Gold Nanoparticle–Modified 6‑Mercaptopurine and Rabies‑Derived Peptide Enhance SH‑SY5Y Neural Cell Proliferation and Neurite Outgrowth

Abstract

Neuroregeneration demands biomaterials that can stimulate neuronal proliferation and axonal extension. Here we present a novel neurophic platform: gold nanoparticles (AuNPs) functionalized with 6‑mercaptopurine (6MP) and a neuron‑penetrating peptide (RDP). When SH‑SY5Y neuroblastoma cells were exposed to 6MP‑AuNPs‑RDP conjugates, metabolic activity and neurite length increased markedly compared with controls. Importantly, cells recovered from cryopreservation retained these growth advantages, indicating robust tolerance to the nanomaterial. These findings support the development of 6MP‑AuNPs‑RDP as a safe and effective neurophic nanomaterial.

Background

Effective promotion of neuronal proliferation and neurite outgrowth is a cornerstone of regenerative strategies for neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and stroke (1–4). Surface chemistry of biomaterials can modulate cell morphology, proliferation, and differentiation, offering a promising route to neurophic agents (5,6). Gold nanoparticles (AuNPs) have become attractive in biomedical applications due to their facile synthesis, surface versatility, low toxicity, and biocompatibility (7,8). Prior work demonstrated that gold nanorods combined with low‑power laser exposure could elongate neurites of NG108‑15 cells by up to 25 µm (9).

6‑Mercaptopurine (6MP) is an anti‑inflammatory drug that binds AuNPs via Au–S bonds, enabling functionalization (10). Although 6MP‑AuNPs have been used for quantitative drug sensing (11), their cellular effects remain unexplored. We therefore investigated 6MP‑AuNPs and a neuron‑targeting peptide (RDP) in the human neuroblastoma SH‑SY5Y model, chosen for its high environmental sensitivity and relevance to neural biomaterial studies.

Methods / Experimental

Synthesis of 6MP‑AuNPs‑RDP Conjugate

20 nm citrate‑coated AuNPs were prepared by the classic Turkevich reduction of HAuCl₄·3H₂O (0.01 %) with sodium citrate (38.8 mM). 6MP (0.046 nM) and AuNPs (0.33 mM) were incubated for 5 h at room temperature, then centrifuged at 17,000 g for 30 min. The pellet was washed thrice with deionized water. For RDP modification, the peptide (0.023 nM) was co‑incubated with the AuNP/6MP mixture under identical conditions. A scrambled FAM‑labeled peptide (FAM‑SP) served as control. Particles were resuspended in 0.1 M NaOH (pH 9.0), filtered (0.22 µm), and stored at 4 °C.

Particle Characterization

UV/Vis spectra were recorded with a Shimadzu UV‑2450. Size distribution and zeta potential were measured by Malvern Zetasizer Nano ZS after dilution. Transmission electron microscopy (TEM, Shimadzu) confirmed spherical morphology.

Cell Culture

SH‑SY5Y cells were maintained in DMEM/F‑12 (1:1) supplemented with 10 % FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C, 5 % CO₂.

Cell Uptake Assay

Cells (5 × 10⁴ cells/well) were incubated with 0.25 µg/mL FAM‑labeled 6MP‑AuNPs‑RDP or 6MP‑AuNPs‑SP for 2 h. Fluorescence microscopy (Olympus) and spectrofluorometry (Hitachi) quantified internalization.

Impact on Neuronal Growth

For proliferation, cells were seeded at 5 × 10⁵ cells/well and treated with 0–1.0 µg/mL 6MP‑AuNPs‑RDP for 24 h. Cell counts (Bio‑Rad automated counter) and MTT metabolic activity (490 nm) were assessed. Neurite length was measured after 3 days of daily 0.25 µg/mL treatment using ImageJ. Surface‑coated dishes (3.5 cm) were also prepared to evaluate neurite extension on a nanoparticle layer.

Statistical Analysis

Data (n = 4) are expressed as mean ± SEM and analyzed by one‑way ANOVA with Dunnett’s test (SPSS 13.0). p < 0.05 denotes significance.

Results

Nanoparticle Appearance and Physicochemical Properties

AuNPs exhibited a red color; upon 6MP addition, the solution turned dark, forming a blue‑black precipitate after 5 h. Dissolving at pH 9.0 yielded a rose‑colored solution, which re‑precipitated at pH 7.4. DLS revealed an average diameter of 20.5 nm for 6MP‑AuNPs and 24.6 nm for 6MP‑AuNPs‑RDP; zeta potentials were −37.2 mV and −25.8 mV, respectively (Figure 2c,d). TEM confirmed spherical morphology.

Enhanced Cellular Uptake via RDP

At pH 7.4, 6MP‑AuNPs‑RDP aggregated less and displayed a prominent pericellular plaque, whereas 6MP‑AuNPs formed extensive aggregates (Figure 3a). Fluorescence imaging showed strong intracellular signal for 6MP‑AuNPs‑RDP compared to weak signal for 6MP‑AuNPs‑SP (Figure 3b‑d), indicating that RDP markedly increases nanoparticle internalization.

Promotion of Proliferation and Metabolic Activity

Both 6MP‑AuNPs and 6MP‑AuNPs‑RDP increased cell numbers and MTT absorbance in a dose‑dependent manner; effects were stronger for the RDP‑conjugated particles (Figure 4a,b). RDP alone did not alter growth.

Neurite Outgrowth Enhancement

After 24 h exposure to 1.0 µg/mL particles, cells treated with 6MP‑AuNPs‑RDP exhibited significantly longer neurites than controls and 6MP‑AuNPs (Figure 5a,b). RDP alone had no effect. When applied as a surface coating, 6MP‑AuNPs‑RDP accelerated neurite extension and facilitated internalization throughout the cytoplasm and nucleus (Figure 6d,e).

Effect of Cryopreservation

Cells pre‑treated with 1.0 µg/mL 6MP‑AuNPs‑RDP retained enhanced proliferation and neurite length after thawing, demonstrating robust tolerance to cryogenic storage (Figure 7a‑c).

Discussion

The data confirm that 6MP‑AuNPs‑RDP combines the biocompatibility of gold nanostructures with the neuronal uptake capability of RDP and the growth‑promoting properties of the exposed purine moiety of 6MP. The acid–base behavior of the conjugate (pI ≈ 7.8) aligns with physiological pH, reducing aggregation in cell media. RDP likely mediates endocytosis via GABA or nicotinic acetylcholine receptors, consistent with prior studies on peptide‑functionalized nanoparticles (15–19). The purine groups exposed on the nanoparticle surface may activate MAPK/ERK and PI3K/Akt pathways, key regulators of neuronal differentiation and neurite outgrowth (21–23). Importantly, SH‑SY5Y cells tolerate repeated exposure and cryopreservation, underscoring the safety profile of the platform.

Conclusions

Gold nanoparticles functionalized with 6‑mercaptopurine and the rabies‑derived peptide RDP constitute a potent neurophic nanomaterial that promotes neuronal proliferation and neurite extension while maintaining biocompatibility. These findings support further development of 6MP‑AuNPs‑RDP for therapeutic strategies in neuroregeneration.

Abbreviations

6MP

6‑Mercaptopurine

AD

Alzheimer’s disease

AuNPs

Gold nanoparticles

DLS

Dynamic light scattering

DMEM

Dulbecco’s modified Eagle’s medium

DMSO

Dimethyl sulfoxide

FCS

Fetal calf serum

MTT

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

PD

Parkinson’s disease

RDP

Rabies virus‑derived peptide

TEM

Transmission electron microscopy