Green and chemical synthesis of PEGylated ginger gold nanoparticles for neuro-nanomedicine applications

Abstract

Background Gold nanoparticles (AuNPs) are promising therapeutic-delivery agents for neuro-nanomedicine, but their cytotoxicity from conventional chemical synthesis remains a challenge. Green synthesis using ginger extract offers a safer, neuroprotective alternative. Methods PEGylated AuNPs were prepared through chemical (CS) and green (GS) methods with and without ginger extract. They were analyzed using SEM, TEM, DLS, and zeta potential. Encapsulation efficiency (EE), loading capacity (LC), drug release profile (pH 7.4, 37 °C), and cytotoxicity in PC12 cells (MTT assay, IC₅₀) were measured. Results Spherical nanoparticles (14–19 nm) were confirmed. GS-PEG-Ginger-AuNPs exhibited superior EE (80.7% vs. 61.8%) and LC (90.7% vs. 77.9%) compared to the CS counterpart. The GS formulation also showed a significantly higher cumulative drug release (~ 85% vs. ~60% at 96 h), with release kinetics indicative of anomalous transport (n = 0.52) versus Fickian diffusion (CS, n = 0.45). Critically, GS formulations were highly biocompatible (IC₅₀ > 100 µg/mL; >70–80% cell viability at 100 µg/mL), whereas CS formulations were toxic (IC₅₀ ≈ 68.7–104.4 µg/mL). Conclusion The green-synthesized PEG-Ginger-AuNPs demonstrate enhanced drug loading, sustained release, and exceptional neuronal biocompatibility, underscoring their strong potential as a translatable and safe nanoplatform for clinical neuro-nanomedicine.

Introduction

The treatment of neurological disorders such as Alzheimer’s and Parkinson’s disease is severely hindered by the blood-brain barrier (BBB). The BBB inhibits the unimpeded flow of therapeutic molecules, leading to poor brain bioavailability and systemic toxicity¹. Nanoscale drug delivery systems in the form of gold nanoparticles (AuNPs) are promising. AuNPs are highly desirable in neuro-nanomedicine due to their tunable size, ease of surface functionalization, biocompatibility, and capacity for crossing the BBB^2,3.

Conventional chemical synthesis of AuNPs, such as the Turkevich process, provides high control over size and shape but involves the use of toxic reducing and capping agents (e.g., trisodium citrate). Residual chemical precursors can make biocompatibility an issue, thus posing a big challenge for medical purposes^4–7. To this end, green synthesis through plant extracts has emerged as a greener and safer alternative. These methods leverage the inherent reducing and capping capacity of phytochemicals, which act as natural stabilizers to prevent particle agglomeration and provide enhanced biocompatibility and other bioactivity⁸.

The importance of these natural capping agents cannot be overstated. Unlike synthetic ligands, phytochemicals in plant extracts form a biocompatible stabilizing corona around nanoparticles. This corona is inherent in nature and often therapeutically active^9,10. This dual function as synthesis reagent and therapeutic payload delivery agent simplifies the production process and optimizes the usefulness of the resulting nanomaterial. Zingiber officinale (ginger) extract is particularly suitable as a dual reducing and stabilizing agent because it contains abundant bioactive constituents such as 6-gingerol, 8-gingerol, 10-gingerol, shogaols, and zingerone. These phenolic compounds exhibit strong antioxidant, anti-inflammatory, and anti-apoptotic activities that help mitigate oxidative stress and neuroinflammation, two critical mechanisms involved in neuronal degeneration^11–13. Recent studies have shown that 6-gingerol and shogaols preserve neuronal viability and cognitive function following ischemic or traumatic brain injury^14–17. The combination of reducing power and intrinsic neuroprotective activity makes ginger extract an effective choice for green synthesis of gold nanoparticles designed for central nervous system delivery Green-synthesized AuNPs are also gaining prominence for central nervous system drug delivery¹⁸. Moosavy et al. (2023) reported enhanced neuronal biocompatibility with Mentha-mediated AuNPs¹⁹. Green-synthesized nanoparticles, such as through Mentha longifolia leaf extracts, also display superb antioxidant and antibacterial properties²⁰. Such bactericidal AuNPs are also hopeful for the management of CNS infections like bacterial meningitis¹⁸.

PEGylation imparts a hydrophilic “stealth” surface that improves colloidal stability and circulation time, facilitating BBB penetration^21,22. When combined with a ginger-derived bioactive corona, the resulting nanocarrier integrates natural antioxidant and anti-inflammatory properties with the extended systemic stability of engineered nanoparticles.

While the current reports have contrasted green and chemical routes to ginger-gold nanoparticles, they were primarily focused on initial characterization and antimicrobial action²³. There is still a critical gap in knowledge regarding how the synthesis route impacts the development of a multifunctional nanocarrier for challenging applications like neuro-nanomedicine. Nanoparticles for effective brain delivery must not only possess natural bioactivity but also engineered properties like increased drug loading, controlled release, and increased neuronal biocompatibility.

This study is therefore designed to provide a novel, integrated comparison that moves beyond simple synthesis route contrasts. We introduce and evaluate a dual-functionalized nanoplatform. This platform uniquely combines the neuroprotective properties of a ginger-derived bioactive corona with the stealth and stability benefits of a PEG coat. We directly contrast two fabrication paradigms: a single-vessel green synthesis, where ginger acts simultaneously as a reducing and capping agent as well as a therapeutic payload, against a conventional chemical reduction with post-synthesis ginger loading, with both routes subsequently PEGylated. Our work is distinctly differentiated from prior reports by its comprehensive focus on key neuro-nanomedicine criteria, including encapsulation efficiency, sustained release profiles, and neuronal cytocompatibility in PC12 cells, to explicitly determine which synthesis strategy yields a superior and safer nanocarrier with high potential for neurological therapy.

