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.