Monitoring Wound Healing with Topically Applied Optical NanoFlare mRNA Nanosensors

Abstract

An effective wound management strategy needs accurate assessment of wound status throughout the whole healing process. This can be achieved by examining molecular biomarkers including proteins, DNAs, and RNAs. However, existing methods for quantifying these biomarkers such as immunohistochemistry and quantitative polymerase chain reaction are usually laborious, resource-intensive, and disruptive. This article reports the development and utilization of mRNA nanosensors (i.e., NanoFlare) that are topically applied on cutaneous wounds to reveal the healing status through targeted and semi-quantitative examination of the mRNA biomarkers in skin cells. In 2D and 3D in vitro models, the efficacy and efficiency of these nanosensors are demonstrated in revealing the dynamic changes of mRNA biomarkers for different stages of wound development. In mouse models, this platform permits the tracking and identification of wound healing stages and a normal and diabetic wound healing process by wound healing index in real time.

Keywords: NanoFlare; diabetic wound; mRNA nanosensors; spherical nucleic acids; wound healing.

Scheme 1

Illustration of NanoSensor (i.e., NFs) to detect cellular mRNA for monitoring wound status.

Figure 1

Screening of biomarkers for fibroblasts (NDF), keratinocytes (HaCaT), and endothelial cells (HUVEC). Cellular expression of the 30 potential RNA biomarkers in A) NDF; B) HaCaT; C) HUVEC. The cellular expression of 10 identified D) NDF biomarkers; E) HaCaT biomarkers; F) HUVEC biomarkers in NDF, HaCaT, and HUVEC respectively. Fold expression of each gene is normalized to the expression levels of housekeeping gene (GAPDH). The candidate gene is marked with a black star, selected gene is a red star respectively (n = 3, Values are means ± s.d.).

Figure 2

Identification of NF sensitivity. A) Illustrationof experiments; NF fluorescence restoration of B) FSP1‐NF, D) KRT14‐NF, F) PECAM1‐NF, and H) GAPDH‐NF over time after the mixing with concentration‐fixed target sequence and mismatched sequence (concentration of target sequence or mismatched target sequence is 2 µm; NF concentration is O.D 0.4, black line = PBS); Fluorescence restoration of C) FSP1‐NF, E) KRT14‐NF, G) PECAM1‐NF, and I) GAPDH‐NF after the mixing with varied concentrations of target sequence and mismatched sequence with a fixed incubation time (NF concentration is O.D 0.4, black line: mismatched sequence = 2 µm. Incubation time is 20 min, n = 3, values are means ± s.d. Excitation and emission of FSP1‐NF are 493 nm and 517 nm. Excitation and emission of KRT14‐NF and PECAM1‐NF are 555nm and 569 nm. Excitation and emission of GAPDH‐NF is 651 and 670 nm).

Figure 3

Evaluation and confirmation of target gene expression under the growth factor stimulation by NFs and qPCR. A) Illustration; B) FSP1 expression in NDF treated with 10 growth factors. The results were derived from FSP1‐NF signal (Figure S11, Supporting Information, red line plot, n = 11) and qPCR (green bar plot); C) KRT14 expression in HaCaT treated with 10 growth factors. The results were derived from KRT14‐NF signal (Figure S12, Supporting Information, green line plot, n = 10) and qPCR (red bar plot); D) PECAM1 expression in HUVEC treated with 10 growth factors. The results were derived from PECAM1‐NF signal (Figure S13, Supporting Information, black line plot, n = 6) and qPCR (blue bar plot). The signals of FSP1‐NFs, KRT14‐NFs, and PECAM1‐NFs were normalized using that of GAPDH‐NFs, values are means ± s.d.

Figure 4

Confocal fluorescence images of 3D spheroid and 2D cell culture with NFs. A) Illustration of experiments; B) 2D and 3D co‐culture of NDF and HaCaT with NFs (white scale bar is 50 µm); and C) KC with NFs: top, DP with NFs: middle, co‐culture of KC and DP with NFs; bottom (scale bar is 200 µm). Concentrations of FSP1‐NF, KRT14‐NF, and GAPDH‐NF were O.D 0.1, 0.0125, and 0.0125, respectively.

Figure 5

Monitoring wound healing by topically applied NFs on normal and diabetic mice. A) Illustration of experiment setup; B) blood glucose level, C) wound area, and D) weight change in normal and diabetic mice through the experiments; Evaluation of three biomarkers by NFs on, values are means ± s.d. E) Normal mice group and F) diabetic mice group (the signal was normalized against that of reference gene, GAPDH, means ± s.d); G) comparison of the date when the highest NF fluorescence signal was observed for each biomarker in two wound healing models from (E) and (F) (*p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns = not significant, n = 6, duplicate); and H) Fluorescence Wound healing Index for normal and diabetic groups (mean value of the highest fluorescence signal from NF was normalized with GAPDH signal in 10 days in both groups respectively, n = 11, values are means ± s.d.).