Biosynthesis of gold nanoparticles using Psilocybe cubensis extracts and their biostimulatory evaluation in Neuro2a cells

In a development that reads like science fiction but is firmly rooted in laboratory bench work, researchers at the University of Concepción in Chile have successfully grown gold nanoparticles using extracts from Psilocybe cubensis, the psilocybin-producing mushroom better known for its psychoactive properties than for its nanotechnological potential. The team, led by Manuel Vergara, Felipe Galleguillos, and José Becerra from the Laboratory Chemistry and Natural Products group in the Department of Botany, has demonstrated that these biologically synthesized gold nanoparticles can significantly stimulate the proliferation of neuronal cells in vitro, while also promoting early morphological changes consistent with the beginning stages of neurite outgrowth. The study, published in Frontiers in Nanotechnology, represents the first report of green synthesis of gold nanoparticles using a psilocybin-producing fungus and the first evaluation of their neuronal bioactivity through real-time kinetic monitoring and three-dimensional holotomographic imaging.

The research sits at a fascinating intersection of myconanotechnology and neuroscience. Gold nanoparticles have long been recognized as versatile platforms for drug delivery, biosensing, bioimaging, and tissue regeneration, owing to their biocompatibility, tunable size and shape, surface functionalizability, and their characteristic Surface Plasmon Resonance. In the context of neuroregeneration, previous studies have shown that gold nanoparticles can promote neurite outgrowth, modulate neuronal electrical activity, enhance synaptogenesis, and facilitate delivery of therapeutic agents across the blood-brain barrier. Their neuroregenerative mechanisms are multifactorial, including modulation of the immune microenvironment through anti-inflammatory signaling, activation of neuroprotective pathways via reactive oxygen species scavenging, and facilitation of neurotrophin integration, particularly brain-derived neurotrophic factor, which is essential for neuronal survival, differentiation, and synaptic plasticity. The Chilean team sought to leverage these established properties while introducing a novel biological matrix: the rich biochemical profile of Psilocybe cubensis fruiting bodies.

The choice of fungal matrix proved to be the decisive variable in the entire study. The researchers compared two different biological sources: mature fruiting bodies and liquid-cultured mycelium. Both were genetically confirmed as Psilocybe cubensis through amplification and sequencing of the internal transcribed spacer region, which yielded 100 percent query coverage and 100 percent identity against reference sequences in the GenBank database, with E-values of 0.0. This molecular validation was critical because recent literature has demonstrated significant inter- and intra-strain variability in secondary metabolite expression within the Psilocybe genus, and because identification based solely on morphology can be unreliable due to the existence of visually similar sympatric species, some of which are highly toxic. The confirmed identity provided a validated and traceable fungal matrix for all subsequent nanoparticle biosynthesis experiments.

Extracts were prepared from both sources. The fruiting body extract was obtained by macerating dried, powdered biomass in ethanol for 24 hours under gentle agitation in the dark, followed by filtration and evaporation to recover the metabolites in concentrated form. The liquid culture extract was prepared by filtering the mycelium from 15-day submerged cultures grown in YMG medium with 2 percent malt extract, then drying the filtrate and scraping the dried contents under sterile conditions. Both extracts were dissolved in distilled water and adjusted to pH 8.0 using sodium hydroxide to promote the deprotonation of reducing groups and colloidal stability. The synthesis itself involved mixing these extracts with chloroauric acid at various concentrations, ranging from 0.1 to 0.9 millimolar, and monitoring the reaction from 0 to 24 hours in complete darkness at room temperature.

The contrast between the two matrices was stark and immediately apparent. The fruiting body extract, at an optimal precursor concentration of 0.3 millimolar chloroauric acid, produced a highly defined, narrow, and symmetrical Surface Plasmon Resonance peak centered at approximately 550 nanometers with an absorbance of 0.303 arbitrary units at 4 hours of reaction. This optical signature corresponds to the homogeneous nucleation of monodisperse, spherical metallic nanoparticles. At 24 hours, the Surface Plasmon Resonance bands for the 0.3 and 0.5 millimolar conditions experienced a subtle bathochromic shift toward 560 to 580 nanometers, indicating controlled growth of the metallic cores as the increasing availability of gold ions slightly outpaced the stabilizing capacity of the bioactive molecules in the extract. In contrast, the liquid culture extract initially showed an absorption band with an intensity of 0.5 arbitrary units at 4 hours, which increased to approximately 0.75 arbitrary units at 24 hours. However, this increase preceded a complete loss of peak definition and eventual macroscopic sedimentation of the material. The liquid medium extract failed to maintain colloidal stability due to a lower content of redox-active metabolites in the mycelium, particularly ergothioneine and tryptamine alkaloids, which reduces both the rate of gold ion reduction and the capacity to stabilize the resulting nanoparticles.