Materials and methods

Materials and reagents

Gold(III) chloride trihydrate (HAuCl₄·3 H₂O, ≥ 99.9%), trisodium citrate dihydrate (≥ 99.0%), and polyethylene glycol (PEG, average MW 20,000) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dried ginger (Zingiber officinale) powder was obtained from a local supplier. Ethanol (70% v/v) and sodium hydroxide (NaOH) pellets were of analytical grade. For cell culture, RPMI-1640 medium, fetal bovine serum (FBS), penicillin-streptomycin solution (10,000 U/mL and 10 mg/mL, respectively), 0.25% trypsin-EDTA, and phosphate-buffered saline (PBS, pH 7.4) were supplied by Gibco (Thermo Fisher Scientific, USA). The MTT reagent [3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide] and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich. Deionized water (DI water, 18.2 MΩ·cm) was obtained from a Milli-Q water purification system (Merck Millipore, USA) and used for all solution preparations.

Cell culture

The rat adrenal pheochromocytoma-derived cell line (PC12 line) is a widely utilized model for investigating neuronal function and differentiation, acquired from the Pasteur Institute in Tehran, Iran. The cells were cultivated in RPMI-1640 media enriched with 10% (v/v) heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were cultivated at 37 °C in a humidified environment with 5% CO₂ and subcultured at 80–90% confluence utilizing trypsin-EDTA.

Preparation of ginger extract

An ethanolic ginger extract was prepared by suspending 10 g dry ginger powder in 100 mL of 70% (v/v) ethanol. The mixture was shaken at room temperature for 24 h. The crude extract obtained was filtered through Whatman No. 1 filter paper and then sterilized using a 0.22 μm syringe filter. The ethanol was removed under reduced pressure at 50 °C using rotary evaporation. The remaining dried extract was weighed and reconstituted to the final stock concentration of 5 mg/mL in DI water. The stock solution was kept at 4 °C in an amber vial.

Preparation of PEGylated ginger extract-loaded gold nanoparticles

Green preparation of PEGylated AuNPs (GS-PEG-Ginger-AuNPs)

A 0.01 M HAuCl₄ solution was prepared by dissolving 0.3938 g of HAuCl₄·3 H₂O in 100 mL of deionized water. In summary, 50 mL of the 0.01 M HAuCl₄ solution was heated to 70 °C while being vigorously stirred magnetically. Subsequently, 10 mL of ginger extract solution (5 mg/mL) was incrementally introduced. The solution was agitated at 70 °C for 30 min, resulting in a color transition from pale yellow to ruby red, signifying the synthesis of AuNPs. Upon completion of the reaction, 10 mL of a 0.1% (w/v) aqueous solution of PEG-20,000 was included into the mixture. The mixture was stirred for a further 2 h at 70 °C to facilitate the conjugation of PEG onto the nanoparticle surface. The GS-PEG-Ginger-AuNPs product was subsequently allowed to cool to ambient temperature.

Chemical preparation of PEGylated AuNPs (CS-PEG-Ginger-AuNPs)

A 50 mL of 0.01 M HAuCl₄ solution was vigorously boiled with stirring. Subsequently, a 5 mL of a 1% (w/v) trisodium citrate solution was injected quickly into the boiling solution. The reaction was allowed to proceed until a typical wine-red coloration formed. The solution was left to cool to 40 °C. To the chemically synthesized AuNPs suspension, 5 mL of the ginger extract stock solution (5 mg/mL) was added, and then 10 mL of the 0.1% (w/v) PEG-20,000 solution was added, and stirring was done at 40 °C for 2 h for simultaneous loading of ginger extract and PEGylation.

Purification of nanoparticles

Both nano-formulations (GS-PEG-Ginger-AuNPs and CS-PEG-Ginger-AuNPs) were purified using the same procedure. The colloidal suspensions were centrifuged at 14,000 rpm for 30 min. The supernatant was systematically decanted and retained for the evaluation of loading efficiency. The pellet was rinsed twice by resuspending in deionized water and then centrifuged under the same conditions to eliminate weakly bound chemicals. The pure nanoparticle pellets were re-suspended in 5 mL of phosphate-buffered saline (PBS, pH 7.4) for characterization and subsequent biological assays.

Characterization of nanoparticles

Physicochemical characterization

FTIR spectroscopy

The efficiency of PEGylation and immobilization of ginger phytochemicals onto the surface of the nanoparticle were verified using FTIR spectroscopy. The spectra were analyzed using a Bruker Alpha spectrophotometer (Germany). Samples were lyophilized and pressed into potassium bromide (KBr) pellets and scanned in the region of 400–4000 cm⁻¹. The common functional groups responsible for the covalent linkage of PEG chains and the occurrence of bioactive compounds of ginger extract were determined by this method.

Morphological analysis by SEM and TEM

The resulting PEGylated ginger–AuNPs size, morphology, and surface properties were characterized by high-resolution electron microscopy. SEM was performed on a TESCAN VEGA3 microscope at 20 kV accelerating voltage. SEM samples were prepared by depositing diluted aqueous nanoparticle suspension on a silicon wafer, allowing it to air-dry, and followed by sputter-coating using a thin gold layer for improved conductivity. Images were obtained with a working distance of 5.75 mm and 100,000× magnification, the optimal conditions to view surface topography and nanoparticle distribution without any charging artifacts. For closer examination of the metal core, TEM was performed on a JEOL JEM-1400Plus microscope at an accelerating voltage of 120 kV. A diluted drop of nanoparticle suspension was applied to a carbon-coated copper grid and air-dried and then imaged. The high-energy electron beam was transmitted through the ultrathin specimens at 200,000× magnifications provided high-resolution data on the distribution of core sizes, inner morphology, and crystallinity of the AuNPs.

DLS and zeta potential assessment

Hydrodynamic diameter, PDI, and zeta potential of the nanoparticles were measured by a Zetasizer Nano ZS (Malvern Instruments) at 25 °C. For each measurement, triplicate results are provided and expressed as the mean ± standard deviation.

Determination of drug encapsulation efficiency and loading capacity

The EE and LC of ginger extract in the nanoparticles were determined indirectly. All measurements were performed in triplicate (n = 3) from three independent synthesis batches. After the final centrifugation step in the purification process, the supernatant was collected. The concentration of unencapsulated ginger extract in the supernatant was measured spectrophotometrically by absorbance at 290 nm, characteristic of gingerols, using a pre-established standard calibration curve. The formulas used for calculation were as follows:

where W_{Initial drug} ​ is the total weight of ginger extract added, W_{Free drug in supernatant} ​ is the weight of free ginger extract in the supernatant, and W_{Nanoparticles}​ is the total weight of dry nanoparticle pellet.

