Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jul 20:13:4189-4206.
doi: 10.2147/IJN.S165966. eCollection 2018.

Chitosan-based nanoformulated (-)-epigallocatechin-3-gallate (EGCG) modulates human keratinocyte-induced responses and alleviates imiquimod-induced murine psoriasiform dermatitis

Jean Christopher Chamcheu #  1   2 , Imtiaz A Siddiqui #  1 , Vaqar M Adhami #  1 , Stephane Esnault  3 , Dhruba J Bharali  4 , Abiola S Babatunde  1   5 , Stephanie Adame  1 , Randall J Massey  6 , Gary S Wood  1 , B Jack Longley  1 , Shaker A Mousa  4 , Hasan Mukhtar  1
Affiliations

Chitosan-based nanoformulated (-)-epigallocatechin-3-gallate (EGCG) modulates human keratinocyte-induced responses and alleviates imiquimod-induced murine psoriasiform dermatitis

Jean Christopher Chamcheu et al. Int J Nanomedicine. .

Erratum in

Abstract

Background: Psoriasis is a chronic and currently incurable inflammatory skin disease characterized by hyperproliferation, aberrant differentiation, and inflammation, leading to disrupted skin barrier function. The use of natural agents that can abrogate these effects could be useful for the treatment of psoriasis. Earlier studies have shown that treatment of keratinocytes and mouse skin with the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) mitigated inflammation and increased the expression of caspase-14 while promoting epidermal differentiation and cornification. However, bioavailability issues have restricted the development of EGCG for the treatment of psoriasis.

Materials and methods: To overcome these limitations, we employed a chitosan-based polymeric nanoparticle formulation of EGCG (CHI-EGCG-NPs, hereafter termed nanoEGCG) suitable for topical delivery for treating psoriasis. We investigated and compared the efficacy of nanoEGCG versus native or free EGCG in vitro and in an in vivo imiquimod (IMQ)-induced murine psoriasis-like dermatitis model. The in vivo relevance and efficacy of nanoEGCG formulation (48 µg/mouse) were assessed in an IMQ-induced mouse psoriasis-like skin lesion model compared to free EGCG (1 mg/mouse).

Results: Like free EGCG, nanoEGCG treatment induced differentiation, and decreased proliferation and inflammatory responses in cultured keratinocytes, but with a 4-fold dose advantage. Topically applied nanoEGCG elicited a significant (p<0.01) amelioration of psoriasiform pathological markers in IMQ-induced mouse skin lesions, including reductions in ear and skin thickness, erythema and scales, proliferation (Ki-67), infiltratory immune cells (mast cells, neutrophils, macrophages, and CD4+ T cells), and angiogenesis (CD31). We also observed increases in the protein expression of caspase-14, early (keratin-10) and late (filaggrin and loricrin) markers of differentiation, and the activator protein-1 factor (JunB). Importantly, a significant modulation of several psoriasis-related inflammatory cytokines and chemokines was observed compared to the high dose of free EGCG (p<0.05). Taken together, topically applied nanoEGCG displayed a >20-fold dose advantage over free EGCG.

Conclusion: Based on these observations, our nanoEGCG formulation represents a promising drug-delivery strategy for treating psoriasis and possibly other inflammatory skin diseases.

Keywords: anti-inflammatory action; chitosan nanoparticles; differentiation; normal human epidermal keratinocytes; phytochemical treatment of psoriasis; psoriasis-like skin inflammation; topical delivery of chitosan nanoformulated EGCG.

