Homogenous photocatalytic decontamination of prion infected stainless steel and titanium surfaces

Figures & data

Figure 1. Photo-Fenton treatment of TiO₂ (rutile) particles contaminated with the RML scrapie strain. 0.2 g of TiO₂ were incubated with 10% w/v RML mouse brain homogenate and were then treated with 56 μg mL⁻¹ Fe³⁺, 600 μg mL⁻¹ h⁻¹, UV-A, pH: 3.5. (A) SDS-PAGE, following AgNO₃ staining of adsorbed proteins after treatment with photo-Fenton at the indicated time points (min). (B) western blotting of adsorbed PrP after treatment with photo-Fenton at the indicated time points (min). Primary antibody: 12F10, visualization: West Femto substrate (Pierce). Arrow: 32.5 kDa relative molecular mass marker.

Table 1. Efficiency of the following photo-Fenton decontamination of scrapie infected TiO₂ particles based on a bioassay employing C57BL/6J mice

Group No	Description	Survival rate (%)	Clinically affected/ Total number	Survival interval ± standard error (days)
1	^aPositive control, 1% brain homogenate	0	4/4	236.8 ± 2.3
2	^aPositive control, contaminated rutile	0	5/5	249.8 ± 8.6
3	^bphoto-Fenton, 180 min	67	2/6^b	269.0 ± 32.0
4	photo-Fenton, 360 min	67	2/6	278.5 ± 22.5

Animals were inoculated with 0.02 g RML-contaminated TiO₂ particles following photo-Fenton treatment (56 μg mL⁻¹ Fe³⁺, 600 μg mL⁻¹ h⁻¹ H₂O₂, UV-A, pH: 3.5). Asymptomatic animals were sacrificed 453 d.p.i. Only clinically affected animals were taken into account in the calculation of the survival interval. ^aThe difference in the incubation period between the two positive control groups was not statistically significant, demonstrating similar initial infectivity titers. ^bAn asymptomatic individual was found with PK-resistant cerebral PrP, 453 d post inoculation (see Fig. S1).

Figure 2. Photo-Fenton treatment of stainless steel and titanium wires contaminated with the 263K scrapie strain. Western blotting of PrP adsorbed on the surface of wires previously incubated with 10% w/v 263K hamster brain homogenate and then, treated with 56 μg mL⁻¹ Fe³⁺, 300 μg mL⁻¹ h⁻¹ H₂O₂, UV-A, pH: 3.5, at the indicated time points (min). Primary antibody: 6Η4 (Prionics, AG), visualization: CDP-Star substrate (New England Biolabs). Arrow: 32.5 kDa relative molecular mass marker.

Figure 3. Scanning Electron Microscopy of stainless steel wires. a: uncontaminated, b-e: incubated with 10% hamster brain homogenate infected with the 263K prion strain and treated with 224 μg mL⁻¹ Fe³⁺, 500 μg mL⁻¹ h⁻¹ H₂O₂, pH = 3.5, UV-A for 0, 240, 360, and 480 min respectively. White bar: 200μm, black bar: 20 μm. Panels to the right (A2-E2) are close ups of respective areas of the left column (A1-E1).

Figure 4. Fluorescence Microscopy of stainless steel wires. (A) uncontaminated, (B–E) incubated with 10% hamster brain homogenate infected with the 263K prion strain and treated with 224 μg mL⁻¹ Fe³⁺, 500 μg mL⁻¹ h⁻¹ H₂O₂, pH = 3.5 and UV-A for 0, 240, 360, and 480 min respectively. Each row of panels (1–2) demonstrates areas of wires of the same group. Bar: 200 μm

Figure 5. Fluorescence Microscopy of titanium wires. (A) uncontaminated, (B–D) incubated with 10% hamster brain homogenate infected with the 263K prion strain and treated with 224 μg mL⁻¹ Fe³⁺, 500 μg mL⁻¹ h⁻¹ H₂O₂, pH = 3.5 and UV-A for 0, 240, and 480 min respectively. Each row of panels (1–2) demonstrates areas of wires of the same group. Bar: 200 μm

Figure 6. Surface protein contamination in groups of wires (n = 8), after densitometric analysis of fluorescence micrographs. (A) Stainless steel, (B) Titanium. Homogenous photocatalytic treatment was performed in the presence of 224 μg mL⁻¹ Fe³⁺, 500 μg mL⁻¹ h⁻¹ H₂O₂, and UV-A (pH = 3.5).

Table 2. Bioassay of wires contaminated with the 263K scrapie strain

Group No	Treatment	Survival interval ± standard error (days)	Clinically affected/ Total
1	Negative controls, stainless steel	>505.0	0/3
2	Positive controls, stainless steel	135.0 ± 8.0	3/3
3	360 min photo-Fenton, stainless steel	302.0 ± 29.3	3/8
4	480 min photo-Fenton, stainless steel	>505.0	0/8
5	Negative controls, titanium	>505.0	0/4
6	Positive controls, titanium	113.8 ± 3.5	4/4
7	360 min photo-Fenton, titanium	202.9 ± 5.8	8/8

After photo-Fenton treatment (224 μg mL⁻¹ Fe³⁺, 500 μg mL⁻¹ h⁻¹ H₂O₂, UV-A) wires were intracranially implanted in male golden Syrian hamsters. The survival interval of group 3 was calculated taking into account only the scrapie affected animals.