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. 2018 Sep 25:13:5771-5786.
doi: 10.2147/IJN.S156206. eCollection 2018.

Carboxymethyl chitosan nanoparticles loaded with bioactive peptide OH-CATH30 benefit nonscar wound healing

Tongyi Sun  1 , Bo Zhan  2   3 , Weifen Zhang  2 , Di Qin  1 , Guixue Xia  2 , Huijie Zhang  1   3 , Meiyu Peng  4 , Sheng-An Li  3 , Yun Zhang  3 , Yuanyuan Gao  2 , Wen-Hui Lee  1   3
Affiliations

Carboxymethyl chitosan nanoparticles loaded with bioactive peptide OH-CATH30 benefit nonscar wound healing

Tongyi Sun et al. Int J Nanomedicine. .

Abstract

Background: Nonscar wound healing is a desirable treatment for cutaneous wounds worldwide. Peptide OH-CATH30 (OH30) from king cobra can selectively regulate the innate immunity and create an anti-inflammatory micro-environment which might benefit nonscar wound healing.

Purpose: To overcome the enzymatic digestion and control release of OH30, OH30 encapsulated in carboxymethyl chitosan nanoparticles (CMCS-OH30 NP) were prepared and their effects on wound healing were evaluated.

Methods: CMCS-OH30 NP were prepared by mild ionic gelation method and properties of the prepared CMCS-OH30 NP were determined by dynamic light scattering. Encapsulation efficiency, stability and release profile of OH30 from prepared CMCS-OH30 NP were determined by HPLC. Cytotoxicity, cell migration and cellular uptake of CMCS-OH30 NP were determined by conventional methods. The effects of prepared CMCS-OH30 NP on the wound healing was investigated by full-thickness excision animal models.

Results: The release of encapsulated OH30 from prepared CMCS-OH30 NP was maintained for at least 24 h in a controlled manner. CMCSOH30 NP enhanced the cell migration but had no effects on the metabolism and proliferation of keratinocytes. In the full-thickness excision animal models, the CMCS-OH30 NP treatment significantly accelerated the wound healing compared with CMCS or OH30 administration alone. Histopathological examination suggested that CMCS-OH30 NP promoted wound healing by enhancing the granulation tissue formation through the re-epithelialized and neovascularized composition. CMCS-OH30 NP induced a steady anti-inflammatory cytokine IL10 expression but downregulated the expressions of several pro-inflammatory cytokines.

Conclusion: The prepared biodegradable drug delivery system accelerates the healing and shows better prognosis because of the combined effects of OH30 released from the nanoparticles.

