Intracellular bio-synthesizing Gold Nanoparticles using bacterial systems and their applications in photothermal therapy (A green approach)

Background: Conventional cancer therapies are heavily constrained by systemic toxicity, off-target side effects, and poor deep-tissue penetration due to the highly heterogeneous tumor microenvironment (TME). Although gold nanoparticles (AuNPs) possess exceptional localized surface plasmon resonance (LSPR) properties ideal for photothermal therapy (PTT), their clinical translation is bottlenecked by inconsistent passive accumulation and chemical toxicity arising from conventional synthetic surfactants. Objective: This study developed a sustainable, biocompatible bio-hybrid platform utilizing the probiotic Escherichia coli Nissle 1917 (EcN) as an autonomous, tumor-homing living carrier integrated with AuNPs synthesized via an eco-friendly intracellular green chemistry approach. Methods: EcN cultures were prepared in Luria-Bertani (LB) medium and subsequently exposed to a 1 mM chloroauric acid (HAuCl4) metallic precursor. A facile, room-temperature green synthesis protocol was established by utilizing ascorbic acid (Vitamin C) as a non-toxic co-reductant to drive a seed-mediated crystallization onto intra-bacterial metallic seeds. The bio-hybrid system (EcN::AuNPs) was characterized via visual inspection, UV-Vis spectroscopy, and in vitro PTT assays under continuous-wave near-infrared (NIR) laser irradiation. Results: Visual confirmation showed an irreversible color shift from pale yellow to a deep dark-brownish hue, serving as a direct diagnostic indicator of successful Au3+ to Au0 reduction. UV-Vis spectroscopic analysis revealed a distinct, broadened surface plasmon resonance (SPR) band centered at 560nm, indicating the formation of stabilized, isotropic gold nanostructures (30-60nm) subjected to near-field electromagnetic coupling within the complex intracellular compartments (periplasm and cytoplasm). Biocompatibility assays demonstrated a significant collapse in cell viability from 10^8CFU/mL down to 10^3CFU/mL, driven by a combination of trivalent gold heavy-metal toxicity, mechanical disruption of the peptidoglycan wall from spatial confinement, and severe metabolic/oxidative stress. Nevertheless, the remaining surviving population maintained required active chemotactic capabilities, while the lysed bacterial matrix provided a natural protein capping layer that prevented nanoparticle aggregation. Under 5 minutes of continuous NIR laser irradiation, the EcN::AuNPs platform exhibited exceptional concentration-dependent hyperthermia kinetics, triggering a drastic thermal escalation from 20 C to an absolute peak of 75.4 C, vastly outperforming all baseline control groups (pure EcN, bare HAuCl4, and standalone ascorbic acid). Conclusion: The engineered EcN::AuNPs bio-hybrid platform represents a highly efficient and biosafe "bacteriobot" paradigm. By shifting the SPR footprint and leveraging the native bacterial cell envelope for colloidal stability, this system achieves an optimal equilibrium between active biological tumor targeting and rapid, localized photothermal ablation, offering a promising alternative for targeted oncological interventions.