PubMed Disclaimer

Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Size characterization and encapsulation and loading efficiencies of chitosan-based nanoEGCG. (A) Size measurement and distribution of nanoEGCG using dynamic light scattering. (B) Zeta potential measurement of nanoEGCG. (C) Representative transmission electron microscopy photomicrographs showing the relative homogeneous size and morphology of (i) diluted nanoEGCG and (ii) undiluted nanoEGCG, and (iii) CHI-Void-NPs. Scale bar=200 nm; the insets represent higher magnification. (D) Encapsulation and loading efficiency of EGCG on to chitosan nanoparticles as monitored with UV-vis spectra for free EGCG (not encapsulated) and total EGCG (encapsulated + free). (E) UV-vis spectra used to construct the standard curve, with EGCG concentrations of 25, 12.5, 6.25, 3.12, and 1.6 µg/mL. Abbreviations: EGCG, (–)-epigallocatechin-3-gallate; nanoEGCG, CHI-EGCG-NPs, chitosan-based polymeric nanoparticle formulation of EGCG; CHI-Void-NPs, chitosan-based void (without EGCG) nanoparticles; UV-vis, ultraviolet–visible.
Figure 2
Figure 2
Effect of nanoparticles on in vitro cellular growth/viability and uptake. (A) Size, PDI, and surface charge of nanoparticles. In both cases the zeta potential is around +33 mV to +38 mV, indicating that both the CHI-Void-NPs and nanoEGCG have positive surface charge, which is due to the presence of cationic chitosan polymer. (B) Dose–response curve of CHI-Void-NPs, nanoEGCG and free EGCG on NHEK growth/viability as assessed with the MTT assay, analyzed as percent viable cell numbers per million. The growth/viability of untreated cells was set as 100%. (C) Histograms showing comparative effects of free EGCG and nanoEGCG treatment on viable NHEK number after 48 h treatment as determined with the trypan blue dye exclusion assay. Results are expressed as percentage cell number harvested (48 h) in relation to the cell number present at the time of initiation of treatment (0 h). (D) Comparative effects of free EGCG versus nanoEGCG on IL-22-induced hyperproliferation (assessed as viability) of keratinocytes in vitro (equivalent doses as indicated in the figure). (E) Cellular uptake and cumulative drug content (in µM) in the cytoplasm of NHEKs after treatment. Data are presented as the mean±SEM of experiments in which each treatment was repeated in 7–10 wells (C), and experiments were performed in triplicate (D, E). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 compared with vehicle-treated controls. Abbreviations: PDI, polydispersity index; EGCG, (–)-epigallocatechin-3-gallate; nanoEGCG, CHI-EGCG-NPs, chitosan-based polymeric nanoparticle formulation of EGCG; CHI-Void-NPs, chitosan-based void (without EGCG) nanoparticles; NHEK, normal human epidermal keratinocyte; ns, not significant.
Figure 3
Figure 3
Cellular uptake of nanoEGCG and its effects on epidermal keratinocyte differentiation markers and TPA-induced inflammatory responses in vitro in keratinocyte cultures. (A–D) Transmission electron microscopy photomicrographs of NHEKs treated with nanoEGCG showing localization of nanoEGCG on the cell surface (A), in the cytoplasm near tonofilaments (B), in cytoplasmic vesicles in the vicinity of multiple mitochondria (C), and in cytoplasmic vesicles within a dense meshwork of tonofilaments (D). (E) Western blot analysis of NHEK lysates showing the effects of free EGCG (10–20 µM) and nanoEGCG (5 µM equivalent concentration) on the expression of early and late differentiation markers as indicated. Blots were stripped and reprobed for β-actin to determine equal protein loading, and the results shown are representative of 3 independent experiments. (F) Effect of a 24 h treatment with free EGCG (20 µM) or nanoEGCG (5 µM) on TPA-induced secretion of pro-inflammatory cytokines in NHEK cultures; cytokines in cultured supernatants were analyzed with human 6-Plex ProcartaPlex mix-matched immunoassay (described in Materials and methods and Supplementary materials section). Data in (F) are expressed as the mean±SEM of experiments in which each treatment was repeated in 10 wells. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 for the indicated 2-way comparisons. Abbreviations: EGCG, (−)-epigallocatechin-3-gallate; nanoEGCG, chitosan-based polymeric nanoparticle formulation of EGCG; NHEK, normal human epidermal keratinocyte; TGase-1, transglutaminase-1; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; CXCL2, C-X-C motif chemokine ligand-2; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; ns, not significant; SEM, standard error of the mean.
