1. Nanoceria-miRNA as a modulator of inflammation in diabetic wounds
Irina Kalashnikova, Junwang Xu, Carlos Zgheib, Soumen Das, Kenneth W Liechty, Sudipta Seal
INTRODUCTION
CNPs AND CNP-miRNA-146a CHARACTERIZATION
Central issue of the wound healing impairment in diabetics is defective immune response.
It has recently been confirmed that correction of miRNA-146a (miR) expression level locally at the wound site regulates inflammation and improve recovery both non-diabetic and diabetics wounds.
Cerium oxide nanoparticles (CNPs) are potent nano-inhibitors of oxidative stress due to their radical scavenging ability, similar to the activity of antioxidant enzymes.
Nanoceria can improve the therapeutical effect of miRNA by protecting it from degradation mediating its entry into the cell.
Fig. 2. a) HRTEM of CNPs; b) UV-Vis spectrophotometry; c) FT-IR; d) DLS.
IN VITRO RESULTS OF CNP-miRNA-146a APPLICATION
HYPOTHESIS
Fig. 3.
A-conjugate activity testing in presence of the controls;
B-time-dependent effect of the conjugate on the fibroblasts;
C-concentration-dependent effect of the conjugate on the fibroblasts.
We hypothesized that synergetic effect of conjugate CNPs, being capable to inhibit ROS, with miRNA-146a, responsible for inflammation, may speed up wounds closure significantly.
METHODS
CNPs were synthesized by simple wet chemistry method. Mature miRNA-146a was attached to hydroxyl groups of CNPs using 1,1-carbonyldiimidazole (Fig. 1). The CNPs and CNP-miR was characterized by different methods (Fig. 2, Table 1).
Two models of wounds were used. Primary dermal murine non-diabetic and diabetic fibroblasts were treated with the conjugate mixed with DMEM at different concentration. CNPs, miRNA-146a, and CNP-scrambled miRNA (CNP-scr) served as the appropriate controls.
10-12-weeks old Db/Db and Db/+ mice underwent 8-mm full-thickness wounding. Postoperatively, the mice received 50ul of 10µM CNPs, CNP-miR, or vehicle by intradermal single injection. RT-PCR analysis and histology were used for inflammation evaluation.
Conjugate increases miR-146a expression level in time-/concentration-dependent manner
IN VIVO DATA OF CNPs-miRNA-146a APPLICATION
Conjugate synthesis
Fig. 4. Slices of wound tissues (7 day of post-wounding) stained with F4/80 antibodies, A-D: Db-vehicle, Db-conjugate, Non-Db-vehicle, Non-Db-conjugate.
Fig. 5. miRNA-146a expression level and macrophages number A-for non-Db mice (*p<0.05) and B-for Db mice (*P<0.04,**p<0.02).
Fig. 6. Gene expression level in wound tissues of: A-non-Db mice (*p<0.02, **p<0.005,***p<2*10-5) and B-Db mice (*p<0.02,**p<0.003).
Conjugate regulates the recruitment of macrophage cells via miR-146a expression and its target genes correcting
CNPs-miRNA-146a EFFECTING ON INFLAMMATION ACCELERATE WOUND HEALING
Fig.1 Scheme of miRNA-146 conjugation with CNPs.
Fig.7. The time course of wound healing in A-non-diabetic and B-diabetic mice that were treated with vehicle and  the conjugate
Conjugate accelerate wound closure via regulation of inflammation phase
CONCLUSIONS
This study has showed that synthesized conjugate nanoceria with miRNA-146a is able to successfully deliver miRNA that improve diabetic wound healing via correction of inflammatory phase of it. The conjugate speeded up the closure of normal and diabetic wounds in skin of mice on 5 and 4 days, respectively, compared to wounds treated with vehicle.
Table1. Characterization of samples using Nanodrop and Zetasizer. Conjugate and physical mixture of CNPs with miRNA was made at ratio 1:100.