Engineering bioactive surfaces on nanoparticles and their biological interactions

Metal nanoparticles possess novel physicochemical properties enabling applications in targeted delivery, sensing, imaging, antimicrobial coatings, wound healing, SERS, photothermal therapy, and implants. Conventional chemical reduction routes can raise stability and safety concerns. Biomolecules such as amino acids, polymers, sugars, and plant extracts provide greener reducing/stabilizing alternatives to synthesize metal nanoparticles. Upon exposure to biological environments, nanoparticles acquire a biomolecule/protein corona that defines their biological identity and governs cellular interactions, internalization, biodistribution, and toxicity. This study aims to engineer defined bioactive coronas on Au and Ag nanoparticles using biological molecules (tyrosine, tryptophan, isonicotinylhydrazide, epigallocatechin gallate, curcumin) that can both reduce metal ions and cap the resulting particles. The hypothesis is that these coronas will impart specific biological activities while minimizing toxicity. The work evaluates physicochemical properties, radical scavenging capacity, peroxidase-like nanozyme activity, cytotoxicity, and genotoxicity, and examines interactions with fibroblasts and macrophages.

Green and biomolecule-assisted syntheses have employed amino acids, biocompatible polymers, sugars, and diverse plant/biological extracts (algae, fungi, bacteria, viruses) to form metal nanoparticles with improved biocompatibility. Au and Ag nanoparticles are attractive due to ease of preparation, biocompatibility (Au), antimicrobial properties (Ag), cost-effectiveness, and modifiable surface stability. The nanoparticle protein/biomolecule corona concept is well established and crucial for biological identity and interactions. Prior reports show amino acids like tyrosine/tryptophan reduce metal ions via phenol/indole groups; INH hydrolyzes under alkaline conditions to hydrazine as a reducing agent; EGCG polyphenols and curcumin’s phenolate/enolate anions can reduce metal ions with pH-dependent solubility/stability. Literature also documents nanoparticle antioxidant (radical scavenging) and nanozyme peroxidase-like behaviors, often size- and composition-dependent, and highlights inconsistencies and gaps in genotoxicity assessments of nanomaterials.

