Her2‑Functionalized Gold‑Nanoshelled Magnetic Hybrid Nanoparticles: Dual‑Modal US/MR Imaging and Targeted Photothermal Therapy for Breast Cancer

Abstract

Early, precise cancer detection and treatment demand integrated theranostic platforms. Here we report Her2‑functionalized gold‑nanoshelled PLGA magnetic hybrid nanoparticles (Her2‑GPH NPs) that combine ultrasound (US) and magnetic resonance (MR) imaging with near‑infrared (NIR) photothermal therapy (PTT) for breast cancer. The nanoparticles consist of a PLGA core encapsulating perfluorooctyl bromide (PFOB) and superparamagnetic iron oxide nanoparticles (SPIOs), coated with a gold nanoshell and conjugated to anti‑Her2 antibodies. Cell‑binding assays confirm receptor‑mediated uptake in Her2‑positive SKBR3 cells (binding rate 86 ± 3.7 % vs 2.3 ± 0.4 % in Her2‑negative MDA‑MBC‑231 cells, P < 0.001). In vitro, the particles provide high‑contrast US (dynamic range up to 2 mg mL^‑1) and T₂‑weighted MR (relaxivity r₂ = 441.47 mM^‑1 s^‑1), surpassing commercial agents. NIR irradiation (808 nm, 1 W cm^‑2) raises local temperature by ~18 °C at 0.2 mg mL^‑1 and selectively kills SKBR3 cells (62 ± 4.6 % viability reduction, P < 0.001). These results demonstrate Her2‑GPH NPs as a promising theranostic tool for non‑invasive breast cancer imaging and therapy.

Introduction

Breast cancer remains the leading cause of cancer‑related mortality in women worldwide [1]. Early, accurate diagnosis is pivotal to reduce deaths [2]. Conventional imaging modalities, including US and MRI, offer complementary strengths: US provides real‑time, safe, cost‑effective imaging with enhanced sensitivity via contrast agents, yet suffers limited spatial resolution; MRI delivers superior soft‑tissue contrast and anatomical detail, but at longer acquisition times and lower sensitivity. Combining US and MR could overcome these limitations and enable precise, multimodal assessment of breast lesions [10].

Theranostics—integrating diagnosis and therapy within a single platform—promises personalized treatment. Photothermal therapy (PTT) using NIR lasers and photoabsorbers offers minimally invasive, controllable tumor ablation [11–12]. Gold nanoshells exhibit strong surface plasmon resonance in the NIR window, enabling efficient heat conversion while being biocompatible [21]. Yet, many nanoplatforms lack specific targeting ligands, limiting their cellular uptake and therapeutic efficacy.

Her2 is overexpressed in ~25–30 % of breast cancers and correlates with aggressive disease and poor prognosis [28–29]. Anti‑Her2 antibody conjugation can enhance tumor selectivity and uptake. We therefore designed Her2‑functionalized gold‑nanoshelled PLGA magnetic hybrids to enable dual‑modal imaging and targeted PTT of Her2‑positive breast cancer.

Materials and Methods

PLGA (MW 24–38 kDa, 50:50 lactide:glycolide), PFOB, SPIOs, and gold precursors were obtained from Sigma‑Aldrich, Shanghai So‑Fe Biomedicine, and Aladdin Chemistry. Anti‑Her2 antibodies (FITC‑labelled) were from Abcam. The synthesis involved an oil‑in‑water emulsion‑solvent evaporation step to encapsulate PFOB/SPIOs within PLGA, followed by surface functionalization with polyallylamine hydrochloride (PAH) to allow attachment of citrate‑stabilized gold nanoparticles (5 nm). Gold nucleation was induced by hydroxylamine reduction, forming a nanoshell that extended to ~30 nm. The final particles were PEGylated (SH‑PEG‑COOH) and activated with EDC/NHS to conjugate anti‑Her2 antibodies, yielding Her2‑GPH NPs.

Characterization included FE‑SEM and TEM for morphology, DLS for hydrodynamic size and zeta potential, UV‑Vis spectroscopy for optical properties, ICP‑AES for elemental composition, and relaxometry for T₂ relaxivity.

In vitro studies used SKBR3 (Her2‑positive) and MDA‑MBC‑231 (Her2‑negative) breast cancer cell lines. Cytotoxicity was assessed by CCK‑8 assays. Targeting was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry. Ultrasound imaging used a Mylab Twice system (LA522 transducer) in 2D gray‑scale and CEUS modes; MR imaging employed a 0.5 T scanner with spin‑echo sequences. PTT efficacy was tested by NIR laser irradiation (808 nm, 1 W cm^‑2) followed by live/dead staining and CCK‑8 viability assays.

