Normal Wound Healing Analysis. An excisional full-thickness mouse model was established to investigate in vivo wound therapeutic efficacy of Hb-PDA nanoparticles. A 6 mm round wound was made on the back of Kunming mice. The animals were randomly divided into saline-treated group (NS), Hb-treated group (Hb) and Hb-PDA-treated group (Hb-PDA). During the healing process, the changes of wounds as illustrated in the digital images were observed and recorded (Figure 1A). The Hb and Hb-PDA presented a relatively smaller wound size than that of the NS from Day 3 to Day 10 (Figure 1B). As illustrated in Figure 1C, a significantly higher wound closure rate of Hb-PDA was measured compared with Hb from Day 3 to Day 10. The wound closure rate of the Hb-PDA reached over 90% on Day 10, while that in Hb was 84.11±1.57%. These results indicate the Hb and Hb-PDA accelerated the wound repair process and the Hb-PDA exhibited a better recovery compared with Hb.
To investigate microscopically therapeutic efficacy of different treatments, the wound pathology was further evaluated. Hematoxylin-eosin (H&E) staining assay was performed from Day 3 to Day 10 after the treatments, as seen from Figure 2. The NS showed a large number of inflammatory cells from Day 3 to Day 10 and granulation tissue was observed until Day 7. By contrast, granulation tissue was found in other groups from Day 3. Nevertheless, Coherent epidermis was observed in Hb-PDA on Day 7, while that was found in Hb on Day 10, indicating Hb-PDA could accelerate healing process with fewer inflammations. Additionally, masson’s trichrome staining was carried out to assess the collagen deposition during tissue remodeling.24 As shown in Figure 3, the Hb-PDA displayed more collagen deposition than the Hb. All the results indicated that the Hb-PDA showed superior efficiency in wound healing in terms of fewer inflammations and more collagen deposition.
Anti-inflammatory effects. Under normal circumstances, inflammatory cells will produce ROS in the wound tissue, which at low concentration are thought as cells messages to stimulate key processes associated with wound healing. While breakdown products further promote oxidative stress occurs when the oxidative capacity exceeds the antioxidant capacity, prolonging the inflammation phase. To validate the ability to suppress the inflammatory, immunohistochemistry staining of interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α), which is known as cytokines with inflammatory property were carried out.25 As shown in Figure 4A, the Hb-PDA presented a relatively lower expression of IL-6 than that of the NS and Hb from Day 3 to Day 10. In addition, in consistency with the trend of IL-6, the expression of TNF-α in Hb-PDA presented a relatively lower level than that in NS and Hb group, reconfirming anti-inflammatory effects due to the PDA modification on Hb surface with a shortened inflammatory phase at the early stage of wound healing.
The existence of oxidative stress should be taken into account in the inflammatory response stage of wound healing, and timely antioxidation treatment will contribute to wound healing. Parameters, such as superoxide dismutase (SOD) and malondialdehyde (MDA) plays a key role in damaged tissue timely caused by oxidative stress.26 In comparison, the pression of SOD in Hb-PDA still remain higher level concentration from Day 3 to Day 10 (Figure 4C). The expression level of MDA was found to significantly decrease in Hb and Hb-PDA from Day 3 to Day 10, especially in the Hb-PDA (Figure 4D). It was reasonably understandable that the PDA modification on surface presented anti-inflammatory effects during wound healing. The distinct free radical character allows PDA to act as radical trap in the biological system. Its interesting abilities to scavenge reactive free radicals have conferred to strong antioxidant effects in the organism.27
Angiogenesis evaluation of the wounds. Angiogenesis is an essential contributor in the process of healing, which is the foundation of nutrient and oxygen supply. Platelet endothelial cell adhesion molecule-31 protein (CD-31) is involved in many pathways, including collagen synthesis and angiogenesis, re-epithelization. Platelet endothelial cell adhesion molecule-34 protein (CD-34) is generally used to indicate the expression in endothelial cell tissue and to assess angiogenesis.28 Immunohistochemical staining analysis shows that the Hb and Hb-PDA accelerates wound closure by upregulating expression of CD-31 and CD-34, indicating the formation of blood vessel increases significantly in the oxygen treatment groups, especially in the Hb-PDA (Figure 5A). The expression of CD-31 and CD-34 was clearly observed in all groups on Day 7. It is worth noted that the expression of CD-31 and CD-34 experienced a time dependent manner. The NS showed higher expression of CD-31 and CD-34 on Day 7 compared to Day 10, demonstrating the wound was still under proliferation phase of wound repair. By contrast, the down-regulation of CD-31 and CD-34 was observed in the Hb and Hb-PDA on Day 10 (Figure 5B, C). This indicated the Hb and Hb-PDA group entered the remodeling phase as evidenced contraction of granulation tissues and thus to prevent extraordinary scar formation.