In vitro drug release study

The release pattern of ginger extract from the PEGylated AuNPs was investigated in phosphate-buffered saline (PBS, pH 7.4) at 37 °C to simulate physiological conditions. A known amount of nanoparticle suspension was placed inside a dialysis bag (MWCO 12–14 kDa) and immersed into 100 mL of PBS under gentle shaking (150 rpm). At predetermined time points (0, 1, 2, 4, 8, 12, 24, 48, and 72 h), 2 mL aliquots of the release medium were taken out and replaced by an equal volume of freshly pre-warmed PBS to maintain sink conditions. Concentration of ginger extract released in the aliquants was measured by a fluorescence spectrophotometer using excitation/emission wavelength most favorable for ginger phytochemicals. The cumulative percentage of drug released was plotted as a function of time.

Kinetic modeling of release

The in vitro release profiles were plotted against different mathematical models to understand the release mechanism of ginger extract from PEGylated AuNPs. Percent cumulative release versus time data were modeled based on the following kinetic²⁴:

1. Zero-order model:

• 2.First-order model:

• 3.Higuchi model:

• 4.Korsmeyer-Peppas model:

• 5.Hixson-Crowell model:

• 6.Baker-Lonsdale model:

(This model is based on drug release from spherical matrices controlled by diffusion.)

For each model, the R² correlation coefficient was calculated. The best fit for the release kinetic description was used as the highest R² value model. For the Korsmeyer-Peppas model, the value of the release exponent n was interpreted to decide the specific transport mechanism (e.g., Fickian diffusion, Case-II transport, or anomalous transport). This served to provide invaluable information on whether or not the release was diffusion-controlled, matrix erosion-controlled, or both.

Cytotoxicity assay

The in vitro cytotoxicity of the PEGylated AuNP formulations was evaluated using the PC12 neuronal cell line, a widely used model for neurotoxicity and neuronal differentiation studies. The employment of PC12 cells was preferable due to their neuron-like phenotype and high sensitivity toward oxidative and inflammatory stress, rendering them a suitable system for determining the neuro-compatibility of nanomaterials. This investigation provides novel insights into the impact of green synthesis and PEGylation on nanoparticle biocompatibility and safety in neuronal environments. The assay included the following control samples¹: untreated cells (negative control)², treatment with pure, non-PEGylated chemically synthesized AuNPs (citrate-capped), and³ treatment with free ginger extract alone at equivalent concentrations to the ones used in the nanoparticle formulations.

PC12 cells were plated in 96-well plates with the density of 1 × 10⁴ cells per well and incubated for 24 h. Cells were exposed to various concentrations (1–100 µg/mL) of the suspensions of nanoparticles or controls for 72 h. Six replicates (n = 6) of each of the concentrations were assayed, and the entire assay was repeated with nanoparticles prepared from three different batches of synthesis. After the 72-hour incubation period with the nanoparticles, 20 µL of MTT reagent (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide) at a concentration of 5 mg/mL in PBS was added to each well. The plates were then incubated for 4 hours at 37 °C to allow formazan crystal formation. The medium was removed gently and the resulting formazan crystals dissolved with 150 µL of DMSO per well. Absorbance was read at 570 nm with a reference wavelength of 630 nm using a microplate reader. Cell viability was expressed as a percentage of the control cells that were not treated. Half-maximal inhibitory concentration (IC₅₀) was derived from dose-response curves using nonlinear regression analysis.

Statistical analysis

All quantitative experiments, including encapsulation efficiency, drug release profiles, and cytotoxicity assays, were performed with a minimum of three independent replicates (n ≥ 3). Data for the biological and loading/release assays are represented as mean ± standard deviation (SD). Physicochemical characterizations (DLS, zeta potential) are reported as single determinations from independent batches of synthesis. Statistical significance of biological assays was determined by one-way or two-way Analysis of Variance (ANOVA), appropriate for experimental design, followed by Tukey’s post-hoc test for comparison between multiple groups. GraphPad Prism software (version 8.4.0) was utilized for analysis. A p-value of < 0.05 (p < 0.05) was utilized to ascertain statistical significance.

Results and discussion

FTIR analysis

FTIR spectroscopy was employed to investigate the surface chemistry and functionalization of the synthesized AuNPs, i.e., successful capping with ginger extract phytochemicals and PEG. The CS-PEG-AuNPs, CS-PEG-Ginger-AuNPs, GS-PEG-AuNPs, and GS-PEG-Ginger-AuNPs spectra of the four formulations are shown in Fig. 1. The spectra contained characteristic absorption bands for PEG and ginger phytochemicals. A broad absorption band in the range of 3200–3600 cm⁻¹ was observed in all samples, and it corresponds to O–H and N–H stretching vibrations. It was highest and broadened in the CS-PEG-Ginger-AuNPs (red spectrum) due to the presence of multiple hydroxyl groups from phenolic compounds in the ginger extract (e.g., gingerols and shogaols) and terminal hydroxyl groups from PEG²³. Weak to moderate absorptions between 2800 and 3000 cm⁻¹ were assigned to aliphatic C–H stretching vibrations characteristic of methylene groups in PEG chains and aliphatic groups of ginger components²⁵.

Fig. 1.

FTIR spectra of CS-PEG-AuNPs (blue), CS-PEG-Ginger-AuNPs (red), GS-PEG-AuNPs (green), and GS-PEG-Ginger-AuNPs (yellow), illustrating surface functionalization

Within the fingerprint region, there were distinguishable peaks at 1501 cm⁻¹ and 1497 cm⁻¹ corresponding to aromatic C = C stretching or conjugated C = O vibrations. The peaks are more prominent in ginger-infused formulations (CS-PEG-Ginger-AuNPs and GS-PEG-Ginger-AuNPs), signifying the presence of aromatic phenolic compounds from ginger on the nanoparticle surface²⁶. An significant absorption was seen at 1294 cm⁻¹, indicating of C–O stretching in alcohols, aryl ethers, and/or C–N stretching vibrations. This peak signifies contributions from both PEG (ether linkages) and ginger phytochemicals (oxygenated groups). Relative intensity of this peak was greater in ginger-loaded samples, which points toward successful adsorption of phytochemicals²⁷. A sharp C–O–C stretching vibration of high intensity was observed in the range 1200–1000 cm⁻¹ characteristic of PEG, thus confirming the successful PEGylation of all the nanoparticle preparations²⁸. A band at 847.6 cm⁻¹ was discovered, indicative of out-of-plane C–H bending in substituted aromatic rings or = C–H bending in conjugated alkenes. The band, primarily in ginger-infused samples, also supports the adsorption of aromatic phytochemicals on AuNP surfaces²⁹.