Materials: HAuCl4, AgNO3, KOH, tyrosine (Tyr), tryptophan (Trp), isonicotinylhydrazide (INH), epigallocatechin gallate (EGCG), curcumin (Cur), ABTS, TMB, H2O2, K2S2O8, trypsin, EtBr, DMSO, Triton X-100, sodium sarcosinate, EDTA, NaCl, methanol, Tris buffers, Na-EDTA, NaOH (Sigma Aldrich; >99% unless stated). Milli-Q water (18.2 MΩ·cm). Cells: m5S mouse skin fibroblasts (RIKEN); cultured in α-MEM with 10% FBS, 2 mM glutamine, penicillin/streptomycin. RAW 264.7 macrophages (Riken BRC); RPMI-1640 with 10% FBS and antibiotics. Assay kits: CCK-8, LDH (Dojindo) and DNA extraction (Qiagen). Synthesis of nanoparticles: In 100 ml aqueous 1 mM KOH, add 0.1 mM of reducing/stabilizing agent (Tyr, Trp, INH, EGCG, or Cur). Under heating with stirring, add 0.1 mM AuCl4− or Ag+ to form Au or Ag nanoparticles. Concentrate solutions to ~50 ml to increase nanoparticle concentration. Particles remained stable, indicating capping by the reducing agent. Samples denoted as Au_Tyr, Au_Trp, Au_INH, Au_EGCG, Au_Cur, and Ag_Tyr, Ag_Trp, Ag_INH, Ag_EGCG, Ag_Cur. Physicochemical characterization: UV–Vis spectroscopy (Shimadzu UV-1800; 2 nm resolution) for SPR. FTIR (Bruker; 4 cm−1 resolution) for functional groups. TEM (Hitachi HT-7700) for morphology; samples drop-cast on copper grids and dried; high-resolution imaging at 100k. DLS and zeta potential (Malvern Zetasizer Ver. 7.10) for hydrodynamic size and surface charge. AAS on aqua regia-digested samples for metal concentration. Radical scavenging capacity (RSC): ABTS•+ generated by mixing 7.4 mM ABTS with 2.45 mM K2S2O8; incubate 16 h in dark. Dilute to absorbance 0.67 ± 0.02 at 734 nm in ethanol. React nanoparticles at 0.1, 0.2, 0.6, and 1.2 ppm metal concentrations with ABTS•+; measure decrease at 734 nm. %RSC = [(Ac − As)/Ac] × 100. Controls included pristine reducing agents and precursor metal ions. Peroxidase nanozyme activity: Incubate nanoparticles with TMB and H2O2 in dark at room temperature; measure absorbance at 650 nm over time. Compare Au vs Ag and effect of surface corona. Controls with reducing agents and free ions. Cell viability/cytotoxicity (m5S fibroblasts): Seed 1×10^5 cells/ml; incubate to confluence. Treat with nanoparticles at 0.5, 1.0, 5.0 µg/ml (metal content) for 16 h. Wash; add CCK-8; incubate 4 h; read 490 nm for viability. For cytotoxicity, measure LDH release: seed 1×10^4 cells/well; treat as above; collect medium; add reaction mix; incubate 30 min dark; read 490 nm. Controls run in parallel. Cytotoxicity (RAW 264.7): Seed in 96-well plates at 1×10^6 cells/ml; treat with 0.5, 1.0, 5.0 µg/ml for 12 h; MTT assay; read 570 nm. Genotoxicity (comet assay): Treat m5S cells in 24-well plates with nanoparticles (0.5, 1.0, 5.0 µg/ml) for 24 h. Harvest with 0.5% trypsin-EDTA; embed cells (1×10^6/ml) in 0.25% low-melting agarose on slides pre-coated with 1% agarose. Lyse (100 mM EDTA, 2.5 M NaCl, 100 mM Tris, 1% sarcosinate, 1% Triton X-100, 10% DMSO; pH 10.0) at 4°C for 1 h. Alkaline treatment (1 mM Na-EDTA, 300 mM NaOH; pH 13.0). Electrophoresis 30 min at 4°C. Neutralize (0.5 M Tris-HCl, pH 7.5), fix with methanol, stain with EtBr. Image 100 cells/slide; analyze %DNA in tail with Komet software. DNA ladder assay: Treat m5S cells (3×10^4/well) 24 h with single-dose panels: Au and Ag nanoparticles (various doses: e.g., Au_Tyr 1 µg/ml; Ag_Tyr 0.5 µg/ml; Au_INH 1 µg/ml; Au_EGCG 5 µg/ml; Au_Cur 5 µg/ml; Ag and Au salts 0.1 µg/ml; pristine reducing agents 0.1–10 µg/ml). Isolate DNA (Qiagen); run on 1.5% agarose with EtBr at 50 V/cm for 60 min; visualize under UV; compare to 1 Kb ladder.

