Green Synthesis of Gold Nanoparticles with Mimosa tenuiflora Bark Extract: Cytotoxicity, Cellular Uptake and Catalytic Degradation of Methylene Blue

Abstract

Gold nanoparticles (AuNPs) produced via plant‐based green chemistry offer broad biomedical prospects. Here we report AuNPs synthesized from Mimosa tenuiflora bark extract (Mt) under controlled precursor concentrations. Mt was extracted in ethanol–water, and its antioxidant activity was quantified by DPPH and total polyphenol assays. The resulting AuMt colloids displayed diverse morphologies (20–200 nm) and were characterized by TEM, XRD, UV‑Vis, FTIR, and XPS. AuMt exhibited catalytic activity in the reduction of methylene blue (MB) by NaBH₄, with the smallest particles (AuMt1) showing a degradation rate constant of 8.24 × 10⁻³ s⁻¹ and 50 % MB removal in 190 s. Cytotoxicity studies on human umbilical vein endothelial cells (HUVEC) revealed moderate effects at 24–48 h, without a clear dose‑response. Confocal microscopy demonstrated cytoplasmic localization of AuMt1 and perinuclear accumulation of AuMt2, indicating efficient cellular internalization without nuclear penetration. These findings position AuMt as promising nanocarriers and catalytic agents in green nanomedicine.

Introduction

One‑pot, plant‑mediated synthesis of metallic nanomaterials is an eco‑friendly alternative to conventional routes, leveraging bio‑reducing and capping agents present in plant extracts. Mimosa tenuiflora bark is rich in condensed tannins, flavonoids, saponins, alkaloids, and starch, all of which can reduce Au³⁺ to Au⁰ and stabilize the resulting nanoparticles. Prior studies have exploited Mt extracts for antibacterial, antiprotozoal, and skin‑regenerative applications, underscoring its antioxidant potential and polyphenolic content. The surface plasmon resonance (SPR) of AuNPs is sensitive to particle size, shape, and the chemical nature of the surrounding stabilizing molecules, influencing both optical and catalytic properties. Green‑synthesized AuNPs have been applied to environmental remediation, including dye degradation, and as probes for metal ion detection or theranostics. The present work evaluates AuNPs produced from Mt bark extract, assessing their physicochemical characteristics, cytotoxicity in HUVEC cells, cellular uptake, and catalytic efficiency in MB degradation.

Materials and Methods

Materials and Extract Preparation

15 g of Mt bark was macerated in 70 mL ethanol (99 %) and 30 mL ultrapure water (18 MΩ) for 15 days at room temperature. The filtrate was collected, centrifuged (14 000 rpm, 1 h), and lyophilized for antioxidant assays. The Mt extract concentration was determined to be 32.5 mg mL⁻¹ via a calibration curve. Gold precursor solutions of 5.3 mM (AuMt1) and 2.6 mM (AuMt2) were prepared in ultrapure water. In each synthesis, 1.6 mL of Mt extract and the gold solution were mixed in a 50 mL tube, vortexed at 3000 rpm for 10 s, and the resulting color change confirmed AuNP formation. The colloids were purified by repeated centrifugation (14 000 rpm, 1 h) and resuspended in ethanol, followed by sonication and a final dry‑heat step at 40 °C to yield AuMt nanocomposites.

Characterization Techniques

UV‑Vis spectra (200–400 nm for extract; 250–875 nm for AuMt) were recorded on a Perkin‑Elmer Lambda 40. SPR kinetics were monitored at 550–560 nm. DPPH (300 µM) and total polyphenol (Folin‑Ciocalteu) assays quantified antioxidant capacity, expressed as % scavenging and gallic acid equivalents, respectively. Zeta potential and dynamic light scattering (DLS) were measured on a Malvern Zetasizer. FTIR spectra (4500–500 cm⁻¹) and XPS (Au 4f, C 1s, O 1s) provided surface chemistry insights. TEM (JEOL 2010F, 200 keV) and HRTEM assessed morphology and lattice spacings; EDS confirmed elemental composition. XRD (Bruker D8 QUEST, CuKα) identified crystalline phases. Cellular uptake was visualized via confocal laser scanning microscopy (CLSM, Zeiss LSM 800) using 640 nm excitation. Cytotoxicity was measured by MTT assay on HUVEC cells (DMEM + 10 % FBS, 37 °C, 5 % CO₂) at concentrations 25–200 µg mL⁻¹ for 24 and 48 h. Live/Dead staining (calcein/ethidium homodimer) complemented viability data.

