Rapid, Green Synthesis of PDMAEMA‑Based Silver‑Containing Copolymer Micelles with Enhanced In Vitro Antibacterial Efficacy

Introduction

Traditional antimicrobials are increasingly challenged by the rapid emergence of multidrug‑resistant pathogens, a threat ranked among the top three by the World Health Organization [1–5]. Silver nanoparticles (AgNPs) have long been prized for their broad‑spectrum activity, low mammalian toxicity, and limited propensity to induce resistance [6–10]. Their antimicrobial mechanism involves membrane disruption, intracellular protein denaturation, and interference with DNA replication [11–13]. However, AgNPs’ high surface energy drives aggregation, undermining their efficacy and stability. Encapsulation within polymer matrices—especially micelles—offers a practical solution by providing steric hindrance and controlled release [14–19]. Common stabilization strategies rely on external reductants (e.g., NaBH₄, ascorbic acid) that introduce toxic by‑products and complicate purification [20–25]. In contrast, amine‑rich polymers can simultaneously coordinate Ag⁺ and reduce it in situ, obviating external reducing agents [26]. Yet, the influence of polymer topology on AgNP nucleation, growth, and antibacterial performance remains underexplored, particularly for star‑shaped architectures that may afford enhanced steric shielding. This work addresses that gap by developing linear and four‑arm star PDMAEMA‑based triblock copolymer micelles, evaluating their ability to generate monodisperse AgNPs in situ, and assessing antibacterial activity and cytocompatibility.

Material and Methods

Materials

All reagents were used as received unless otherwise noted. Key monomers included DMAEMA (>98 %), HEMA (99 %), and PEGMA (Mn = 300 Da, 99 %) (Aldrich). Pentaerythritol, EBiB, and BIBB were purified by alumina column chromatography. Silver nitrate (AgNO₃, 99.9 %) was used as the Ag⁺ source.

General Characterization and Instrumentation

¹H NMR spectra were recorded on a Bruker 400 MHz spectrometer in CDCl₃ or D₂O at 25 °C. FTIR spectra were obtained with a Nicolet Nexus spectrophotometer (4000–400 cm⁻¹, 32 scans, 8 cm⁻¹ resolution). Zeta potentials were measured on a Malvern Zetasizer Nano S. TEM images were acquired on a FEI Tecnai‑G20 (200 kV). UV‑Vis spectra were recorded on a Shimadzu UV‑2450. Thermogravimetric analysis (TGA) employed a NETZSCH STA409PC under nitrogen (25–600 °C, 10 °C min⁻¹).

Synthesis of PDMAEMA‑b‑PHEMA‑b‑PPEGMA

Polymerization was performed via ARGET ATRP in toluene (70 °C) with CuBr₂/HMTETA as catalyst, Sn(Oct)₂ as reducing agent, and EBiB as initiator. DMAEMA, HEMA, and PEGMA were sequentially polymerized to yield a triblock copolymer with Mn ≈ 19 kDa (DMAEMA), 12 kDa (PHEMA), and 25 kDa (PPEGMA). The product was precipitated in cold n‑hexane, filtered, and dried under vacuum (48 h, 35 °C).

Synthesis of (PDMAEMA‑b‑PHEMA‑b‑PPEGMA)₄

A four‑arm star initiator was prepared by esterifying pentaerythritol with BIBB in THF/TEA, yielding a tetrabrominated core. Subsequent ARGET ATRP, following the same conditions as the linear copolymer, produced the star‑shaped triblock copolymer. Yield and purity were confirmed by ¹H NMR and SEC.

Preparation of AgNPs Using Linear or Star‑Shaped Copolymer Micelles

Copolymer solutions (4.8 mM DMAEMA) were stirred in acetone (5 mL) for 4 h, then diluted with water (20 mL) and stirred overnight to form micelles. AgNO₃ (220 µL, 48 mM) was added dropwise, and the mixture was stirred at 25 °C in the dark for 48 h. Micelles stabilized with AgNPs were isolated by centrifugation, freeze‑dried, and stored at –20 °C.

Antibacterial Assay

Growth inhibition against E. coli DH5α was assessed by mixing 1 × 10⁵ CFU mL⁻¹ bacteria with serial dilutions of micelles or micelles‑stabilized AgNPs. After 16 h incubation at 37 °C, optical density at 600 nm (OD₆₀₀) was measured with a microplate reader. Six replicates were performed for each concentration.