FTIR spectra demonstrated significant disparities between chemically manufactured AuNPs and those produced via green synthesis. For CS-PEG-Ginger-AuNPs (red spectrum), O–H stretching band was broad and intense, while aromatic/conjugated vibrations at 1501–1497 cm⁻¹ were enhanced in comparison to GS-PEG-Ginger-AuNPs (yellow spectrum). This indicates higher richness or stronger ginger phytochemicals interaction in the chemically prepared samples, where ginger extract was loaded after citrate reduction²³. Conversely, GS-PEG-Ginger-AuNPs (yellow range), where ginger extract served as reducing and stabilizing agent prior to PEGylation, presented conventional ginger-related peaks but of relatively lower intensity. This is indicative of shifts in phytochemical direction or partial substitution by PEG during post-reduction conjugation. Both PEG-alone formulations (CS-PEG-AuNPs and GS-PEG-AuNPs) exhibited the typical PEG-specific bands (C–H stretching, C–O–C stretching, and CH₂ deformations) that reflect the capping of PEG^19,30. However, none of them indicated the strong aromatic and phenolic features of ginger-loaded samples, attesting to the participation of ginger extract in additional surface functionalization. In general, FTIR verifies successful ginger phytochemical loading and PEGylation of AuNPs synthesized by both chemical and green routes. The PEG-related bands (C–H and C–O–C stretching) define uniform PEG conjugation in all samples, while ginger-specific absorptions (O–H stretching, aromatic C = C, C–O, and aromatic bending) define effective phytochemical adsorption on the surface of nanoparticles. The differences observed between CS-PEG-Ginger-AuNPs and GS-PEG-Ginger-AuNPs reflect the effect of synthesis route on surface chemistry of nanoparticles. Chemical synthesis with subsequent ginger loading resulted in stronger signatures of phytochemical binding, while green synthesis with ginger extract as reducing agent produced weaker but consistent ginger-related features most likely due to different binding orientations or surface coverage.

In conclusion, FTIR analysis validated the effective dual functionalization of AuNPs with ginger phytochemicals and PEG. The spectrum variations between CS-PEG-Ginger-AuNPs and GS-PEG-Ginger-AuNPs underscore the crucial influence of the synthesis pathway on the surface chemistry and phytochemical characteristics of the final product.

SEM and TEM analysis

The size distribution and morphology of the produced nanoparticles were also studied by TEM and SEM (Fig. 2). Spherical morphology with homogeneous distribution were observed in all formulations CS-PEG-AuNPs, CS-PEG-Ginger-AuNPs, GS-PEG-AuNPs, and GS-PEG-Ginger-AuNPs. No aggregation was observed, which suggests successful PEGylation and stabilization of the nanostructures by both synthesis routes. SEM-based mean diameters were 14.79 ± 2.61 nm for CS-PEG-AuNPs, 15.45 ± 4.78 nm for CS-PEG-Ginger-AuNPs, 15.49 ± 3.90 nm for GS-PEG-AuNPs, and 19.17 ± 4.29 nm for GS-PEG-Ginger-AuNPs (Fig. 2C). TEM analysis, which provides high-resolution metal core imaging, showed consistently smaller sizes: 9.61 ± 0.81 nm (CS-PEG-AuNPs), 12.57 ± 1.57 nm (CS-PEG-Ginger-AuNPs), 11.02 ± 2.59 nm (GS-PEG-AuNPs), and 12.24 ± 1.23 nm (GS-PEG-Ginger-AuNPs) (Fig. 2D). The consistent rise in size in ginger-loaded preparations in both SEM and TEM evidence the efficient adsorption of ginger phytochemicals on the nanoparticle surface. The spherical morphology and non-aggregation in all the formulations demonstrate stabilization efficacies, accomplished through PEGylation and, in the case of GS-PEG-Ginger-AuNPs, in-situ capping ability of ginger phytochemicals upon synthesis.

Fig. 2.

Morphological and size characterization of PEGylated gold nanoparticle formulations. SEM and TEM images of CS-PEG-AuNPs, CS-PEG-Ginger-AuNPs, GS-PEG-AuNPs, and GS-PEG-Ginger-AuNPs showing spherical morphology and uniform dispersion (scale bars = 50 nm). Representative particle size measurements are indicated in SEM images. (C) SEM-derived size distribution and (D) TEM-derived size distribution of nanoparticles, revealing statistically significant size differences between formulations (*p < 0.05, **p < 0.01). Ginger incorporation and green synthesis moderately increased particle size, consistent with phytochemical-mediated stabilization

As a factor in neuro-nanomedicine, particle size is a critical feature that influences BBB penetration, neuronal cell endocytosis, and cytocompatibility. Particles in the range of 10–20 nm are considered optimal for neural uptake. This size range offers a favorable balance between cellular permeability and low cytotoxicity. The TEM-derived size distribution of all formulations (9–13 nm) is within this optimum window with relevance to future neuronal delivery. The slight size increase in ginger-conjugated groups could prove advantageous through bioactivity by added antioxidant and anti-inflammatory moieties by ginger phytochemicals³¹. Moreover, the greener GS-PEG-Ginger-AuNPs, although marginally larger in SEM profiles, had a good TEM size of around ~ 12 nm, indicating that green synthesis routes do not compromise biomedical utility. The secondary advantage of green synthesis is minimal risk of chemical contamination, which supplements translational safety protocols for neuro-nanomedicine.