Catalytic Degradation of Methylene Blue

MB (3.33 × 10⁻⁵ M) was reduced by NaBH₄ (100 mM) in the presence of 90 µL AuMt (2 mg mL⁻¹). UV‑Vis monitoring at 660 nm yielded degradation percentages (%D). The rate constant K was derived from the Langmuir–Hinshelwood linear plot of ln(A/A₀) vs. time.

Results and Discussion

Synthesis and Antioxidant Characterization

The Mt extract displayed a prominent UV‑Vis peak at 280 nm, indicative of polyphenols. DPPH inhibition reached 50 % at 12.5 µg mL⁻¹, comparable to vitamin C and catechins. Total polyphenol content was 425 mg g⁻¹ gallic acid equivalents, confirming the extract’s reducing capacity.

AuNP Morphology and Size Distribution

TEM images revealed AuMt1 particles averaging 40 nm with diverse shapes, while AuMt2 particles averaged 150 nm and were more spherical. DLS measurements in water yielded hydrodynamic diameters of 38 nm (AuMt1) and 150 nm (AuMt2), with negative zeta potentials (≤ −30 mV) that favor colloidal stability. In s‑DMEM, both systems retained stability (ζ ≈ −25 mV) but exhibited modest size increases due to protein corona formation (AuMt1: +34 nm; AuMt2: +43 nm).

Surface Chemistry

FTIR spectra showed characteristic O–H stretching (≈ 3250 cm⁻¹) and C=O/C–O vibrations (≈ 1705 cm⁻¹) for both AuMt samples, confirming adsorption of tannins and flavonoids. XPS analysis indicated complete reduction of Au³⁺ to Au⁰ (Au 4f₇/₂ at 84.9 eV). The C 1s spectra displayed C=O, C–O, and C–C contributions, with AuMt2 exhibiting a higher C–O fraction, consistent with greater surface oxidation of phenolic groups during synthesis.

Cytotoxicity and Cellular Uptake

MTT assays showed a moderate decline in viability (10–30 %) at 24–48 h for concentrations ≥ 50 µg mL⁻¹, without a clear dose‑dependent trend. Live/Dead staining confirmed high cell viability at 50 µg mL⁻¹. CLSM revealed cytoplasmic dispersion of AuMt1 and perinuclear localization of AuMt2 after 24 h; 3‑D reconstructions confirmed absence of nuclear penetration, suggesting minimal genotoxicity. Quantitative fluorescence intensity indicated a ~3:1 uptake ratio for AuMt1 over AuMt2, attributable to smaller size, higher zeta potential, and thinner protein corona.

Catalytic Performance

AuMt1 achieved 50 % MB degradation in 190 s with a rate constant K = 8.24 × 10⁻³ s⁻¹, outperforming AuMt2 (K = 3.54 × 10⁻³ s⁻¹). The size‑dependent activity aligns with increased surface area of smaller nanoparticles.

Conclusions

We demonstrate a scalable, room‑temperature green synthesis of AuNPs using Mimosa tenuiflora bark extract. By tuning the precursor/extract ratio, particle size can be precisely controlled. AuMt nanoparticles exhibit favorable colloidal stability, moderate cytotoxicity, efficient cellular uptake without nuclear entry, and robust catalytic degradation of methylene blue. Their intrinsic fluorescence at low excitation power further enhances their potential as fluorescent probes and drug delivery vehicles. Future work will focus on mitigating toxicity through surface engineering and exploring biomedical applications.

Availability of Data and Materials

All datasets are presented in the main paper.

Abbreviations

ANOVA

Analysis of variance

AuMt

Colloids formed by AuNPs and Mt molecules

AuNPs

Gold nanoparticles

CLSM

Confocal laser scanning microscopy

DLS

Dynamic light scattering

DMEM

Dulbecco’s modified Eagle medium

DMSO

Dimethyl sulfoxide

DPPH

2,2‑Diphenyl‑1‑picrylhydrazyl

EDS

Energy dispersive X‑ray spectroscopy

FTIR

Fourier transform infrared spectroscopy

HAuCl₄

Tetraclo­ra­tic acid

HRTEM

High‑resolution TEM

HUVEC

Human umbilical vein endothelial cells

MB

Methylene blue

Mt

Mimosa tenuiflora

MTT

3-(4,5‑Dimethylthiazol‑2‑yl)-2,5‑diphenyltetrazolium bromide

NaBH₄

Sodium borohydride

PBS

Phosphate‑buffered saline

PDI

Polydispersity index

PMT

Photomultiplier tube

ROI

Region of interest

SD

Standard deviation

SPR

Surface plasmon resonance

TEM

Transmission electron microscopy

UV‑Vis

Ultraviolet‑visible spectroscopy

XPS

X‑ray photoelectron spectroscopy

XRD

X‑ray diffraction