3.4.2. Cellular Uptake Mechanisms

Folic acid coated on bi-HNTs was designed to bind to folate receptors expressed by cancer cells. In the binding process, folic acid initiates cellular uptake mechanisms in two different ways: clathrin-mediated endocytosis or caveolae-mediated endocytosis. Chlorpromazine (CPZ) has the ability to disrupt clathrin-mediated endocytosis; while filipin inhibits caveolae formation. CT26WT cells were pretreated with these inhibitors and co-cultured in the presence of bi-HNTs. Their final fluorescent intensity was recorded, and the results are shown in Figure 7C. The addition of CPZ promoted HNT uptake in first 12 h, then the cell uptake decreased over time. On the other hand, filipin provided uptake inhibition during the entire testing time period. This indicates cellular absorption of bi-HNTs may mainly depend on caveolae-mediated endocytosis assist by clathrin-mediated endocytosis.

#### 3.4.3. In Vitro Targeted Drug Release-Cellular Specificity

The bi-HNTs drug delivery system was designed to deliver drugs to specific cancer cells. Methotrexate is one of the most widely used drugs in treating osteosarcoma. Like many cancer drugs, it does have adverse side effects. In this experiment, the specific targeting of cancer cells versus normal cells was addressed. A pre-osteoblast cell line (MC3T3-E1), and two cancer cell lines, a colon cancer cell (CT26WT) and an osteosarcoma cell line (K7M2-WT), were selected to assess the targeting potential of bi-HNTs, and drug effectiveness in inhibiting cellular proliferation, the main effect of MTX (Figure 8A). The results suggest that MTX-loaded bi-HNTs (MTX-bi-HNTs) significantly inhibited osteosarcoma cell proliferation, and, at a low concentration (50 μg/mL). The other two cell types were not significantly affected by exposure to MTX (Figure 8B).

**Figure 8.** (**A**) Graphic depiction of osteosarcoma cells (K7M2WT), murine colon carcinoma cells (CT26WT) and preosteoblast cells (MC3T3) co-cultured with bi-HNTs. (**B**) Cell proliferation data after all three cell types were co-cultured with 50 μg/mL drug loaded bi-HNTs for 24 h. (**C**) Cell proliferation of above 3 types of cells after co-cultured with 50 μg/mL bi-HNTs for 24 h. (\* represents *p* < 0.05, \*\* represents *p* < 0.005).

In order to clarify that inhibition of cell growth was caused by MTX instead of bi-HNTs, we analyzed cell viability by co-culturing cells with bi-HNTs but without MTX. The results showed that none of the osteosarcoma (K7M2-WT) or pre-osteoblast cells (MC3T3-E1) were affected by bi-HNTs (Figure 8C). This finding is consistent with our previous study using CT26WT; a low dose of bi-HNTs did not elicit any cytotoxicity (Figure 3). Collectively, these results demonstrate the effectiveness of MTX loaded bi-HNTs in selectively targeting osteosarcoma cells in delivery of MTX, inhibiting cell proliferation.

#### **4. Discussion**

Many studies have exploited halloysite as a nanocontainer loading chemotherapeutic agents into the HNT lumen [19–22] and adding doped HNTs to a range of polymers for anti-cancer drug delivery [23–26]. Other studies have focused on using surface modification of HNTs for use as a nanocarrier for anti-cancer drug delivery [20,27–29]. A commonly used strategy is to functionalize HNTs with an –NH2 group by using aminopropyltriethoxysilane (APTES). In a study by Guo et al. (2012), FA and magnetite nanoparticles (Fe3O4) were successfully grafted onto the HNT surface. The coated Fe3O4@HNTs exhibited a pH-sensitive drug release under the electrostatic interaction between the cationic and HNTs [20]. Coating nanotubes with a polymer shell is another mechanism, as shown by Li et al. (2018). They examined the potential of chitosan grafted onto HNTs as a nano-formulation for the anti-cancer drug curcumin [27].

Fewer studies have attempted to use modified HNTs as a mechanism for the intracellular delivery of anti-cancer agents [16,28–30] Dzamukova et al. (2015) used physically adsorbed dextrin end stoppers to secure the intracellular release of brilliant green [28]. Tagged halloysite nanotubes were also used as carriers for intercellular delivery to brain microvascular endothelium [29]. Kamalieva et al. (2018) studied the intracellular pathway of HNTs for potential application for antitumor drug delivery using human adenocarcinoma epithelial cells (A549) [30].