As suggested from Fig. 2C,D SEM-derived profiles revealed statistically significant elevation of GS-PEG-Ginger-AuNPs compared to CS-PEG-AuNPs (p < 0.05). TEM results also revealed significant differences between CS-PEG-AuNPs and ginger-functionalized groups (p < 0.01), confirming ginger biomolecules’ function in determining nanoparticle size and surface topology. The collective findings support the hypothesis that green synthesis under ginger functionalization yields distinct nanoscale features with potential biological relevance²³.

Hydrodynamic size distribution and colloidal stability

DLS confirmed distinct hydrodynamic size distributions for ginger-functionalized samples (Fig. 3A,B). The CS-PEG-Ginger-AuNPs possessed a mean hydrodynamic diameter of 14.91 nm with the calculated PDI of 0.038, which corresponds to a moderately polydisperse distribution (Coefficient of variation (CV) = 19.5%). As opposed to this, the GS-PEG-Ginger-AuNPs exhibited a mean hydrodynamic diameter of 16.84 nm with a very low PDI of 0.0034 (CV = 5.8%), indicating a highly monodisperse system.

Fig. 3.

DLS and zeta potential analysis of ginger-functionalized PEGylated gold nanoparticles. (A) DLS size distribution of chemically synthesized CS-PEG-Ginger-AuNPs (mean hydrodynamic diameter = 14.91 nm, PDI = 0.038). (B) DLS size distribution of green-synthesized GS-PEG-Ginger-AuNPs (mean hydrodynamic diameter = 16.84 nm, PDI = 0.0034). (C) Zeta potential of CS-PEG-Ginger-AuNPs (− 14.01 mV) and (D) GS-PEG-Ginger-AuNPs (− 16.34 mV) confirming moderate surface charge stability. Together, the results highlight the narrower distribution and enhanced colloidal stability of green-synthesized nanoparticles compared to chemically synthesized counterparts

The sizes obtained using DLS reflect the hydrodynamic diameter, which includes the gold core, the surface coatings (PEG and ginger phytochemicals), and the attached solvation layer. This explains why DLS measurements are larger than the core diameters from TEM and the surface-influenced diameters from SEM for the two preparations. GS-PEG-Ginger-AuNPs possessed the highest hydrodynamic size (16.84 nm) and very low PDI (0.0034). This indicates that the green one-pot method facilitated the formation of a dense and homogeneous phytochemical corona on the surface of nanoparticles, demonstrating improved stability and monodispersity compared to the chemical path of synthesis, in which ginger was added following reduction. Zeta potential analysis (Fig. 3C,D) also aided the stability of green-synthesized nanoparticles. GS-PEG-Ginger-AuNPs possessed a more negative surface charge (−16.34 mV) than CS-PEG-Ginger-AuNPs (−14.01 mV). This augmented negativity, combined with the steric shield of PEG and the phytochemical corona, creates a firm electrosteric barrier against aggregation. The synergy between ginger’s inherent capping agents and the synthetic PEG polymer in the green synthesis route is thus directly responsible for the remarkable colloidal stability.

Encapsulation efficiency and drug loading

EE and LC were analyzed spectrophotometrically in relation to the calibration curve of reference gingerol at 290 nm. Both formulations presented various encapsulation profiles. GS-PEG-Ginger-AuNPs possessed a significantly higher EE of 80.72 ± 4.81% compared to CS-PEG-Ginger-AuNPs of 61.82 ± 6.58% (p < 0.05) (Fig. 4A). This discovery indicates that the green synthesis route allows for higher drug loading. The principal advantage is that during green synthesis, ginger phytochemicals like gingerols act as both reducers and capping agents during the nucleation of gold nanoparticles. The carbonyl (C = O) and phenolic hydroxyl (-OH) groups of the phytochemicals bind directly to gold ions (Au³⁺) and form strong Au-O bonds that immobilize the phytochemicals as an integral part of the nanoparticle matrix. In this in-situ method, there is a denser and more stable phytochemical corona by better encapsulation. In contrast, in the chemical method, ginger extract is adsorbed after citrate reduction. This post-synthesis loading is due to weaker physisorption and hydrogen bonding onto the pre-existing, citrate-capped AuNP surface, resulting in lower and less stable encapsulation³². Conversely, in the chemical synthesis method, ginger extract is introduced after citrate reduction, potentially restricting the degree of drug–nanoparticle interaction and leading to diminished encapsulation efficiency.

Fig. 4.

EE and LC of PEGylated ginger-gold nanoparticles. (A) EE of CS-PEG-Ginger-AuNPs (61.82 ± 6.58%) and GS-PEG-Ginger-AuNPs (80.72 ± 4.81%). The green-synthesized formulation showed a statistically significant increase in EE (*p < 0.05, unpaired t-test). (B) LC of CS-PEG-Ginger-AuNPs (77.85 ± 15.79%) and GS-PEG-Ginger-AuNPs (90.69 ± 9.47%). The difference between the two formulations was not statistically significant (ns, p > 0.05, unpaired t-test). Data are expressed as mean ± SD from three independent replicates (n = 3)

GS-PEG-Ginger-AuNPs exhibited the capacity for drug loading as 90.69 ± 9.47%, and CS-PEG-Ginger-AuNPs exhibited as 77.85 ± 15.79% (Fig. 4B). GS-based nanoparticles possessed numerically greater LC, but the two drug values were not significantly different (ns, p > 0.05). Both synthesis processes are capable of delivering sufficient surface accessibility for loading the drug, and the more homogeneous LC values of GS-PEG-Ginger-AuNPs correlate with a narrower size distribution and a more homogenous phytochemical surface coating. The provision of high energy efficiency and stable liquid crystal nature along with other benefits further enhances the value of green synthesis for multifunctional nanocarriers. Similar findings have also been observed for other AuNPs synthesized green. Green-synthesized chitosan-capped AuNPs had a drug loading capability of 71% of rifampicin³³. Gold nanoparticles synthesized with Cannabis sativa L. leaf extract were also conjugated with doxorubicin, which showed enhanced nanoparticle size and activity along with an enhanced antiproliferative effect, which proves that there is good drug loading³⁴. While quantitative results for encapsulation efficiency and drug-loading are not always provided in all studies, there is some evidence available to demonstrate that nanoparticles prepared through green synthesis pathways possess good drug-loading capacity³⁵.

