**5. Conclusions**

bi-HNTs were cytocompatible in an appropriate dosage range, permitting a decrease in MTX dose when used with size reduced bi-HNTs. The results further showed that caveolae-mediated endocytosis is the main uptake pathway of bi-HNTs, and multi-photon images confirmed cellular uptake of bi-HNTs. This modified nanocarrier also showed excellent MTX drug loading capability. Most importantly, drug-loaded MTX-bi-HNTs exhibited an excellent selectively targeting ability in vitro, as both colon carcinoma and osteosarcomas cells demonstrated bi-HNT uptake. Due to the drug specificity, the inhibition of cell proliferation was restricted to osteosarcomas, while murine colon carcinoma cells and pre-osteoblasts were not affected. Surface modification of HNTs with two ligands—FA and FITC—provided a selective targeting ability for osteosarcoma cells and an imaging vehicle for tracking bi-HNTs, and may have potential as an alternative treatment for osteosarcoma.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/12/10/962/s1. Figure S1: A. Flow cytometer observation of each treatment. B. The selected dot were continued applied with FITC filter for apoptosis (first row) and PE filter for necrosis (second row).

**Author Contributions:** The authors all contributed to the writing of the manuscript. Y.L. conducted the experiments under the direction of D.K.M. Both authors reviewed and analyzed the data. All authors have read and agreed to the published version of the manuscript.

**Funding:** Funding for this study was provided by a grant (to DKM) from the Louisiana Biomedical Research Network (through an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103424-17.

**Acknowledgments:** The authors also wish to acknowledge the support of the College of Applied and Natural Sciences (Louisiana Tech University) Matching Grant Program. In addition, the authors appreciate Mengcheng Liu and William Clower for thier help in this study.

**Conflicts of Interest:** The authors declare no conflict of interest.
