Characterization of usAgNPs-PL
Figure 2A showed the TEM images of synthesized usAgNPs, and the spherical nanoparticle morphology with diameter was around 13 nm. In addition, the powder XRD patterns of usAgNPs (Fig. 2B) showed the obvious diffraction peaks at 2θ = 13.2° and 16.5°, indicating obvious crystalline and better peak shape [25]. To determine the chemical composition of usAgNPs and identify the chemical state of the Ag element in our sample, monochromic X-radiation were used to detect the samples, and the inner-shell electrons property was to be explored.
Based on the XPS spectrum of usAgNPs sample (Fig. 2C(a)), 4 kinds of elements (Ag, O, C and N) were examed in usAgNPs samples. The spectrum of the Ag 3d level was displayed in Fig. 2C(b). The Ag 3d3/2 and Ag 3d5/2 spin–orbital photoelectrons were located at binding energies of 368.5 eV and 374.5 eV, respectively. According to previous report[26], the peaks at 368.5 and 374.5 eV were attributed to metallic silver, and these two peaks with a spin energy separation of 6.0 eV further showed the existence of Ag in usAgNPs sample. Figure 2D showed that TGA curves of usAgNPs reflected two steps of degradation: when water was evaporated at 260℃, the decomposition started at 300℃, accomplished at 400℃ for hydroxyl, carboxylic, and pyridyl rings, mass loss was about 25%. After reached 400℃, the usAgNPs could not be further degrade.[27] The infrared absorption peaks showed in IR spectrum (Fig. 2E) were summarized as follows: the observed peak at 1517 cm− 1 was attributed to C − N stretching, N − H stretching was observed at 1729 cm− 1. The two peaks identified at 1288 and 1350 cm− 1 most likely corresponded to C = C and C ≡ O stretching, respectively. The peak at 1288 cm− 1 is caused by C − O−C symmetric stretching.Futherly, the peak at 1082 cm− 1 was related to C − O stretching. In addition, several peaks were in the range of 881 − 762 cm− 1 might be assigned to C − H bonding, especially rocking vibrations. 512cm− 1 belonged to the fine peak between 500 and 577 cm− 1, which may be related to the vibration of Ag − O[28]. 779cm− 1 was also attributed to Ag − O vibration. Figure 2F showed UsAgNPs had strong adsorption bands at 252–310 nm, that indicated the π → π* transition of pyridine ring[29].
To prove that usAgNPs could adsorb PL, the BET experiment was used in vitro. In Fig. 2G, the usAgNPs showed a proper surface area with a typical IV isotherm. N2 adsorption of usAgNPs gave the BET surface area of 29.003 m2/g and pore volume of 0.062970 cm3/g. So, as nanomaterials, usAgNPs exhibited good absorption capacity.
When usAgNPs were mixed with PL, the pellet was collected to get usAgNPs-PL. The residual supernatant was used as negative control. As shown in Fig. 2H, It revealed core-shell structural characteristics and usAgNPs were absorbed at the surface of PL. To further elucidate the above speculation, the distribution of usAgNPs of platelets was detected by TEM (Fig. 2I). The platelets displayed well-developed structure[30]. Almost all usAgNPs in platelet was adsorbed.[31] TEM clearly showed usAgNPs in the (unstained) vesicles (Fig. 2I)[32]. At the same time, western blotting (Fig. 2J) showed that compared with usAgNPs, usAgNPs-PL was confirmed to contain PDGF-β, TGF-β, VEGF and bFGF.
To explore whether the usAgNPs is fit as an therapeutic agent, the biocompatibility usAgNPs of was detected in vivo and in vitro. The hemolysis test (Supplementary Fig. 1) showed that usAgNPs had compatibility in blood. The red blood cells at the positive control group which treated by ddH2O were all broken. The usAgNPs treated red blood cells still kept a normal cell structure and morphology, the haemolysis rate was less than 5%. On the contrary, when incubated with usAgNPs at the concentration from 0.5mg/ml to 4mg/mL, red blood cells still maintained normal structure and morphology, indicating usAgNPs was a safe nanomaterial. MTT results (Supplementary Fig. 2) further confirmed usAgNPs did not have significant cytotoxicity, and survival rate of the red blood cells was greater than 85% at 100 µg/mL usAgNPs. When usAgNPs-PL concentration reached 250 µg/mL, the survival rate was also greater than 85%. HE results (Supplementary Fig. 3) showed the rats which exposed to usAgNPs-PL at the concentration of usAgNPs at 2 mg/kg ,15 d had no obvious toxic and side effects in the main organs. Compared with PBS treated group (sham operation), usAgNPs and usAgNPs-PL did not cause significant histological changes and toxic effect at the main organs.
