Optimizing Magnetofection of MG‑63 Osteoblasts with Uniform Magnetic Field and PEI‑SPIO Nanoparticles

Abstract

This study demonstrates that a novel uniform magnetic field significantly improves the magnetofection of MG‑63 osteoblasts using low‑molecular‑weight linear PEI (MW 20 kDa) modified superparamagnetic iron oxide nanoparticles (PEI‑SPIO‑NPs). The PEI‑SPIO‑NPs exhibit optimal size, zeta potential, plasmid DNA (pDNA) binding capacity, and DNA‑protection ability, making them suitable non‑viral gene carriers. Under the uniform field, PEI‑SPIO‑NP/pDNA complexes rapidly and evenly coat the cell surface, avoiding local transfection hotspots and membrane disruption that can arise from the clustering of positively charged carriers. This uniform distribution increases the effective coverage of magnetic gene carriers and enhances transfection efficiency. The uniform field also provides a controlled environment to fine‑tune the PEI‑SPIO‑NP:pDNA ratio and screen optimal carrier formulations. Consequently, this approach offers a new strategy for targeted delivery of therapeutic genes to osteosarcoma tissues and may be extended to other tumor models.

Background

Osteosarcoma remains the most common malignant bone tumor in children and adolescents, and current therapies yield limited survival benefits. Gene therapy has emerged as a promising adjunct, yet effective, safe, and non‑viral delivery systems are still lacking. Conventional viral vectors, while highly efficient, raise safety concerns including immunogenicity and insertional mutagenesis, pushing research toward non‑viral carriers. Unfortunately, non‑viral methods typically exhibit low transfection efficiencies in osteosarcoma cells.

Physical transfection aids—gene guns, ultrasound, electroporation—can enhance delivery but often inflict cellular damage. Magnetic‑assisted transfection, or magnetofection, offers a gentler alternative: magnetic nanoparticles (MNPs) are attracted to target cells by an external field, concentrating the gene complexes at the cell surface and promoting uptake. Over the past decade, advances such as oscillating fields, rotating magnets, and dynamic gradient fields have increased magnetofection efficiency, yet the uniformity of the magnetic field remains a limiting factor. Most commercial devices produce heterogeneous fields, leading to uneven nanoparticle distribution, localized toxicity, and variable transfection outcomes.

To address this, our group collaborated with the Electrical Engineering College of Chongqing University to design a Halbach‑type magnetic field generator that creates a highly uniform field across a 50 mm × 50 mm area (field strength 0.0739 T, uniformity 1.3 × 10⁻³). This uniformity is >100‑fold better than conventional 96‑well magnetic plates. We hypothesized that such a field would enable even distribution of PEI‑SPIO‑NP/pDNA complexes and enhance magnetofection of MG‑63 cells.

Methods

Materials and Reagents

SPIO‑NPs, linear PEI (MW 20 kDa), EDC, NHS, plasmid DNA, and all cell culture reagents were sourced from standard suppliers (Sigma‑Aldrich, Invitrogen, etc.). The Halbach magnetic field generator was fabricated in-house, and 96‑well magnetic plates were purchased from Chemicell. All chemicals were analytical grade.

Synthesis of PEI‑SPIO‑NPs

Carboxyl‑modified SPIO‑NPs (5 mg mL⁻¹) were activated with EDC (0.1 g) and NHS (0.5 g) at pH 5 for 4–6 h, then reacted with PEI (20 mg mL⁻¹) for several hours. The conjugate was dialyzed (MWCO 20 kDa) for 48 h, freeze‑dried, and stored.

Formation and Characterization of PEI‑SPIO‑NP/pDNA Complexes

PEI‑SPIO‑NPs and plasmid DNA were mixed at N/P ratios (NH₂/PO₄) ranging from 2.5 to 25. Complexes were characterized by TEM, dynamic light scattering (DLS), zeta potential, vibrating sample magnetometry (VSM), inductively coupled plasma optical emission spectrometry (ICP‑OES), and atomic force microscopy (AFM). Gel electrophoresis assessed pDNA binding and protection against DNase I.

Magnetic Field Generation

The Halbach generator consists of nine 40 × 40 × 200 mm³ Nd‑Fe‑B blocks and two passive shims. Field uniformity was measured with a Gaussmeter; the resulting field was 0.0739 T with <0.13 % variation across the 50 mm × 50 mm plane.

Cell Culture

Human osteosarcoma MG‑63 cells were cultured in DMEM + 10 % FBS + antibiotics at 37 °C, 5 % CO₂.

