Supplementary Materials

Table S1. Constituents of the Vehicles used for NP penetration study.

Eucerin Smoothing Essentials (Fast Absorbing Lotion for Dry Skin) Beiersdorf Inc. (Based in Germany) Ingredients: Gluco-Glycerol Enhanced Formula Water, Glycerin, Caprylic/Capric Triglyceride, Cetearyl Alcohol, Urea, Hydrogenated Cocoglycerides, Isopropyl Stearate, Octyldodecanol, Sodium Lactate, Dimethicone, Arginine HCL, Glyceryl Stearate SE, Myristyl Myristate, Carnitine, Chondrus Crispus (Carrageenan), Sodium Cetearyl Sulfate, Lactic Acid, Sodium Citrate, Citric Acid, Acrylates/C10–30 Alkyl Acrylate Crosspolymer, Phenoxyethanol, Benzyl Alcohol, Propylparaben, Potassium Sorbate. Eucerin Calming (Itch Relief Treatment) Beiersdorf Inc. (Based in Germany) Ingredients: Active Ingredient: Menthol Inactive Ingredients: Water, Glycerin, Octyldodecanol, Caprylic/Capric Triglyceride, Isopropyl Stearate, Myristyl Myristate, Cetyl Alcohol, Colloidal Oatmeal, Cetearyl Alcohol, Dimethicone, Glyceryl Stearate SE, Oenothera Biennis (Evening Primrose) Oil, PEG-40 Castor Oil, Caprylyl Glycol, , Sodium Cetearyl Sulfate, Sodium Citrate, Citric Acid, Polyglyceryl-3 Methyl Glucose Distearate, Benzyl Alcohol, Phenoxyethanol, DMDM Hydantoin, Potassium Sorbate. Eucerin Daily Protection SPF 15 Sunscreen Lotion Beiersdorf Inc. (Based in Germany) Ingredients: Active Ingredients (sunscreens): Homosalate 5.0%, Octinoxate 5.0%, Octisalat 4.0%, Titanium Dioxide 1.0%. Other Ingredients: Water, Glycerin, Butylene Glycol, Cetearyl Alcohol, Urea, C18-36 Acid Triglyceride, Caprylic/Capric Triglyceride, Dimethicone, Octyldodecanol, Sodium Lactate, Arginine HCL, Tocopheryl Acetate, Glyceryl Stearate SE, Chondrus Crispus (Carrageenan), PEG-40 Castor Oil, Sodium Cetearyl Sulfate, Disodium EDTA, Sodium Citrate, Lactic Acid, Carbomer, Citric Acid, Trimethoxycaprylysilane, Benzyl Alcohol, Phenoxyethanol, Methylparaben, Propylparaben, Potassium Sorbate. Vaseline Intensive Rescue Clinical Therapy Lotion Unilever Ingredients: Water, Glycerin, Petrolatum, Stearic acid, Glycol stearate, Dimethicone, Isopropyl isostearate, Dihydroxypropyltrimonium chloride, Hydroxyethyl urea, Tapioca starch, Cetyl alcohol, Glyceryl stearate, Magnesium aluminum silicate, Stearamide amp, Carbomer, Isopropyl myristate, Cedrol, triethanolamine, Disodium edta, Phenoxyethanol, Ethylparaben, Propylparaben. Dermovan Healthpoint Ltd Ingredients: Water, Glyceryl stearate (and) stearanidoethyl diethylamine, glycerin, mineral oil, cetyl esters, cetyl alcohol, butylparaben, methylparaben, propylparaben.

a b c

Figure S1. Comparison between the exposure models show that the mouse skin swells up after a 24 h duration in the Franz diffusion cell. (a) Mouse skin placed in the Franz Chamber for swelled up after a 24 h exposure. (b) Mouse skin removed from the Franz chamber after a 24 h exposure shows that the portion of the skin exposed to the bottom chamber with the circulating media swells up. (c) No gross physical changes were observed in the mouse skin treated using the petri dish protocol.

Cosmetics 2016, 3, 6; doi:10.3390/cosmetics3010006 www.mdpi.com/journal/cosmetics Cosmetics 2016, 3, 6 S2 of S7

Vaseline Eucerin Smoothing Lotion

0 Hours after 24 Hours after 0 Hours after 24 Hours after application application application application Dermovan Glycerol

0 Hours after 24 Hours after 0 Hours after 24 Hours after application application application application Figure S2. Samples under a hand-held UV Lamp. Mouse skin samples were photographed under a hand-held UV lamp immediately after and 24 h after the application of the vehicle with QDs in the Petri dish protocol. The vehicles do not affect the QD intensity in the 24 h duration. Lowest QD penetration was observed in the Vaseline group after confocal imaging, however it can be observed that QD intensity is intact in this vehicle. Pictures are not to scale.

Human Skin-Control Mouse Skin-Control

1.40

) 1.20

D 1.00

(

t 0.80

t 0.60

a t 0.40

0.20

0.00 0 5 10 15 20 25 30 35 40 Depth from Stratum Corneum (µm)

Figure S3. No autofluorescence was measured in untreated ex vivo mouse and human skin using CLSM. No autofluorescence was measured in the two skin types using the QD605 filter.

