gH625 Peptide vs. Folic Acid: Enhancing Fe3O4 Magnetic Nanoparticle Uptake Across the Blood–Brain Barrier

Abstract

We developed a robust, two‑step synthesis of Fe₃O₄ magnetic nanoparticles (MNPs) pre‑functionalized with a bifunctional phosphonic acid monolayer. This platform enabled covalent attachment of the membranotropic peptide gH625, known for its efficient cell‑penetrating properties, and, as a reference, PEG/folic acid/rhodamine (PEG,FA@MNPs) coatings were prepared. Confocal laser‑scanning microscopy of immortalized human brain microvascular endothelial cells (HBMECs) revealed that, after 24 h, gH625‑decorated MNPs are internalized nearly twice as efficiently as FA‑functionalized particles. Both surface chemistries were readily taken up by glioblastoma A‑172 cells, indicating that gH625 does not compromise tumor‑cell targeting. These findings demonstrate that gH625 functionalization is a promising strategy for delivering magnetic nanostructures across the blood‑brain barrier (BBB) and into brain tumor cells.

Background

The BBB is a highly selective interface that protects the central nervous system but also hampers drug delivery. Endothelial cells, astrocytes, and pericytes form a continuous, tight‑junctioned monolayer that excludes ~98 % of small molecules and all large therapeutics from the brain. Nanoparticle‑based carriers, particularly magnetic iron oxide MNPs, offer unique advantages—magnetic manipulation, imaging, and biocompatibility—but their ability to cross the BBB remains limited.

Cell‑penetrating peptides (CPPs) such as the 20‑residue gH625 (derived from HSV‑1 glycoprotein H) have shown remarkable membrane‑crossing capabilities and have been successfully used to ferry cargoes across the BBB. In contrast, folic acid (FA) is a well‑established ligand for targeting folate receptors overexpressed on many tumors, including glioblastoma, but its efficacy for BBB penetration is lower.

Methods

Materials

All reagents, including FeCl₂·4H₂O, FeCl₃·6H₂O, 3‑aminopropylphosphonic acid, PEG‑NHS (MW 5 kDa), FA, NHS, and Rhod‑NHS, were purchased from Sigma‑Aldrich and used as received. Milli‑Q water (18.2 MΩ cm) was employed throughout.

Synthesis of Fe₃O₄ MNPs

Fe₃O₄ nanoparticles were obtained by alkaline co‑precipitation (Fe³⁺:Fe²⁺ = 2:1) at 80 °C under N₂. The resulting brownish suspension was washed, magnetically decanted, and dried.

Phosphonic Acid Functionalization (NH₂@MNPs)

200 mg of bare MNPs were dispersed in 25 mL water, sonicated for 30 min, and reacted with 100 mg 3‑aminopropylphosphonic acid (NH₂-PA) for 2 h at room temperature. After magnetic separation and thorough washing, NH₂@MNPs were obtained.

gH625 Conjugation (gH625@MNPs)

300 mg NH₂@MNPs were mixed with 1.2 mg NBD‑labeled gH625 in 20 mL DMSO and incubated overnight at 25 °C. Magnetic purification and washing yielded gH625@MNPs.

PEG/FA/Rhodamine Conjugation (PEG,FA@MNPs)

300 mg NH₂@MNPs were reacted with 30 mg PEG‑NHS, 3 mg FA‑NHS, and 3 mg Rhod‑NHS in 15 mL DMSO overnight. After magnetic separation and washing, PEG,FA@MNPs were collected.

Characterization

XRD confirmed the mixed magnetite/maghemite phase (a = 8.389 Å, crystal size ≈ 11 nm). XPS verified phosphonate anchoring and preserved Fe³⁺/Fe²⁺ ratios. FT‑IR showed characteristic P–O–Fe bands (1040 cm⁻¹) and amide signals (1650 cm⁻¹). UV/Vis of PEG,FA@MNPs displayed the FA absorption peak at 274 nm, confirming successful conjugation.

Dynamic light scattering (PBS, pH 7.4) gave hydrodynamic diameters of 73 ± 3 nm (NH₂@MNPs), 104 ± 4 nm (gH625@MNPs), and 51 ± 2 nm (PEG,FA@MNPs). All systems exhibited highly negative zeta potentials (< −30 mV), ensuring colloidal stability for at least 72 h.

Cell Viability

MTT assays on HBMECs and A‑172 cells (10–20 µg mL⁻¹, 24–72 h) showed no significant cytotoxicity for either functionalized MNPs.

Cellular Uptake

HBMECs incubated with 15 µg mL⁻¹ of each MNP type for 24 h displayed markedly stronger green fluorescence (gH625@MNPs) than red (PEG,FA@MNPs), confirming superior uptake. Quantification revealed a ~2× increase in intracellular fluorescence for gH625@MNPs versus PEG,FA@MNPs at 24 h. After 72 h, the difference diminished, likely due to non‑specific internalization of FA‑decorated particles.

In A‑172 glioblastoma cells, uptake of both gH625@MNPs and PEG,FA@MNPs after 24 h was comparable, indicating that gH625 does not impair tumor‑cell targeting.

Conclusions

Fe₃O₄ MNPs functionalized with the gH625 peptide via a phosphonic acid linker exhibit markedly enhanced uptake by BBB endothelial cells compared to FA‑functionalized counterparts. The phosphonic acid platform ensures stable, highly negative surfaces and preserves nanoparticle integrity over time. gH625‑decorated MNPs therefore represent a potent vehicle for magnetic drug delivery across the BBB and into brain tumors, offering a safer and more efficient alternative to traditional ligand strategies.