Using GaN/Fe Nanoparticles to Magnetically Guide Endothelial Cells in Vitro

Background

Nanotechnology has emerged as a powerful tool in oncology, largely through nanoparticle-based drug carriers ^[1,2]. However, ligand coating, drug encapsulation, and covalent attachment can limit delivery efficiency and raise safety concerns. An alternative strategy involves using nanoparticles as direct biological modulators—targeting and guiding cells themselves ^[3]. For instance, magnetic nanoparticles can direct endothelial cells to sites of arterial injury when exposed to an external magnetic field. Beyond therapeutic applications, such guided cells can aid in in‑vitro cell sorting and scaffold functionalization ^[4]. Here we present the first evidence that endothelial cells can internalize GaN/Fe nanoparticles and that this uptake enables controlled spatial positioning of the cells.

Methods

Nanoparticle Synthesis

Thin GaN layers were grown on ZnO nanoparticles alloyed with Fe₂O₃ using a two‑step HVPE process. An initial nucleation layer was deposited at 600 °C for 5 min, followed by a growth phase at 800 °C for 10 min to decompose the ZnO core and improve GaN crystalline quality. The process employed metallic gallium, NH₃, HCl, and H₂ as carrier gases at flow rates of 20, 600, and 3500 sccm, respectively ^[5,6].

Cell Culture

Primary porcine aortic endothelial cells were isolated by gentle scraping of the aortic wall, then cultured in EGM™-2 (Lonza) at 37 °C, 5% CO₂. Passages 3–8 were used, and cells were transduced with GFP via lentivirus ^[7]. Cell detachment for experiments was performed with TrypLE™Select(1X) (Gibco®).

XTT Assay

Cell viability was assessed 24 h after adding nanoparticles. XTT reagent (0.1 ml in 5 ml XTT) was mixed with fresh EGM2 medium (2:1) and incubated for 4 h at 37 °C, 5% CO₂. Absorbance was measured on a Paradigm multi‑mode plate reader.

Cell Counting

After 48 h of nanoparticle exposure, cells were fixed in 4% paraformaldehyde, washed with PBS, and stained with DAPI (1:7500). Images were captured from six wells per condition using a Zeiss fluorescence microscope and quantified with DotCount v1.2 ^[8].

Transmission Electron Microscopy

Cells incubated for 24 h with 50 µg/ml GaN/Fe nanoparticles were fixed in 2% glutaraldehyde/2% formaldehyde, post‑fixed in 1% OsO₄, dehydrated, and embedded in EPON. Ultra‑thin sections (~50 nm) were stained with 4% uranyl acetate and lead citrate, then examined on an FEI Tecnai 20 (200 kV).

Results and discussion

GaN/Fe nanoparticles were engineered by coating ZnO/Fe₂O₃ cores with GaN, followed by core removal. The resulting particles exhibit a GaN shell (~20–100 nm) embedded with Fe, conferring magnetic responsiveness while retaining the piezoelectricity of GaN. SEM, TEM, XRD, Raman, and EDX analyses confirm core decomposition, GaN growth, and a Fe content of ~50% ^[Figure 1].

Analysis of nanoparticles. a SEM of GaN on ZnO/Fe₂O₃ cores. b TEM of GaN/Fe particles. c XRD of ZnFe₂O₄ vs. GaN/ZnFe₂O₄. d Raman spectra before and after GaN growth. e EDX of ZnO/Fe₂O₃. f EDX after GaN growth.

When incubated with porcine aortic endothelial cells, the nanoparticles were predominantly internalized into vesicles, with no cytoplasmic or nuclear localization (Fig. 3a). Uptake occurred via classical pathways—micropinocytosis, clathrin‑mediated, and caveolin‑mediated endocytosis—likely dominated by caveolae in endothelial cells ^[9,10,11]. Viability assays revealed a modest, concentration‑dependent reduction in metabolic activity at 50 µg/ml, while lower doses (<10 µg/ml) maintained >90% viability (Fig. 2).

Impact of nanoparticles on cell viability. XTT reduction after 1 day of incubation with various concentrations. Results are expressed relative to untreated controls (mean ± SD, n = 6).

Given the high Fe loading, the particles exhibit strong ferromagnetism, while the GaN shell preserves piezoelectricity. These dual properties enable remote actuation via ultrasound‑induced polarization and magnetic manipulation. To validate magnetic guidance, endothelial cells loaded with 50 µg/ml GaN/Fe were detached, re‑suspended, and plated onto EGM™-2. When the culture plate was positioned beneath permanent magnets, the cells displayed a highly non‑random distribution that matched the magnetic field map, whereas control cells showed random placement (Fig. 4). This demonstrates that GaN/Fe nanoparticles can be used to direct cells in a predictable manner.

TEM images of a single endothelial cell after 1 day with GaN/Fe nanoparticles. a Vesicular distribution. b–d Uptake pathways (red arrows denote high‑density particles).

Guiding of nanoparticle‑laden endothelial cells with magnetic fields. a Cells with nanoparticles, no field. b Cells without nanoparticles, field applied. c Cells with nanoparticles under seven neodymium magnets. d Cells with nanoparticles under a ring magnet.

Conclusions

We have established that GaN/Fe nanoparticles are safely internalized by endothelial cells and stored within vesicles. Their magnetic responsiveness allows precise, field‑driven positioning of the cells, opening avenues for targeted cell delivery and the fabrication of engineered tissues. Additionally, the intrinsic piezoelectricity of GaN offers a platform for remote electrical stimulation of cellular functions. Ongoing work will explore in vivo applications and the integration of piezoelectric activation for therapeutic modulation.

Abbreviations

EDX:

Energy‑dispersive X‑ray analysis

EGM™-2:

Endothelial Growth Medium 2

Fe:

Iron

Fe₂O₃:

Iron(III) oxide

GaN:

Gallium nitride

GFP:

Green fluorescent protein

H₂:

Hydrogen

HCl:

Hydrogen chloride

NH₃:

Ammonia

OsO₄:

Osmium tetroxide

PBS:

Phosphate‑buffered saline

SEM:

Scanning electron microscopy

TEM:

Transmission electron microscopy

XRD:

X‑ray diffraction

ZnO:

Zinc oxide