Figure 3: Visualizing oxygen within liver spheroids using RuDPP as an oxygen sensing dye, which quenches fluorescence emission in the presence of sufficient oxygen. A) Reconstruction of a 3D spheroid and intensity variance throughout the spheroid cultured for 5 days. PFC- MPs resulted in lower fluorescence intensity compared to the control which did not have any MPs. B) Measurement of intensity of individual cells along a circular line in a central plane of spheroids with quantitative data. The average intensity of bright dots around each line was quantified and converted to O2% via a calibration curve. Data represented as mean ± SD. n = 3 independent spheroids, one-way ANOVA and Tukey’s post hoc .

Covalent immobilization of liver ECM adhesive ligands to PFC-MPs

Next, we modified MP surfaces by covalently binding ECM proteins to overcome the limitations associated with simple physical adsorption of ECM proteins, including their undesired conformational changes and release from surfaces over time.[34] In this study, biofunctionalization with the ECM proteins plasma fibronectin, laminin-111, 511, and 521 to the surface of PFC-MPs was accomplished by a coupling reaction between the carboxyl residues of proteins to the free amine residues of chitosan using EDC as crosslinker and NHS ester as an enhancer of coupling (Figure. 4A) . The EDC/NHS coupling reaction is a selective method for preservation of biological activity of the protein and has previously been applied in the production of protein functionalized chitosan derivatives.[35–37] Since the pH of the reaction solution is critical for maximizing the amination reaction, we tested different pH levels keeping all other conditions the same, to determine the favored pH to maximize attachment. The recommended pH conditions for the carbodiimide reaction via EDC/NHS is in the range of pH 4.5–5.5.[38] However, due to the presence of amine groups, chitosan based materials become protonated at a pH below pKa 6.0.[39] Between a pH of 5.9 and 6.8 we achieved ligand conjugation on MPs ranging from 2 to 3.5 µg.ml-1, as quantified by BCA kit (Figure 4B) . We can attribute the poorer yield at the lowest pH (5.9) to high chitosan protonation, resulting in a slower reaction. Poor yield at the highest pH (6.8) might be due to the formation of macroscopic aggregates resulting in reduced availability of surface area for reactions. The formation of aggregates at pH >6.5 where chitosan particles are weakly charged and less stable was reported previously.[40,41] We further characterized the uncoated and coated MPs modified at different pH levels via XPS.Figure 4C presents atomic compositions of MPs modified at different pHs. This result shows an increase in atomic nitrogen percentages (N1s%) as a reliable marker to detect additional amide groups (N–C=O) from amino acids in added ECM proteins compared to non-modified MPs. Interestingly, we observed an increase from 0.9% to 6.0% at pH=6.2 which confirms the data obtained from colorimetric quantification. Similarly, Huang and coworkers evaluated the grafting efficiency of laminin (derived from Engelbreth-Holm-Swarm mouse sarcoma basement membrane) on poly(lactic-co-glycolic acid) (PLGA) film using XPS and observed an increase in N1s from 0.5 to 1.1% after modification with laminin.[42] We opted to focus on only N1s because C1s and O1s peaks can originate from the MP composition itself and might lead to misleading data.