As I already reported (please see Fig. 4.3), spheroid formation involves three subsequent steps: initial aggregation, compaction and spheroid maturation. In order to quantify the changes occurring in the ECM structure throughout the process of spheroid formation, I measured the diffusivity of PEG-coated S34(1) nanoparticles in HeLa spheroids. I choose this probe since its size (rp = 20.6 nm) is close to the correlation length of the ECM in HeLa spheroids.
I began the experiment one hour after seeding the spheroids and proceeded for the following 13 days, assessing the correlation length (ξ) at various time intervals to identify changes in the ECM mesh size. Based on Eq. 5.2, we get: values for Rh and A were taken from Tab. 5.2 (HeLa spheroids), and a = 1.
90 5.8. The time-related changes of the ECM structures
Figure 5.10: The variations in the interfibrillar spacing (characterised by ξ) of the ECM network over time during HeLa spheroid formation. Five hours after seeding, the aggregates have a loose and irregular structure, as seen in A. After around 3 days, aggregates became more compact, as seen in panel B, which corresponded to a reduction in the size of the mesh in the ECM network. The structural properties of the matrix do not alter as spheroids further mature, however the spheroid structure is more compacted, as can be noticed in panel C. Scale bar corresponds to 250 µm. The lower panel presents mean ξ values ± SD. Each bar was calculated based on at least 15 correlation functions that were recorded per spheroid. The measurements were performed for 3 different spheroids [10].
The changes of the correlation length, calculated based on Eq. 5.8, throughout the process of HeLa spheroid formation are shown in Fig. 5.10.
According to the presented data, in the first three days of HeLa spheroid culture, the average half-distance between the points of entanglement in the ECM is ξ = (31.59 ± 7.86) nm. After one hour from seeding the spheroids, the interfibrillar spacing was unexpectedly narrow and remained at a comparable value over the following 67 hours.
The fact that ξ has quite small values in such a short period of time
after cell seeding may be due to the presence of surface proteins. For instance, fibronectin is a glycoprotein present at the cell surface, which forms a fibrillar network between adjacent cells [138]. According to the formation mechanism of spheroids, dispersed cells initially are drawn closer to form loose aggregates thanks to, e.g. fibronectin that can bind tightly to the integrin on the cell membrane surface [7]. I observed that aggregates were forming immediately due to cell-cell contact. Moreover, it has been shown that procollagen is secreted with a half-life within the cell of fewer than 30 minutes [139].
After 72 h, there is around a two-fold decrease in ξ = (17.97 ± 6.65) nm, which does not change upon further maturation of spheroids. The formation of compact spheroids from loose aggregates through tight ag-gregates is a macroscopic process. Apparently, the change of interfibrillar spacing at the nanoscale does not decrease monotonically. Thus, it can be concluded that it takes approximately 60–70 hours for the cells in the spheroids to create the network of collagen in the ECM [10].
Figure 5.11: Histogram showing the distribution (N = 60) of a measured diffusion time for PEG-coated S34(1) silica nanoparticles within the ECM of HeLa spheroids. The solid blue line corresponds to kernel density estimation (KDE) of the histogram (right y-axis) [10].
92 5.8. The time-related changes of the ECM structures
The occurring large deviations in the results (range from 72 h to 304 h of the spheroid culture) can be related to the heterogeneity of the ECM structure. The diffusion times of the analysed probe was in the range from 1 ms up to 4 ms. The short diffusion times suggest the presence of large free extracellular spaces. On the contrary, slow-moving probes (long diffusion times) probe fibrous-rich areas. However, if there were only fibrous-rich and fibrous-free areas, the bimodal distribution would be obtained. In our case, the distribution, presented in Fig. 5.11, is wide and quite uniform, what points at the heterogeneity of the ECM structure - the presence of the different matrix pore sizes, an intrinsic disorder of the fibre network, and uneven distribution of the ECM components. The heterogeneity of the ECM structure was additionally illustrated in Fig. 5.12.
Figure 5.12: Confocal image of the ECM structure (in HeLa spheroid) after staining with Col-F (green). The nuclei were counterstained with Hoechst 33342 (blue). Panel A represents the fibrous-rich extracellular space. In contrast, panel B depicts a large free extracellular space. The scale bar is 10 µm [10].
Additionally, Fig. 5.12 indicated that the dense network of the ECM is predominantly localised around the cells. Moreover, the density of the matrix around the cell varies significantly throughout the spheroid cell population, as could be shown in Fig. 5.13.
Figure 5.13: The confocal image of HeLa GFP spheroid with PEG-coated silica nanoparticles. The green fluorescence signal is from HeLa cells expressing EGFP, while the red signal corresponds to PEG-coated silica nanoparticles with a hydrodynamic radius of 22.72 nm. The dense structure of extracellular fibres can cause accumulation of the nanoprobes in their network (intense red signal around some of the cells). The imaging was performed 20 minutes after adding nanoparticles. Because of this short incubation time of spheroid with nanoparticles, they do not penetrate the cells, ensuring that the signal does not originate from the cytoplasm. z = 0 µm corresponds to the bottom of the glass.
5.9 Method to test factors influencing the ECM structure
As I demonstrated, the extracellular matrix acts as a transport barrier, slowing the movement of nanoparticles and so reducing their effective penetration. Therapies targeting the extracellular matrix are among the approaches that aim to overcome this obstruction [140–142]. Goodman et al. [143] assessed the effect of collagenase (enzyme that breaks the peptide bonds in collagen) treatment on nanoparticles penetration into spheroids. Collagenase treatment increased delivery efficiency for tested nanoparticles. However, since the conclusions were drawn only on the