Results and Discussion

**Nanoparticle Characterization** – The core–shell structure displayed uniform spherical morphology with a mean diameter of 248 nm and polydispersity 0.037. After gold coating, size increased to 282 nm (PDI 0.18) and zeta potential shifted to −31 mV, indicating good colloidal stability. EDS confirmed the presence of C, O, Fe, Br, and Au, while ICP‑AES quantified Au (67.7 wt %) and Fe (2.1 wt %). UV‑Vis spectra showed a broad NIR absorption (600–900 nm) after nanoshell growth, essential for efficient photothermal conversion.

**Photothermal Performance** – Under 808 nm irradiation, a 0.2 mg mL^‑1 Her2‑GPH solution rose by 18.5 °C after 10 min, compared to 1.2 °C for water. The absorption spectrum remained unchanged post‑irradiation, confirming photothermal stability.

**Cytotoxicity** – Her2‑GPH NPs exhibited negligible intrinsic toxicity in both cell lines (≥90 % viability up to 200 μg mL^‑1), demonstrating biocompatibility.

**Targeting Efficacy** – CLSM showed bright green fluorescence on SKBR3 membranes with Her2‑GPH, absent in MDA‑MBC‑231 cells. Flow cytometry revealed a 86 % binding rate to SKBR3 versus 2.3 % to MDA‑MBC‑231; pre‑blocking with free antibody reduced binding to 3.2 %. These results confirm receptor‑mediated, specific targeting.

**Ultrasound Imaging** – In vitro, Her2‑GPH solutions produced strong CEUS signals, with a 2.5‑fold intensity increase at 2 mg mL^‑1 compared to 0.1 mg mL^‑1. Time‑intensity curves showed signal retention up to 5 min. In cell phantoms, SKBR3 cells treated with Her2‑GPH displayed significantly higher gray‑scale values than controls, indicating enhanced molecular imaging.

**MR Imaging** – T₂ relaxivity of Her2‑GPH was 441.47 mM^‑1 s^‑1, >2× that of Feridex (152 mM^‑1 s^‑1). In cell imaging, SKBR3 treated with Her2‑GPH showed a 48 % signal drop relative to controls; MDA‑MBC‑231 remained unchanged, confirming selective contrast enhancement.

**Targeted PTT** – Calcein‑AM/PI staining and CCK‑8 assays revealed that only Her2‑GPH + NIR significantly reduced SKBR3 viability (62 % reduction), whereas GPH + NIR or Her2‑GPH alone had minimal effects. Increasing nanoparticle concentration further lowered viability (<20 % at 200 μg mL^‑1 with NIR). Co‑culture experiments mimicking heterogeneous Her2 expression showed proportional cell death relative to SKBR3 proportion.

Conclusions

We have engineered Her2‑functionalized gold‑nanoshelled magnetic hybrid nanoparticles that integrate dual‑modal US/MR imaging with targeted NIR photothermal therapy. The platform demonstrates high‑specificity targeting, robust contrast in both modalities, and effective tumor cell ablation in vitro. These findings lay the groundwork for future in vivo studies and potential clinical translation of a precision breast cancer theranostic system.

Availability of Data and Materials

All datasets generated and analyzed are included in this article.

Abbreviations

Au NPs

Gold nanoparticles

CCK‑8

Cell‑counting kit‑8

DAPI

4′,6‑Diamidino‑2‑phenylindole

DI

Deionized

DLS

Dynamic laser scattering

DMEM

Dulbecco’s modified Eagle’s medium

EDC

Ethyl‑3-(3‑dimethylaminopropyl) carbodiimide

EDS

Energy dispersive X‑ray spectroscopy

FCM

Flow cytometry

FE‑SEM

Field emission scanning electron microscopy

FITC

Fluorescein isothiocyanate

GPH NPs

Gold‑nanoshelled magnetic hybrid nanoparticles

Her2

Human epidermal growth factor receptor 2

ICP‑AES

Inductively coupled plasma atomic emission spectroscopy

LSCM

Laser scanning confocal microscopy

MRI

Magnetic resonance imaging

NIR

Near‑infrared

PAH

Polyallylamine hydrochloride

PFOB

Perfluorooctyl bromide

PLGA

Poly lactic‑co‑glycolic acid

PTT

Photothermal therapy

PVA

Polyvinyl alcohol

SPIOs

Superparamagnetic iron oxide nanoparticles

TIC

Time‑intensity curve

UCA

Ultrasound contrast agent

US

Ultrasound imaging