Cosmetics 2016, 3, 6 S3 of S7

9000 a *

8000 40 µm 40 - 7000

6000

5000

4000

3000

2000 * *

Total Intensity (QD 605) between 0 between 605) (QD Intensity Total 1000 *

0 * * Eucerin Dermovan Glycerol Eucerin Eucerin Vaseline Water Only Lotion- Smoothing Calming SPF15 Intensive No QD Lotion Itch Relief Lotion Care 40000 b

35000 *

40 µm 40 - 30000

25000

20000

15000

10000

5000 Total Intensity (QD 605) between 0 between 605) (QD Intensity Total

0 * Eucerin Dermovan Glycerol Water Only Lotion-No Smoothing QD Lotion Figure S4. Eucerin Smoothing lotion enhances QD penetration in ex vivo mouse skin using the petri- dish protocol. (a) Integrated QD fluorescence signal in the Eucerin smoothing lotion group was 2 fold higher than water. QD penetration in the Dermovan group was comparable to water. QD presence between 0 and 40 µm depth in all other vehicle groups was significantly lower compared to water alone; (b) Integrated QD fluorescence signal in the Eucerin smoothing lotion group was 2.2 fold higher than water. QD penetration in the Dermovan and glycerol test groups was comparable to water. Means ± SEM (N = 4, n = 3). * p < 0.05 vs. Water. Lotion with no QD is the Control.

Figure S5 Cosmetics 2016, 3, 6 S4 of S7

Side View-Section a Top View-Stack 630 um Epidermis Epidermis Epidermis 40 um Dermis Dermis Dermis

b 1 3 2

i 630 630 µm µm ii

iii v

40 µm 40 µm

c Injection QD 605 Diffusion QD 605 600000

500000

)

6 400000

(

s 300000

t 200000

100000

0 0 5 10 15 20 25 30 35 40 45 50 Depth from the Stratum Corneum (µm) Figure S5. Injection and diffusion of QDs in ex vivo human skin demonstrate that epidermal scattering and absorption generate a decay in the intensity profile with an increase in depth from the stratum corneum. (a) QDs were either injected intradermally into ex vivo human skin or the skin was immersed in a QD solution for 24 h prior to CLSM imaging. The top view was obtained using the stacks from 0 to 50 µm. Skin was cryosectioned and imaged to obtain the side profile views. (b) 1 & 2. Cryosection of human skin showing high QD presence at the periphery of the tissue. The QD diffusion front produced over the 24 h immersion was sufficient to yield homogenous QD presence to depth exceeding 40 µm. (b) 3. Cryosection of human skin showing high QD presence in the dermis after a sub-dermal injection. Scale Bars (i) 50 µm (ii) 120 µm (iii) 170 µm (iv) 40 µm (v) 150 µm. (c) Stacks obtained using CLSM were quantified using ImageJ. Similar trends were observed for both exposure conditions with intensity values peaking around 10 µm followed by a subsequent decay in the intensity profile; Means ± SEM (N = 4, n = 3).

Cosmetics 2016, 3, 6 S5 of S7

3 Hours 6 Hours 24 Hours 3500

3000

2500

2000

1500

1000 Total Intensity 605) (QD Intensity Total *

500

0 0 5 10 15 20 25 30 35 40 Depth from Stratum Corneum (µm)

Figure S6. Less QD presence is observed in the ex vivo mouse skin treated using the petri dish protocol at 3 h compared to a 6 and 24 h exposure. Significantly lower QD presence was observed in mouse skin treated for 3 h compared to 24 h. There were no significant differences between the 6 and 24 h exposure group; Means ± SEM (N = 4, n = 3). * p < 0.05 vs. 24 h time point.

3 Hours 6 Hours 24 Hours 1400

1200 *

1000 * 800

600

400 * Total Intensity 605) (QD Intensity Total

200

0 0 5 10 15 20 25 30 35 40 Depth from Stratum Corneum (µm)

Figure S7. Less QD presence is observed in the ex vivo mouse skin treated using the Franz diffusion cell at 24 hours compared to a 3 and 6 h exposure. Significantly higher QD presence was observed in mouse skin treated for 3 and 6 h compared to 24 h. There were no significant differences between the 3 and 6 hour exposure group; Means ± SEM (N = 4, n = 3). * p < 0.05 vs. 24 h time point.

Cosmetics 2016, 3, 6 S6 of S7

a Petri Dish Protocol 24 Hours Franz Chamber Protocol 24 Hours

b * 30.00 *

25.00

20.00

15.00

10.00

5.00 Epidermal Thickness (µm) Thickness Epidermal

0.00 Control Skin Petri-Dish 24 Franz Chamber (No Treatment) Hour Exposure 24 Hour Exposure

Figure S8. Quantification of epidermal thickness of mouse skin using ImageJ. (a) Mouse skin was exposed to the vehicle Eucerin Smoothing lotion and GSH QDs using the Petri dish protocol and Franz diffusion chamber. 24 h after exposure the vehicle was wiped off and the skin was cryosectioned. Brightfield images obtained at 20× magnification were used to quantify the epidermal thickness in the two exposure protocols. (b) The epidermal thickness of skin exposed to the vehicle using the petri dish protocol was significantly lower than the skin exposed to the lotion using the Franz diffusion chamber and was of similar thickness to control skin—measured immediately post- euthanasia—no treatment. * p < 0.05, 2-talied t-Test, unpaired with unequal variances. Means ± SEM (N = 3, n = 3).

Cosmetics 2016, 3, 6 S7 of S7

30000 Human Skin (Rest) Human Skin (Rest) Franz Diffusion Cell Petri Dish Protocol *

40 µm 40 25000 -

20000 ) between 0 between ) 15000

10000

5000 Total Intensity 605 (QD Intensity Total 0 Eucerin Smoothing Glycerol Water Only Lotion-no QD Lotion Figure S9. Comparable QD presence was observed in ex vivo human skin (rest t = 24 h) in the Eucerin smoothing lotion and water test groups treated using the Franz diffusion cell and petri dish protocol. No significant differences were observed in the two treatment protocols in the Eucerin smoothing lotion and water test groups. A significantly higher QD presence (1.7 fold) was observed in skin treated using the petri dish protocol compared to the Franz diffusion cell in the glycerol treatment group; Means ± SEM (N = 4, n = 3). * p < 0.05.