Shape‑Dependent Uptake of Gold Nanostructures in Glioblastoma Cells: From Synthesis to Cellular Interaction

Abstract

Gold nanostructures (Au NSs) with controlled size and shape are emerging as versatile tools for nanomedicine. However, the cellular fate of different geometries remains poorly understood, particularly in aggressive cancers such as glioblastoma. Here we synthesized five distinct Au NSs—nanorods (NRs), tetrahexahedra (THH), bipyramids (BPs), spherical particles (SPs), and a novel shape termed nano‑makura (NM)—using seed‑mediated growth with binary surfactants and a modified Turkevich route. All particles were surface‑functionalized with PEG‑SH and 11‑mercaptoundecanoic acid (MUA) to impart a negative charge and steric stability. Cytotoxicity assays (LIVE/DEAD) on U‑87 MG glioblastoma cells revealed shape‑dependent effects: NM exhibited the highest cell death (~20 % at 2 mg mL⁻¹) while SPs and BPs remained largely non‑toxic. Transmission electron microscopy showed that NM enters cells via receptor‑mediated endocytosis within 2 h and later triggers macropinocytosis, leading to vesicular accumulation and eventual detachment of cells. These findings demonstrate that particle morphology, rather than size alone, governs cellular uptake pathways and cytotoxic outcomes, positioning NM as a promising candidate for targeted photothermal therapy and drug delivery in glioblastoma.

Background

Gold nanostructures are prized for their shape‑ and size‑dependent optical and electronic properties, enabling applications from biosensing to photothermal therapy. Localized surface plasmon resonance (LSPR) in the visible–near‑infrared (NIR) region makes anisotropic particles, especially high‑aspect‑ratio nanorods, excellent contrast agents for computed tomography and photoacoustic imaging. Smaller nanostructures exhibit superior absorption, enhancing photothermal efficiency. Moreover, anisotropic shapes can self‑assemble into superstructures that generate intense electromagnetic fields, advantageous for surface‑enhanced Raman scattering and targeted drug delivery.

Seed‑mediated growth, often supplemented with silver ions and surfactant mixtures (e.g., CTAB with oleic acid or DDAB), has become the preferred method for tailoring Au NSs, as it allows precise control over aspect ratio and facet growth. Despite advances in synthesis, systematic studies on how different geometries interact with cells—particularly glioblastoma, a highly proliferative and invasive brain tumor—are limited. Understanding these interactions is essential for designing nanomedicines that selectively target cancer cells while minimizing off‑target effects.

Glioblastoma multiforme (GBM) remains the most lethal primary brain tumor, with a median survival of less than 15 months despite aggressive therapy. U‑87 MG glioblastoma‑astrocytoma cells proliferate rapidly (doubling time ~32 h) and readily internalize foreign particles, making them ideal for uptake studies. Here, we present a comprehensive comparison of five Au NS shapes, focusing on synthesis, surface functionalization, cytotoxicity, and uptake mechanisms in U‑87 MG cells.

Experimental

Materials

All reagents (oleic acid, silver nitrate, CTAB, HAuCl₄·3H₂O, ascorbic acid, NaBH₄, PEG‑SH, MUA, etc.) were purchased from Sigma‑Aldrich or Merck and used without further purification. Solutions were prepared with ultrapure water (18.2 MΩ cm).

Synthesis of anisotropic Au NSs

Seed particles (~5 nm) were prepared by reducing HAuCl₄ with NaBH₄ in CTAB. For nanorods, tetrahexahedra, and bipyramids, a growth solution containing CTAB, a co‑surfactant (oleic acid or DDAB), AgNO₃, and ascorbic acid was heated to 80 °C. After cooling, seed solution and HAuCl₄ were added, and the mixture was stirred at 35 °C for 24 h. Adjusting the oleic acid/CTAB ratio yielded the desired morphology: low ratios produced dog‑bone nanorods, higher ratios gave elongated tetrahexahedra, and the CTAB/DDAB combination produced bipyramids. For the novel nano‑makura shape, a two‑step growth was employed: an initial seed‑mediated reaction was followed by a second growth stage using the intermediate solution, producing a three‑dimensional, irregular particle resembling a pillow (pillow = makura in Japanese).

Synthesis of spherical Au NSs

SPs were fabricated by a modified Turkevich method: a 10 mM sodium citrate solution (10 mL) was heated to 70 °C, followed by the dropwise addition of 1.5 mM HAuCl₄ (10 mL). The mixture turned purplish‑red after ~8 min and was cooled to room temperature before centrifugation (14 500 rpm, 10 min).

Surface functionalization

Particles were first incubated with PEG‑SH (1 mg mL⁻¹) for 2 h to replace CTAB and provide steric stabilization. After centrifugation, the PEGylated NSs were treated with MUA (250 µL 10 mM in EtOH/H₂O) for 1 h at 55 °C, achieving a complete CTAB removal and imparting a negative surface charge. Size and zeta potential were monitored by DLS; XPS confirmed the removal of bromine from the surface.

Cell culture and viability assay

U‑87 MG cells were maintained in EMEM supplemented with 10 % FBS, 2 mM L‑glutamine, 1 % NEAA, 1 mM sodium pyruvate, and 1.25 % gentamicin. For cytotoxicity, cells at 70 % confluence were incubated with Au NSs at 100, 200, 500 µg mL⁻¹, and 2 mg mL⁻¹ for 24 h. Cell death was quantified by a LIVE/DEAD assay (calcein AM and ethidium homodimer‑1) and Hoechst nuclear staining, imaged on a Zeiss Axiovert 200 M microscope. Three independent wells per concentration were analyzed.

