Intradermal air pouch leukocytosis as an in vivo test for nanoparticles

Figures & data

Table 1 The effect of different sets of nanoparticles according to the air pouch test

Production	Nanoparticle†††	LAL assay (pg/mg)	Cells (×10^6) per air pouch	% Neutrophils
	PBS	≤12.5*	0.77±0.39†	37.8±6.9††
	COAM	13.3	1.53±0.51†	60.7±7.7††
BIU, Israel	CANsmaghemite NPs^1a	28.4	2.77	76
BIU, Israel	Type I PEI-CAN-maghemite^1b	≤9.3	0.91	88
BIU, Israel	Type II PEI-CAN-maghemite^1c	42.5	2.83	55
CIDETEC, Spain	PDMAEMA-SCPNs^2a	562.5	1.75	89
CIDETEC, Spain	PMAAc-SCPNs^2b	762.5	0.45	56
UNIBO, Italy	PLGA-COOH^3a	≤3.1	0.9	34
UNIBO, Italy	PLGA-b-PEG-COOH^3b	12.5	1.1	39
UNIBO, Italy	Magh@PNPs^3c	41.7	1.22	44
GU, Germany	LNP LII^4a	12	0.89	59
GU, Germany	CAN CIII^4b	≤1.3	0.74	39

Notes:

† The amount of cells per air pouch in the PBS and COAM conditions was calculated over a set of seven experiments with three mice per condition. The values are shown as mean ± SD. The PBS and COAM groups were significantly different as calculated with a paired t-test (n=7, P=0.0043).

†† The percentage of neutrophils per air pouch in the PBS and COAM conditions were calculated over a set of seven experiments with three mice per condition. The values are shown as mean ± SD. The PBS and COAM groups were significantly different as calculated with a paired t-test (n=7, P=0.0032).

††† Identification and characteristics of nanoparticles: 1a–c: (1a) CAN-γ-Fe₂O₃ NPs: average NP diameter 7.61 ± 2.33 nm (TEM), weight ratio Ce/Fe: 0.029; (1b) Type I PEI₂₅-CAN-γ-Fe₂O₃ NPs: average NP diameter 6.50 ± 2.15 nm (TEM), 71.2–96.56 nm (DLS average hydrodynamic NP diameter), ζ potential: +23.0–24.0 mV; weight ratio Ce/Fe: 0.095; (1c) Type II PEI₈-CAN-γ-Fe₂O₃ NPs: average NP diameter 7.65 ± 2.64 nm (TEM), 58.0–62.0 nm (DLS average hydrodynamic NP diameter), ζ potential: +56.3 mV; weight ratio Ce/Fe: 0.100. 2a and b: (2a) PDMAEMA-SCPNs: N,N′-dimethylaminoethyl methacrylate-based single chain polymer nanoparticles, ±13 nm (DLS), ζ potential: +11.5, (2b) PMAAc based SCPNs: polymethacrylic acid-based single chain polymer nanoparticles, ±17 nm (DLS); 3a–c: (3a) PLGA-COOH: average NP diameter 160 nm, ζ potential: −55.1 mV, PDI: 0.078, (3b) PLGA-b-PEG-COOH: average NP diameter 74 nm, ζ potential: −11.5 mV, PDI: 0.095; (3c) Magh@PNPs: average NP diameter 92.3 nm, ζ potential: −7.56 mV, PDI: 0.098. 4a and b: (4a) LNP lII: average NP diameter 193 ± 1 nm, ζ potential: −41.9 ± 0.7 mV, PDI: 0.023 ± 0.021, (4b) CAN CIII: average NP diameter 191 ± 1 nm, ζ potential: −52.7 ± 0.3 mV, PDI: 0.027 ± 0.035.

* For PBS the endotoxin analysis was obtained per mL volume. For all other products we calculated the endotoxin level/mg pure product.

Abbreviations: BIU, Bar-Ilan University; CAN, ceric ammonium nitrate; CIDETEC, Fundación CIDETEC; COAM, chlorite-oxidized oxyamylose; DLS, dynamic light scattering; GU, Goethe-Universität; LAL, Limulus amebocyte lysate; LNP, lipid nanoparticle; Magh, Maghemite; n, number; NP, nanoparticle; PBS, phosphate-buffered saline; PDI, polydispersity index; PDMAEMA, poly(N,N-dimethylaminoethyl methacrylate); PEG, polyethylene glycol; PEI, polyethyleneimine; PLGA, poly(D,L-lactide-co-glycolide); PMAAc, polymethacrylic acid; PNP, polymeric nanoparticles; SCPN, single-chain polymeric nanoparticle; TEM, transmission electron microscopy; UNIBO, University of Bologna; SD, standard deviation.

Figure 1 Optimization of the air pouch model.

