**4. Conclusions**

We studied the drug delivery efficacy and therapeutic efficacy of HIFU-triggered drug-loaded nanoparticles on ECM-rich tumor models. Doxorubicin-loaded glycol chitosan nanoparticles (DOX-CNPs) could be well accumulated in ECM-rich tumor tissues through HIFU treatment. The in vitro HIFU-triggered DOX-CNPs showed enhanced cellular uptake and rapid drug release from drug-loaded nanoparticles in cultured cells, resulting in increased cytotoxicity, compared to DOX-CNPs without HIFU treatment. In addition, when ECM-rich A549 tumor tissues were treated with HIFU, DOX-CNP could be effectively accumulated at targeted tumor tissues via deep tumor penetration, wherein the dense ECM is broken by external HIFU. Based on these results, HIFU treatment on ECM-rich tumor tissues could increase the accumulation efficiency of DOX-CNPs and inhibit efficiently tumor growth. Therefore, the improved antitumor efficacy of HIFU-triggered DOX-CNPs can be successfully applied to heterogeneous cancer treatments.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/12/10/974/s1, Figure S1: TEM size histogram and volume-weighted size distribution of CNPs and DOX-CNPs, Figure S2: Quantitative analysis of cellular uptake of free DOX and Cy5.5-labeled DOX-CNPs in A549 cancer cells, Figure S3: Collagen matrix in SCC7 and A549 tumor tissues that were stained with Masson's trichrome staining, Figure S4: Masson's trichrome staining images of A549 tumor tissues without or without HIFU treatment, Figure S5: Liver and kidney H&E staining images to demonstrate organ toxicity of DOX-CNPs after HIFU treatment.

**Author Contributions:** Conceptualization, H.Y.Y., H.K. and K.K.; methodology, Y.C. and H.H.; validation, Y.C. and H.H.; formal analysis, S.J. and H.Y.Y.; resources, H.K. and K.K.; data curation, S.J. and H.Y.Y.; writing—original draft, Y.C. and S.J.; writing—review and editing, H.Y.Y.; supervision, K.K.; project administration, H.K., I.C.K. and K.K.; funding acquisition, K.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grants from the National Research Foundation (NRF) of Korea, funded by the Ministry of Science (NRF-2019R1A2C3006283), the KU-KIST Graduate School of Converging Science and Technology (Korea University) and the Intramural Research Program of KIST.

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

#### **References**


**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
