Evaluation of a Highly Skin Permeable Low-Molecular-Weight Protamine Conjugated Epidermal Growth Factor for Novel Burn Wound Healing Therapy  Ji Hae Lee, Il-Hong Bae, Jin Kyu Choi, Jin Woo Park  Journal of Pharmaceutical Sciences  Volume 102, Issue 11, Pages 4109-4120 (November 2013) DOI: 10.1002/jps.23725 Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 1 (a) SDS-PAGE analysis after various purification steps of recombinant low-molecular-weight protamine conjugated epidermal growth factor (rLMWP-EGF). Markers (M), total cell lysate after induction with 1mM IPTG (lane 1), total cell lysate after cell lysis by sonication (lane 2), loading sample on ion-exchange chromatography (lane 3), blank (lane 5); eluted fractions from ion-exchange chromatography and loading sample on gel filtration chromatography (lanes 7 and 8), and purified and concentrated rLMWP-EGF after ion-exchange and gel filtration (lanes 4 and 6). (b) Migration and proliferation of NIH3T3 cells versus the control group after incubation with 1, 10, 100, and 1000nM rEGF or rLMWP-EGF for 24h. Each value is the mean±standard deviation (n=6). *p<0.05 versus control. Journal of Pharmaceutical Sciences 2013 102, 4109-4120DOI: (10.1002/jps.23725) Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 2 In vitro permeation through artificial skin constructs and hairless mouse skin. (a) Permeated concentrations of rEGF, physical mixture of LMWP with rEGF, and rLMWP-EGF through the artificial skin construct after applying concentrations equivalent to 12 and 60μg/mL rEGF. (b) Time-course of cumulative permeated concentrations of rEGF or rLMWP-EGF through the skin PAMPA; (c) comparison of cumulative permeation of rEGF or rLMWP-EGF through the excised hairless mouse skin after 24h. Each value is the mean±standard deviation (n=6). *p<0.05 versus rEGF. Journal of Pharmaceutical Sciences 2013 102, 4109-4120DOI: (10.1002/jps.23725) Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 3 rLMWP-EGF gel formulation accelerated healing of laser-induced burn wounds. (a) Burn wound closure over time, measured as percent of original wound area. (b) Representative photographs of wounds treated with vehicle [poloxamer 188, 6% (w/v)], rEGF (10μg/mL), or rLMWP-EGF (10μg/mL). Bar indicates 10mm. (c) Eschar size over time, measured as percent of original eschar area. Each value is the mean±standard deviation (n=10). *p<0.05 versus vehicle; **p<0.01 versus vehicle. Journal of Pharmaceutical Sciences 2013 102, 4109-4120DOI: (10.1002/jps.23725) Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 4 rLMWP-EGF gel formulation accelerates wound reepithelialization and activates keratinocytes around the burn wound margin. Wound sections stained with hematoxylin and eosin (H&E) for general observation of reepithelialization and immunohistochemical staining for keratin protein, K6 (brown) as a marker of activated keratinocytes via binding with rEGF or rLMWP-EGF. Representative light microscopy images of sections from the wound tissues at day 3. Arrows indicate newly formed epithelial layer within the burn wound legion. Bar=200μm. Journal of Pharmaceutical Sciences 2013 102, 4109-4120DOI: (10.1002/jps.23725) Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 5 Effect of RF microporation with topical application rLMWP-EGF gel formulation on scar reduction in the hypertrophic burn scar model. (a) Percentages of scar area measured over 22days. (b) Representative images of burn scar treated with vehicle, RF microporation+vehicle, RF microporation+rEGF, or RF microporation+rLMWP-EGF. Bar indicates 10mm. (c) Effect of RF microporation treatment with or without topical application of rEGF or rLMWP-EGF on the TEWL in the burn scar area on day 22. (d) Difference in skin color between normal and burn scar area on day 22. The difference in skin color was evaluated by analyzing digital images. Each value is the mean±standard deviation (n=10). *p<0.05 versus control. Journal of Pharmaceutical Sciences 2013 102, 4109-4120DOI: (10.1002/jps.23725) Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 6 In vivo skin sensitization effects of rEGF or rLMWP-EGF. (a) Ear swelling response to 300, 100, or 10μg/mL rEGF or rLMWP-EGF. (b) Proliferative activity of lymph nodes after application of 300, 100, or 10μg/mL rEGF or rLMWP-EGF. Each value is the mean±standard deviation (n=10). *p<0.05, versus control. Journal of Pharmaceutical Sciences 2013 102, 4109-4120DOI: (10.1002/jps.23725) Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions