Preparation and in vivo evaluation of a topical hydrogel system incorporating highly skin-permeable growth factors, quercetin, and oxygen carriers for enhanced diabetic wound-healing therapy

Abstract

Purpose

We created and evaluated an enhanced topical delivery system featuring a combination of highly skin-permeable growth factors (GFs), quercetin (QCN), and oxygen; these synergistically accelerated re-epithelialization and granulation tissue formation of/in diabetic wounds by increasing the levels of GFs and antioxidants, and the oxygen partial pressure, at the wound site.

Methods

To enhance the therapeutic effects of exogenous administration of GFs for the treatment of diabetic wounds, we prepared highly skin-permeable GF complexes comprised of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), platelet-derived growth factor-A (PDGF-A), and basic fibroblast growth factor (bFGF), genetically attached, via the N-termini, to a low-molecular-weight protamine (LMWP) to form LMWP-EGF, LMWP-IGF-I, LMWP-PDGF-A, and LMWP-bFGF, respectively. Furthermore, quercetin (QCN)- and oxygen-carrying 1-bromoperfluorooctane (PFOB)-loaded nanoemulsions (QCN-NE and OXY-PFOB-NE) were developed to improve the topical delivery of QCN and oxygen, respectively. After confirming the enhanced penetration of LMWP-GFs, QCN-NE, and oxygen delivered from OXY-PFOB-NE across human epidermis, we evaluated the effects of combining LMWP-GFs, QCN-NE, and OXY-PFOB-NE on proliferation of keratinocytes and fibroblasts, and the chronic wound closure rate of a diabetic mouse model.

Results

The optimal ratios of LMWP-EGF, LMWP-IGF-I, LMWP-PDGF-A, LMWP-bFGF, QCN-NE, and OXY-PFOB-NE were 1, 1, 0.02, 0.02, 0.2, and 60, respectively. Moreover, a Carbopol hydrogel containing LMWP-GFs, QCN-NE, and OXY-PFOB-NE (LMWP-GFs/QCN-NE/OXY-PFOB-NE-GEL) significantly improved scratch-wound recovery of keratinocytes and fibroblasts in vitro compared to that afforded by hydrogels containing each component alone. LMWP-GFs/QCN-NE/OXY-PFOB-NE-GEL significantly accelerated wound-healing in a diabetic mouse model, decreasing wound size by 54 and 35% compared to the vehicle and LMWP-GFs, respectively.

Conclusion

LMWP-GFs/QCN-NE/OXY-PFOB-NE-GEL synergistically accelerated the healing of chronic wounds, exerting both rapid and prolonged effects.

Keywords:

• diabetic wound-healing
• topical hydrogel
• highly skin-permeable growth factors
• quercetin
• 1-bromoperfluorooctane
• oxygen delivery

Acknowledgments

This research was supported by the Basic Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2017R1D1A1B03032283).

Disclosure

The authors report no conflicts of interest in this work.

Supplementary materials

Figure S1 Cumulative oxygen release from OXY-PFOB-NE at different PFOB-NE concentrations in 5% CO₂ (air) under static conditions at 37°C.

Notes: All data are expressed as mean ± standard deviation (n=6).

Abbreviations: PFOB, 1-bromoperfluorooctane; NE, nanoemulsion; OXY-PFOB-NE, oxygen-carrying PFOB-loaded NE.

Figure S2 Fibroblast proliferation in response to treatment with LMWP-GFs, QCN-NE, and OXY-PFOB-NE. Relative fibroblast (CCD-986sk cells) proliferation after 24 h incubation with either (A) LMWP-GFs (***P<0.001 compared to the control group; ^###P<0.001 compared to LMWP-GFs), (B) QCN (**P<0.01, ***P<0.001 compared to the control group; ^###P<0.001 compared to QCN [0.1 µg/mL]; ^$$$P<0.001 compared to QCN [1 µg/mL]), (C) OXY-PFOB-NE (***P<0.001 compared to the control group; ^#P<0.05, ^###P<0.001 compared to OXY-PFOB-NE [3 µg/mL]) alone, or (D) all combined. ***P<0.001 compared to the control group. ^###P<0.001 compared to all LMWP-GFs, QCN, and OXY-PFOB-NE combined (LMWP-EGF [500 ng/mL] + LMWP-IGF-I [500 ng/mL] + LMWP-PDGF-A [10 ng/mL] + LMWP-bFGF [10 ng/mL] + QCN [0.1 µg/mL] + OXY-PFOB-NE [30 µg/mL]).

Notes: Cell viability was measured using WST-1 and the growth of CCD-986sk cells compared to the control group. All data are expressed as mean ± standard deviation (n=6).

Abbreviations: LMWP, low-molecular-weight protamine; GFs, growth factors; LMWP-GFs, LMWP-fused GFs; EGF, epidermal growth factor; IGF-I, insulin-like growth factor-I; PDGF-A, platelet-derived growth factor-A; bFGF, basic fibroblast growth factor; LMWP-EGF, LMWP-fused EGF; LMWP-IGF-I, LMWP-fused IGF-I; LMWP-PDGF-A, LMWP-fused PDGF-A; LMWP-bFGF, LMWP-fused bFGF; QCN, quercetin; PFOB, 1-bromoperfluorooctane; NE, nanoemulsion; OXY-PFOB-NE, oxygen-carrying PFOB-loaded NE.

Figure S3 In vitro scratch-wound healing in fibroblasts following treatment with LMWP-GFs, QCN-NE, and OXY-PFOB-NE. (A) Relative scratch-wound recovery of CCD-986sk cells at 24 h. (B) Time-course showing relative scratch-wound recovery. (C) Representative microscopic images of CCD-986sk-cell scratch-wounds at 0, 8, 12, 20, 24, 36, and 48 h after incubation with serum-free medium (control) or Carbopol hydrogel containing either LMWP-GFs, QCN-NE, OXY-PFOB-NE alone, or all combined.

Notes: All data are expressed as mean ± standard deviation (n=6). ***P<0.001 compared to the control group. ^##P<0.01, ^###P<0.001 compared to all LMWP-GFs, QCN-NE and OXY-PFOB-NE combined (LMWP-EGF [500 ng/mL] + LMWP-IGF-I [500 ng/mL] + LMWP-PDGF-A [10 ng/mL] + LMWP-bFGF [10 ng/mL] + QCN-NE [0.1 µg/mL QCN] + OXY-PFOB-NE [30 µg/mL]). The scale bar in (C) equals 300 µm.

Abbreviations: LMWP, low-molecular-weight protamine; GFs, growth factors; LMWP-GFs, LMWP-fused GFs; EGF, epidermal growth factor; IGF-I, insulin-like growth factor-I; PDGF-A, platelet-derived growth factor-A; bFGF, basic fibroblast growth factor; LMWP-EGF, LMWP-fused EGF; LMWP-IGF-I, LMWP-fused IGF-I; LMWP-PDGF-A, LMWP-fused PDGF-A; LMWP-bFGF, LMWP-fused bFGF; QCN, quercetin; NE, nanoemulsion; QCN-NE, QCN-loaded NE; PFOB, 1-bromoperfluorooctane; OXY-PFOB-NE, oxygen-carrying PFOB-loaded NE.