Time‑dependent uptake study (NM)

Cells were exposed to 2 mg mL⁻¹ NM for 2, 6, 12, and 24 h. After incubation, cells were fixed with 2 % paraformaldehyde and 2.5 % glutaraldehyde, post‑fixed with osmium tetroxide (± potassium ferrocyanide), dehydrated, and embedded for ultrathin (70 nm) TEM sections. TEM images were used to track localization and morphology of NM within cellular compartments.

Characterization

Bright‑field STEM and HRTEM (JEOL 2100, 200 kV) assessed morphology and crystallinity. UV‑Vis spectra (200–800 nm) were recorded on a Shimadzu UV‑2401PC. Dynamic light scattering and zeta potential were measured on a Malvern Zetasizer Nano‑ZS. XPS was performed with a Kratos Axis Ultra DLD spectrometer (Al Kα, 1486.6 eV).

Results and Discussion

Synthesis and characterization of anisotropic Au NSs

Seed‑mediated growth with binary surfactants produced high‑yield nanorods (~20 nm × 50 nm), tetrahexahedra (~50 nm edge length), and bipyramids (~200 nm). The nano‑makura particles displayed a unique, three‑dimensional, irregular morphology, confirmed by TEM at multiple orientations. HRTEM revealed single‑crystalline cores with smooth facets for NRs, while NM showed jagged surfaces and a broader LSPR band, reflecting their polydispersity. UV‑Vis spectra exhibited distinct longitudinal and transverse plasmon peaks: NRs (516, 679, 796 nm), THH (≈568 nm), BPs (≈593 nm), NM (≈557 and 760 nm). SPs showed a single peak at 520 nm.

Surface functionalization outcomes

DLS measurements showed incremental size increases after PEG‑SH and MUA coating, except for BPs where a slight decrease was observed due to tighter packing. Zeta potential shifted from positive (CTAB‑coated) to negative (MUA‑coated) values, confirming successful ligand exchange. XPS spectra exhibited negligible bromine, indicating near‑complete CTAB removal. Optical spectra remained largely unchanged, with only minor peak broadening for anisotropic shapes, attributable to size enlargement and surface roughness.

Shape‑dependent cytotoxicity

LIVE/DEAD assays revealed that NM induced the highest cell death (~20 % at 2 mg mL⁻¹), whereas SPs and BPs remained <5 % cytotoxic across all concentrations. NRs, despite their high aspect ratio and positive surface charge, did not increase cell death relative to other shapes, suggesting that surface charge measurement by zeta potential may not accurately reflect the true local charge on anisotropic particles. Overall, size played a minor role; both 15 nm SPs and 650 nm BPs displayed comparable uptake and toxicity.

Uptake mechanisms of nano‑makura

Time‑resolved TEM showed NM associating with the plasma membrane after 2 h, followed by internalization via receptor‑mediated endocytosis. At 12 h, vesicles containing NM indicated a switch to macropinocytosis, likely due to aggregation and protein adsorption. NM preferentially localized at the periphery of endosomes, aligning with the vesicular membrane, and trafficked toward the perinuclear region. At 24 h, extensive vesicular accumulation correlated with morphological changes: cells lost filopodia, became rounded, and detached from the substrate, indicating cytoskeletal disruption and apoptosis.

Discussion

These results highlight the critical influence of particle morphology on cellular uptake pathways. While anisotropic shapes can enhance optical and photothermal properties, their irregular geometry may also increase protein corona formation, promoting macropinocytosis and cytotoxicity. The nano‑makura particles, with their pillow‑like structure, achieved efficient internalization and significant cell death, making them attractive candidates for glioblastoma therapy. Future studies should explore in‑vivo biodistribution and therapeutic efficacy, as well as functionalization with targeting ligands to improve selectivity.

Conclusions

We synthesized five gold nanostructures of distinct shapes and sizes, uniformly functionalized them with PEG‑SH and MUA to yield stable, negatively charged particles. Shape, rather than size alone, dictated cellular uptake and cytotoxicity in U‑87 MG glioblastoma cells. The novel nano‑makura shape demonstrated superior internalization, leading to significant cell death via receptor‑mediated endocytosis and subsequent macropinocytosis. These findings position nano‑makura as a promising platform for combined photothermal therapy and drug delivery in aggressive brain tumors.

Abbreviations

AgNO₃

Silver nitrate

Au

Gold

BF

Bright field

BPs

Bipyramids

CTAB

Cetyltrimethylammonium bromide

DDAB

Didecyldimethylammonium bromide

DLS

Dynamic light scattering

EMEM

Eagle’s Minimal Essential Medium

EthD-1

Ethidium homodimer‑1

GBM

Glioblastoma multiforme

HAuCl₄

Chloroauric acid

HRTEM

High‑resolution transmission electron microscopy

IR

Infrared

LSPR

Localized surface plasmon resonance

MUA

11‑mercaptoundecanoic acid

Na‑citrate

Sodium citrate dihydrate

NM

nano‑makura

NRs

Nanorods

Ns

Nanostructure

OA

Oleic acid

PEG‑SH

O‑[2‑(3‑mercaptopropionylamino) ethyl]-O‑ethylpolyethylene glycol

STEM

Scanning transmission electron microscopy

TEM

Transmission electron microscopy

THH

Tetrahexahedra

UV‑Vis

Ultraviolet‑visible spectroscopy

XPS

X‑ray photoelectron spectroscopy