Lipid nanoparticles as delivery vehicles for the Parietaria judaica major allergen Par j 2

Figures & data

Figure 1 Western blot analysis of recombinant Par j 2 with ten sera from Parietaria judaica allergic patient (lanes 1–10). Lane NA shows incubation with a nonallergenic control serum. Lane C displays a Coomassie Brilliant Blue stained sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified recombinant Par j 2. Molecular weights are indicated in lane M.

Figure 2 Reversed phase high-performance liquid chromatography elution profile of recombinant Par j 2.

Table 1 Enzyme-linked immunosorbent assay inhibition assay performed on plate-bound Parietaria judaica extract

Coated antigen Inhibitor	Parietaria judaica extract
	P. judaica extract	Recombinant Par j 2
Patient 1	94%	80%
Patient 2	98%	85%
Patient 3	91%	65%
Patient 4	94%	58%
Mean value	94.25%	72%

Note: Numbers indicate the percentage of inhibition.

Figure 3 Schematic diagram of solid lipid nanoparticle (SLN) preparation using double emulsion/evaporation method. Lipid heated to ~10°C of its melting point and organic solution of dichloromethane (CH₂Cl₂) containing Epikuron™ 200 added. Aqueous phase or aqueous dispersion of recombinant Par j 2 (rPar j 2) added under stirring by Ultra-Turrax^® T125 at 13,500 rpm (A), water in oil (w/o) emulsion formed (B), aqueous solution of Tween 80^® at 2% and second one at 1% added under stirring to obtain double emulsion water-in-oil-in-water (w/o/w) (C), organic solvent removed by evaporation (D), and freeze-dried (E).

Table 2 Chemical-physical characterization of empty and recombinant Par j 2-loaded solid lipid nanoparticles

Sample	Dispersing medium	Mean size (nm)	PDI	Zeta potential (mV) (± SD)	EE% (w/w)
Empty-SLN	PBS pH 7.4	224.7	0.282	–10.1	–
	NaCl 0.9 %	222.8	0.230	–5.34	–
	H2O	242.7	0.270	–44.9 ± 6.99	–
rPar j 2-loaded SLN	PBS pH 7.4	228.9	0.366	–7.27	40.0%
	NaCl 0.9 %	249.0	0.286	–6.74	40.0%
	H2O	252.5	0.311	–38.3 ± 5.50	40.0%
Empty-SLN (4 months)	H2O	247.3	0.287	–43.8 ± 5.88	–
Empty-SLN (10 months)	H2O	249.7	0.295	–44.9 ± 5.76	–
rPar j 2-loaded SLN (4 months)	H2O	251.5	0.383	–38.1 ± 4.18	38.7%
rPar j 2-loaded SLN (10 months)	H2O	230.5	0.364	–29.6 ± 3.26	39.5%

Note: Mean size, polydispersity index, and zeta potential values of empty and recombinant Par j 2-loaded solid lipid nanoparticles in twice-distilled water, phosphate buffered saline at pH 7.4, and sodium chloride 0.9% weight-in-volume aqueous solution before and after storage.

Abbreviations: EE%, encapsulation efficiency; H₂O, water; NaCl, sodium chloride; PBS, phosphate buffered saline; PDI, polydispersity index; rPar j 2, recombinant Par j 2; SD, standard deviation; SLN, solid lipid nanoparticles; w/v, weight-in-volume; w/w, weight-in-weight.

Figure 4 Basophil activation assay. Basophils from one representative healthy subject were stimulated with an increasing concentration of solid lipid nanoparticles produced under standard procedures (panels B and C) and with an equimolar concentration of SLN-pyrogen free particles (panels E and F). Panels A and D show the unstimulated sample.

Note: Numbers inside the graph express the percentage of cells with upregulated CD203c.

Abbreviations: LAL, limulus amebocyte lysate; PBS, phosphate buffered saline; SLN, solid lipid nanoparticles; SSC, side-scatter characteristics.

Figure 5 Basophil activation assay. Basophils from one representative Parietaria judaica healthy subject were stimulated with increasing concentration of solid lipid nanoparticles (SLN)-recombinant Par j 2 (panels B and C) and with an equimolar concentration of the recombinant Par j 2 (panels E and F). Panels A and D show the unstimulated sample.

Note: Numbers inside the graph express the percentage of cells with upregulated CD203c.

Abbreviations: PBS, phosphate buffered saline; SSC, side-scatter characteristics.