PEGylation of zinc nanoparticles amplifies their ability to enhance olfactory responses to odorant

Abstract

Olfactory responses are intensely enhanced with the addition of endogenous and engineered primarily-elemental small zinc nanoparticles (NPs). With aging, oxidation of these Zn nanoparticles eliminated the observed enhancement. The design of a polyethylene glycol coating to meet storage requirements of engineered zinc nanoparticles is evaluated to achieve maximal olfactory benefit. The zinc nanoparticles were covered with 1000 g/mol or 400 g/mol molecular weight polyethylene glycol (PEG). Non-PEGylated and PEGylated zinc nanoparticles were tested by electroolfactogram with isolated rat olfactory epithelium and odorant responses evoked by the mixture of eugenol, ethyl butyrate and (±) carvone after storage at 278 K (5 oC), 303 K (30 oC) and 323 K (50 oC). The particles were analyzed by atomic force microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and laser Doppler velocimetry. Our data indicate that stored ZnPEG400 nanoparticles maintain physiologically-consistent olfactory enhancement for over 300 days. These engineered Nanoparticles support future applications in olfactory research, sensitive detection, and medicine.

Fig 1. Physical properties of non-PEGylated and PEGylated zinc nanoparticles.

(a) AFM images, and (b) histograms showing the size distribution of the nanoparticles. A: Zinc nanoparticles, B: ZnPEG400, C: ZnPEG1000.

Fig 2. Electron microscopy images of Zn, ZnPEG400, and ZnPEG1000 nanoparticles.

TEM images (a, c, d) are showing the primarily-elemental zinc nanoparticles at different magnifications, (b) ZnPEG400 nanoparticle showing the metal core and the PEG passivation layer, (e) ZnPEG1000 nanoparticles with negative staining, and (f) ZnPEG1000 magnified from (e). The numbers 1, 2, 3 point to the nanoparticles with visible lattice fringe patterns, indicating their crystallinity.

Fig 3. XPS spectra.

Representative high resolution XPS spectra showing the (a) C 1s and (b) Zn 2p3/2 core lines for the freshly prepared bare zinc and PEGylated zinc nanoparticles stored 1 day at 278 K (5 ^oC), with the spectra offset to facilitate viewing. The Zn 2p spectra are shown in log-scale. The solid curves indicate the experimentally obtained spectra, with the dotted curves underneath indicating their best-fit chemical components. For the C 1s spectra, from lower to higher binding energy, the components are C-C, C-O, and C = O. For the Zn 2p spectra, the components are Zn and ZnO. For the (a) C 1s plots, the solid vertical line represents the position of the C-C peaks, to which all spectra were calibrated to, with the dotted lines showing the spectral shift of the C-O peaks, which were 286 eV, 286.4 eV, and 286.2 eV for the Zn, ZnPEG400, and ZnPEG1000 systems, respectively. For (b) Zn 2p plots, the solid line represents the position of the Zn peak for the bare Zn system, while the dotted lines show the spectral shift of the ZnO peaks, which were 1024.6 eV, 1025.6 eV, and 1026.0 eV for the Zn, ZnPEG400, and ZnPEG1000 systems, respectively. Each spectrum represents an average of six spectral runs.

Fig 4. Representative EOG recordings from rat olfactory epithelium.

a The stimuli were of 0.25 s pulses of (1) odorant mixture, (2) odorant mixture +1.2 nm zinc nanoparticles, (3) odorant mixture + 0.25 mmole/L PEG400, (4) odorant mixture + 0.10 mmole/L PEG1000, (5) water vapor + PEG400, (6) water vapor + PEG1000, and (7) water vapor. The concentration of zinc nanoparticles and odorant mixture were 0.02 nmole/L and 1.6 mmole/L, respectively. The representative set of traces was obtained from 50 EOG traces.

Fig 5. The relative EOG signals as a function of time of storage of zinc nanoparticles.

The difference between peak values of EOG evoked by odorant and by non-PEGylated and PEGylated zinc nanoparticles was normalized by the EOG peak evoked by an odorant alone. The relative enhancement by zinc nanoparticles was calculated as described in Materials and methods.

Fig 6. The relative EOG signals evoked by non-PEGylated and PEGylated zinc nanoparticles.

a. The relative enhancement by non-PEGylated zinc nanoparticles as a function of ZnO concentration. The difference between peak values of EOG evoked by odorant and by zinc nanoparticles was normalized by the EOG peak evoked by an odorant alone as a function of ZnO concentration that was determined by XPS. The first bar at 3% of ZnO, corresponds to zinc nanoparticles stored one day at 278 K (5 ^oC). The second and third bars reflect zinc nanoparticles stored for two days at 303 K (30 ^oC) and 323 K (50 ^oC), at 7.1% and 7.8% respectively. The forth bar at 11.5% of ZnO corresponds to the negative enhancement (inhibition) that was observed with zinc nanoparticles oxidized by percolating air. b. The relative EOG enhancement produced by ZnPEG400 nanoparticles after they were stored for one day at 278 K (5 ^oC), two days at 303 K (30 ^oC) and 323 K (50 ^oC), respectively. c. The relative EOG enhancement produced by ZnPEG1000 nanoparticles after they were stored for one day at 278 K (5 ^oC), two days at 303 K (30 ^oC) and 323 K (50 ^oC), respectively.