QbD assisted development of hyaluronic acid and TPGS incorporated DOX liposome(s): in vitro assessment of cytotoxicity, uptake, ROS, and NF-κB potential on MDA-MB-231 cells

Lakshmi Kumari; Rajveer Singh; Preeti Patel; Amrinder Singh; Dilpreet Singh; Nitin Sharma; Ghanshyam Das Gupta; Balak Das Kurmi

doi:10.1007/s00210-025-04053-7

QbD assisted development of hyaluronic acid and TPGS incorporated DOX liposome(s): in vitro assessment of cytotoxicity, uptake, ROS, and NF-κB potential on MDA-MB-231 cells

Naunyn Schmiedebergs Arch Pharmacol. 2025 May 1. doi: 10.1007/s00210-025-04053-7. Online ahead of print.

Authors

Lakshmi Kumari¹, Rajveer Singh², Preeti Patel³, Amrinder Singh⁴, Dilpreet Singh⁵, Nitin Sharma⁶, Ghanshyam Das Gupta¹, Balak Das Kurmi⁷

Affiliations

¹ Department of Pharmaceutics, ISF College Pharmacy, GT Road, Moga, 142001, Punjab, India.
² Department of Pharmacognosy, ISF College Pharmacy, GT Road, Moga, 142001, Punjab, India.
³ Department of Pharmaceutical Chemistry, ISF College Pharmacy, GT Road, Moga, 142001, Punjab, India. ppatelpharma@gmail.com.
⁴ Chitkara College of Pharmacy, Chitkara University, Rajpura, 140401, Punjab, India.
⁵ School of Pharmaceutical Sciences, CT University, Ferozepur Road, Sidhwan Khurd, 142024, Punjab, India.
⁶ Department of Pharmaceutics, Amity Institute of Pharmacy, Amity University, Noida, Uttar Pradesh, India.
⁷ Department of Pharmaceutics, ISF College Pharmacy, GT Road, Moga, 142001, Punjab, India. bdkurmi@gmail.com.

PMID: 40310527
DOI: 10.1007/s00210-025-04053-7

Abstract

This work aimed to develop Doxorubicin (DOX), D‑alpha‑tocopheryl polyethylene glycol 1000 succinate (TPGS), and hyaluronic acid (HA) liposomal delivery system using the Quality by Design (QbD) methodology. Developed liposomal formulations show sustained release behaviour as compared to free DOX (96.7 ± 1.81% release in 12 h), as indicated by the release kinetics metrics (82.8 ± 2.82% & 69.8 ± 2.25% releases in 72 h of HA-DOX-TPGS-LIPO and DOX-TPGS-LIPO respectively). In vitro cytotoxicity by MTT assay on MDA-MB-231 and MCF-7 breast cancer cells demonstrated that HA-DOX-TPGS-LIPO had significantly higher cytotoxicity and a clear synergistic impact over free drug or DOX-TPGS-LIPO respectively. Higher cell internalization of HA-DOX-TPGS-LIPO was seen in an in vitro cellular uptake assay in contrast to free drug and DOX-TPGS-LIPO investigated by FACS analysis. ROS production was also assessed using 2', 7'-Dichlorofluorescin diacetates (DCFDA) in the FACS study. Upon treatment with HA-DOX-TPGS-LIPO, triple-negative breast cancer (TNBC) cells showed a higher ROS generation than free DOX and DOX-TPGS-LIPO. Moreover, the expression of the NF-κB protein was also analyzed as a possible substitute mechanism for apoptosis. Free DOX treatment resulted in the maximum expression of NF-κB protein, whereas the HA-DOX-TPGS-LIPO formulation showed the minimum expression of NF-κB protein at the same concentration of DOX-TPGS-LIPO. The study suggested that HA-tethered developed liposomal formulation could be an effective tumor treatment and may be a valuable targeted nanocarrier.

Keywords: CD44 receptor; Cellular uptake; Cytotoxicity study; Doxorubicin; Hyaluronic acid; Liposome; TNBC.