Transmission electron microscopy physically validated the optical predictions from the fruiting body system, revealing highly uniform, non-aggregated spherical gold cores with an average diameter of 18.94 plus or minus 5.28 nanometers, measured across 100 individual particles using ImageJ software and fitted with a nonlinear Gaussian regression. These dimensional characteristics, falling within the 10 to 120 nanometer range, and their quasi-spherical morphology are critical for successfully crossing the blood-brain barrier and maximizing the safe interaction area with neuronal receptors, presenting significantly lower toxicity profiles than other structures. Dynamic light scattering yielded a Z-average hydrodynamic diameter of 19.41 nanometers, showing an exceptional correlation with the transmission electron microscopy core size. This negligible difference mathematically proves that the nanoparticles are individual, non-aggregated, and perfectly dispersed in the liquid medium. The zeta potential of minus 31.0 millivolts exceeds the critical thermodynamic threshold of plus or minus 30 millivolts required to ensure robust, long-range electrostatic repulsion, physically preventing van der Waals-driven self-assembly and conferring superior biosafety against the oxidative stress typically induced by cationic nanomaterials on cell membranes.

Fourier-transform infrared spectroscopy confirmed that the gold core is encapsulated by a pre-assembled organic biocorona derived from the fungal extract. The spectroscopic analysis revealed a complex biogenic matrix dominated by protein peptide bonds, with Amide I at 1630 inverse centimeters and Amide II at 1598 inverse centimeters, aliphatic chains at 2926 to 2853 inverse centimeters, and carbohydrate structures at 1079 to 1027 inverse centimeters. Crucially, comparing the free extract with the functionalized nanoparticles revealed the persistence of the 1598.16 inverse centimeters signal, corresponding to the indole ring of tryptamine derivatives, and the 1027.58 to 990.18 inverse centimeters doublet, corresponding to phosphate ester stretching. This retention provides direct spectroscopic evidence that key neuroactive alkaloids, specifically psilocybin, remain structural components of the pre-assembled biocorona encapsulating the gold nanoparticles. This dense, negatively charged organic envelope is the direct cause of the highly stable zeta potential and provides robust electrostatic repulsion that prevents aggregation.

The biological evaluation was conducted using the Neuro2a neuronal model, a murine neuroblastoma cell line widely used as a standard in vitro platform for tracking both proliferation and early neuritogenesis. Cells were exposed to a concentration gradient across three distinct seeding densities and monitored continuously for 48 hours using the automated IncuCyte S3 platform, which enables live-cell analysis through phase contrast and fluorescence imaging without the optical interference that frequently compromises traditional static endpoint assays when plasmonic nanoparticles are present. The results revealed a clear biphasic, dose-dependent response consistent with the phenomenon of hormesis, defined as a biphasic dose-response in which low doses produce stimulation and high doses produce inhibition. Concentrations between 22.91 and 183.25 micrograms per milliliter induced a significant increase in cell proliferation and viability without morphological compromise, while doses at or above 733.0 micrograms per milliliter resulted in acute cytotoxicity. At the optimal density of 2,500 cells per well, the concentration of 91.63 micrograms per milliliter emerged as the optimal neurostimulatory dose, triggering the most significant confluency increase in the study, reaching a mean surface occupation of 19.00 percent compared to 12.17 percent in the untreated control group, effectively doubling the confluency at the 48-hour endpoint while preserving cell death at basal, non-toxic levels.