Figure 4
Figure 4
Effect of topically applied free EGCG and nanoEGCG on IMQ-induced psoriasis-like ear/skin lesions. (A–L) Mouse ears and back skins were shaved, then topically treated with either control cream (IMQ(−)) (A, E, I) or IMQ alone (IMQ(+)) (B, F, J) for 14 days. Two additional IMQ+ groups were co-treated with either free EGCG (C, G, K) or nanoEGCG (D, H, L) starting at day 5 for 9 additional days (depicted in treatment protocol diagram, Figure S5). Photomicrographs show: (A–D) ears with IMQ-induced erythema and scaling; (E–H) H&E-stained histological sections of skin showing pathological features (arrows in panels F, G, and H represent the thickness of the back skin epidermis in the respective groups); (I–L) Ki67-stained sections of skin showing (hyper)proliferation. For panels E–L, magnification ×200. Scale bar=50 µM. Arrows represent the thickness of the back skin epidermis in the respective groups. (M–Q) Quantitative assessments of changes reflecting the following pathological hallmarks of psoriasis: (M) inflamed ear erythema, (N) degree of scaling, (O) changes in ear thickness over time, (P) epidermal thickness at the end of the experiment, and (Q) proportion of Ki67+-cells in total skin and epidermis. For (M–Q), each data point represents the mean of 6 random fields per mouse per treatment group with 5 mice/group. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Abbreviations: EGCG, (−)-epigallocatechin-3-gallate; nanoEGCG, chitosan-based polymeric nanoparticle formulation of EGCG; IMQ, imiquimod.
Figure 5
Figure 5
Effect of topically applied free EGCG and nanoEGCG on infiltrating immune cells and expression of differentiation markers in IMQ-treated mouse skin lesions: Mice were treated in 4 groups as described in the legends to Figure 4 and Figure S5. (A–D, F–I, K–N, P–S) Photomicrographs showing immunohistological features of: (A–D) mast cells (toluidine blue staining); (F–I) epidermis/dermis (NE, brown staining), and microabscesses (arrow); (K–N) macrophages (F4/80, red staining); and (P–S) double immunofluorescence staining for loricrin (green) and T-lymphocytes (CD4+, red staining). Nuclei were counterstained blue with DAPI. Magnification for all panels ×200. (E, J, O, T, U) Quantitative analyses of changes in immune cells: (E) mast cells; (J) NE+ cells; (O) F4/80+ cells; and (U) loricrin in the 4 treatment groups. Each data point represents the mean±SD of 4 random fields/mouse from 5 mice/group. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 for the indicated 2-way comparisons. Abbreviations: EGCG, (–)-epigallocatechin-3-gallate; nanoEGCG, chitosan-based polymeric nanoparticle formulation of EGCG; IMQ, imiquimod; NE, neutrophil elastase; LPV, low-power view.
Figure 6
Figure 6
Effect of topically applied free EGCG and nanoEGCG on expression of epidermal differentiation markers and JunB in IMQ-treated mouse skin. (A–P) Photomicrographs showing expression of various epidermal differentiation markers and the activator protein-1 factor JunB. Mice (6–8 weeks old) underwent treatments in 4 groups as described in the Supplementary materials and shown in Figure S5. After the end of the treatment period, all mice were euthanized, skin samples were taken, and sections were processed for immunostaining of various proteins using specific antibodies as described in Materials and methods and Table S1. Immunohistochemical labeling of differentiation markers and JunB expression are shown for control (IMQ(−)) (A, E, I, M), IMQ(+) (B, F, J, N), IMQ+free EGCG (C, G, K, O), and IMQ+nanoEGCG (D, H, L, P) samples from treated skin. Expression of the following proteins was analyzed in brown staining and counterstained in blue: (A–D) caspase-14, (E–H) filaggrin, (I–L) keratin-10, and (M–P) JunB. Magnification ×400. Scale bar=100 nm. Abbreviations: EGCG, (–)-epigallocatechin-3-gallate; nanoEGCG, chitosan-based polymeric nanoparticle formulation of EGCG; IMQ, imiquimod.
Figure 7
Figure 7
