To prepare a hybrid pyrolysis liquid hydrogel, lysates from M1 macrophages and pancreatic cancer Panc02 cells were extracted and mixed in different ratios to create three-dimensional hydrogels. Two different ratios were designed based on protein quantification: 1:4 for M1 macrophages and Panc02 cell lysates, and 3:2 for M1 macrophages and Panc02 cell lysates, respectively. These hydrogels were named Sample-1 and Sample-2. Gelation was achieved by adding NHS and EDC catalysts at -20°C, resulting in the formation of stable three-dimensional hydrogels. The appearance and size of the hydrogels could be controlled according to the experimental conditions. Furthermore, scanning electron microscopy revealed a porous structure in both hydrogels; however, there were significant differences in pore sizes within them. The pore size of Sample-2 was notably larger than that of Sample-1 (Figure 1). The distinct internal structures indicate different biological functionalities and potential characteristic differences between the two hydrogel samples.
To further investigate the differences in gel properties between the two different cell lysate sources, we first examined their degradation characteristics. Cylindrical hydrogel samples with a diameter of 2.5 cm and a height of 1 cm were placed in 2 mL of distilled water. At different time points, the hydrogel samples were removed from the water, freeze-dried, and weighed to evaluate their degradation performance. The results depicted in Figure 2A demonstrate that compared to Sample-1, Sample-2 exhibited a slightly faster degradation rate. However, both hydrogels had degradation periods lasting up to 12 days, ensuring a prolonged functional effect when applied in vivo. Additionally, we further investigated the swelling ratio and porosity of the two different hydrogels. The results indicated that although the differences were not significant, Sample-2 possessed higher swelling intensity and pore ratio (Figure 2B and 2C). These findings, along with the faster degradation rate and larger pore size observed in Sample-2, may reflect potential differences in their biological functionalities.
In order to further validate the biological functionality and cell regulatory effects of the well-characterized hydrogels, we conducted toxicity assessments on these hydrogels towards relevant cell types. Considering the potential target cells of the hydrogels, we selected L929 cells as representative normal tissue cells, Panc02 pancreatic tumor cells, and M2 macrophages isolated from tumor tissues as the target cells. These three cell types represent the main cell populations that the hydrogels would come into contact with in vivo. The cells were seeded in 24-well plates and placed in Transwell chambers, with the prepared hydrogels added to the chambers. The plates were then incubated in a CO2 culture environment for 12 and 24 hours. Subsequently, cell viability was assessed using the CCK-8 assay. The results showed that, at both the 12-hour and 24-hour time points, there was no significant cell death observed in cells treated with either of the hydrogels (Figure 3). The cells maintained a healthy growth status. These experimental findings confirm that the prepared hydrogels do not cause significant cytotoxic effects on cells and demonstrate a certain level of safety for in vivo applications.
Upon confirming the absence of significant cytotoxicity on cells, we proceeded to investigate the content of regulatory factors within the prepared hydrogels, as well as their release kinetics. The two hydrogel formulations were placed in 500 μL of pure water, and at different time intervals, aliquots of the solution were collected for enzyme-linked immunosorbent assay (ELISA) analysis. In this study, we selected several representative cytokines, including TNF-α, IL-12, and IFN-γ, which are highly characteristic of M1 macrophages, for investigation. The polarization of M2 macrophages is closely associated with the functionality of these factors, and their effective concentration and optimal release kinetics contribute to enhancing M2 macrophage polarization efficiency. Based on the experimental results shown in Figure 4, it can be observed that during the initial 2 days, there was no significant difference in the released levels of these factors between the two samples. However, as time progressed, similar changes in the release profiles of the three different cytokines were observed. Notably, the release of factors from Sample-2 hydrogel was significantly higher than that from Sample-1, indicating that Sample-2 possesses stronger biological functionality and exhibits greater potential in polarizing M2 macrophages.
To assess the differential polarization-inducing capability of the two prepared hydrogels, M2 macrophages isolated from tumor tissues were co-cultured with these hydrogels. The treated macrophages were then extracted, and the proportion of M1 macrophages was analyzed using flow cytometry. We observed a significant increase in the proportion of M1 macrophages in both groups treated with the two different hydrogels. Particularly, the experimental group treated with Sample-2 showed a ~3-fold increase in the proportion of M1 macrophages compared to the control group (Figure 5). The results indicate that Sample-2 hydrogel possesses a strong ability to induce polarization of M2 macrophages towards M1 macrophages. This suggests that in future tumor treatments, it may be possible to convert "cold" tumors into "hot" tumors, thereby enhancing the effectiveness of tumor immunotherapy.
The above experimental results confirm that the prepared functional hydrogel stimulates the polarization of M2 macrophages into M1 macrophages, with Sample-2 hydrogel demonstrating the most optimal effect. Furthermore, M1 macrophages theoretically play a role in antigen presentation. Therefore, the tumor cell lysate present in the hydrogel provides a rich source of antigens, facilitating the uptake and presentation by M1 macrophages, thereby activating T lymphocytes for tumor cell killing (Figure 6A). To further investigate whether the M1 macrophages induced by this hydrogel have tumor antigen presentation capabilities and whether activated T cells exhibit tumor specificity, we co-cultured the hybrid hydrogel of Sample-2 derived from Panc02 tumor cell lysate with M2 macrophages, followed by co-culturing the treated M2 macrophages with freshly isolated, non-activated T cells extracted from the body. The resulting T cells were then co-cultured with CT26 and Panc02 cells to assess their cytotoxicity against these two tumor cell lines. The results from Figure 6B demonstrate that T cells not treated with Sample-2 showed no significant killing effect on either tumor cell line. However, T cells treated with Sample-2 exhibited significant inhibition against Panc02 cells, while the inhibitory effect on CT26 cells was not significant. Therefore, the results indicate that Sample-2 can induce the polarization of M2 macrophages and efficiently present tumor antigens within the hydrogel, leading to the generation of tumor antigen-specific T cells capable of selectively killing tumor cells.