Characterization of silver particles in the stratum corneum of healthy subjects and atopic dermatitis patients dermally exposed to a silver-containing garment

Figures & data

Figure 1. AFM particle characterization of silver garment: (A) AFM height image of a silver coated fiber (vertical scale of 600 nm); (B) height profile along indicated lines. Arrow indicates particle height analysis taking into account the fiber curvature. (C) Histogram of particle height distribution (117 particles were analyzed). Red curve (Line 1) represents empirical distribution and the blue curve (Line 2) represents theoretical Gumbel distribution with parameters a = 36.2 and b = 15.9.

Figure 2. SEM image of the fiber surface on the silver containing garment: (A) low (1000×) magnification illustrating overall appearance of fibers (white square indicates location of EDX spectra collection); (B) increased (5000×) magnification (area inside yellow rectangle in panel A) illustrating rough appearance of coating on fiber surfaces; (C) increased (50 000×) magnification (area inside yellow rectangle in panel B) illustrating roughness is due to presence of nanoparticles coating fibers; and (D) SEM-EDX spectra from SEM image A (white square) demonstrating that the nanoparticles coating the fibers are composed of silver (purple tags = silver, green tag = oxygen, orange tag = carbon).

Table1. The summary of the SEM-EDX analysis of the SC samples after exposure to silver containing garment.

Subject	Tape no. (depth)	Location Ag aggregates	Presence of other inorganic particles	Other comments
1 AD	1 (0.39 μm)	On ridge or near edge of skin cell	No particles detected	Only silver aggregates were detected (size: about 1.0 to about 1.5 μm)
	2 (1.16 μm)	On skin cell	No particles detected	Only silver aggregates were detected (size: about 1.0 μm)
	7 (4.89 μm)	On ridge of skin cell	No particles detected	Only silver aggregates were detected (size: about 500 nm)
	10 (6.96 μm)	On skin cell	No particles detected	Only silver aggregates were detected (size: about 1.0 μm)
2 AD	1 (0.19 μm)	Mostly onto keratinocytes surface	Few particles (about 1 μm) containing titanium were found in the intercellular space (tape 10)
	3 (0.95 μm)
	10 (3.68 μm)
5 AD	1 (0.27 μm)	Mostly on cells, rarely between them	The deeper a layer, the more non-silver elements are found	• On surficial layers of the SC only pure Ag, while in deeper layers more Cl, K and S. • Silver mostly is at the apical side of the corneocyte, also in deeper layers (tape 5). The number of Ag-clusters on the second and fifth strip is approx. 10–30% less as compared to strip 1
	2 (0.79 μm)
	5 (2.31 μm)
6 AD	10 (5.81 μm)	Mostly onto keratinocytes surface, few in the intercellular space	Few particles (about 1 μm) containing titanium were found in the intercellular space (tape 15)	Silver aggregates were the most abundant form, but also few aggregates with Ag–Cl–K–Na were found
	15 (7.65 μm)
7 AD	1 (0.32 μm)	Mostly onto keratinocytes surface, few in the intercellular space	Submicrometrical particles of KCl were found on the corneocytes surface and in the intercellular space (tape 1)	Silver aggregates were the most abundant form, but also few aggregates with Ag–Cl–K–Na were found
	20 (8.89 μm)
8 H	3 (1.77 μm)	Mostly onto keratinocytes surface, few in the intercellular space	No particles detected	Silver aggregates were the most abundant form, but also few aggregates with Ag and Cl were found
9 H	1 (0.37 μm)	On skin cell	Numerous dull gray particles detected on skin cell ridges and in furrows containing K–C–-Ca–S	Numerous particles containing only Ag detected (size: about 500 nm)
	2 (1.19 μm)	On skin cell	No particles detected	Particles containing only Ag detected (size: about 500 nm)
	6 (3.89 μm)	No Ag particles detected	No particles detected
	10 (5.93 μm)	On skin cell; on ridge or in the center	Large jelly bean shaped particle on skin cells containing Cl	Particle size: few hundred nm to over 1 μm
	15 (7.86 μm)	On skin cell; near edge of skin cell	No particles detected	Particles containing only Ag detected (size: about 1 μm to about 2 μm)
	20 (9.21 μm)	On skin cell: near center	No particles detected	Particles containing only Ag detected (size: about 500 nm)
10 H	3 (1.78 μm)	Mostly onto keratinocytes surface, few in the intercellular space	No particles detected	Only silver aggregates were found
	9 (5.13 μm)
	19 (10.25 μm)
11 H	3 (1.79 μm)	Mostly onto keratinocytes surface, few in the intercellular space	No particles detected	Silver aggregates were the most abundant form, but also few aggregates with Ag and Cl were found
	7 (4.84 μm)
	9 (5.73 μm)
	19 (10.42 μm)
12 H	1 (0.36 μm)	On skin cell	No particles detected	1 Ag particle
	6 (3.50 μm)	No Ag particles detected	No particles detected
13 H	3 (1.39 μm)	Mostly onto keratinocytes surface	Submicrometrical particles containing K–Cl–Ca–Al–Ti were found on the corneocytes surface and in the intercellular space (tape 1)	The aggregates in this volunteer were more abundant than in other subjects. Silver aggregates were the most abundant, but also few aggregates with Ag–Ca–Al were found
	15 (6.83 μm)
	20 (8.65 μm)

H: healthy controls; AD: atopic dermatitis patients.

