**3. Conclusions**

This study highlights the importance of paclitaxel delivery using PLHNPs to maximize the efficacy of the drug for overcoming paclitaxel tolerance in metastasizing lung cancer cells. The anoikis resistance property of the A549 floating cells was confirmed by the absence of caspase-3 activation, in contrast to the anoikis induction observed in HMEC floating cells. The self-assembly of PLGA-core and lipid-shell hybrid nanoparticles were formulated through a modified nanoprecipitation technique leading to an average particle diameter of 103.0 nm and strongly negative surface charge with a zeta potential of −52.9 mV. The paclitaxel-PLHNPs showed the ability to increase the therapeutic effect of paclitaxel in A549 floating cells with lower IC50 values compared with free paclitaxel.

Our results sugges<sup>t</sup> that the PLHNP platform has grea<sup>t</sup> potential for delivering hydrophobic drugs to treat floating cancer cells during metastasis in the circulatory systems

of the body. Further studies in animal models are required for validating the capability of paclitaxel-PLHNPs to reduce metastasis and recurrence.

However, the development of PLHNPs encapsulating other drugs is also necessary, because anoikis-resistant cells of different cancer types might acquire tolerance to distinct drugs. We also sugges<sup>t</sup> that a targeted delivery system or combination with other treatments is required to improve effectiveness for overcoming drug tolerance of anoikis-resistant cancer cells, in order to reduce the metastatic rate and prevent cancer recurrence.

## **4. Materials and Methods**

## *4.1. Materials and Chemicals*

Poly(D,L-lactide-co-glycolide) (PLGA) with a 50:50 monomer ratio, soybean lecithin consisting of 95% phosphatidylcholine, paclitaxel, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and poly(2-hydroxyethyl methacrylate) or polyHEMA were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1,2-Distearoyl-sn-glycero-3- phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG-COOH) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), and antibiotic–antimycotic solution were supplied by Gibco (Grand Island, NY, USA). All other reagents were of analytical grade and used as received without further purification. Ultrapure water purified by Milli-Q-plus system (Millipore, MA, USA) was used throughout the study.

## *4.2. Nanoparticle Preparation and Characterization*

PLGA-lipid hybrid nanoparticles or PLHNPs were prepared through a previously reported nanoprecipitation method combined with self-assembly [38]. In brief, 5 mg of PLGA polymer was dissolved in 2 mL acetonitrile. Lecithin and DSPE-PEG-COOH (3:1, molar ratio) at 20% PLGA weight were dissolved in 10 mL of 4% ethanol. Polymer solution was then slowly dropped into preheated lipid aqueous solution (65 ◦C) under stirring. The mixture was subsequently stirred at room temperature for 1.5 h. Paclitaxel-PLHNPs were formulated with a similar method where 0.5 mg of paclitaxel was added to 2 mL of PLGA/acetonitrile solution. The resulting nanoparticles were collected and washed three times with water through an Amicon Ultra-15 centrifugal filter, 10 kDa MWCO (Millipore, MA, USA). The nanoparticles were filter-sterilized and stored at 4 ◦C for later use.

Hydrodynamic size and size distributions were analyzed by dynamic light scattering (DLS). The zeta potential was determined via electrophoretic mobility. Measurements were performed on samples appropriately diluted with using Zetasizer Nano ZS90 (Malvern Instruments, UK). Paclitaxel content in the nanoparticles was quantified by analyzing the absorbance of paclitaxel in nanoparticles and comparing it to the standard calibration curve of the drug. Nanoparticle stability tests were performed to investigate the effect of storage temperature and duration of storage on the nanoparticle stability in terms of particle size.

## *4.3. Cell Culture*

A549 human lung adenocarcinoma cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Normal human mammary epithelial cells (HMECs) were purchased from LONZA (Walkersville, MD, USA). A549 cells were grown as monolayer cultures in RPMI 1640 supplemented with 10% (v/v) FBS and 1% (v/v) antibiotic–antimycotic. HMEC cells were cultured in Mammary Epithelial Cell Growth Medium (MEGM). All cells were maintained at 37 ◦C in a humidified incubator with 5% CO2.

Floating cells were obtained by cultivating the cells under non-adherent culture conditions using polyHEMA-coated culture plates which were prepared according to a previous report with some modifications [15]. Briefly, 96-well plates were coated with a solution of 30 mg/mL polyHEMA in 95% ethanol, followed by drying at 37 ◦C for 48 h in a non-CO2 incubator for ethanol evaporation. The dried coated plates were sterilized by exposure to UV light for 20 min prior to beginning each test.

## *4.4. Caspase-3 Activity Assay*

Anoikis cell death was assessed by determining caspase-3 activation using Caspase-Glo 3/7 Assay (Promega, Medison, WI, USA). Briefly, 1 × 10<sup>4</sup> cells (100 μL/well) were seeded into non-coated or polyHEMA-coated 96-well plates. After 24 h, Caspase-Glo 3/7 reagen<sup>t</sup> (100 μL) was added to each well. Then, the plates were incubated at room temperature in the dark for 1 h. The resulting luminescence was measured with a luminescence microplate reader (Molecular Devices, Sunnyvale, CA, USA).