Characterization of usAgNPs-PL/CMC hydrogel
In Fig. 3A, the three hydrogels (CMC hydrogel, usAgNPs/CMC hydrogel and usAgNPs-PL/CMC hydrogel) showed a uniform and porous three-dimensional network shape by SEM, the addition of usAgNPs-PL did not influence the microstructure in the gel system. usAgNPs-PL/CMC hydrogel showed rheological behaviour which is like that of CMC hydrogel or usAgNPs/CMC hydrogel. To investigate the rheological and shear stress of hydrogel, dynamic temperature scanning rheological test was carried out to calculate the change of storage modulus (G') and loss modulus (G") of hydrogel. Figure 3B showed that the G' and G" of the three hydrogels were relatively stable between 20℃ and 40℃, and the G' value of the hydrogel group was always greater than G". So, the gel remained an elastic network. Flow curves were detected at variable shear rates and temperatures. The viscosities of the gels declined sharply with slight rise at shear rate. For the gradually increased shear rate, the gel viscosity declined slowly, exhibiting shear thinning behavior (Fig. 3C). Figure 3D showed swelling rates of the CMC hydrogel, usAgNPs/CMC and usAgNPs-PL/CMC after 80 min were 907.3%, 870.7% and 836% respectively. Figure 3E showed the state of the hydrogel at room temperature, indicating that all hydrogels were in gel state.
Comparison of antibacterial effect between usAgNPs and other nanosized silver nanoparticles
It is well known that owing to the larger superficial area and higher surface charge density, silver nanocomposite with smaller particle size demonstrated stronger its antibacterial effect[33]. So, We compared the bacteriostatic effect of usAgNPs with that of silver nanoparticles with different particle sizes AgNPs at 20nm, AgNPs at 50nm, AgNPs at 100nm, AgNPs at 200nm, AgNPs at 500nm (Supplementary Fig. 4). It was evident in Fig. 5A that the smaller the particle size of silver nano particles, the more effective the antibacterial activity in the growth of bacteria (E. coli and S. aureus). The antibacterial activities were detected at OD600nm. Furthermore, the diameters of bacteriostatic inhibition zone of silver nanoparticles with different particle sizes were analyzed and compared. Figure 5B showed with the decrease of particle size, silver nanoparticles gradually enhanced their antibacterial activities by increasing diameter of bacteriostatic inhibition zone. Futhermore, diameter of bacteriostatic circles of prepared usAgNPs with the particle size at 13 nm was the largest, indicating that the bacteriostatic effect was the strongest, indicating that the bacteriostatic effect of the prepared usAgNPs was better than that of ordinary Ag nanoparticles. It can be observed from Fig. 4D that the control group formed a relatively complete biofilm, but Ag nanoparticles treated group formed a nearly transparent lavender. Meanwhile, quantitative analysis showed the absorbance was gradually reduced with the decrease of particle size of NPs. XTT results demonstrated that destruction effect of Ag nanoparticles on the biofilm of E. coli and S. aureus was enhanced with the decrease of particle size of NPs. The usAgNPs with smaller particle sizes had stronger destruction effect on biofilm of Escherichia coli and Staphylococcus aureus as evidenced by the lowest optical density (OD) measured at 490 nm. This proved that usAgNPs had a obvious inhibitory and scavenging effect on biofilm formation in E. coli and S. aureus owing to their high permeability on biofilm. To further verify usAgNPs induced antibacterial effect, PI staining were used to identify the dead bacteria and found the usAgNPs resulted in the maximum bacterial death by showing strongest red fluorescence in bacteria, indicating that usAgNPs induced the strongest antibacterial ability (Fig. 4E).
To explore the mechanisms of usAgNPs on bacteria, ROS, MDA and ATP levels were carried out. ROS were detected by DCFH-DA (2,7-dichloro-dihydrofluorescein diacetate) method. As shown in the Fig. 4F, Ag nanoparticles enhanced the generation of ROS in bacteria with the decreased particle size. Especially, usAgNPs induced the highest green fluorescence intensity among all groups, indicating that usAgNPs induced a large amount of reactive oxygen species for causing bacterial cell death. MDA (malondialdehyde) were continued to exam the cell lipid peroxidation to reflect the damage extent of membrane. We can see the differences of MDA among different treatment groups were significant (Fig. 4G) and the MDA in bacteria treated with usAgNPs was significantly increased compared with the control group. usAgNPs also induced a significant decrease in ATP levels (Fig. 4H). So, the usAgNPs affected bacterial cell metabolism.
