In this current work, we describe an effective approach for synthesis of PEG/TA-Se NPs hydrogel for cardiac tissue engineering application. Se NPs was prepared by ionic liquid mediated synthesis, then the particle formation was confirmed by UV-Visible spectroscopy. An absorption spectrum of sodium Selenite and Se NPs (after the reduction) was provided in Fig. 1
An overlapped spectrum displayed a peak at around 200 nm for precursor and a peak present at 265 nm which is due to formation of Se NPs using reducing agent NaBH4. Comparing the spectrum for Na2SeO3 and absorption spectrum obtained for Se NPs in Fig. 1, the peak was shifted into 265 (red shift) which indicating the formation of nano selenium. Structural morphology of as prepared Se NPs was characterised by TEM studies. A small portion of colloidal solution of nano selenium was centrifuged at 12000 rpm, washed with ethanol in order to remove excess the ILs present in the solution. The reddish orange precipitate was settled and dispersed in ethanol for TEM analysis. Figure 1b showed a TEM image of Se NPs, the particles are almost spherical shape ∼ 10 to15 nm respectively. Further, the crystalline nature of as prepared Se NPs was confirmed X-ray studies. The particles showed the peaks at 2 (value 23.3°, 28.5°, 41.2°, 43.5°and 44.4° which is corresponds to a plane lattice (100), (101), (110), (102) and (111) was a good match to the literature as shown in Fig. 1d respectively. In addition to that, SAED pattern of Se NPs depicted in Fig. 1c revealed the crystalline nature of NPs.
Besides, the FTIR spectra of tetra aniline (TA), PEG, PEG-Se NPs and PEG/TA-Se NPs was depicted in Fig. 2. Spectrum 2a showed a sharp peak at around 3500 cm− 1 due to presence N-H stretching for the amine group in TA. The characteristic peak at 1568 cm− 1 due to C = C stretching vibration of benzeniod structure. Moreover, the sharp observed at 1128 cm− 1 was attributed to C-N bending vibration. Figure 2b displayed FT-IR spectrum for PEG, it showed a broad band at around 3500 cm− 1 was predicted as OH stretching of the hydroxyl group. The peak present at wavenumber 1450 cm− 1 to 1292 cm− 1is owing to scissor and bending vibration. Further the peak at 1256 cm− 1 was attributed to C-O stretching from Alcohol present in PEG. The peak observed at 1180 cm− 1 is C-O-C stretching, vibration which confirmed the presence of the ether linkage.
The FT-IR spectrum of PEG/TA-Se NPs hydrogel composite is closely resembled to a spectrum of PEG and TA is given in Fig. 2D, the main characteristic peaks for OH, C-O is stretching for Alcohol, ether was obtained in the spectrum D, Also a small sharp peak 1158 cm− 1 for C-N bending vibration which revealed the existence of TA in ternary hydrogel composite. Based on theses FTIR results, we conclude the conjugation of PEG/TA in Se NPs vividly supported that PEG forms the ternary composite hydrogel. In addition to chemical characterisation, hydrogel composite formation was further confirmed by UV-Visible spectroscopy. Figure 2b exhibited an overlapped UV-visible spectrum of Se NPs, PEG/TA, and PEG/TA-Se NPs. We observe a peak at 265 nm for Se NPs and according to previous reports, an absorption band at around 300 nm in spectrum b for PEG-TA respectively. On the other hand, the spectrum c displayed an intense peak at 265 nm and a shoulder peak at 320 nm revealed grafting of Se NPs in PEG/TA hydrogel network.
Surface morphology of PEG hydrogel was examined in SEM analysis. Figure 3A showed the hydrogel surface was highly porous and fibrous in texture. However, this porosity will enhance the swelling behaviour of hydrogel. As seen in figure, shape of pores is spherical and ellipsoid with different diameter, it has a tendency to absorb high permeability for nutrients, oxygen and water soluble metabolites. After the incorporation of SeNPs in to hydrogel, the particles are embedded in the pores of hydrogel network considerably it will improve the mechanical properties. In addition to SEM studies, Se NPs doping was further confirmed through TEM analysis. Spherical shaped Se NPs were embedded on the surface of PEG/TA hydrogel composite is displayed in Fig. 4A and B showed the existence of NPs in hydrogel in another portion of grid.
Toxicity analysis of prepared hydrogel samples on cardiomyocytes
The cytocompatibility of the prepared nanoformulated hydrogel samples was primarily analysed by the treatment of dose-depended manner treatment of SeNPs (6.25, 12.5, 25 and 50 µg/mL) on the human cardiomyocytes (AC16). The cell viability results on dose-depended manner treatment of prepared nanoformulated samples are displayed in Fig. 5. The presented results of cell survival exhibited that Se NPs in lower concentrations and control samples was not significantly affected the cell viability ratio.