Effect of topically applied free EGCG and nanoEGCG on tissue vascularization and cytokine expression in IMQ-treated mouse skin. Skin samples were processed for immunofluorescence staining with antibodies. (A–D, F–I, K–N) Photomicrographs showing representative skin sections for protein expression of: (A–D) angiogenesis marker CD31 (green staining), (F–I) psoriasis-related pro-inflammatory cytokines IL-1β (green staining) and (K–N) TNF-α (red staining), all at a magnification of ×400. Scale bar=50 µm. (E, J, O) Histograms of quantitation of IMQ+ sections stained for (E) CD31+-positive blood vessels (number per 400 µM section), (J) fluorescence intensity for IL-1β, and (O) fluorescence intensity for TNF-α. Quantitative analyses using the Nuance imaging and Inform software were as detailed in Materials and methods. Statistical significance is shown for all samples (±SEM) and comparisons meeting significance criteria are shown: *p<0.05, **p<0.01, and ***p<0.001. Abbreviations: EGCG, (−)-epigallocatechin-3-gallate; nanoEGCG, chitosan-based polymeric nanoparticle formulation of EGCG; IMQ, imiquimod; TNF-α, tumor necrosis factor-α.
Figure 8
Figure 8
Effect of topical application of free EGCG and nanoEGCG on the expression of signature inflammatory cytokines and chemokines in IMQ-treated mouse skin. (A, B) Expression levels of secreted pro-inflammatory and anti-inflammatory cytokines and chemokines using the mouse-specific 36-Plex ProcartaPlex multiplex immunoassay as described in Materials and methods and Supplementary materials. Total back skin lysates were isolated from the 4 treatment groups and equal protein aliquots were processed for expression levels of: (A) pro-inflammatory or anti-inflammatory Th1 and Th2 cytokines as well as activators of granulocytes and/or monocytes/macrophages, and (B) Th17/Th22/Treg cytokines as well as type 1 and type 2 chemokines along with granulocyte and monocyte/macrophage chemokines. Bars are mean±SEM for each cytokine and chemokine, and statistical significance for comparisons is shown as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Abbreviations: EGCG, (–)-epigallocatechin-3-gallate; nanoEGCG, chitosan-based polymeric nanoparticle formulation of EGCG; IMQ, imiquimod; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α; LIF, leukemia inhibitory factor; G-CSF, granulocyte–colony-stimulating factor; M-CSF, macrophage–colony-stimulating factor; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein; IP, interferon-inducible protein.
Figure 8
Figure 8
Effect of topical application of free EGCG and nanoEGCG on the expression of signature inflammatory cytokines and chemokines in IMQ-treated mouse skin. (A, B) Expression levels of secreted pro-inflammatory and anti-inflammatory cytokines and chemokines using the mouse-specific 36-Plex ProcartaPlex multiplex immunoassay as described in Materials and methods and Supplementary materials. Total back skin lysates were isolated from the 4 treatment groups and equal protein aliquots were processed for expression levels of: (A) pro-inflammatory or anti-inflammatory Th1 and Th2 cytokines as well as activators of granulocytes and/or monocytes/macrophages, and (B) Th17/Th22/Treg cytokines as well as type 1 and type 2 chemokines along with granulocyte and monocyte/macrophage chemokines. Bars are mean±SEM for each cytokine and chemokine, and statistical significance for comparisons is shown as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Abbreviations: EGCG, (–)-epigallocatechin-3-gallate; nanoEGCG, chitosan-based polymeric nanoparticle formulation of EGCG; IMQ, imiquimod; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α; LIF, leukemia inhibitory factor; G-CSF, granulocyte–colony-stimulating factor; M-CSF, macrophage–colony-stimulating factor; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein; IP, interferon-inducible protein.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Stenderup K, Rosada C, Dam TN, Salerno E, Belinka BA, Kachlany SC. Resolution of psoriasis by a leukocyte-targeting bacterial protein in a humanized mouse model. J Invest Dermatol. 2011;131:2033–2039. - PubMed
    1. Nestle FO, Kaplan DH, Barker J. Psoriasis. N Engl J Med. 2009;361:496–509. - PubMed
    1. Gelfand JM, Dommasch ED, Shin DB, et al. The risk of stroke in patients with psoriasis. J Invest Dermatol. 2009;129:2411–2418. - PMC - PubMed
    1. Hoffjan S, Stemmler S. On the role of the epidermal differentiation complex in ichthyosis vulgaris, atopic dermatitis and psoriasis. Br J Dermatol. 2007;157:441–449. - PubMed
    1. Lowes MA, Bowcock AM, Krueger JG. Pathogenesis and therapy of psoriasis. Nature. 2007;445:866–873. - PubMed

MeSH terms

-