For neuro-nanomedicine, successful encapsulation and drug loading stability are essential to obtain the highest therapeutic payload at the lowest carrier dose. The greatly improved EE of GS-PEG-Ginger-AuNPs (> 80%) shows that increased gingerol is effectively encapsulated by the nanoparticle system, which may improve transport efficacy through the blood-brain barrier. While both preparations contain high LC content (> 75%), the superior encapsulation efficiency of the GS preparation can provide reduced nanoparticle dosage for administration, thus minimizing systemic toxicity and enhancing translational utility.

In conclusion, while both methods of synthesis synthesized PEGylated ginger-AuNPs with high drug loading capacity, green synthesis considerably surpassed chemical synthesis in encapsulation efficiency, highlighting the functional merits of phytochemical-catalyzed nanoparticle synthesis within the context of neurotherapy applications.

In vitro drug release study

Release profiles of gingerol from CS-PEG-Ginger-AuNPs and GS-PEG-Ginger-AuNPs were evaluated in physiologic conditions (PBS, pH 7.4, 37 °C) for 96 h to simulate systemic circulation (Fig. 5). Both systems showed biphasic release profiles with burst release during the initial 12 h followed by sustained and slow release. This biphasic release is typical of nanocarrier systems where the initial rapid phase is characteristic of desorption of loosely associated surface drug and the latter slow phase is characteristic of drug molecule diffusion encapsulated from the inner side of the PEGylated nanoparticle matrix.

Fig. 5.

In vitro cumulative release of gingerol from PEGylated ginger-gold nanoparticles at pH 7.4 (37 °C). CS-PEG-Ginger-AuNPs (■) and GS-PEG-Ginger-AuNPs (■) both exhibited biphasic release profiles with an initial burst followed by sustained release. GS-PEG-Ginger-AuNPs demonstrated significantly higher cumulative release (~ 85% at 96 h) compared to CS-PEG-Ginger-AuNPs (~ 60% at 96 h), indicating enhanced diffusion and controlled release capacity of green-synthesized nanoparticles. Data are expressed as mean ± SD (n = 3)

Quantitatively, GS-PEG-Ginger-AuNPs also showed much higher cumulative release compared to CS-PEG-Ginger-AuNPs throughout the experiment. Cumulative release at 96 h for GS-PEG-Ginger-AuNPs was approximately 85%, whereas CS-PEG-Ginger-AuNPs released minimal amounts of gingerol entapped, i.e., as low as 60%. The enhanced release profile of GS-PEG-Ginger-AuNPs is due to the synthesis process. The green synthesis-induced in-situ formation of a dense, hydrophilic phytochemical corona results in a matrix that efficiently hydrates and swells in PBS (pH 7.4). This swelling, along with the high initial drug loading, enhances the effective and sustained diffusion of gingerol. Conversely, post-synthesis adsorption of ginger extract in CS-PEG-Ginger-AuNPs demonstrates reduced phytochemical interactions and reduced permeability of the matrix, which results in limited diffusion and lessened release profile. For chemically synthesized nanoparticles (EE = 61.82%), greater percentages of the drug were entrapped in the gold–PEG matrix, resulting in reduced cumulative release.

Improved release profile of GS-PEG-Ginger-AuNPs is also consistent with their larger encapsulation and narrower polydispersity index (PDI = 0.0034), reflecting more uniform nanostructure. Increased colloidal stability and negative surface charge (− 16.34 mV) of GS nanoparticles can also cause controlled release, which prevents premature aggregation and maintains drug diffusion kinetics. Conversely, CS-PEG-Ginger-AuNPs with larger size distribution (PDI = 0.038) and low encapsulation had poor release potential, which can decrease therapeutic payload availability in vivo.

Extended and controlled drug release is paramount in neuro-nanomedicine, with therapeutic levels being maintained through the blood–brain barrier. Sustained release in the case of GS-PEG-Ginger-AuNPs (~ 85% after 96 h) means greater hope for extended neuroprotection with reduced need for repeat dosing. The intermediate release rate of CS-PEG-Ginger-AuNPs (~ 60%) can still be therapeutically effective but at the expense of decreased bioavailability. Remarkably, biphasic release from the two systems corroborates their effectiveness as controlled delivery systems, though the green-synthesized nanoparticles exhibit a superior and more sustained release profile, proportional to their high encapsulation efficacy.

Kinetic modeling of gingerol release

To further clarify gingerol release mechanisms from PEGylated AuNP formulations, release profiles were curve-fitted to different kinetic models (Zero-order, First-order, Higuchi, Korsmeyer–Peppas, Hixson–Crowell, and Baker–Lonsdale) (Fig. 6; Table 1). The determination coefficient (R²) was utilized to establish goodness of fit such that release mechanisms of green-synthesized and chemically synthesized nanoparticles could be compared directly. For CS-PEG-Ginger-AuNPs, the greatest fitting was with Higuchi model (R² = 0.9558), followed by Korsmeyer–Peppas (R² = 0.936), while Zero-order and First-order models were less correlated with R² = 0.8093 and 0.8229, respectively. The predominance of Higuchi model indicates that release was primarily governed by Fickian diffusion of gingerol from the nanoparticle matrix to the aqueous medium. Korsmeyer–Peppas exponent n (0.45) also approved a Fickian-controlled release mechanism, in concurrence with the low encapsulation efficiency (EE = 61.82%) and cumulative release (~ 60% at 96 h), demonstrating that most of the drug was entrapped in diffusion-limited PEG–AuNP structures.

Fig. 6.

Kinetic modeling of gingerol release from PEGylated ginger-gold nanoparticles

Table 1.

Goodness-of-fit (R²) values for different kinetic models describing Gingerol release from PEGylated ginger-gold nanoparticles.