The most visually striking finding came from the three-dimensional holotomographic microscopy, which complemented the quantitative kinetic data with high-resolution structural details in living cells without the need for fluorescent dyes or chemical fixatives, thereby preventing phototoxicity and structural distortion. Cells treated at the optimal biostimulatory concentration of 91.63 micrograms per milliliter transitioned from the typical rounded neuroblastic form to a notably elongated, fusiform, and pear-shaped morphology featuring thinned somatic poles. The three-dimensional reconstructions confirmed the active projection of long, well-defined cytoplasmic extensions extending from the cell body in multiple directions. This elongated phenotype is morphologically compatible with cytoskeletal reorganization processes associated with active cell extension, corresponding to the initial stages of neuritogenesis. The interpretation of these cytoplasmic extensions as indicators of incipient neurite formation is reinforced by the convergence toward basal eccentricity values at the endpoint, which rules out that the elongated phenotype is a response to pathological cytoskeletal stress and instead confirms that morphological reorganization occurs in structurally intact cells.

The mechanistic interpretation proposed by the Chilean team centers on the dual functionality of the fungal metabolites retained on the nanoparticle surface. A recent molecular finding of high relevance is that psilocin directly binds to the TrkB tyrosine kinase receptor, the high-affinity receptor for brain-derived neurotrophic factor, with affinities approximately 1,000 times higher than those of other classical antidepressants, acting as positive allosteric modulators that potentiate endogenous neurotrophic signaling independently of 5-HT2A receptor activation. This action on the BDNF/TrkB pathway activates the mTORC1 and MAPK/ERK signaling cascades, which are the master regulators of the cell cycle, protein synthesis, and survival in proliferative cell lines such as Neuro2a neuroblasts. The most plausible hypothesis is that the biosynthesized nanoparticles act as a nanoplatform that stabilizes and enhances the bioavailability of the psychoactive components in the cellular microenvironment, while the gold core provides the nanoscale properties that facilitate interaction with neuronal receptors. Additionally, the retention of ergothioneine on the surface would confer an antioxidant shield to the cellular microenvironment, as this amino acid possesses an exceptional capacity to neutralize free radicals and protect neuronal tissue from oxidative damage, explaining the marked reduction in basal cell mortality observed at biostimulatory doses compared to the untreated control.

The study does acknowledge important limitations. The experimental design does not allow dissociating the effects of the gold core, the metabolites alone, or the synergy between both, given that controls with conventionally chemically synthesized gold nanoparticles or with Psilocybe cubensis extract without gold were not included. This constitutes the main limitation and a priority for future research. The team also notes the absence of molecular differentiation markers such as TUJ-1 or TrkB activation, and the lack of quantitative holotomographic analysis of parameters such as dry mass and cell volume. Future work should implement high-performance liquid chromatography techniques to accurately characterize the profile of metabolites retained in the organic corona and transition toward quantitative biophysical analysis. Nevertheless, the findings position these fungal-derived gold nanoparticles as a promising nanotherapeutic system with neuroprotective and pro-proliferative capabilities, laying the groundwork for future applications in regenerative medicine and neurorehabilitation aimed at evaluating their capacity to induce neuroplasticity and functional differentiation in the central nervous system.

As the field of green nanotechnology continues to expand, this innovative approach demonstrates that the traditional view of nanoparticles as inert vehicles can be transcended by endowing the metallic core with a biocorona that possesses intrinsic pharmacological properties. The pre-assembled and stable fungal biocorona, composed of tryptamine alkaloids and antioxidants, prevents nonspecific opsonization and directly presents neuroactive ligands to neuronal receptors. Consequently, the cellular response transitions from a stress event due to metallic internalization to a robust process of biostimulation. This research fills a critical gap in the literature and responds to the urgent need to explore more efficient alternative carriers for central nervous system therapeutics, suggesting that the humble mushroom may hold keys to unlocking new frontiers in nanomedicine.

Subject of Research: gold nanoparticles

Article Title: Biosynthesis of gold nanoparticles using Psilocybe cubensis extracts and their biostimulatory evaluation in Neuro2a cells

Article References: Vergara, M., Galleguillos, F., & Becerra, J. (2026). Biosynthesis of gold nanoparticles using Psilocybe cubensis extracts and their biostimulatory evaluation in Neuro2a cells. Frontiers in Nanotechnology, 8, Article 1939102. https://doi.org/10.3389/fnano.2026.1939102

Image Credits: AI Generated

DOI: 10.3389/fnano.2026.1939102

Keywords: Biosynthesis, gold, nanoparticles, Psilocybe, cubensis, extracts, biostimulatory, evaluation, Neuro2a, cells, scientific research