(T0930-01-01) Enhanced Conjugated Linoleic Acid Paclitaxel-Loaded Superparamagnetic Iron Oxide Nanoparticles for Anti-proliferative Activity in Non-Small-Cell Lung Carcinoma

Tuesday, October 18, 2022

9:30 AM – 10:30 AM ET

Presenting Author(s)

Samson A. Adeyemi, PhD

Post-doctoral Scientist
University of the Witwatersrand
Johannesburg, Gauteng, South Africa

Main Author(s)

LN

Lindokuhle M. Ngema, MA

University of the Witwatersrand
Johannesburg, Gauteng, South Africa

Purpose: Non-small-cell lung carcinoma (NSCLC) is the prevalent type of lung cancer affecting millions of people worldwide [1, 2]. NSCLC management continues to be a daunting challenge, with combinational chemotherapy as first-line treatment reportedly at its therapeutic plateau, owing to intolerable toxicity and side effects. Paclitaxel (PTX) is a first-line anticancer drug used in platinum-based combination chemotherapy [3], however, it presents with poor bioavailability and side effects [4]. Consequently, numerous studies have proposed the use of drug delivery nanosystems, including nanoparticles, to improve the efficacy of PTX in NSCLC therapy [5-7]. The use of superparamagnetic iron oxide nanoparticles (SPIONs) as nanovectors has shown enormous success over the years [8, 9], with promising application in cancer nanotherapies [10, 11]. Meanwhile, trans10, cis12 conjugated linoleic acid (10E, 12Z CLA) has been studied to have anticancer benefits [12, 13], and has been implicated in suppression of proliferation of various cancer cells [14-18]. Thus, the present study set to formulate a novel anticancer nanosystem comprising SPIONs, 10E, 12Z CLA and PTX for potential NSCLC management. We hypothesized that the combination of SPIONs, PTX, and a natural fatty acid (CLA) bearing anticancer properties could yield a potent NSCLC therapeutic nanosystem, exhibiting enhanced antiproliferative activity and biocompatibility.

Methods: SPIONs were fabricated via co-precipitation and coated with 10E, 12 Z CLA in a single-step reaction. PTX was loaded on 10E, 12Z coated SPIONs, to formulate CLA-PTX-SPIONs. Fourier Transform Infrared (FT-IR) spectroscopy was employed to assess chemical transformations and confirm the formation of CLA-PTX-SPIONs. The size and surface charge of CLA-PTX-SPIONs were measured using the ZetaSizer, and the overall morphology was analysed using Scanning Electron Microscope (SEM). The magnetic properties of CLA-PTX-SPIONs were analysed using a vibrating sample magnetometer (VSM). The drug entrapment efficiency (%EE) and loading capacity (%DLC) were determined, and the in vitro drug release profile of CLA-PTX-SPIONs was evaluated at pH 6.8 and pH 7.4 for 24 hours. A cell viability study was conducted on lung adenocarcinoma cell line (A549) for 72 hours, using 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The experimental cells were treated in triplicates (n=3) with SPIONs and CLA-PTX-SPIONs at varying concentrations (3.125 – 100 µg/mL). PTX was used as a positive control (0.156 – 10 µg/mL).

Results: Purely magnetite SPIONs (Fe₃O₄) were successfully synthesized, coated with 10E, 12Z CLA and loaded with PTX (Figure 1a-e). CLA-PTX-SPIONs exhibited a hydrodynamic size of 96.5±0.6 nm and zeta potential of -27.3±1.9 mV (Figure 2a, b), with a polydispersity index of 0.1±0.02. This revealed that CLA-PTX-SPIONs had good dispersion and stability in aqueous media, suggesting suitability as a nanovector in biomedical applications [19]. CLA-PTX-SPIONs had a spherical shape with roughness on the surface (Figure 2d, e). Moreover, CLA-PTX-SPIONs exhibited superparamagnetic behaviour, with saturation magnetization of 60 emu/g and 29 Oe coercivity. The %EE and %DLC of CLA-PTX-SPIONs was found to be 98.5% and 8.9%, respectively. The surface coating of SPIONs with 10E, 12Z CLA allowed efficient loading and encapsulation of PTX via hydrophobic interactions. CLA-PTX-SPIONs demonstrated a sustained in vitro release of PTX over 24 hours, with maximum release of 94% at pH 6.8 (mimicking tumor microenvironment) (Figure 3a), evidencing the ability of the nanosytem to release PTX at tumor microenvironment. Treatment with CLA-PTX-SPIONs significantly suppressed A549 cell proliferation after 72 hours in a dose-dependent manner (Figure 3b). Treatment with the highest (100 µg/mL) and lowest (3.125 µg/mL) concentrations resulted in 17.1% and 24.2% cell viability, respectively. Interestingly, the lowest CLA-PTX-SPIONs concentrations of 3.125 and 6.25 µg/mL with corresponding % cell viability of 24.2 and 21.6%, respectively, demonstrated enhanced anti-proliferative activity than the highest concentration of naked PTX (10 µg/mL) with cell viability of 25.3% (Figure 3c). Essentially, this showed that only a small dose of CLA-PTX-SPIONs is required to elicit equivalent anti-proliferative activity as that exerted by ~3-fold dose of PTX. The enhanced anti-proliferative activity of CLA-PTX-SPIONs observed could be attributed to the presence of 10E, 12Z CLA in the nanosystem. It is presumed that, in addition to acting as a surface coating agent that allowed maximal partitioning of PTX on SPIONs, 10E, 12Z CLA imparted additional anticancer action, resulting in increased CLA-PTX-SPIONs anti-proliferative activity.

Conclusion: Herein, we successfully formulated a potent nanosystem which exhibits superlative anti-proliferative activity against lung adenocarcinoma. The nanosystem validates the proof-of-concept for the use of CLA, a natural compound, to enhance the anticancer activity of hydrophobic anticancer drugs. Fundamentally, CLA-PTX-SPIONs could offer various potential benefits in NSCLC therapy, including biocompatibility, site-specific drug release, and cost-effective formulation. Further modifications to CLA-PTX-SPIONs such as functionalization with homing peptides, and in vivo studies could be explored in the future for potential application of the nanosystem in targeted NSCLC drug delivery.

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Acknowledgements: National Research Foundation (NRF) of South Africa for funding the study.

Figure 1. FT-IR spectra of (a) SPIONs, (b) CLA, (c) CLA-SPIONs, (d) PTX, and (e) CLA-PTX-SPIONs.

Figure 2. A visualization of morphological attributes of CLA-PTX-SPIONs, showing (a) average hydrodynamic size of 96.5±0.6 nm, (b) zeta potential of -27.3±1.9 mV, and (c) a vacuum dried sample with corresponding SEM images showing overall morphology at (d) 300 nm magnification, and (e) 100 nm (isolated particle), clearly showing the roughness of the surface and the particle size of < 100 nm.

Figure 3. Representation of (a) in vitro drug release profile and cell viability of A549 cells treated with (b) SPIONs and CLA-PTX-SPIONs, and (c) PTX (positive control) after 72 hours.