MTX is an FDA approved anti-cancer drug commonly used in the treatment of osteosarcoma. In a recent study, MTX-doped HNTs were coated with polyelectrolytes (PE), polyvinylpyrrolidone, and polyacrylic acid, and methotrexate [31] was infused within the coated layers. MTX release

and cytotoxicity studies showed effectiveness in inhibiting osteosarcoma cell growth, and inhibition continued after PE/MTX-coated halloysite nanotubes were added to a polymer, Nylon-6.

Due to high chemical similarity in the structure between MTX and FA, several studies have shown that MTX modified nanoparticles have specificity for tumor cells [32–34]. Folic acid has a high binding affinity for the folate receptor, which is overexpressed in numerous cancers, including ovarian, endometrial, and renal carcinoma, lung, breast, and brain cancers [35]. MTX-loaded PEGylated chitosan nanoparticles had a higher cellular uptake efficiency compared to the FA-tagged group [36]. The high receptor affinity and overexpression enables folate-based nanoparticles to be highly specific, targeting tumor sites, and have great potential as a therapeutic application. FA-conjugated chitosan oligosaccharide-magnetic HNTs were studied as a delivery system for camptothecin, an anti-cancer drug [37]. Wu et al. (2018) also functionalized the HNT surface with APTES for conjugation of PEG and folic acid and subsequently loaded HNTs with doxorubicin [38]. The limitation of their study was the reported low drug loading efficiency, which was only 3%. In contrast, in this study, the loading efficiency of methotrexate was over 30%. In a recent report, HNTs had a loading efficiency of indocyanine green (ICG) as high as above 60% [39]. The variation in drug loading capability may be related to the different modification strategies used and the molecular size of drugs loaded.

The intracellular uptake pathway of HNTs was previously studied by Liu et al. [40]. HNTs were modified with APTES and labeled with FITC. Cells were treated with four different inhibitors, respectively, and then co-cultured with the FITC functionalized HNTs. They found both clathrin- and caveolae- dependent endocytosis were involved in the internalization of HNTs [40]. Liu's group also found that microtubules and actin microfilaments transported HNTs with the involvement of the Golgi apparatus and lysosome. Our results are consistent with their study. However, we observed that caveolae-mediated endocytosis was the primary mechanism of cellular import, and clathrin-mediated endocytosis was a secondary mechanism. Different surface modification strategies and cell types may be the possible explanation for the different results obtained in these studies and those reported here.

Even after pretreatment with chlorpromazine (CPZ), cytoplasmic accumulation of bi-HNTs was much more significant than the control group after the initial 12 h incubation time. One possible explanation is that disruption of clathrin-mediated endocytosis caused by CPZ induced caveolae to expand or more endocytic vesicles formed to internalize bi-HNTs. However, the overloading capability of caveolae reached capacity after several hours. Cells were unable to process the bi-HNTs loaded vesicles, so some were released from the cells. There is no report on the interaction between clathrin-and caveolae-mediated endocytosis during HNT uptake. Therefore, the detailed mechanism behind this phenomenon remains to be determined.

Our data further confirmed that bi-HNTs served well as a drug carrier and provided sustained MTX release time and showed selective binding to osteosarcoma cells. In contrast, non- osteosarcoma cells exposed to MTX were unaffected. We further showed that HNT size was also played a critical role in cellular uptake. Cellular uptake of smaller sized bi-HNTs was observed in greater amounts than unmodified HNTs. Increased intracellular accumulation may contribute to cell death through disruption of normal cellular metabolism resulting in cell death. Therefore, the application dosage of bi-HNTs should decrease with the size diminution; in other words, the smaller size of bi-HNTs has a higher working efficiency.

Even though the drug-loaded bi-HNTs has targeted FA receptors and successfully inhibited cell proliferation, this study lacks the comparison between MTX and MTX-loaded bi-HNTs. As an FDA approved anti-cancer drug, MTX would inhibit cell proliferation for sure. As a drug delivery system, bi-HNTs could extend the drug release time. We hypothesis MTX-loaded bi-HNTs would limit cell proliferation for a longer time compared to pure MTX treatment.
