The viability of the MCTSs treated with 150 pM Au NRs with different surface
modifications for 24 h was assessed by acid phosphatase (APH) assay. Conventional cell
viability assays need to digest the MCTSs in order to disperse cells before the assays. This
step would damage cells and cause inaccuracy in the results. Hence, the APH assay was
employed to avoid the disadvantages. This assay could be run without any pretreatment of
the MCTSs and is more accurate in reflecting the cellular viability in the 3D cell culture
model.24, 25 As is shown in Figure 3A, the viability of the MCTSs was not affected
significantly after 24 h of Au NRs treatment in different groups. Thus, the following
experiments were conducted with 24 h Au NRs treatment to rule out the impact of cellular
toxicity on the thermal therapy test result.
A quantitative, inductively coupled plasma mass spectrometry (ICP-MS) measurement was
conducted to estimate the amount of Au NRs internalized by the spheroids. The ICP-MS
results showed that PDDAC-coated Au NRs had the greatest amount of retention (Fig. 3B).
It has been reported that in a monolayer cell culture, the PDDAC-coated Au NRs had the
greatest cellular uptake amount, while the PSS-coated Au NRs had the least cellular uptake
amount. The uptake dosage of the PDDAC-coated Au NRs was more than 16-fold compared
with the CTAB-coated Au NRs and 30-fold compared with the PSS-coated Au NRs.12 The
tendency of Au NRs uptake in MCTSs is consistent with that of a monolayer cell culture;
however, the difference is not that obvious. The number of Au NRs per spheroid treated
with the PDDAC-coated Au NRs was only twice that of the PSS-coated Au NRs treated
spheroids. The difference in the uptake of Au NRs between the PDDAC-coated and the
CTAB-coated Au NRs treated groups was minimal. In a monolayer cell culture, cationic
surface coatings will enhance the uptake of nanoparticles greatly.26 While in MCTSs, this
kind of surface coatings had effect only on the surface cells. Penetration is another key
factor which is only present in MCTSs. Consequently, the results obtained in different cell
culture models varied.
Thermal therapy is one of the major applications of Au NRs in cancer treatment; therefore,
the bio-distribution of the Au NRs in tumor tissue would affect the therapeutic efficiency.
The MCTSs were radiated under the NIR laser for 4 min after incubation with Au NRs for
24 h. The thermal therapy efficiency of each kind of Au NRs is shown in Fig. 3C and
calculated according to the following formula:
After laser radiation, the Au NR-treated MCTSs suffered great viability loss. The thermal
therapy efficiency of the PSS-coated Au NRs was the highest, followed by the PDDACcoated
Au NRs. The CTAB-coated Au NRs had the lowest efficiency, which was less than
40 % of that of the PSS-coated Au NRs.
Hematoxylin-eosin (HE) staining was employed to observe the morphological change of
MCTSs after thermal therapy. The HE staining result showed that radiation rarely had any
effect on the control group, while the MCTSs treated with Au NRs showed a variety of
structural changes. In the PSS-coated Au NRs group, a mass of inner cells were killed, and
the skeleton was destroyed; hence, cavities appeared, the structure became loose, and the
shape became irregular. The damage of inner cells was not obvious in the other two Au NRs
treated groups, and cavities could only be seen at the border of the MCTSs (Fig. 3D).
To show the distribution of Au NRs in the MCTSs directly, DF microscopy was used to
examine the penetration of Au NRs in the tumor spheroids. The DF microscope illuminated
the samples with oblique beam and collected the reflected and scattered light for imaging.
The viability of the MCTSs treated with 150 pM Au NRs with different surfacemodifications for 24 h was assessed by acid phosphatase (APH) assay. Conventional cellviability assays need to digest the MCTSs in order to disperse cells before the assays. Thisstep would damage cells and cause inaccuracy in the results. Hence, the APH assay wasemployed to avoid the disadvantages. This assay could be run without any pretreatment ofthe MCTSs and is more accurate in reflecting the cellular viability in the 3D cell culturemodel.24, 25 As is shown in Figure 3A, the viability of the MCTSs was not affectedsignificantly after 24 h of Au NRs treatment in different groups. Thus, the followingexperiments were conducted with 24 h Au NRs treatment to rule out the impact of cellulartoxicity on the thermal therapy test result.A quantitative, inductively coupled plasma mass spectrometry (ICP-MS) measurement wasconducted to estimate the amount of Au NRs internalized by the spheroids. The ICP-MSresults showed that PDDAC-coated Au NRs had the greatest amount of retention (Fig. 3B).It has been reported that in a monolayer cell culture, the PDDAC-coated Au NRs had thegreatest cellular uptake amount, while the PSS-coated Au NRs had the least cellular uptakeamount. The uptake dosage of the PDDAC-coated Au NRs was more than 16-fold comparedwith the CTAB-coated Au NRs and 30-fold compared with the PSS-coated Au NRs.12 Thetendency of Au NRs uptake in MCTSs is consistent with that of a monolayer cell culture;however, the difference is not that obvious. The number of Au NRs per spheroid treatedwith the PDDAC-coated Au NRs was only twice that of the PSS-coated Au NRs treatedspheroids. The difference in the uptake of Au NRs between the PDDAC-coated and theCTAB-coated Au NRs treated groups was minimal. In a monolayer cell culture, cationicsurface coatings will enhance the uptake of nanoparticles greatly.26 While in MCTSs, thiskind of surface coatings had effect only on the surface cells. Penetration is another keyfactor which is only present in MCTSs. Consequently, the results obtained in different cellculture models varied.Thermal therapy is one of the major applications of Au NRs in cancer treatment; therefore,the bio-distribution of the Au NRs in tumor tissue would affect the therapeutic efficiency.The MCTSs were radiated under the NIR laser for 4 min after incubation with Au NRs for24 h. The thermal therapy efficiency of each kind of Au NRs is shown in Fig. 3C andcalculated according to the following formula:After laser radiation, the Au NR-treated MCTSs suffered great viability loss. The thermaltherapy efficiency of the PSS-coated Au NRs was the highest, followed by the PDDACcoatedAu NRs. The CTAB-coated Au NRs had the lowest efficiency, which was less than40 % of that of the PSS-coated Au NRs.Hematoxylin-eosin (HE) staining was employed to observe the morphological change ofMCTSs after thermal therapy. The HE staining result showed that radiation rarely had any
effect on the control group, while the MCTSs treated with Au NRs showed a variety of
structural changes. In the PSS-coated Au NRs group, a mass of inner cells were killed, and
the skeleton was destroyed; hence, cavities appeared, the structure became loose, and the
shape became irregular. The damage of inner cells was not obvious in the other two Au NRs
treated groups, and cavities could only be seen at the border of the MCTSs (Fig. 3D).
To show the distribution of Au NRs in the MCTSs directly, DF microscopy was used to
examine the penetration of Au NRs in the tumor spheroids. The DF microscope illuminated
the samples with oblique beam and collected the reflected and scattered light for imaging.
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