Cytotoxicity Assessment

CCK‑8 assays measured viability after 24–96 h exposure to PEI‑SPIO‑NP/pDNA, PEI‑NP/pDNA, and PolyMag‑200 controls under uniform or non‑uniform fields.

Confocal Microscopy

RBITC‑labelled complexes were incubated with MG‑63 cells under uniform or non‑uniform fields. Cells were stained with Hoechst 33342 and LysoTracker Green; images were captured on a Zeiss LSM 700.

Flow Cytometry

Transfection efficiency was quantified by flow cytometry 48 h post‑transfection using GFP‑expressing plasmid.

Statistical Analysis

Data (n = 5) were analyzed by one‑way ANOVA and Student’s t‑test; p < 0.05 indicated significance.

Results and Discussion

Principle of Magnetofection

Magnetofection relies on magnetic gradients to pull PEI‑SPIO‑NP/pDNA complexes onto target cells, achieving near‑complete surface coverage within minutes. This rapid sedimentation bypasses the slow diffusion barrier typical of non‑magnetic transfection.

Characterization of PEI‑SPIO‑NP/pDNA Complexes

PEI‑SPIO‑NPs were ~10 nm spheres; complexes aggregated to ~180–200 nm with positive zeta potentials (+11.9 mV at N/P = 10). VSM showed a saturation magnetization of 21.5 emu g⁻¹, sufficient for magnetic manipulation. Gel shift assays confirmed complete DNA condensation at N/P ≥ 5, and DNase protection assays demonstrated robust protection at N/P ≥ 5.

Magnetic Field Uniformity and Nanoparticle Distribution

Under the Halbach field, PEI‑SPIO‑NPs dispersed uniformly across the plate, whereas the conventional 96‑well magnetic plate produced clumped aggregates along magnetic lines. Uniform distribution minimized local charge density, reducing membrane disruption and cytotoxicity.

Cytotoxicity

CCK‑8 results indicated that PEI‑SPIO‑NP/pDNA complexes had lower toxicity than unmagnetized PEI‑NP/pDNA and PolyMag‑200/pDNA controls. The uniform field further reduced cytotoxicity relative to the non‑uniform field (p < 0.05). Nanoparticles containing pDNA exhibited less toxicity than empty particles, likely due to charge neutralization by the negatively charged plasmid.

Cellular Uptake

Confocal imaging showed extensive co‑localization of RBITC‑labelled complexes with LysoTracker at 6 h, followed by escape into the cytoplasm and nuclei by 12–24 h. Uptake was highest under the uniform field, correlating with greater transfection efficiency.

Transfection Efficiency

GFP expression under the uniform field reached 42.1 % (flow cytometry), roughly twice the efficiency of the non‑uniform field (≈ 21 %). PEI‑SPIO‑NP/pDNA outperformed PEI‑NP/pDNA, and while PolyMag‑200 achieved high transfection, it remained cytotoxic. The data suggest that rapid sedimentation, uniform coverage, and efficient endosomal escape collectively drive the enhanced magnetofection.

Conclusions

Our Halbach‑type magnetic field generator produces a highly uniform field that enables even distribution of PEI‑SPIO‑NP/pDNA complexes on MG‑63 osteoblasts. This configuration reduces local toxicity, increases cell surface coverage, and markedly improves magnetofection efficiency. The platform offers a controllable, scalable approach for targeted gene delivery to osteosarcoma and potentially other solid tumors. Further in‑vivo studies are warranted to validate therapeutic efficacy.

Abbreviations

AFM

Atomic force microscopy

CLSM

Confocal laser scanning microscopy

DLS

Dynamic light scattering

DMEM

Dulbecco’s modified Eagle’s medium

EDC

1‑Ethyl‑3‑[3-(dimethylamino)propyl] carbodiimide

FBS

Fetal bovine serum

GFP

Green fluorescent protein

ICP‑OES

Inductively coupled plasma optical emission spectrometry

MF

Magnetic field

MG‑63

Human osteosarcoma cell line

MRI

Magnetic resonance imaging

MWCO

Molecular weight cut‑off

NHS

N‑hydroxy succinimide

PBS

Phosphate‑buffered saline

pDNA

Plasmid DNA

PEI

Polyethylenimine

PEI‑NPs

Polyethylenimine nanoparticles

PEI‑SPIO‑NPs

Polyethylenimine‑modified superparamagnetic iron oxide nanoparticles

RBITC

Rhodamine B isothiocyanate

SPIO‑NPs

Superparamagnetic iron oxide nanoparticles

TEM

Transmission electron microscopy

VSM

Vibrating sample magnetometer