The cell survival experimental results were greatly used to measure the dose-depended influencing factors and favourable interactions between the prepared samples and treated cells (Fig. 5a and 5b). In addition, we have examined the damage of cardiomyocytes cell membranes through measurement of LDH level after treatment of prepared samples as exhibited in Fig. 5e. The observed data have been exhibited that significant increasing LDH level at higher concentrations when compared to lower concentration and control, which demonstrates appropriate concentration of Se nanoparticles could be highly suitable with cardiomyocytes cell survival. Importantly, the examination of ROS generation and oxidative stress efficiency is highly needful for the cardiac regeneration applications. As showed in Fig. 5c, the results from flow cytometry analysis of intracellular ROS generations exhibited that composited group of PEG-TA/Se was suggestively greater that bare group of Se NPs and control group. These investigations demonstrated that enhanced ROS generations was persuaded by interactions between Se NPs and TA/PEG macromolecular structure. The anti-oxidant activity of the prepared samples on the AC16 was investigated by the SOD as shown in Fig. 5d. The analysis results demonstrated that intracellular SOD and GSH-Px level was decreased by inducing interactions between the Se NPs and TA-PEG.
In vivo examinations of MI-induced inflammatory responses after treatment of prepared hydrogel
To investigate therapeutic potential of the prepared hydrogel materials after MI, we examined an animal MI model with presence of permanent occlusion of coronary artery (LAD). The prepared Se NPs and Se hydrogel samples were individually injected on the myocardial infarction site after MI occlusion to observed therapeutic potential and regeneration ability. The microscopic observation of large occupation of collagen zone on the LV wall and infarct site as shown in Fig. 7 at control (PBS), which signifying the existence of extensively spread scar tissue. On the other hand, the MI models injected with Se hydrogel samples have exhibited that large myocardial tissue at MI site as displayed in Fig. 7 (A). Meanwhile these observations are strongly consistent with the results of LV wall thickness and relative scar thickness. The quantitative data showed LV wall thickness was conserved significantly from the value of 235.6 µm to 390 µm (Fig. 7B). Meanwhile, the relative scar thickness (33.6 %) and infarct size (17.1 %) of the MI model was enormously reduced after injection of Se hydrogel when compared to the Se NPs and control (MI) sample, respectively, as displayed in Figure (7C & 7D), which confirmed that Se introduced hydrogel have greatly influenced on the restoration of infarcted heart.
Though post-infarction inflammatory processes are played essentially in the heart healing mechanism, which mainly demonstrates excessive and upregulated inflammation provides negative effects and causes adversative pathological remodelling. During inflammation period (at beginning of 1–48 h after MI), the pro-inflammatory cytokines (TNF-α and IL-18) level have been significantly increasing and subsequently the levels of anti-inflammatory cytokines (IL-10 and TGF-b) also enthused to suppress over-expressions of inflammatory expressions. In the present study, we have examined those inflammatory cytokines that are possibly related to macrophage recruitment at infarcted regions to establish the activations of macrophages and functional heart regeneration as showed in Fig. 6. Primarily, we have analysed the chemokine and cytokines at infarcted heart regions, which are capable of engaging macrophages. On day 3, the pro-inflammatory factors and anti-inflammatory factors were upregulated in control samples (MI heart). Nevertheless, the hydrogel treated samples exhibited to specifically downregulated levels of mRNA level (Fig. 6a) and protein expressions (Fig. 6b) of pro-inflammatory factors (TNF-α and IL-18) and chemokines (CCL2) without influencing the anti-inflammatory factors, demonstrating that prepared hydrogel samples have probable anti-inflammatory efficiency and favourable pro-inflammatory effect on in vivo MI treatment. And we further examined the infiltration of macrophages in the infarcted hearts after macrophages recruitments and activation of CCL2 as exhibited in Fig. 6d and 6d1. The present investigation established that number of macrophages was significantly decreased after treatment of prepared hydrogel when compared to the control sample, which displays clear evidence of hydrogel has been prevents macrophages activations, which is possibly under downregulating CCL2.
In vivo analysis of pro-inflammatory cytokines (TNF-α, IL-18 and CCL2) expressions with treatment of hydrogels
The in vivo observations of inflammatory cytokines determined that prepared hydrogel samples greatly influenced for anti-inflammatory effects as exhibited in Fig. 8. In addition, we have examined the inflammatory cytokines responses on the cell types including NIH3T3 (Fig. 8a) and iPSC-CMs (Fig. 8b), which are involved in the MI-based healing process. The present investigation exhibited that hydrogel treatment on cell types have been significantly suppressed the mRNA and protein expressions of pro-inflammatory factors (TNF-α, IL-18 and CCL2) as showed in Fig. 8 (a). These in vitro analysis results provided additional support that role of inflammatory production of cytokines by in vivo treatment. To examination the hydrogel role in the inflammation-endorsed production of cytokines, we have observed in vitro inflammation analysis under LPS-induced inflammatory process. In this study, mouse macrophages (RAW 264.7) (Fig. 8c1-c3) were selected for inflammation examination because these similar types of cell type are involved in the myocardial healing process and also momentously responded LPS-based inflammatory investigations. The treatment of LPS onto the RAW 264.7 macrophages cells promisingly producing inflammatory cytokines (TNF-α, IL-18 and CCL2), which also established that production and upregulation of inflammatory cytokines levels on RAW 264.7 have been reduced after treatment of prepared hydrogel. The microscopic visualization of Raw 264.7 macrophages was presented in Fig. 8 (d) to establish regeneration ability of the prepared hydrogels. The observed results demonstrated that the treatment of hydrogel samples onto the LPS-induced macrophages cells have blocked the further accessibility of LPS with the cells. The presented results indicated that presented selenium nanoformulations into the hydrogel greatly influenced into the LPS-induced inflammatory response and provided strong anti-inflammation role.