3.2. mTHPC Delivery to the Tumor Cells In Vitro
As stated above, the main purpose for encapsulating of mTHPC in the form of inclusion complexes in liposomes is to control PS release by β-CDs after liposome destruction in the medium. In order to investigate the validity of this hypothesis, we analyzed intracellular localization of mTHPC in HT29 human colon adenocarcinoma monolayer cells and PS distribution in HT29 MCTSs after pre-treatment with DCLs (Figure 4
). Preliminary studies have shown that cellular uptake strongly depends on the absolute mTHPC concentration loaded in DCLs (data not shown). We demonstrated that DCLs containing less mTHPC (DCL2 and DCL3) exhibited lower accumulation in HT29 cells when compared with DCL1. Therefore, for our further in vitro studies we selected only HDCL1, MDCL1 and TDCL1.
a displays the fluorescence images of HT29 cells treated with mTHPC and Foslip®
for 3 h. All formulations deliver mTHPC into the cells and we did not observe an obvious difference in intracellular localization between Foslip®
and DCLs. mTHPC is predominantly localized in the perinuclear region with diffuse fluorescence in the cytoplasm, as consistent with literature data related to Foslip®
]. It is worth to note that fluorescence intensity of TDCL1 in HT29 cells is significantly lower compared with other mTHPC formulations, perhaps because of the tight binding of mTHPC in TM-β-CD complexes.
Understanding the PS distribution processes in target tissues is a primary factor that is responsible for prediction of antitumor efficiency of photodynamic agents. MCTSs represent avascular regions found in many solid tumor tissues and allow for simulating the penetration and intratumor transport of anticancer nanomedicines, including photoactive NPs [38
]. In the present work, HT-29 MCTSs were generated by spinner technique and filtered by the size from 380 to 520 µM for further experiments. Figure 4
b displays epifluorescence imaging of the intact spheroids treated by Foslip®
and mTHPC-DCLs for 24 h. As seen on these images, the red fluorescence pattern of mTHPC was mainly localized on the periphery of Foslip®
-treated spheroids, consistent with the literature data [40
]. It is worth noting that HT-29 MCTSs that were treated with free mTHPC demonstrated similar peripherical profiles of fluorescence distribution [9
]. The corresponding linear profile of mTHPC fluorescence across MTCSs, as presented in Figure 4
c, demonstrates the presence of intensity peaks in the outer rim of spheroids that were treated with Foslip®
. MCTSs exposed to HDCL1 also display high fluorescence in the outer rim of spheroids. The significant changes of mTHPC distribution in HT-29 MCTSs were observed for DCLs that contained mTHPC inclusion complexes with methylated β-CDs (MDCL1 and TDCL1). In the case of MDCL1, the intensity peaks on the periphery were smoothed, displaying an increase in the penetration depth of mTHPC. Similar changes of mTHPC distribution in spheroids were reported for free mTHPC/CD complexes in our recent paper [9
]. However, the strongest changes of the mTHPC fluorescence pattern in MCTSs were observed for TDCL1. Linear profiles obtained after incubation of MCTSs with TDCL1 display spherical pattern with the maximal fluorescence intensity in the center. Taking into the account distortions due to the limited penetration of excitation light and spherical geometry of spheroids one can conclude that such fluorescence pattern corresponds to the almost complete penetration of mTHPC in spheroid depth and probably homogeneous PS distribution between cells of spheroids.
To confirm the DCLs-induced alterations of mTHPC distribution in spheroids we used a flow cytometry technique (Figure 4
d). The spheroids treated with various mTHPC formulations were trypsinized after 24 h incubation and analyzed by flow cytometry to assess the heterogeneity of mTHPC distribution between spheroid cells. Foslip®
-treated spheroids exhibited a strong heterogeneity of mTHPC distribution between the cells. The distribution is broad and consists of several peaks. Obviously, mTHPC accumulates insufficiently in the deep layers of spheroids. Application of HDCL1 for mTHPC delivery in MCTSs does not significantly affect its distribution. Meanwhile, in the case of MDCL1, the distribution histogram slightly changes, still conserving strong heterogeneity similarly to Foslip®
and HDCL1. Finally, the incubation of MCTSs with TDCL1 results in an almost homogeneous distribution of mTHPC, supposing similar PS bioavailability for peripherical cells as well as for cells in the core of spheroid.
Thus, our data clearly demonstrate that DCLs significantly alter mTHPC distribution in spheroids. The alteration strength strongly depends on the type of used β-CD and increases in function of the affinity of β-CD to mTHPC in the following order (HDCL1 < MDCL1 < TDCL1). Taking into the account the influence of free CDs on mTHPC distribution in spheroids [9
], one can suppose that the higher affinity of β-CD to mTHPC leads to the longer life-time of complex and as a matter of fact to the deeper delivery of mTHPC in the tumor tissue after the destruction of liposomes. In turn, deeper PS penetration results in its more homogeneous distribution of between cells [9
]. Thus, in the case of TM-β-CD, the affinity is so high (>107
] that mTHPC remains in the complex, even in the deep cell layers, resulting in almost homogeneous PS distribution in spheroid.