- Successful one-pot synthesis of Au and Ag nanoparticles using Tyr, Trp, INH, EGCG, and Cur as dual reducing/stabilizing agents; stable after concentration and on storage (>1.5 years in DI water). - UV–Vis SPR confirmed nanoparticle formation with single plasmon peaks indicative of predominantly spherical morphology: Au_Tyr ~520 nm, Au_Trp ~528 nm, Au_INH ~532 nm, Au_EGCG ~503 nm, Au_Cur ~531 nm; Ag_Tyr ~406 nm, Ag_Trp ~417 nm, Ag_INH ~395 nm, Ag_EGCG ~406 nm, Ag_Cur ~412 nm. - TEM showed spherical to quasi-spherical particles; DLS hydrodynamic radii and zeta potentials (mean ± SD): Au_Tyr 58.26±0.68 nm, −12.80±0.34 mV; Au_Trp 21.60±0.96 nm, −27.63±0.65 mV; Au_INH 98.86±1.32 nm, −17.96±0.49 mV; Au_EGCG 41.39±1.47 nm, −32.40±0.81 mV; Au_Cur 56.80±0.83 nm, −24.50±0.27 mV; Ag_Tyr 38.14±0.93 nm, −41.10±0.18 mV; Ag_Trp 50.04±0.81 nm, −33.76±0.54 mV; Ag_INH 11.65±1.12 nm, −39.83±0.45 mV; Ag_EGCG 33.14±0.72 nm, −27.56±0.39 mV; Ag_Cur 83.22±0.69 nm, −7.48±0.63 mV. - FTIR identified characteristic functional group vibrations from the capping molecules on nanoparticle surfaces (e.g., INH N–H stretch ~3277/3263 cm−1; C=O ~1634 cm−1; Tyr/Trp carbonyl/carboxylate ~1635–1639 cm−1), confirming engineered surface coronas. - Radical scavenging capacity (ABTS assay) increased with dose (0.1–1.2 ppm). At 1.2 ppm: Au_Tyr 4.88%, Au_Trp 1.41%, Au_INH 0.67%, Au_EGCG 2.46%, Au_Cur 2.31%; Ag_Tyr 3.93%, Ag_Trp 6.20%, Ag_INH 7.78%, Ag_EGCG 11.06%, Ag_Cur 7.56%. Ordering: Au: Tyr > EGCG > Cur > Trp > INH; Ag: EGCG > INH > Cur > Trp > Tyr. Ag generally outperformed Au in RSC at equal metal dose. - Peroxidase-like nanozyme activity (TMB/H2O2): Ag nanoparticles generally showed higher activity than Au at equal metal concentration (0.6–1.2 ppm), consistent with literature; exception: Cur systems where Au_Cur > Ag_Cur at 1.2 ppm, indicating surface corona effects beyond core composition. Among Ag systems, surface-dependent ordering observed (e.g., EGCG > Tyr > INH > Trp > Cur). - m5S fibroblast viability (16 h): Dose-dependent decreases. Examples: Au_Tyr 86.27%, 85.60%, 82.63% at 0.5, 1.0, 5.0 µg/ml; Au_Trp 89.87%, 88.23%, 84.27%; Ag_Trp 92.8% to 81.27% (0.5 to 5.0 µg/ml). At 5.0 µg/ml: Au_Tyr 82.63%, Au_Trp 83.57%, Ag_Tyr 84.27%, Ag_Trp 81.27%. Strong corona and core effects: Au_Cur 61.37% vs Ag_Cur 80.50% at 5.0 µg/ml; Au_INH 68.10%; Au_EGCG 79.10%. - Cytotoxicity (LDH) mirrored viability trends; pristine reducing agents and free metal ions assessed separately supported the role of nanoparticle formulation in modulating toxicity. - RAW 264.7 macrophages: Tyr-, EGCG-, and Cur-synthesized nanoparticles increased viable cell numbers at 1.0 and 5.0 µg/ml, indicating possible stimulatory effects dependent on corona. - Genotoxicity (comet assay, 24 h): Generally low DNA damage across systems; only Au_Tyr at 5.0 µg/ml showed notable (~4-fold vs control) increase in %DNA tail intensity. Nanoparticles exhibited reduced genotoxicity compared to corresponding free metal ions and pristine INH. - DNA ladder assay: No apoptotic laddering with Au or Ag nanoparticles; clear fragmentation observed with pristine INH and free metal ions, but not when INH was incorporated as a corona (Au_INH, Ag_INH), indicating mitigation of genotoxic effects through nanoparticle formulation.

The study demonstrates that engineering biomolecule-derived coronas on Au and Ag nanoparticles modulates their physicochemical behavior and biological interactions. Surface coronas from Tyr, Trp, INH, EGCG, and Cur confer stability (negative zeta potentials, steric effects) and impart distinct functional activities such as radical scavenging and peroxidase-like catalysis, with performance contingent on both surface chemistry and metal core. Biological assays reveal that cytotoxicity and genotoxicity can be minimized by appropriate corona selection, and potentially harmful components (e.g., INH, free Au/Ag ions) exhibit mitigated effects when integrated into nanoparticle constructs. Differences between Au and Ag cores at equal metal doses underscore core-dependent contributions, while variations among surface coronas highlight the importance of nano-bio interface design. The size distribution differences may further influence activity and must be considered when attributing effects to specific properties.

A single-step, biomolecule-mediated strategy was established to synthesize Au and Ag nanoparticles with tailored bioactive surface coronas using amino acids (Tyr, Trp), an antibiotic (INH), and plant polyphenols (EGCG, Cur). The engineered coronas controlled colloidal stability, antioxidant capacity, nanozyme-like peroxidase activity, and cellular interactions, enabling limited cytotoxicity and genotoxicity relative to free ions and pristine reagents. The complementary roles of the metallic core and the surface corona determine biological responses. These findings provide avenues to rationally engineer nanoparticle surfaces and compositions for biomedical applications. Future work should investigate interactions with biological fluids and perform in vivo studies to better predict and optimize activities.

- Experiments were conducted in vitro; no in vivo validation was performed. - Interactions with complex biological fluids were not directly assessed; authors note the need to study corona formation in such environments to predict in vivo behavior. - Nanoparticle samples exhibited differing hydrodynamic radii, which may confound direct attribution of biological effects solely to surface chemistry or core composition. - Single or limited time points and dose ranges were used for some assays, and mechanistic pathways were not exhaustively dissected.