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

Figures & data

Figure 1 Schematic illustration of LMWP-GFs, QCN-NE, OXY-PFOB-NE, and a hydrogel comprising LMWP-GFs, QCN-NE, and OXY-PFOB-NE.

Abbreviations: LMWP, low-molecular-weight protamine; GFs, growth factors; LMWP-GFs, LMWP-fused GFs; QCN, quercetin; NE, nanoemulsion; QCN-NE, QCN-loaded NE; PFOB, 1-bromoperfluorooctane; OXY-PFOB-NE, oxygen-carrying PFOB-loaded NE.

Figure 2 Characterization of LMWP-EGF, LMWP-IGF-I, LMWP-PDGF-A, and LMWP-bFGF. (A) Molecular weights of LMWP-EGF, LMWP-IGF-I, LMWP-PDGF-A, and LMWP-bFGF determined by liquid chromatography-electrospray ionization-tandem mass spectrometry. (B) Confocal laser scanning microscopy images of full-thickness human skin after 12 h treatment with 0.1% (w/w) Carbopol hydrogel containing fluorescent dye (Alexa Fluor 647) conjugated to either native GFs or LMWP-GFs: EGF and LMWP-EGF; IGF-I and LMWP-IGF-I; PDGF-A and LMWP-PDGF-A; bFGF and LMWP-bFGF.

Notes: The scale bars in the upper (20×) and lower (40×) images equal 50 and 20 µm, respectively.

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.

Figure 3 Characterization of QCN-NE and OXY-PFOB-NE. TEM images of (A) QCN-NE, (B) OXY-PFOB-NE, (C) QCN-NE dispersed in 0.1% (w/w) Carbopol hydrogel, and (D) OXY-PFOB-NE dispersed in 0.1% (w/w) Carbopol hydrogel.

Notes: The scale bars in (A), (B), (C), and (D) equal 100, 500, 50, and 100 nm, respectively.

Abbreviations: TEM, transmission electron microscopy; QCN, quercetin; NE, nanoemulsion; QCN-NE, QCN-loaded NE; PFOB, 1-bromoperfluorooctane; OXY-PFOB-NE, oxygen-carrying PFOB-loaded NE.

Figure 4 Keratinocyte proliferation in response to treatment with LMWP-GFs, QCN-NE, and OXY-PFOB-NE. Relative keratinocyte (HaCaT 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<0.001 compared to the control group; ###P<0.001 compared to QCN [1 µg/mL]; $$$P<0.001 compared to QCN [10 µg/mL]), (C) OXY-PFOB-NE (**P<0.01, ***P<0.001 compared to the control group; #P<0.05 compared to OXY-PFOB-NE [30 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 HaCaT cells compared to the control group. All data are expressed as means ± 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 5 Collagen synthesis in response to treatment with LMWP-GFs, QCN-NE, and OXY-PFOB-NE. Synthesis of procollagen type I C-peptide in CCD-986sk cells after 24 h incubation with either LMWP-GFs, QCN, OXY-PFOB-NE alone, or all combined.

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

Abbreviations: GFs, growth factors; LMWP, low-molecular-weight protamine; 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.

Figure 6 In vitro penetration of LMWP-GFs, QCN, and oxygen through human epidermis. Time-course showing cumulative penetration of (A) EGF and LMWP-EGF; (B) IGF-I and LMWP-IGF-I; (C) PDGF-A and LMWP-PDGF-A; (D) bFGF and LMWP-bFGF through human epidermis after incubation with each native GF or LMWP-GF in 0.1% (w/w) Carbopol hydrogel. ***P<0.001 compared to each native GF. (E) Time-course showing cumulative penetration of QCN through human epidermis after incubation with QCN dispersed in 0.3% (w/v) NaCMC or QCN-NE dispersed in 0.1% (w/w) Carbopol hydrogel. ***P<0.001 compared to QCN in 0.3% (w/v) NaCMC. (F) Time-course showing oxygen penetration though human epidermis (1 atm, 32°C) after treatment with PFOB-NE dispersed in 0.1% (w/w) Carbopol hydrogel (blank control) or OXY-PFOB-NE dispersed in 0.1% (w/w) Carbopol hydrogel.