The usAgNPs-PL enhanced angiogenesis via the mediation of PL in vitro
To prove usAgNPs-PL could promote the angiogenesis in vitro, HUVECs were used to exam the ability of vessels formation. First, wound healing assay showed wound of Hg-HUVECs was larger than normal HUVECs (Fig. 5A). The wound closure rate of Hg-HUVECs treated with usAgNPs-PL was significantly increased after 12 hours and at 24 hours (Supplementary Fig. 5A). The usAgNPs and usAgNPs-PL treatment can accelerate the wound healing ability of Hg-HUVECs. Second, Transwell assay showed usAgNPs-PL enhanced migration of Hg-HUVECs by inducing more cells to migrate through transwell (Fig. 5B). Hg inhibited significantly HUVECs migration ability (Supplementary Fig. 5B). On the contrary, the migration ability was enhanced and the tube formation was accelerated in usAgNPs-PL treated group (Fig. 5C). And correspondingly, the usAgNPs and usAgNPs-PL also promoted the migration and tube formation in HUVEC (Supplementary Fig. 5C). To verify whether usAgNPs-PL influence the proliferation of HUVEC, western blotting was carried out to detect the expression of Erk, Akt and p-Akt, p-Erk in Hg-HUVECs and Hg-HUVECs treated with usAgNPs-PL. Compared to other groups, usAgNPs-PL significantly increase the phosphorylation level of Akt and Erk, and did not influence the expression of total ERK and AKT, indicating that the two signaling of Hg-HUVECs was activated by usAgNPs-PL (Fig. 5D). In addition, usAgNPs-PL significantly promoted the expression of PI3K and VEGFR (Supplementary Fig. 6). Together, the usAgNPs-PL could exert pro-angiogenesis function.
Optimization of usAgNPs-PL/CMC hydrogel
UsAgNPs and usAgNPs-PL was usually administrated as the liquid form for wound healing. However, it is difficult for these traditional dosage forms to stay in the wound surface and there are defects such as the loss and discharge of drugs after smearing. The poor availability and short retention of usAgNPs and usAgNPs-PL in the wound surface largely compromised its therapeutic effect on wound healing[34]. Moreover, large volume and multiple administrations regime are usually required for liquid preparation to exert the effective therapeutic response, which reduce the sensitivity of the wound. The poor availability and short retention of usAgNPs and usAgNPs-PL in the wound surface largely compromised their therapeutic effect on wound healing.
Here, we selected hydrogel as the carrier to load the usAgNPs-PL complex. To find out the best antibacterial effect of usAgNPs-PL/CMC hydrogel, usAgNPs with different concentrations were added into hydrogel for studying their antibacterial activities on Gram-negative and Gram-positive bacteria. usAgNPs/CMC hydrogel obviously inhibited E. coli and S. aureus (Fig. 6A). As high concentration of Ag has significant toxicity, the biggest bactericidal concentration of usAgNPs was 2.5 mg/ml in hydrogel. We can see that usAgNPs, usAgNPs-PL and usAgNPs-PL/CMC hydrogel all obviously inhibited the growth of bacteria, and usAgNPs-PL and usAgNPs-PL/CMC hydrogel treated bacteria grew more than usAgNPs at 2.5 mg/ml. So, to a certain extent, PL and CMC hydrogel could decrease the toxicity of Ag. Though compared with usAgNPs, usAgNPs-PL and usAgNPs-PL/CMC hydrogel has lower antibacterial effect, usAgNPs-PL and usAgNPs-PL/CMC hydrogel all have effective inhibition rate. And usAgNPs/CMC hydrogel significantly inhibited bacteriostatic ring range of E. coli and S.aureus (Fig. 6B). Moreover, XTT assay was undertaken to examine the effect of usAgNPs-PL/CMC hydrogel on viability in E. coli and S. aureus within biofilm forming and the metabolic activity of this structure. The statistic figure showed usAgNPs/CMC hydrogel inhibited E.coli and S.aureus reproduction (Fig. 6C). Then, biomasses of biofilms were determined by a crystal violet staining. It can be observed from Fig. 6D that the control group are dark purple, indicating the bacteria formed a relatively complete biofilm. However, usAgNPs-PL/CMC hydrogel group formed an almost transparent lavender. This proves that usAgNPs-PL/CMC hydrogel can inhibit bacteria to form biofilm and reproduct. PI staining were used to detect the dead bacteria (Fig. 6E). The control group showed red fluorescence weaker than the usAgNPs-PL/CMC hydrogel group, indicating that usAgNPs-PL/CMC hydrogel had the more vital antibacterial ability.