Model	Equation	R² (CS-PEG-Ginger-AuNPs)	R² (GS-PEG-Ginger-AuNPs)
Zero-order		0.8093	0.8698
First-order		0.8229	0.9486
Higuchi		0.9558	0.9809
Korsmeyer-Peppas		0.936	0.9539
Hixson-Crowell		0.7925	0.9063
Baker-Lonsdale		0.6655	0.8295

For comparison, GS-PEG-Ginger-AuNPs exhibited a good fit to Higuchi (R² = 0.9809), followed by First-order (R² = 0.9486), Korsmeyer–Peppas (R² = 0.9539), and Hixson–Crowell (R² = 0.9063). The good fit to Higuchi again points to diffusion as the dominant mechanism. However, the higher correlation with First-order and Hixson–Crowell models compared to CS nanoparticles suggests an additional dependence on concentration gradients and negligible changes in surface area upon release. Importantly, the Korsmeyer–Peppas exponent n for GS-PEG-Ginger-AuNPs was 0.52, which fell in the range of the anomalous transport (0.43 < n < 0.85). This is indicative of a combined mechanism involving Fickian diffusion along with matrix relaxation/surface desorption processes. These results are consistent with the higher encapsulation efficiency (EE = 80.72%), greater cumulative release (~ 85% at 96 h), and lower size distribution (PDI = 0.0034) achieved for GS-based nanoparticles. The Baker–Lonsdale model provided the worst fits for both formulations (R² = 0.6655 for CS, 0.8295 for GS), again confirming that drug release did not follow strictly the assumptions of homogeneous spherical matrix diffusion.

The kinetic analysis reveals a fundamental difference between the two synthesis routes. The Fickian diffusion mechanism (n = 0.45) of CS-PEG-Ginger-AuNPs indicates a release process slowed down by the dense PEG-AuNP matrix. This results in slower kinetics and less overall release. Anomalous transport (n = 0.52) for GS-PEG-Ginger-AuNPs points to a combination mechanism of diffusion and matrix relaxation, corroborated by the dynamic phytochemical corona formed under green synthesis. This provides a more efficient and longer-lasting drug release profile. These differences have direct translational significance. Finite, diffusion-controlled release from the chemical formulation can reduce CNS drug delivery effectiveness, potentially requiring higher doses. Conversely, the improved encapsulation and hybrid release mechanism of GS-PEG-Ginger-AuNPs provide a more viable neuro-nanomedicine platform with the capability to sustain therapeutic gingerol levels over extended periods, which could reduce dosing frequency.

Cytotoxicity assay

Cytotoxicity of PEGylated AuNP formulations in PC12 cells was also assessed using the MTT assay and concentration–response data (1–100 µg/mL) to obtain the half-maximal inhibitory concentration (IC₅₀). There was visible dose-dependent loss of viability with chemically synthesized nanoparticles (Fig. 7), whereas green-synthesized samples displayed outstanding biocompatibility at all concentration levels. IC₅₀ values were calculated from viability data for experiments by nonlinear regression (four-parameter logistic model) and linear interpolation/extrapolation as a cross-validation. The used four-parameter logistic equation was:

Fig. 7.

Cytotoxicity of PEGylated AuNP formulations in PC12 cells determined by MTT assay. PC12 cells were exposed to increasing concentrations (1–100 µg/mL) of CS-PEG-AuNPs, CS-PEG-Ginger-AuNPs, GS-PEG-AuNPs, and GS-PEG-Ginger-AuNPs for 72 h. Chemically synthesized formulations (CS-PEG and CS-PEG-Ginger) induced significant, dose-dependent reductions in cell viability (**** p < 0.0001 vs. control), whereas green-synthesized formulations (GS-PEG and GS-PEG-Ginger) maintained high cell viability (> 70% at 100 µg/mL) with no significant difference from untreated controls. Data are expressed as mean ± SD (n = 3)

where Y represents the percentage of viable cells, X is the log concentration of nanoparticles, and Top and Bottom represent the upper and lower plateaus of the dose–response curve. The regression analysis was performed using GraphPad Prism 8.4.0.

IC₅₀ values derived from linear extrapolation and interpolation indicated that CS-PEG-AuNPs were most cytotoxic, having an IC₅₀ of approximately 68.7 µg/mL, followed by CS-PEG-Ginger-AuNPs, which was slightly more tolerant with an IC₅₀ value greater than 100 µg/mL (extrapolated ≈ 104.4 µg/mL). On the other hand, both the green-synthesized formulations were significantly less toxic, with GS-PEG-AuNPs and GS-PEG-Ginger-AuNPs exhibiting IC₅₀ values far in excess of 100 µg/mL (extrapolated ≈ 189.4 and 190.4 mg µg/mL/mL, respectively). Four-parameter logistic curve fitting was also confirming these observations to reflect uniform trends across formulations: chemically synthesized nanoparticles recorded the greatest viability drop (CS-PEG-AuNPs ≈ 99 µg/mL, Hill slope ≈ 0.7), whereas green-synthesized formulations recorded stretched curves with minimal inhibition, reflecting high biocompatibility. Altogether, these results show that green synthesis and PEGylation greatly improve neuronal safety and minimize cytotoxic reactions (Table 2).

Table 2.

Cytotoxicity of PEGylated AuNP formulations in PC12 cells: IC₅₀ values and overall significance compared with control (Tukey’s test).

Formulation	IC₅₀ (µg/mL)	Mean viability trend	Significance vs. control	Interpretation
CS-PEG-AuNPs	≈ 68.7	↓↓	**** (p < 0.0001)	Highest cytotoxicity
CS-PEG-Ginger-AuNPs	> 100 (≈ 104.4)	↓↓	**** (p < 0.0001)	Moderate cytotoxicity
GS-PEG-AuNPs	> 100 (≈ 189.4)	↓	ns (p > 0.05)	Low cytotoxicity
GS-PEG-Ginger-AuNPs	> 100 (≈ 190.4)	~	ns (p > 0.05)	Lowest cytotoxicity, highest safety

Arrows indicate the relative strength of cytotoxicity: ↓↓ (strong), ↓ (mild), ~ (negligible).

Statistical comparison using one-way ANOVA and post-hoc Tukey’s multiple comparison test confirmed these results. CS-PEG-AuNPs and CS-PEG-Ginger-AuNPs significantly lowered PC12 cell viability (**** p < 0.0001 vs. control), whereas GS-based samples were statistically not different from untreated control cells (ns, p > 0.05). The repeatedly observed very high cell viability reported at 100 µg/mL (> 70–80%) for GS-PEG-Ginger-AuNPs is a reflection of their higher neuronal compatibility and safety profile.