Keywords: OH-CATH30; antimicrobial peptide; nanoparticles; skin destruction; wound healing.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1
TEM images of CMCS-OH30 NP releasing in SWF solution (pH 7.4). (A) Initial shape in SWF, (B) 4 h in SWF, (C) 12 h in SWF, and (D) 24 h in SWF. Abbreviations: CMCS-OH30 NP, carboxymethyl chitosan nanoparticles; SWF, simulated wound fluid.
Figure 2
Figure 2
In vitro drug release curve and antimicrobial activity assay. Notes: (A) In vitro release profile of OH30 (plotted as a function of % cumulative release vs time) from CMCS-OH30 NP (mean ± SD, n = 3). (B) Representative antibacterial activities of CMCS-OH30 NP and CMCS NP against the E. coli (mean ± SD, n = 3). Three independent experiments were carried out and each tested points was triplicated in all experiments. Abbreviations: CMCS-OH30 NP, carboxymethyl chitosan nanoparticles; E. coli, Escherichia coli.
Figure 3
Figure 3
Internalization of OH30 in RAW264.7 cells were detected by confocal laser scanning microscope. Notes: Cells were treated with CMCS-OH30 NP at 37°C for 1 h (A) and 4 h (B). Cells were treated with free OH30 at 37°C for 1 h (C) and 4 h (D). Nuclei were stained with Hoechst 33258 (blue) and OH30 labeled by Cy5 (red). Abbreviation: CMCS-OH30 NP, carboxymethyl chitosan nanoparticles.
Figure 4
Figure 4
In vitro HaCaT cell migration assays. Notes: (A) Effects of untreated, CMCS NP, OH30, and CMCS-OH30 NP were assessed by microscope images. (B) Cell migration rates of each group were expressed as percentage of initial blank area. Statistical significance compared with other groups (mean ± SD; n = 4; *p < 0.05 and **p < 0.01). Abbreviation: CMCS-OH30 NP, carboxymethyl chitosan nanoparticles.
Figure 4
Figure 4
In vitro HaCaT cell migration assays. Notes: (A) Effects of untreated, CMCS NP, OH30, and CMCS-OH30 NP were assessed by microscope images. (B) Cell migration rates of each group were expressed as percentage of initial blank area. Statistical significance compared with other groups (mean ± SD; n = 4; *p < 0.05 and **p < 0.01). Abbreviation: CMCS-OH30 NP, carboxymethyl chitosan nanoparticles.
Figure 5
Figure 5
Distribution of CMCS-OH30 NP in the nude mouse skin tissue observed by fluorescence microscope. Notes: Images of CMCS-OH30 NP at 1 h (A) and 4 h (B) posttreatment; images of free OH30 at 1 h (C) and 4 h (D) posttreatment. OH30 used was labeled with Cy5 (red). Abbreviation: CMCS-OH30 NP, carboxymethyl chitosan nanoparticles.
Figure 5
Figure 5
Distribution of CMCS-OH30 NP in the nude mouse skin tissue observed by fluorescence microscope. Notes: Images of CMCS-OH30 NP at 1 h (A) and 4 h (B) posttreatment; images of free OH30 at 1 h (C) and 4 h (D) posttreatment. OH30 used was labeled with Cy5 (red). Abbreviation: CMCS-OH30 NP, carboxymethyl chitosan nanoparticles.
Figure 6
Figure 6
Wound healing effects on mice models. Notes: (A) Macroscopic observations of wound closure for different treated groups at day 0, 5, 10, and 15 postinjury. The initial wounds were round (ID = 7 mm). (B) Wound closure rate of all groups. Data were presented as mean ± SD, n = 6. *p < 0.05 and **p < 0.01, contrast with other groups. Abbreviation: CMCS-OH30 NP, carboxymethyl chitosan nanoparticles.
Figure 7
Figure 7
Histological examination of the healing wounds skin sections at day 15 postinjury in four tested groups. Notes: MT staining (A) and H&E staining (B). The green arrow indicated the multiple invaginated epidermises. Scale bars = 200 μm. Abbreviations: CMCS-OH30 NP, carboxymethyl chitosan nanoparticles; MT, Masson’s trichrome; H&E, hematoxylin and eosin.
Figure 8
Figure 8
Quantification of collagen I and III content in skin homogenate 15 days after injury. Notes: The content of collagen I (A), the content of collagen III (B), the ratio of collagen I and collagen III (C), and the total content of collagen I plus collagen III (D). Statistical significance of CMCS-OH30 NP group was compared with other groups (mean ± SD; n = 6, Student’s t-test. *p < 0.05 and **p < 0.01). Abbreviation: CMCS-OH30 NP, carboxymethyl chitosan nanoparticles.
Figure 9
Figure 9
Quantification of cytokines in skin lysates. Notes: IL-6 (A), IL-8 (B), IL-10 (C), TGF-β1 (D), and TNF-α1 (E). The black boxes ■ on the y-axis refer to the quantification level of cytokines in normal mouse skin. Statistical significance compared with the untreated group was indicated (mean ± SD; n = 6, Student’s t-test. *p < 0.05 and **p < 0.01). Abbreviations: CMCS-OH30 NP, carboxymethyl chitosan nanoparticles; d, days.
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