Base on the results of antibacterial experiments results, we tried to explore the antibacterial mechanism of usAgNPs-PL/CMC hydrogel. Such as above (Fig. 4), ROS, MDA and ATP levels were carried out. Figure 6F showed that usAgNPs-PL/CMC hydrogel induces a large quantity of ROS. Figure 6G showed usAgNPs-PL/CMC hydrogel MDA induced by ROS reached 149.94% in E. coli and 127.08% in S. aureus. Figure 6H showed ATP levels decrease significantly in the usAgNPs-PL/CMC hydrogel group. So, usAgNPs-PL/CMC hydrogel inhibit ATP level in the bacterias (E. coli and S. aureus).
The usAgNPs-PL/CMC hydrogel promoted diabetic wound healing in vivo
We further explored the effect of usAgNPs-PL/CMC hydrogel on potential regulation of diabetic wound healing in SD rats with type 1 diabetes by observing the closure ratio and healing quality in the wound of diabetes process. Figure 7A showed our schedule. As shown in Fig. 7B, we photographed diabetic wounds size in the five groups at 0, 3, 7, 10, and 14 days after surgery. We can see wound closure in all experimental groups decreased significantly at days 7, day10. And wound healing velocity of negative control group was slower. Furthermore, in model group, the wound was not completely closed at 14 days, incomplete epithelization and thin epithelium was still observable. The wound healing rates of other treated groups were higher than model group (Supplementary Fig. 7), usAgNPs-PL/CMC hydrogel can induce the highest wound closure rate by showing basically complete closure within 14 days (Fig. 7C). After covered with CMC hydrogel, usAgNPs/CMC hydrogel and usAgNPs-PL/CMC hydrogel, bacteria in diabetic wound tissue were isolated and fostered on agar mediums. The results (Fig. 7D) showed that being consistent with in vitro antibacterial results of usAgNPs, usAgNPs/CMC hydrogel and usAgNPs-PL/CMC hydrogel could inhibit the survival and reproduction of bacteria of the wound.
The usAgNPs-PL/CMC hydrogel hydrogel improved the quality of diabetic wound healing by facilitating tissue generation improving collagen deposition and enhancing angiogenesis in vivo
Pathological structure change of diabetic wound in rats treated with different administrations were investigated by HE staining. As shown in Fig. 8A, the wounds treated with usAgNPs/CMC hydrogel and usAgNPs-PL/CMC hydrogel appeared more reepithelialization and intact epidermis while, the model group did not have a shaped epidermis. In the usAgNPs-PL/CMC hydrogel treated group, newborn hair follicle formation was notable, fibroblast cells proliferated and collagen deposition was orderly and sufficient. But, all the characters could not be examed in the usAgNPs/CMC hydrogel treated groups. HE staining analysis supposed that usAgNPs-PL/CMC hydrogel was more effective in the repair and regeneration process in diabetic wound healing.
As proper collagen deposition and remodeling could modify tensile strength of tissues and advanced therapeutic effects, we further evaluated collagen synthesis by Masson staining. It was observed that the collagen has been stained blue fiber, and collagen deposition could be found in all groups. Furthermore, densely packed and parallel mature collagen fibers in the dermis of the usAgNPs-PL/CMC hydrogel treated group was more organized than other treatment groups. The results of Massons staining showed that usAgNPs-PL/CMC hydrogel can promote collagen deposition, thereby facilitate skin regeneration and repair wounds in diabetes (Fig. 8B).
Angiogenesis is pivotal throughout the entire process of wound repair. Blood vessels provide progenitor cells, oxygen and nutrients to maintain proliferation and remodeling at the wound site[35]. Angiogenesis is essential for wound healing and many studies suggesting impaired angiogenesis result in chronic non-healing wounds in diabetic conditions[36]. CD-31 is an angiogenesis marker, which expresses on the surface of endothelial cells[37]. Here, we detected the expression of CD-31 by IHC and evaluate the treatment effect of usAgNPs-PL/CMC hydrogel on angiogenesis. Fewer number of new vessels was observed at day 7 and represented by higher expression of CD-31 whereas usAgNPs-PL/CMC hydrogel induced high number of CD-31 positive cells (Fig. 8C).
To investigate how the topical application of usAgNPs-PL/CMC hydrogel affect the cells in the granulation tissues, Ki67 were detected by immunofluorescence staining (Fig. 8D). We can see Ki67 expressed higher in wounds treated with usAgNPs-PL/CMC hydrogel at day 7 than CMC hydrogel group and the control groups.
Taken together, usAgNPs-PL/CMC hydrogel demonstrated the better wound healing effects with higher quality of diabetic wound healing by promoting collagen evelopment, angiogenesis and proliferation of fibroblasts from granulation tissue.