Engineering Monodisperse GFP‑Doped Silica Nanoparticles for Efficient Intracellular Protein Delivery and Bioimaging

Abstract

We report a streamlined, one‑pot synthesis that yields monodisperse green fluorescent protein (GFP)‑doped silica nanoparticles with diameters ranging from 15 to 35 nm. The method employs L‑arginine‑catalysed sol‑gel chemistry in a biphasic cyclohexane/water system, allowing GFP to be selectively incorporated into the core, shell, or both without compromising protein fluorescence. Detailed characterisation—TEM, DLS, zeta‑potential, fluorescence spectroscopy, and quantum‑yield analysis—demonstrates that the GFP retains its photophysical properties and achieves a quantum yield of 0.62 versus 0.38 for free GFP. Stability studies show negligible protein leakage, enhanced thermal resilience, and photostability, while protease assays confirm protection against enzymatic degradation. Cellular uptake assays in A549 lung carcinoma cells reveal efficient endocytic internalisation and cytosolic delivery of GFP, with minimal lysosomal quenching. These findings establish GFP‑doped silica nanoparticles as robust, multifunctional platforms for intracellular protein delivery and high‑resolution bioimaging.

Background

Encapsulation of proteins within nanoparticles is a cornerstone of modern biomedical technology, enabling biosensing, controlled delivery, and tissue engineering. Maintaining protein conformation and activity during encapsulation is essential, particularly for therapeutic antibodies and enzymes. Silica nanoparticles, prized for their biocompatibility and tunable chemistry, offer an attractive matrix for protein entrapment. Previous approaches—reverse emulsion, sol‑gel, and mesoporous silica—often require harsh conditions that can denature sensitive proteins or yield broad size distributions. Here, we present a gentle, scalable protocol that preserves GFP fluorescence while producing narrowly distributed particles suitable for intracellular delivery.

Methods

Materials

All reagents were purchased from Sigma‑Aldrich and used without further purification. Ultrapure water (18.2 MΩ) served for all aqueous steps.

GFP Preparation

GFP with an N‑terminal His₆‑tag was expressed in E. coli XL1‑Blue using a pQE vector, purified by Ni‑affinity chromatography, and buffer‑exchanged into 7.2 mM L‑arginine (pH 10.3) or 10 mM NaHCO₃ (pH 9.2). Final concentration was 1 mg mL⁻¹.

Nanoparticle Synthesis

Core particles were formed by hydrolysing 5.5 mL TEOS in 69 mL water containing 91 mg L‑arginine and 4.5 mL cyclohexane at 40 °C for 20 h. Subsequent shell growth involved 10 mL core dispersion, 5 mL cyclohexane, and 3.52 mL TEOS, again at 40 °C for 20 h. GFP (200 µg) was added 30 min after TEOS addition to embed the protein during nucleation.

Purification

Nanoparticles were dialysed (MWCO 10 kDa) against water for 4 h with intermittent exchanges, then sterile‑filtered (0.22 µm).

Characterisation

Transmission electron microscopy (JEM‑2100F) quantified core diameters. Dynamic light scattering (Malvern Zetasizer) provided hydrodynamic diameters and zeta potentials. Fluorescence spectra (Fluoromax‑3) were recorded with 488 nm excitation; quantum yields were calculated relative to rhodamine 6G and Atto488 using the Williamson method. Stability was probed by thermal incubation (20 °C vs 60 °C), photobleaching under green LED illumination, and protease‑K digestion. Analytical ultracentrifugation (Beckman‑Coulter XL‑80 K) assessed GFP retention. Cellular uptake was evaluated in A549 cells via confocal microscopy after 24 h exposure to 37 µg mL⁻¹ SiO₂ nanoparticles (5 µg mL⁻¹ GFP) or free GFP.

Results and Discussion

Particle Morphology and Size

Three generations of particles were obtained: core (15.5 ± 1.1 nm), core + shell 1 (23.5 ± 2.0 nm), core + shell 1 + shell 2 (35.3 ± 2.0 nm). Size distribution remained <10 % polydispersity, independent of GFP loading or buffer choice. Unlabelled controls exhibited identical diameters, confirming that protein incorporation does not perturb growth.

Zeta Potential

All formulations displayed highly negative zeta potentials (−28 to −36 mV), indicating robust electrostatic stability and that GFP inclusion does not alter surface charge.

Fluorescence Properties

Emission maxima consistently centered at 508 nm, matching free GFP. Normalised fluorescence intensity increased proportionally with the number of GFP‑doped shells, reflecting volumetric scaling. GFP incorporated from L‑arginine buffer yielded a 1.3‑fold higher intensity than from NaHCO₃, likely due to higher pH (10.3 vs 9.2). Quantum yield measurements revealed 0.62 for GFP‑doped particles versus 0.38 for free GFP, demonstrating that the silica matrix enhances photonic efficiency, possibly by restricting protein motion and shielding from quenchers.

Stability

• Protein leakage: Ultrafiltration through 100 kDa membranes showed no detectable GFP in the filtrate.
• Thermal stability: After 24 h at 60 °C, GFP‑doped particles retained 20 % of initial fluorescence, whereas free GFP lost all signal.
• Photostability: After 20 min of LED exposure, nanoparticles preserved 89 % of fluorescence, compared to 81 % for free GFP.
• Protease resistance: After 90 min with proteinase K, GFP‑doped particles maintained 52 % of fluorescence, versus <7 % for free protein.

Cellular Uptake

Confocal imaging of A549 cells revealed perinuclear fluorescence after nanoparticle treatment, indicative of endocytic uptake and cytosolic delivery. No intracellular GFP signal was observed for free protein, consistent with limited cellular internalisation or rapid lysosomal degradation.

Conclusions

We have developed a versatile, low‑temperature synthesis that produces monodisperse GFP‑doped silica nanoparticles (15–35 nm) with preserved fluorescence, high quantum yield, and superior stability. The ability to control protein placement within core or shell layers enables the design of multifunctional carriers. Importantly, the silica matrix protects the cargo from thermal, photonic, and enzymatic degradation, and facilitates efficient intracellular delivery. These particles represent a promising platform for targeted protein therapeutics and advanced bioimaging.