Notes: All data are expressed as means ± 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; NE, nanoemulsion; QCN-NE, QCN-loaded NE; NaCMC, sodium carboxymethyl cellulose; PFOB, 1-bromoperfluorooctane; OXY-PFOB-NE, oxygen-carrying PFOB-loaded NE.

Figure 7 In vitro scratch-wound healing in keratinocytes following treatment with LMWP-GFs, QCN-NE, and OXY-PFOB-NE. (A) Relative scratch-wound recovery of HaCaT cells at 10 h. (B) Time-course showing relative scratch-wound recovery. (C) Representative microscopic images of HaCaT cell scratch-wounds at 0, 2, 4, 6, 8, 10, and 12 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 means ± standard deviation (n=6). ***P<0.001 compared to the control group. #P<0.05, ###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.

Figure 8 In vivo diabetic wound closure following treatment with hydrogels containing LMWP-GFs, QCN-NE, and OXY-PFOB-NE. Progression of wound closure in a full-thickness excisional diabetic mouse wound model following treatment with Carbopol hydrogel containing either LMWP-GFs, QCN-NE, OXY-PFOB-NE alone, or all combined. (A) The wound area was measured over 12 days. (B) Representative photographs of non-diabetic and diabetic wounds treated with vehicle (0.1% [w/w] Carbopol hydrogel), and diabetic wounds treated with LMWP-GFs-GEL (0.1% [w/w] Carbopol hydrogel containing LMWP-EGF [100 µg/mL], LMWP-IGF-I [100 µg/mL], LMWP-PDGF-A [2 µg/mL], and LMWP-bFGF [2 µg/mL]), QCN-NE-GEL (0.1% [w/w] Carbopol hydrogel containing QCN-NE [20 µg/mL QCN]), PFOB-NE-GEL (0.1% [w/w] Carbopol hydrogel containing oxygen-carrying PFOB-NE [6 mg/mL]), and LMWP-GFs/QCN-NE/OXY-PFOB-NE-GEL (0.1% [w/w] Carbopol hydrogel containing LMWP-EGF [100 µg/mL], LMWP-IGF-I [100 µg/mL], LMWP-PDGF-A [2 µg/mL], LMWP-bFGF [2 µg/mL], QCN-NE [20 µg/mL QCN], and OXY-PFOB-NE [6 mg/mL PFOB-NE]).

Notes: All data are expressed as means ± standard deviation (n=15). ^aP<0.05, ^bP<0.01, ^cP<0.001 compared to the control (non-diabetic). ^dP<0.05, ^eP<0.01, ^fP<0.001 compared to the control (diabetic). ^gP<0.05, ^hP<0.001 compared to the LMWP-GFs-GEL. ⁱP<0.001 compared to the QCN-NE-GEL. ^jP<0.001 compared to the PFOB-NE-GEL. The scale bar in (B) equals 10 mm.

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.

Figure 9 Histological analysis during wound closure. The Carbopol hydrogel containing LMWP-GFs, QCN-NE, and OXY-PFOB-NE enhanced re-epithelialization and granulation tissue-formation in full-thickness skin wounds of diabetic mice within 12 days. Representative light micrograph images of sections from the wounds stained with (A) H&E to assess re-epithelialization, (B) CK6 (brown) as a marker for activated keratinocytes around the wound edge, (C) α-SMA (brown) to identify differentiated myofibroblasts in the wound, and (D) MT (blue) to identify collagen fibers in the wound at 3, 6, 9, and 12 days after treatment.

Notes: The scale bar equals 50 µm.

Abbreviations: LMWP, low-molecular-weight protamine; GFs, growth factors; LMWP-GFs, LMWP-fused GFs; QCN, quercetin; NE, nanoemulsion; QCN-NE, QCN-loaded NE; PFOB, 1-bromoperfluorooctane; OXY-PFOB-NE, oxygen-carrying PFOB-loaded NE; H&E, hematoxylin & eosin; CK6, cytokeratin 6; α-SMA, α-smooth muscle actin; MT; Masson’s trichrome.

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.