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. 2018 Oct;22(10):5008-5019.
doi: 10.1111/jcmm.13768. Epub 2018 Jul 20.

Collagen peptide modified carboxymethyl cellulose as both antioxidant drug and carrier for drug delivery against retinal ischaemia/reperfusion injury

Hua Mu  1 , Yeqing Wang  1 , Haiying Wei  1 , Hong Lu  1 , Zhuolei Feng  1 , Hongmin Yu  1 , Yue Xing  1 , Haijing Wang  1
Affiliations

Collagen peptide modified carboxymethyl cellulose as both antioxidant drug and carrier for drug delivery against retinal ischaemia/reperfusion injury

Hua Mu et al. J Cell Mol Med. 2018 Oct.

Abstract

Oxidative stress can cause injury in retinal endothelial cells. Carboxymethyl cellulose modified with collagen peptide (CMCC) is of a distinct antioxidant capacity and potentially a good drug carrier. In this study, the protective effects of CMCC against H2 O2 -induced injury of primary retinal endothelial cells were investigated. In vitro, we demonstrated that CMCC significantly promoted viability of H2 O2 -treated cells, efficiently restrained cellular reactive oxygen species (ROS) production and cell apoptosis. Then, the CMCC was employed as both drug and anti-inflammatory drug carrier for treatment of retinal ischaemia/reperfusion (I/R) in rats. Animals were treated with CMCC or interleukin-10-loaded CMCC (IL-10@CMCC), respectively. In comparisons, the IL-10@CMCC treatment exhibited superior therapeutic effects, including better restoration of retinal structural thickness and less retinal apoptosis. Also, chemiluminescence demonstrated that transplantation of IL-10@CMCC markedly reduced the retinal oxidative stress level compared with CMCC alone and potently recovered the activities of typical antioxidant enzymes, SOD and CAT. Therefore, it could be concluded that CMCC provides a promising platform to enhance the drug-based therapy for I/R-related retinal injury.

Keywords: IL-10; antioxidant carrier; drug delivery; retinal ischaemia/reperfusion injury.

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Figures

Figure 1
Figure 1
Characterization of rRECs and the effects of antioxidative gels, CMCC, on their intracellular ROS levels and apoptosis. A, Morphological image of isolated rRECs was attained by the inverted microscopy. Bar scale = 100 μm. B, Flow cytometry of rREC stained by PI. C, Immunofluorescence staining with antibodies against factor VIII‐related antigen showed positive staining in rRECs. Bar scale = 100 μm. D, The cell viability was dose‐dependently related to H2O2. E, The H2O2‐mediated decrease in cell viability was attenuated by add CMCC. F, Intracellular ROS levels in rRECs were analysed by flow cytometry. G and H, The quantitative detection of DHE staining‐positive rRECs and fluorescence intensity of DHE. (n = 5). * P < .05 compared with control; # P < .05 compared with H2O2 group
Figure 2
Figure 2
Representative TUNEL images of rRECs. The rRECs were pre‐treated with or without H2O2 and antioxidative gels. The blue was nucleus staining with DAPI and the green was TUNEL staining, representing apoptotic rRECs. Three groups. Bar scale = 100 μm
Figure 3
Figure 3
Assessment of apoptosis‐related proteins in rRECs. A, Apoptosis of rRECs treated with or without H2O2 or following antioxidative gels was analysed by flow cytometry. B, The levels of apoptotic proteins in H2O2‐treated rRECs were analysed by Western blotting (n = 5). ** P < .01 compared with control; ## P < .01 compared with H2O2 group
Figure 4
Figure 4
IL‐10 releasing measurement in vitro. Upper panel) The schematic image of IL‐10@CMCC structure. Bottom panel) the releasing progress was recorded at 1‐day interval and up to 10 d
Figure 5
Figure 5
Effects of IL‐10@CMCC on retinal histology at 21 d post‐I/R injury. A, Representative retinal section images of control, I/R + PBS, I/R + Gel or I/R + Gel/IL‐10 group were attained by H&E staining and the corresponding quantitative total retinal thickness (from the inner limiting membrane to the pigment epithelium). B, Representative immunofluorescence images specified for Csp3 in the 4 groups. C, Western blotting analysis of apoptotic proteins of rRECs, for example Csp3, Bcl‐2 and Bax, in the 4 groups. n = 5, bar scale = 50 μm, a P < .05 compared with I/R + PBS group; aa P < .01 compared with I/R + PBS group; b P < .05 compared with I/R + Gel group; bb P < .01 compared with I/R + Gel group
Figure 6
Figure 6
Effects of IL‐10@CMCC on the expression of inflammatory mediators in retinal cells after I/R injury. At day 3, the mRNA expression of inflammatory mediators, for example A, TNF‐α, B, IL‐1β, C, iNOS, D, ICAM‐1 and E, MCP‐1 in retinas of control, I/R + PBS, I/R + Gel or I/R + Gel + IL‐10 group, was investigated via qPCR based on 2^‐Ct (n = 5). F) Western blotting images of the corresponding mediators in the 4 groups. * P < .05 compared with I/R + PBS group; ** P < .01 compared with I/R + PBS group; ^ P < .05 compared with I/R + Gel group; ^^ P < .01 compared with I/R + Gel group
Figure 7
Figure 7
Effects of IL‐10@CMCC on retinal ROS and anti‐oxidase activity post‐I/R injury. A, Luminol‐ and (B) lucigenin‐enhanced chemiluminescence as well as the activity of (C) SOD and (D) CAT enzymes as well as E) GSH levels in retinas of the control, I/R + PBS, I/R + CMCC or I/R + IL‐10@CMCC group were investigated at days 3, 7 and 14, respectively (n = 5). aa P < .01 compared with I/R + PBS group; b P < .05 compared with I/R + Gel group; bb P < .01 compared with I/R + Gel group
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