Hydrophilic HBP‑Modified rGO Enables Dual pH/NIR‑Triggered DOX Release for Synergistic Chemo‑Photothermal Tumor Therapy

Abstract

We report a facile route to synthesize hydrophilic reduced graphene oxide (NrGO) by reducing graphene oxide (GO) with amino‑terminated hyperbranched polymer (NHBP). NrGO exhibits excellent aqueous dispersibility, strong near‑infrared (NIR) absorption, efficient photothermal conversion, and chemical stability. Loading the chemotherapeutic agent doxorubicin (DOX) onto NrGO yields a dual‑responsive drug carrier that releases DOX more rapidly under acidic tumor‑like conditions or upon NIR laser irradiation. In vitro studies confirm that NrGO is biocompatible, and DOX@NrGO achieves superior tumor cell inhibition compared with free DOX, demonstrating effective chemo‑photothermal synergy. These findings highlight DOX@NrGO as a promising platform for combined cancer therapy and other biomedical applications.

Introduction

Near‑infrared (NIR) photothermal therapy (PTT) has emerged as a minimally invasive strategy for cancer treatment because NIR light (700–1100 nm) penetrates tissue with low absorption, sparing healthy cells while enabling localized heating.1–3 For PTT, the therapeutic agent must combine high photothermal conversion efficiency, good biocompatibility, and colloidal stability in aqueous environments.

Graphene‑based materials have attracted attention as PTT agents owing to their two‑dimensional structure, large surface area, and tunable optical properties.4–14 However, conventional reduced graphene oxide (rGO) produced via chemical or hydrothermal methods often retains hydrophobic character, limiting its applicability in biological systems.16

We addressed this limitation by employing a water‑soluble, reductive amino‑terminated hyperbranched polymer (NHBP) to convert GO into a hydrophilic, stable rGO (NrGO). Previous work demonstrated NHBP’s ability to functionalize metal nanoparticles while preserving dispersibility.17,18

In addition, loading cytotoxic drugs onto photothermal carriers can synergize chemotherapy with PTT, as heat not only ablates tumor cells but also accelerates drug release.19–21 Here, we describe the synthesis of NHBP‑modified rGO, its photothermal properties, DOX loading, pH/NIR‑triggered drug release, and the resulting chemo‑photothermal efficacy against HeLa cells.

Materials and Methods

Materials

Graphene oxide (GO, 0.8–1.2 nm thick, 0.5–5 µm wide) was obtained from XFNANO Co., Ltd. Doxorubicin (DOX) was purchased from HuaFeng United Technology Co., Ltd. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin, penicillin (100 U/mL), and streptomycin (100 µg/mL) were supplied by Thermo Fisher Scientific. MTT, DAPI, and PI reagents were from Beyotime Biotechnology. All other chemicals were analytical grade from Sinopharm Chemical Reagent Co., Ltd.

Preparation of Amino‑Terminated Hyperbranched Polymer (NHBP)

The amino‑terminated hyperbranched polymer (NHBP) was synthesized following our previous protocol.16 In brief, tetraethylenepentamine (94 mL, 0.5 mol) was stirred under nitrogen in a 250‑mL three‑neck round‑bottom flask. A methanol solution of methyl acrylate (43 mL, 0.5 mol) was added dropwise, then the mixture was heated and cooled alternately, followed by 4 h of stirring at room temperature. The product was isolated by rotary evaporation at 150 °C, yielding a yellowish viscous polymer with a weight‑average molecular weight of ~7759.

Preparation of NHBP‑Reduced GO (NrGO)

GO was dispersed in deionized water and ultrasonicated with NHBP at weight ratios of 1:10, 1:20, and 1:30 for 10 min, then stirred at 90 °C for 1 h. The resulting suspensions (NrGO‑10, NrGO‑20, NrGO‑30) were centrifuged and washed thrice with water.

Preparation of DOX‑Loaded NrGO (DOX@NrGO)

NrGO suspension was mixed with DOX solution at a 1:1 weight ratio and stirred at room temperature for 24 h. The composite was collected by centrifugation and washed to obtain DOX@NrGO.

Characterization and Measurements

Transmission electron microscopy (TEM, JEOL JEM‑2100) was used to examine morphology. Fourier‑transform infrared (FTIR, Nicolet iS10) spectra were recorded over 400–4000 cm⁻¹ (4 cm⁻¹ resolution). Zeta potential and hydrodynamic size were measured with a NanoBrook 90plus Zeta analyzer. UV‑vis–NIR absorption (400–900 nm, 1 cm⁻¹ resolution) was recorded on a Thermo Fisher Evolution 300. Photothermal performance was assessed using an 808‑nm laser (SFOLT Co.) and a thermocouple thermometer (DT‑8891E). The sample (0.2 mL) in a 0.25 cm² spot was irradiated at 1 W cm⁻² for 5 min, and temperature changes were recorded in real time.