Notes: (A) schematic outline of the air pouch model. At the start of the test, 3 mL air is injected through a sterile 0.20 μm filter at a dermal site of a mouse to form a pouch. On the third day, this action is repeated. Products are injected on day 6 after the start and their cellular and molecular effects are measured 24 hours later by retrieval of cells and molecules within the pouch exudates. (B–D) COAM induces a dose-dependent recruitment of neutrophils and can be used as a standard for the air pouch model. The air pouch model was executed in the standard setting of 6 day formation and 24 hour contact time for test samples. COAM was used at different doses. Three possible read-outs are shown: (B) Increase (and saturation) of percentage neutrophils, (C) decrease of macrophage percentages and (D) decrease of percentage lymphocytes (n=5 for all test samples). The positive control reached saturation at 1 mg COAM per pouch. (E) Analysis of the absolute numbers of retrieved cells per pouch. (F and G) correlation between FACS and cytospin data from mouse air pouches. (F) Correlation (two-tailed parametric correlation analysis) between percentages of neutrophils (polymorphonuclear cells) as measured by FACS analysis and cytospin analysis (r=0.80, n=45). Linear regression was performed and found to be significant (P<0.0001). (G) Correlation (two-tailed parametric correlation analysis) of percentages mononuclear cells as measured by FACS analysis and cytospin analysis (r=0.58, n=79). Linear regression was performed and found to be significant (P<0.0001).

Abbreviations: COAM, chlorite-oxidized oxyamylose; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline.

Figure 2 Analysis of polystyrene particles with different sizes in air pouches.

Notes: Particles were either endotoxin-free polystyrene nanoparticles (100 nm) or polystyrene microspheres with a diameter of 1 μm. (A) Analysis of the absolute numbers of retrieved cells. (B) The relative numbers of leukocytes as determined by hemocytometry and as corroborated by FACS analysis. (C) Illustration of cytospin preparations stained with hemacolor and higher magnifications of the cells in the insets. (D) Illustration of gelatin zymography analysis of air pouch fluids. Gelatinase A/matrix metalloproteinase-2 (MMP-2) is constitutively expressed in all samples and gelatinase B/MMP-9 is up regulated after injection of COAM or polystyrene nanoparticles. (E) Scanning densitometry analysis of gelatin zymograms. The MMP-9 content is expressed as the ratios of MMP-9 versus constitutive MMP-2. The results illustrate the acute inflammatory context of air pouches, after COAM treatment, at the molecular level. Clearly, cellular infiltration is correlated with the induction of MMP-9.

Abbreviations: COAM, chlorite-oxidized oxyamylose; FACS, fluorescence-activated cell sorting; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline.

Figure S1 CAN-γ-Fe₂O₃, type I PEI₂₅-CAN-γ-Fe₂O₃ and type II PEI₂₅-CAN-γ-Fe₂O₃ NPs TEM and size distribution.

Notes: (A) CAN-γ-Fe₂O₃ NPs (left) TEM microphotograph; (right) size distribution histogram of 7.61±2.33 nm-sized CAN-γ-Fe₂O₃ NPs. (B) Type I PEI₂₅-CAN-γ-Fe₂O₃ NPs (left) TEM microphotographs; (right) size distribution histogram of averaged 6.50±2.15 nm-sized Type I PEI₂₅-CAN-γ-Fe₂O₃ NPs. (C) Type II PEI₈-CAN-γ-Fe₂O₃ NPs (left) TEM microphotographs; (right) size distribution histogram of averaged 7.65±2.64 nm-sized Type II PEI₂₅-CAN-γ-Fe₂O₃ NPs.

Abbreviations: NP, nanoparticle; PEI, polyethylene imine; TEM, transmission electron microscopy.

Figure S2 PDMAEMA and PMAAc SCPNs dynamic light scattering (DLS) and atomic force microscopy (AFM) images.

Notes: (A) PDMAEMA SCPNs (left) DLS measurement data: average size of 13±2.9 nm, and (right) AFM picture of dry nanoparticles around 3 nm in size; (B) PMAAc SCPNs (left) DLS measurement data: average size of 16±4.2 nm; and (right) AFM picture of dry nanoparticles around 5 nm in size.

Abbreviations: PDMAEMA, poly(N,N-dimethylaminoethyl methacrylate); PMAAc, polymethacrylic acid; SCPN, single-chain polymeric nanoparticle.

Figure S3 PLGA-COOH NPs, PLGA-b-PEG-COOH NPs and Magh@PNPs size distribution histograms, zeta potentials and transmission electron microscopy images.

Notes: (A) PLGA-COOH NPs (top) size distribution histogram; (bottom) ζ potential of PLGA-COOH NPs; (B) PLGA-b-PEG-COOH NPs (top) size distribution histogram; (bottom) ζ potential of PLGA-b-PEG-COOH NPs; (C) Magh@PNPs (top) size distribution histogram, (middle) ζ potential, and (bottom) TEM microphotographs of Magh@PNPs.

Abbreviations: NP, nanoparticle; PEG, polyethylene glycol; PLGA, poly(D,L-lactide-co-glycolide); PNPs, polymeric nanoparticles; TEM, transmission electron microscopy.