The improved cytocompatibility of GS-PEG-Ginger-AuNPs results from the biomimetic nature of the green synthesis route. In this method, ginger extract phytochemicals serve as non-toxic, plant-derived reducing and stabilizing agents, thereby eliminating the need for toxic chemical reductants. Phytochemicals also impart other antioxidant and neuroprotective properties to nanoparticles. Moreover, PEGylation confers a hydrophilic “stealth” surface that minimizes nonspecific protein adsorption and oxidative stress reduction. Synergistic interaction among PEG and ginger phytochemicals is responsible for increased neuronal viability observed and justifies the use of GS-PEG-Ginger-AuNPs as a biocompatible nanocarrier platform for neuro-nanomedicine applications where biocompatibility over long time periods is essential.

The enhanced safety of GS-PEG-Ginger-AuNPs can be mechanistically explained by the biomimetic synthesis pathway and the inherent bioactivity of ginger. Green synthesis utilizes phytochemicals from plants, such as polyphenolic and flavonoid compounds from ginger extract, which serve as natural reductants and stabilizers. This approach avoids the use of harsh chemical reductants commonly employed in CS-PEG-AuNPs. This eliminates the primary source of cytotoxicity associated with chemical synthesis. More importantly, the ginger phytochemicals (e.g., gingerols and shogaols) that form the stabilizing corona on the nanoparticles are potent antioxidants. This antioxidant-dusted surface can directly scavenge reactive oxygen species (ROS) and alleviate oxidative stress in the PC12 cells, a primary mechanism of nanoparticle-induced cytotoxicity. By preventing oxidative damage, ginger-capped nanoparticles not only block cell death but potentially actively induce an overall protective cellular setting^36,37.

Moreover, PEGylation grafts on a hydrophilic “stealth” surface that inhibits nonspecific protein adsorption and cellular uptake, reducing passive toxicity³⁸. The complementary action of the active antioxidant defense from the ginger corona and the passive coating by the PEG layer collectively creates an extremely biocompatible interface¹⁴. This dual action underlies the improved cell viability of GS-PEG-Ginger-AuNPs, suggesting their suitability for further investigation in neuro-nanomedicine applications where minimizing cytotoxic load is vital for long-term therapeutic safety.

Conclusion, limitations, and future direction

This study conclusively demonstrates that the synthesis route is a critical determinant of the physicochemical and biological properties of PEGylated ginger-gold nanoparticles. While chemically synthesized nanoparticles (CS-PEG-AuNPs and CS-PEG-Ginger-AuNPs) exhibited smaller core diameters (14.8 ± 2.6 to 15.5 ± 4.8 nm), they were characterized by modest zeta stability (− 14.0 mV), poor encapsulation efficiency (EE = 61.8%, LC = 77.9%), and an inferior cumulative release profile (~ 60% at 96 h). In contrast, the green-synthesized formulations (GS-PEG-AuNPs and GS-PEG-Ginger-AuNPs) displayed relatively larger mean sizes (15.5 ± 3.9 to 19.2 ± 4.3 nm) alongside greater colloidal stability (− 16.3 mV) and superior drug loading capacity (EE = 80.7%, LC = 90.7%). Furthermore, the drug release from GS-PEG-Ginger-AuNPs was more sustained and efficient, reaching ~ 85% cumulative release at 96 h and following an anomalous diffusion mechanism (n = 0.52), unlike the Fickian diffusion (n = 0.45) observed for the CS formulations.

The biological evaluation unequivocally confirmed the superior safety profile of the green synthesis approach. As detailed in Table 2, the CS-PEG-AuNPs formulation showed the highest cytotoxicity (IC₅₀ ≈ 68.7 µg/mL), while the incorporation of ginger extract slightly improved the tolerance of the chemical formulation (CS-PEG-Ginger-AuNPs, IC₅₀ ≈ 104.4 µg/mL). Most importantly, both green-synthesized formulations exhibited exceptional biocompatibility. GS-PEG-AuNPs and GS-PEG-Ginger-AuNPs maintained high cell viability (> 70–80% at 100 µg/mL) with extrapolated IC₅₀ values far exceeding 100 µg/mL (≈ 189.4 and ≈ 190.4 µg/mL, respectively), showing no significant toxicity compared to untreated cells. Collectively, these results identify GS-PEG-Ginger-AuNPs as the most promising nanocarrier candidate, combining a high drug payload, controlled release kinetics, and superior neuronal compatibility.

Several limitations of this study should be acknowledged. The in vitro PC12 cell model, while informative, necessitates subsequent in vivo validation in relevant neurodegenerative models. The static PBS environment (pH 7.4) used for drug release studies cannot fully replicate the dynamic complexity of the brain microenvironment. Furthermore, the analysis focused on gingerol, whereas other neuroprotective ginger phytochemicals were not individually assessed for their encapsulation or release. Finally, critical long-term pharmacokinetic data, including BBB penetration, biodistribution, and clearance, are yet to be investigated.

Future work will focus on translating these promising findings into in vivo models of neurodegenerative disease to demonstrate therapeutic efficacy and safety. A thorough investigation of pharmacokinetics and biodistribution is essential to characterize nanoparticle circulation and brain penetration. To enhance specificity, the functionalization of GS-PEG-Ginger-AuNPs with targeting ligands will be explored to achieve site-specific delivery to neurons. Expanding encapsulation studies to include a broader spectrum of ginger bioactives could further improve the therapeutic potential. Additionally, integrating diagnostic functionalities could transform this platform into a multifunctional theranostic system, enabling concurrent drug delivery and imaging for clinical neuro-nanomedicin.

Author contributions

Elham Hatami Monfared: Data curation, writing—original draft & editing. Sonia Fathi-karkan: Conceptualization, Investigation, Writing – original draft & editing, Supervision. Zakieh Keshavarzi: Conceptualization, Investigation, Writing—original draft, Supervision.

Funding

The current study was approved & supported by North Khorasan University of Medical Sciences with Reg. No. 4030077 was approved by ethical committee of North Khorasan University of Medical Sciences. The Ethic approval Cod is IR.NKUMS.REC.1403.105.

Data availability

All data supporting the findings of this study are available within the paper.

Declarations

Competing interests

The authors declare no competing interests.