enlarged pictures.

Note: Scale bars = 20 μm.

Abbreviations: Rhod, rhodamine B; PLGA, poly (lactide-co-glycolide); PF68, Pluronic^® F68.