Drug Release Assay

DOX@NrGO was placed in a dialysis bag (MWCO 8000) containing PBS (pH 7.4 or 4.0) and incubated at 37 °C with 100 rpm agitation. For the NIR group, the bag was irradiated for 5 min after each sampling time point. At each interval, 10 mL of PBS was withdrawn, replaced with fresh buffer, and DOX concentration was determined by UV‑vis spectrophotometry.

Cellular Cytotoxicity and Chemo‑Photothermal Evaluation

HeLa cells were seeded at 5 × 10³ cells per well in 96‑well plates and incubated until ~80 % confluence. Cells were treated with NrGO (3.125–50 µg mL⁻¹) for 24 and 48 h, then viability was measured by MTT assay according to Equation (1). After 4 h incubation with DOX@NrGO (3.125–50 µg mL⁻¹), HeLa cells were irradiated with the 808‑nm laser (0.5 W cm⁻²) for 5 min and cultured for an additional 20 h. Viability was assessed by MTT, and cell morphology was examined by DAPI and PI staining under confocal microscopy.

Cell viability (%) = (OD_sample / OD_control) × 100%.

Results and Discussion

Physical and Chemical Characterization

Reduction of GO with NHBP transformed the brown suspension into a black, water‑dispersible solution, confirming successful conversion. TEM images (Fig. 2) show that NrGO‑30 retains a monolayer sheet structure without aggregation, indicating that NHBP does not disturb the graphene morphology. FTIR spectra (Fig. 3) reveal disappearance of the 1725 cm⁻¹ C=O peak from GO and appearance of a 1633 cm⁻¹ C–N amide signal, confirming covalent attachment of NHBP. Zeta potential measurements (Fig. 4) demonstrate a shift from negative (GO) to positive values (NrGO), further supporting surface functionalization. UV‑vis‑NIR spectra (Fig. 5) display strong absorption across the NIR window for all NrGO samples, whereas GO and NHBP alone show negligible NIR response. Hydrodynamic size (Fig. 6) remains consistent across different NHBP ratios, indicating size stability.

Photothermal Properties

Under 808‑nm irradiation, water showed negligible heating, while GO raised <5 °C. In contrast, NrGO samples reached 40–45 °C at 200 µg mL⁻¹, with higher NHBP ratios producing greater temperature elevations (Fig. 7). Temperature rose proportionally with NrGO concentration and laser power, achieving the therapeutic window (~41–43 °C) without damaging normal cells. Photothermal cycling (Fig. 7d) and post‑irradiation UV‑vis (Fig. 8) confirm excellent stability and retention of optical properties.

Drug Delivery Behavior

DOX release from DOX@NrGO was markedly enhanced in acidic (pH 4.0) and NIR‑irradiated conditions (Fig. 9). Acidic pH ionizes NHBP amine groups, reducing electrostatic binding to DOX and promoting release. NIR heating accelerates molecular motion, further accelerating drug diffusion. Thus, DOX@NrGO offers dual responsiveness suitable for tumor microenvironments.

Cytotoxicity of NrGO

MTT assays (Fig. 10a) indicate >80 % cell viability at 50 µg mL⁻¹ after 24 h, confirming low intrinsic toxicity and suitability as a drug carrier.

Synergistic Tumor Cell Inhibition

Treatment with DOX@NrGO alone reduced HeLa viability in a dose‑dependent manner (Fig. 10b). When combined with NIR irradiation (0.5 W cm⁻², 5 min), cell viability decreased significantly more, evidencing chemo‑photothermal synergy. Confocal images (Fig. 11) show dispersed nuclei with DOX@NrGO, while irradiation induces extensive cell detachment. PI staining (Fig. 12) reveals increased dead cells after combined therapy, corroborating the enhanced cytotoxic effect.

Conclusions

In summary, we developed a straightforward, water‑soluble NHBP‑modified rGO with robust photothermal conversion and colloidal stability. DOX loading confers pH‑ and NIR‑triggered release, and the DOX@NrGO system achieves superior chemo‑photothermal tumor cell inhibition in vitro. These results demonstrate the promise of DOX@NrGO as a versatile platform for cancer therapy and other biomedical applications.

Abbreviations

CLSM:

Confocal laser scanning microscopy

DAPI:

4′,6‑diamidino‑2‑phenylindole

DOX:

Doxorubicin

DOX@NrGO:

DOX‑loaded NrGO

FTIR:

Fourier‑transform infrared

GO:

Graphene oxide

HBP:

Hyperbranched polymer

MTT:

Methyl thiazolyl tetrazolium

NHBP:

Amino‑terminated HBP

NIR:

Near infrared

NrGO:

Amino‑terminated hyperbranched polymer reduced graphene oxide

PI:

Propidium iodide

PTT:

Photothermal therapy

rGO:

Reduced graphene oxide

SEM:

Scanning electron microscopy

TEM:

Transmission electron microscope