Figure 3. SEM image and silver elemental mapping from three spots on the SC tape no.15 of a healthy subject after wearing of silver garment. Two bright spots on the corneocyte surface (yellow squares in images A and D) correspond to the spots with high silver electron density in the elemental maps (yellow squares in images B and E) and were identified as silver in the EDX spectrum (C and F).

Figure 4. SEM image of SC tape no. 5 after wearing of silver garment. Micrometric aggregates with high electron density (white rectangles 1 and 2) on the corneocyte surface were identified as silver in the EDX spectrum (Spectrum 1 and 2). The EDX did not detect silver on the “black” spot (white rectangle 3) (Spectrum 3).

Figure 5. (A). SEM image of SC tape strip after wearing of silver garment. Micrometric aggregates on the corneocyte surface found on tape strip no. 5. (B): Corresponding EDX spectra of five bright spots (white rectangles) in the SEM image (A) showing different composition of the particles.

Figure 6. AFM imaging of the 15th SC tape of a healthy subject revealing cluster of nanoparticles: (A) height image with vertical scale of 400 nm; (B) 3D view of zoomed area (white box in A); (C) height image of zoomed area in A with height profile of the cluster along indicated line.

Figure 7. AFM imaging of the 12th SC tape of of an AD patient revealing particles of micrometric dimensions: (A) height image of the crystallites (vertical scale 1.2 mm); (B) 3D view; (C) height profile of the large crystallite along indicated line in A.

Table 2. The summary of the AFM results of SC samples analysis.

ID volunteer	Exposure	Tape no. (depth)	Description and location of Ag NPs	Presence of aggregates/agglomerates	Max. height
3 AD	Ag	1 (0.37 μm)	Ag NPs – not conclusive. Three single NPs found, height 20, 26 and 30 nm. Mostly onto keratinocytes surface	Not detected
		7 (4.28 μm)	• More single NPs detected, but not conclusive to be Ag NPs. • Five single NPs detected, height 30–60 nm, possibly Ag NPs covered with lipid layer. Mostly onto keratinocytes surface	Three aggregates: height 70 nm, 200 nm width; height 500 nm, 220 nm width; height 1000 nm, 600 nm width	1000 nm
		12 (7.09 μm)	2 Ag NPs, height 45 nm. More single NPs detected, but not conclusive to be Ag NPs. Mostly onto keratinocytes	Small cluster/agglomerate (3 NPs): height 45 nm, 400 nm width
5 AD	Ag	1 (0.24 μm)	• Large population of Ag NPs, height range 20–30 nm. • One particle 70 nm. Mostly onto keratinocytes surface	Not detected
		6 (2.57 μm)	Single NPs detected, but not conclusive to be Ag NPs. Mostly onto keratinocytes surface	Not detected
		12 (5.59 μm)	No single Ag NPs detected	Three large aggregates (Figure 8): height 750 nm, 1 μm width; height 1100 nm, 2 μm width; height 1220 nm, 3 μm width	1220 nm
6 AD	Ag	1 (0.34 μm)	Few Ag NPs, height 10–20 nm. Several NPs but not conclusive to be Ag NPs	Not detected
		6 (3.26 μm)	Single NPs detected, possibly Ag NPs covered with lipid layer, height 20–100 nm	Not detected
		12 (6.77 μm)	Some NPs detected, possibly Ag NPs covered with lipid layer (difficult to measure height precisely)	Not detected
		20 (10.94 μm)	Very few NPs not conclusive to be Ag NPs	Several agglomerates composed of up to 30 agglomerated NPs, elongated shape, height 100 nm, width 300 nm, length up to 1.5 μm. One aggregate encountered; after applying high force to peel off the lipid cover, height 200 nm, 1.2 μm width	200 nm
13 H	Ag	1 (0.30 μm)	Numerous single Ag NPs, height 10–30 nm	Not detected
		6 (2.88 μm)	Single NPs detected, possibly Ag NPs covered with lipid layer, height 30, 50 and 100 nm	Not detected
		12 (5.91 μm)	Numerous Ag NPs, heights 20, 30, 50, 70 nm. On edge of the cell	Not detected
		20 (9.55 μm)	Single NPs detected, possibly Ag NPs, heights 30–45 nm	Small agglomerate: height 80 nm, 400 nm width	80 nm
14 H	Ag	1 (0.33 μm)	NPs detected, but not conclusive to be Ag NPs (images not well resolved)	Small agglomerate: height 100 nm, 550 nm width	100 nm
		15 (8.65 μm)	Few single NPs detected, but not conclusive to be Ag NPs	• Well defined isolated sharp cluster of Ag NPs (agglomerate) maximum height 100 nm, 350 nm width (cca 10 NPs). • Numerous small agglomerates composed of several NPs (possible Ag NPs), height predominantly 60 nm	100 nm
4 AD	Placebo	1 (0.40 μm)	No Ag NPs detected (nor other NPs)	Not detected
		10 (6.93 μm)	Only few NPs detected, estimated height 30–40 nm	Not detected
6 AD	Placebo	1 (0.35 μm)	Few NPs detected, height 20, 30 and 50 nm	Not detected
		10 (6.81 μm)	No NPs detected	Not detected
14 H	Placebo	1 (0.36 μm)	Only few NPs detected (not well resolved)	Not detected
		10 (5.17 μm)	2 Ag NPs detected, height 35 and 52 nm (high-resolution imaging, 2 μm × 2 μm scan size) and three more of the same height (5 μm × 5 μm size scan)	Not detected

H: healthy controls; AD: atopic dermatitis patients.