NP characteristics found in this study were similar to those reported previously (13
). The positive zeta potential of gelatin NPs can be explained by using a pH lower than the isoelectrical point of gelatin during NP preparation. The positively charged primary amine groups (−NH3+
) result in a positive zeta potential. Mannosylated gelatin NPs were bigger than non-mannosylated NPs and had less positive charge on the surface (Table ). This can be attributed to the formation of a Schiff’s base (−C
) between the primary amine groups on the surface of NPs and the aldehyde groups of the ring-opened mannose moieties (13
). An IR spectrum of mannosylated gelatin NPs confirms the success of the synthesis. The stretches between 1,465–1,500 and 1,505–1,550 cm−1
correspond to Schiff’s bases (−C
) and secondary amine groups, respectively (13
). Please refer to the Electronic Supplementary Material (ESM 1
) for the IR spectrum of mannosylated gelatin NPs.
The thermal activity profile of macrophages alone, Fig. , is different from what was reported previously (30
). The thermal activity of living cells is comprised of an ascending phase followed by a descending phase. Previous reports describe both phases to be linear with no exponential subphase. The observations in this study can be explained by a difference in experimental settings. In the previous reports, experiments were performed on macrophages suspended in cell culture media with no surface to adhere to; this is not optimal. In the present study, macrophages were seeded on special cell culture slabs that provided optimum conditions for the expression of biological functions. The ascending phase of the thermal activity profile represents a period in which the macrophages are metabolically active, with oxygen and nutrients still abundant in the cell culture medium. The exponential increase in heat exchange can be attributed to growth and multiplication of macrophages. Later, due to the increasing number of macrophages, oxygen and other essential nutrients will be depleted and cellular death will follow. This is represented by a decrease in thermal activity during the descending phase.
The aim of this study was not to compare different types of NPs prepared from different polymers, but to investigate the impact that pharmaceutical manipulation of NPs, such as surface decorating or coating, might have on biological interactions such as phagocytosis monitored by ITMC.
Prephagocytosis, phagocytosis, and postphagocytosis processes may explain the initial fast increase in heat production after the titration of uncoated PIBCA NPs (Fig. ). The observation that the heat exchange was slower in the beginning of the exponential subphase of the ascending phase might be explained by the transient conversion in the metabolic energy source required to finish the phagocytosis process. The source may have changed from oxidative phosphorylation to glycolysis, as it is known that glycolysis is associated with less heat production than oxidative phosphorylation (37
). Heat production associated with uncoated PIBCA NPs was significant compared with heat production of macrophages alone, 37.6
5.6 J and 9.8
2.3 J, respectively. Since macrophage count, NP concentration, and volume titrated were constant, the relative interactive coefficient of different NP formulations was used to evaluate the phagocytosis process and compare different NP formulations.
Titration with polysorbate-80-coated PIBCA NPs resulted in a significant decrease in the total heat production as shown in Fig. . The polysorbate-80 coating material decreased the macrophage cellular response to PIBCA NPs. Polysorbate alone or in a physical mixture with NPs had no effect on the thermal activity profiles. This suggests that the inhibition of NP phagocytosis by polysorbate-80 is limited to the formation of a surfactant layer on the NP surface.
Cyto B is a mycotoxin with high cell permeability properties. It inhibits the formation of contractile actin microfilaments which results in inhibition of cellular movement and phagocytosis functionality (38
). Cyto B has been used as a phagocytosis inhibitor to study this cellular function (36
). Cyto B was included in this study design to confirm that the increase in heat exchange observed after NP titration is a direct result of the phagocytosis process. If the increase in heat exchange observed after NP titration is inhibited by Cyto B, this would imply that phagocytosis is responsible for the heat exchange. As shown in Fig. , in the presence of Cyto B the macrophage thermal activity profile was significantly lower and there was a significant decrease in the total heat exchange in the medium compared with experiments performed without Cyto B, 3.9
1.3 J and 9.8
2.3 J, respectively. In addition to its effect on phagocytosis, Cyto B could affect other aspects of cellular function such as inhibition of glucose transportation and induction of nuclear extrusion (39
), and this would impact the thermal activity profile. However, Cyto B totally inhibited an increase in heat production after NP titration, as shown in Fig. (b, c). On the other hand, Cyto B had no effect on the ligand–receptor reaction of mannose and mannose receptor as shown in Fig. . Therefore, it could be concluded that the observed increase in heat exchange induced by NP titration (Figs. and (b, c)) was directly connected to the phagocytosis process.
Targeting mannose receptors on the macrophage cellular surface using mannose-decorated NPs is an established strategy to enhance phagocytosis (19
). Therefore, mannosylated NPs are expected to be associated with enhanced phagocytosis and, consequently, with higher heat production as detected by ITMC. The initial sharp increase in heat production associated with mannosylated gelatin NPs (Fig. ) can be explained by ligand–receptor interaction between mannose moieties and mannose receptors on the surface of macrophages. The similarity between the heat activity profiles of mannosylated gelatin NPs and the non-chemically bonded mixture of mannose and gelatin NPs (Figs. and ) suggests that the presence of mannose in solution is enough to provoke and enhance the phagocytotic functionality of macrophages. This observation is in contrast to what was noticed with polysorbate-80-coated PIBCA NPs. The fact that mannose solution induced an initial sharp increase in heat production implies that the similar sharp increase observed with mannosylated gelatin NPs is a result of interaction between mannose moieties and mannose receptors. Moreover, the initial sharp increase in the heat production observed in the presence of Cyto B after titrating 100 μL (Fig. ) implies that Cyto B affects phagocytosis process, yet has not effect on ligand–receptor interaction.
Flow cytometry analysis was used to validate the data obtain from ITMC. Macrophages were exposed to different NP formulations and the results were expressed in relative to the untreated macrophages. The locations of the macrophages on the dot plot along with the value of MFI were used to assess the uptake of different NP formulations. The percentage of macrophages that relocated from LL to LR quadrant represents the portion of macrophages that has been taken up NPs, however, the MFI value represents the extent of uptake.
Mannosylated gelatin NPs were associated with the highest uptake. 58.69% of the macrophages were detected in LR quadrant Fig. . These portion of macrophages showed a high MFI values, 46.08. Polysorbate-80-coated PIBCA showed the lowest uptake. Only 1% of macrophages expressed an increase in the fluorescence properties with low MFI value of 5.07. Macrophages treated with uncoated PIBCA and gelatin NPs showed similar MFI values of 16.8 and 18.39, respectively. However, gelatin NPs were associated with higher percentage of macrophages in the LR quadrant and, consequently, a higher uptake.
The results of the flow cytometry highly confirm the data collected via ITMC. The higher uptake of mannosylated gelatin NPs detected by flow cytometry was associated with the highest heat exchange and relative interactive coefficient as discussed earlier. Moreover, titrating polysorbate-coated NPs into the ITMC was associate with insignificant increase in the heat exchange detected via ITMC as shown in Fig. . This fact was supported by flow cytometry as these NPs showed almost no cellular uptake. Flow cytometry showed that gelatin NPs had a higher uptake compared with uncoated PIBCA NPs and a lower uptake in comparison with mannosylated gelatin NPs. These results are in agreement with what was observed using ITMC. ITMC showed that heat exchange and relative interactive coefficient of gelatin NPs falls in between compared with uncoated PIBCA and mannosylated gelatin NPs as shown in Table .