Cell Viability Evaluation

HepG2 cells were seeded in 96‑well plates (1 × 10⁴ cells mL⁻¹) and cultured 24 h. Test solutions (10–400 µg mL⁻¹) were added for 24 h, followed by MTT assay (4 h incubation with 5 mg mL⁻¹ MTT, 200 µL DMSO). Absorbance at 570 nm quantified cell viability. Experiments were performed in sextuplicate.

Dissipative Particle Dynamics Simulation

Coarse‑grained DPD simulations (Materials Studio 8.0) modeled copolymer and AgNP interactions at a PDMAEMA/Ag⁺ ratio of 1:1. A 30 × 30 × 30 rₙ³ box with 100 000 steps (0.05 ns) captured micelle self‑assembly and AgNP encapsulation. Interaction parameters were taken from previous work [31, 32].

Statistical Analysis

Data were analyzed by two‑sample Student’s t‑test (unequal variance); p < 0.05 was considered significant.

Results and Discussion

Synthesis and Characterization of Linear/Star Copolymers

Both linear and star triblock copolymers were successfully synthesized by ARGET ATRP. ¹H NMR confirmed the expected block composition: PDMAEMA₁₉.₃–b–PHEMA₁₂.₅–b–PPEGMA₂₄.₆ and (PDMAEMA₅.₀–b–PHEMA₅.₆–b–PPEGMA₅.₀)₄. The star core (pentaerythritol) was fully brominated, as evidenced by the disappearance of the –CH₂–OH signal and the appearance of new bromine‑bearing methylene peaks.

Preparation and Characterization of the Linear/Star Copolymer Micelles Stabilized AgNPs

In aqueous media, the triblock copolymers self‑assembled into core–shell micelles. The PDMAEMA core binds Ag⁺ via tertiary amine coordination and reduces it in situ, generating Ag atoms that nucleate into nanoparticles. The hydrophilic PHEMA/PPEGMA shell imparts steric protection, preventing aggregation. DPD simulations showed that Ag beads rapidly localize within micelle cores, confirming efficient encapsulation. UV‑Vis spectra displayed a surface plasmon resonance (SPR) peak at 437 nm for linear micelles and 422 nm for star micelles, indicating monodisperse, near‑spherical AgNPs. TEM images revealed particle diameters of 11.1 nm (linear, 1:1 ratio) and 3.7 nm (star, 1:1 ratio), expanding to 25.7 nm and 6.4 nm, respectively, at a 6:1 ratio. Zeta potentials ranged from +15 mV to +23 mV, increasing with Ag content, reflecting successful surface coverage.

Stability of the Linear/Star Copolymers Micelles Stabilized AgNPs

UV‑Vis monitoring over 30 days showed negligible shift for star micelles even after 1:3 dilution, whereas linear micelles exhibited a modest red‑shift, suggesting less robust colloidal stability. TGA indicated onset degradation temperatures of 213 °C for star micelles and 172 °C for linear micelles with AgNPs, underscoring superior thermal resilience of the star architecture.

Antibacterial Activity and Cell Viability

OD₆₀₀ measurements revealed concentration‑dependent inhibition of E. coli growth by micelles‑stabilized AgNPs. Star micelles achieved >90 % inhibition at 50 µg mL⁻¹, whereas linear micelles required 200 µg mL⁻¹ for comparable effect, attributable to smaller particle size and higher surface area. Cytotoxicity assays with HepG2 cells showed viability above 90 % across all tested concentrations, confirming low mammalian toxicity.

Conclusion

This study demonstrates that PDMAEMA‑based linear and star triblock copolymer micelles can be used as dual‑function platforms—simultaneously reducing and stabilizing silver nanoparticles—via a single, green synthetic step. Star‑shaped micelles produce smaller, more stable AgNPs and exhibit superior antibacterial activity while maintaining excellent biocompatibility. The topology‑tuned approach offers a versatile strategy for designing advanced antimicrobial nanomaterials.

Availability of Data and Materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

AgNPs:

Silver nanoparticles

DMAEMA:

2‑(dimethylamino) ethyl methacrylate

HEMA:

2‑hydroxyethyl methacrylate

PEGMA:

Poly(ethylene glycol) methyl ether methacrylate

CuBr₂:

Cupric bromide

¹H NMR:

Proton nuclear magnetic resonance

FTIR:

Fourier‑transform infrared spectroscopy

KBr:

Potassium bromide

UV‑Vis:

Ultraviolet‑visible spectroscopy

MTT:

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

HepG2:

Liver hepatocellular carcinoma cell line

DPD:

Dissipative particle dynamics

SPR:

Surface plasmon resonance

XRD:

X‑ray diffraction