Chitosan-Based Nanoparticles for Twist1 Knockdown in 4T1 Cells

Abstract

Bone metastasized breast cancer reduces the quality of life and median survival. Targeted delivery of twist1-siRNA using nanoparticles (NPs) is a promising strategy to overcome current limitations in treating such metastatic breast cancers. This research evaluates two types of chitosan (CHI)-based NPs for the delivery of twist1-siRNA. Alendronate conjugated PEG functionalized chitosan (ALD-PEG-CHI) NPs are developed for active targeting while PEG functionalized CHI (mPEG-CHI) NPs are fabricated for passive targeting. The size of twist1-siRNA-loaded NPs is below 70 nm and the zeta potential is near neutral for both types of NPs. Based on gel retardation assay, complete encapsulation of twist1-siRNA is achieved in both NP systems. The ALD-PEG-CHI-siRNA and mPEG-CHI-siRNA NPs display serum protection for 6 and 4 h, respectively, compared to the immediate degradation of naked twist1-siRNA. The NPs can knockdown twist1 in 4T1 cells as demonstrated through protein expression as well as by phenotypic change in directional cell migration by wound healing assay. Overall, these in vitro results illustrate the potential of the NPs as an effective therapeutic system for bone metastasized breast cancer.

Keywords: SiRNA delivery; chitosan; nanomedicine; poly(ethylene glycol); twist1.

Scheme 1

A) Chemical structures of mPEG‐CHI and ALD‐PEG‐CHI; B) Schematic illustration of the fabrication of twist1‐siRNA NPs prepared from mPEG‐CHI and ALD‐PEG‐CHI, respectively. Part of this figure was generated using BioRender.

Figure 1

Gel retardation assay of siRNA in CHI, mPEG‐CHI, and ALD‐PEG‐CHI NPs systems along with the free siRNA in the filtrate (running buffer of pH 7.2–7.4). Labels represent i) naked siRNA, ii) CHI‐siRNA NPs, iii) mPEG‐CHI‐siRNA NPs, iv) ALD‐PEG‐CHI‐siRNA NPs, v) CHI NP filtrate, vi) mPEG‐CHI NP filtrate, vii) ALD‐PEG‐CHI NP filtrate. The labels ii to iv indicate the retention of siRNA‐loaded NPs in the wells and no band of free siRNA, while the labels v to vii show no band for filtrate in the wells and no free siRNA band. Collectively, this confirms maximum encapsulation of siRNA.

Figure 2

Size distribution data of A) mPEG‐CHI‐siRNA NPs, B) ALD‐PEG‐CHI‐siRNA NPs by number weighted distribution using DLS. n = 3. Errors represent the standard deviation. The ζ‐potential distribution plots are provided in Figure S1 (Supporting Information). Part of this figure was generated using BioRender.

Figure 3

Cytotoxicity (MTT assay) of twist1‐siRNA loaded mPEG‐CHI‐siRNA and ALD‐PEG‐CHI‐siRNA NPs against A) 4T1 cells and B) MDA‐MB‐231 cells for 48 h. Seeding density = 3000 cells/well in a 96‐well plate, n = 3.

Figure 4

Electrophoretic mobility of A) control naked siRNA B) mPEG‐CHI‐siRNA NPs C) ALD‐PEG‐CHI‐siRNA NPs after incubating with cell culture medium DMEM containing 50% FBS. The same amount of free siRNA was used as a control. Samples were collected at different time points and stored at −20 °C before running them on the gel (running buffer pH 8.3). The faint bands of naked siRNA from 0 h timepoint show rapid degradation of siRNA in the presence of 50% FBS, while mPEG‐CHI‐siRNA NPs and ALD‐PEG‐CHI‐siRNA NPs protected the siRNA form serum nucleases up to 6 and 4 h, respectively. The presence of very faint or no bands at some time points is due to the full degradation of released siRNA. The faint bands toward the top of the gel relate to components from FBS. Part of this figure was generated using BioRender.

Figure 5

Western blot analysis of 4T1 cells. Cells were treated with twist1‐siRNA‐loaded NPs for 24 h. 1, 2, and 3 indicate repeats of the experiment (n = 3). Control represents untreated cells.

Figure 6

Wound healing effect of A) mPEG‐CHI‐siRNA NPs (seeding density = 2.8 × 10⁴ cells cm⁻², n = 3) and B) ALD‐PEG‐CHI‐siRNA NPs (seeding density = 3.3 × 10⁴ cells cm⁻², n = 3) against 4T1 cells. Part of this figure was generated using BioRender.