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Evaluation of Nanoparticle Penetration in the Tumor Spheroid Using Two-Photon Microscopy

Mesoporous silica nanoparticles (MSNs) have emerged as a prominent nanomedicine platform, especially for tumor-related nanocarrier systems. However, there is increasing concern about the ability of nanoparticles (NPs) to penetrate solid tumors, resulting in compromised antitumor efficacy. Because the physicochemical properties of NPs play a significant role in their penetration and accumulation in solid tumors, it is essential to systematically study their relationship in a model system. Here, we report a multihierarchical assessment of the accumulation and penetration of fluorescence-labeled MSNs with nine different physicochemical properties in tumor spheroids using two-photon microscopy. Our results indicated that individual physicochemical parameters separately could not define the MSNs’ ability to accumulate in a deeper tumor region; their features are entangled. We observed that the MSNs’ stability determined their success in reaching the hypoxia region. Moreover, the change in the MSNs’ penetration behavior postprotein crowning was associated with both the original properties of NPs and proteins on their surfaces.

with FITC labeled-MSN. A z-sectioned two-photon scanning was performed to evaluate MSN's ability (green) targeting the hypoxia region (red) in the spheroid at an excitation wavelength of 900 nm. The images obtained were then analyzed, and the colocalization percentage between these two fluorescences was calculated based on the Mander coefficient using Fiji Image J software.

Evaluation NP uptake in individual cells in spheroid
After 16 h of incubation with 0.5 mg/mL RITC-MSNs, the spheroids stained with Hoeschest were washed at least three times to remove the excess of NPs. The spheroids were dissociated by adding 50 uL 0.25% trypsin-EDTA and incubated for 15 min. Then, the solution was gently pipetted to separate individual cells. The live-cells were immediately imaged using a confocal microscope with a 10x objective at excitation wavelengths of 405 and 543 nm for Hoeschest and MSN, respectively. The percentages of cells contained MSN were obtained by calculating the numbers of cells containing MSNs( red color) over the total number of cells (blue).

a. NP's entrapment in the lysosome
First, the 50 µg/mL of RITC-MSNs were incubated with 1µM of lysosome tracker (LysoTracker® Green DND-26, Thermo Scientific Inc., Rochester, NY) in the spheroid containing Hoeschest for 16 h to evaluate the NP entrapment in the lysosomes. After 16 h of incubation, the spheroids were washed at least three times to remove the excess of NPs and dyes. The living spheroid was then imaged using a confocal microscope with a 40x oil objective at excitation wavelengths of 405, 488, and 543 nm for Hoeschest, lysotracker, and MSN, respectively. The images obtained were then analyzed, and the colocalization percentage between these two fluorescences was calculated based on the Mander coefficient using Fiji Image J software.

b. NP's in the recycling endosome
After 16 h of incubation with 50 µg/mL RITC-MSNs, the spheroids were washed at least three times to remove the excess of NPs and dyes and were fixed with 4% PFA for 1h. The spheroids were then permeabilized with 0.25% Triton X-100 in PBS for 15 minutes and blocked with 3% BSA in PBS for another 1-h at room temperature. The spheroids were then treated with Rab 11A rabbit polyclonal antibody (Invitrogen) at a concentration of 5µg/mL overnight at 4º C.
After the washing steps; the spheroids were incubated with goat anti-rabbit secondary antibody conjugate Alexa-fluor ® 488 (1:1000 dilution) for 1-h at room temperature. The spheroid's images were obtained using a confocal microscope with the same setup described in the previous experiment.

Quantitative image analysis
For example, diameter (D) of spheroid ~320 µm, therefore: ra~100 µm, rb~127 µm, rc~145 µm, rd~160 µm Region 1 is the volume in rb, therefore, the thickness is about 100 µm (core region) Region 2 is the rim volume in between ra and rb, therefore the thickness is about 27 µm Region3 is the rim volume in between rb and rc, therefore, the thickness is about 18 µm Region 4 is the rim volume in between rc and rd, therefore the, thickness is about 15 µm Figure S1. The immunostaining of cell spheroid with various protein marker commonly found in ECM such as fibronectin (red), collagen (green) and vitronectin (green). Scale bar 25 µm