VEGF promotes multilevel processes of the angiogenic activity and vascular permeability. The immunohistochemistry staining indicated that Hb-PDA secretes much more VEGF to promote angiogenesis, demonstrating Hb-PDA enhances angiogenesis to promote the vascular stabilization stage and accelerate wound healing (Figure 5A). Elisa and Real-time quantitative PCR (qRT-PCR) was carried out to measure the expression of VEGF.29 The Level of VEGF in the Hb and Hb-PDA group rapidly reached above 100 pg/mL on Day 3 and remained higher level until Day 7. As shown in Figure 5B, the Hb-PDA group presented a significant higher VEGF production than that of the Hb group on Day 7. In contrast, the expression of VEGF in the NS showed a slowly increasing trend and reached above 100 pg/mL on Day 10. In consistency with the trend of results from Elisa, the expression of VEGF in Hb-PDA presented a relatively higher level than that in NS and Hb group from Day 3 to Day 7 (Figure 5C). Based on the above results, due to the lack of oxygen delivery without vascular, it’s insufficient to provide energy critical for angiogenesis in the wound site. It should be noted that the addition of Hb with the oxygen loading and unloading capacity contributes to effectively enhance oxygen concentration to show the better therapeutic effects.
Oxygenation effects of Hb-PDA. It should be mentioned that adequate oxygen is prerequisite for successful wound healing, which can promote cell proliferation and tissue remodel. Therefore, attempts to promote wound healing for tissue repair can tend to focus on the study of oxygen-delivering.30 Due to the vascular rupture in the wound, the blood supply to the skin tissue was affected, leading to the lack of oxygen in the wound area. In addition, the increased oxygen consumption in the process of wound repair, such as normal operation of inflammatory cells, proliferation of fibroblasts and synthesis of collagen, leads to hypoxia at the wound site. Thus, providing adequate oxygen supply to the wound tissue is the key factor in wound treatment. Three different groups have been compared through measuring the changes of fluorescent intensity by VISISENS fluorescence imaging device. Oxygen saturation was further calculated with time. Compared to the pristine wound on Day 0, a clear rise of oxygen saturation in each group was observed from Day 1 to Day 10, which was attributed to the inflammatory mediators and immune response cells inducing the dilatation of blood vessels during the inflammatory phase of wound healing (Figure 7A). It was obvious that oxygen saturation shows enlacement from Day 1 to Day 8, while it began to decline on Day 10. Compared with other groups, a higher level of oxygen saturation of Hb-PDA group was also measured from Day 1 to Day 10 (Figure 7B).
Hypoxia-inducible factor 1 (HIF-1) is an oxygen-regulated transcriptional activator that plays essential roles in mammalian development, physiology and disease pathogenesis. Due to the oxygen delivery, the immunohistochemistry staining demonstrated that Hb-PDA secretes lower HIF-1α (Figure 7C). The expression of HIF-1α in the Hb and Hb-PDA group showed a decreasing trend on Day 7 and Day 10 (Figure 7D). The expression of HIF-1α in Hb-PDA presented a relatively lower level than that in NS and Hb group on Day 10. Tsuyoshi applied liposomal-coated hemoglobin (m-LEH) with high oxygen affinity to the skin ulcer wound in mice, which could improve the aerobic metabolism of skin ulcer in mice and accelerate wound healing. Using hemoglobin to carry oxygen and release oxygen can improve the utilization rate of oxygen and promote wound healing, and become a potential application of oxygen therapy. In addition, the expression of HIF-1α in the Hb-PDA group was significantly lower than that in Hb group on Day 10. The reason may be that the ability to carry and release oxygen of Hb in the Hb-PDA nanoparticles was maintained for a longer period of time by the antioxidant activity of PDA than the free Hb. The PDA surface modification technology could maintain its oxygen-carrying and oxygen-releasing function, which is essential for cell proliferation, antimicrobial activity, angiogenesis and collagen generation.