Lactoferrin-conjugated poly(ethylene glycol)-coated Fe3O4 nanoparticles


Shan L.

Publication Details



In vitro Rodents



The lactoferrin (Lf)-conjugated poly(ethylene glycol) (PEG)-coated Fe3O4 nanoparticles, abbreviated as Fe3O4-Lf, was synthesized by Qiao et al. for use as a contrast agent for magnetic resonance imaging (MRI) of the brain (1). As a ligand, Lf acts to enhance the blood–brain barrier (BBB) penetration and Lf receptor-targeting of Fe3O4-Lf.

BBB is composed of tight junction-sealed brain capillary endothelial cells (BCECs) and supporting pericytes and astrocytic endfeet (2). Exogenous compounds are prevented by the BBB from reaching the brain by passive transport or through the paracellular route (3). Because many proteins including Lf could effectively cross the BBB through transcytosis, an active transport mechanism of BCECs, this mechanism has been actively used to design brain-targeted delivery systems for targeted imaging and therapy of neurological diseases (4, 5).

Mammalian Lf is a cationic iron-binding glycoprotein (80 kDa), and its receptor expresses on the endothelial cells of BBB (6). Upon binding with its receptor, Lf could cross the BBB through receptor-mediated transcytosis (5). The transport of Lf across the BBB is unidirectional, from the apical side to the basolateral side, with no apparent intraendothelial degradation (1, 7). Studies with membrane preparations of mouse brains have shown that the Lf receptor in the BCECs has two classes of binding sites: a high-affinity site that has a dissociation constant (Kd) of 10.61 nM and a Bmax of 410 fmol bound/µg protein; and a low-affinity site that has a Kd of 2,228 nM and a Bmax of 51,641 fmol bound/µg protein (5). The plasma concentration of endogenous Lf (~5 nM) is lower than the Kd of Lf receptors in the BBB, which avoids the competitive inhibition of endogenous Lf to exogenous Lf-conjugated agents (1). Lf receptor has also been shown to be overexpressed in various tumors including brain glioma (8). Because of these features, Lf has been applied as a ligand in designing brain-targeted delivery systems.

Xie et al. conjugated Lf to superparamagnetic iron oxide nanoparticles (SPIONs) to develop the contrast agent Lf-SPION (8), while Qiao et al. developed the agent Fe3O4-Lf by conjugating Lf to Fe3O4 nanoparticles through PEG (1). For the former agent, Lf was designed to target Lf-SPIONs to tumors expressing the Lf receptor. For the latter agent, Lf served to enhance the BBB-penetrating ability of Fe3O4-Lf by targeting Lf receptors expressed in the BCECs. Studies with the two agents showed that Lf-SPIONs were able to effectively enhance the glioma contrast, and Fe3O4-Lf could effectively penetrate the BBB in healthy rats because of the conjugation of Lf (1, 8). This chapter summarizes the data obtained by Qiao et al. with Fe3O4-Lf.



Qiao et al. described the synthesis of Fe3O4-Lf in detail (1). Briefly, the PEG-coated Fe3O4 nanoparticles were synthesized with a “one-pot” synthetic reaction of Fe(acac)3, oleylamine, and HOOC-PEG-COOH in the diphenyl ether solution. Ether was then used to precipitate and purify the Fe3O4 nanocrystal. The final product, PEG-coated Fe3O4, was formulated in either Milli-Q water or phosphate-buffered saline.

The average size of PEG-coated Fe3O4 nanoparticles was 16.5 ± 1.6 nm in diameter, as measured with transmission electron microscopy. The molar transversal relaxivity was 231 mM–1s–1. The organic content accounted for ~16.6% of the particles. A room-temperature magnetization curve confirmed that the PEG-coated Fe3O4 nanocrystals were superparamagnetic, and the particles exhibited a saturation magnetization of 66.0 emu/g, corresponding to 79.1 emu/g Fe3O4.

The Fe3O4-Lf conjugate was prepared with the classic EDC/sulfo-NHS-mediated amidation reaction. The hydrodynamic size of PEG-coated Fe3O4 as determined with dynamic light scattering was 43.6 nm, and this value increased to 48.9 nm after Lf conjugation, suggesting that Lf was effectively coupled to the PEG-coated Fe3O4 nanoparticles. The polydispersity index of Fe3O4-Lf (0.386) was similar to that of PEG-coated Fe3O4 (0.348), indicating that particles did not coagulate during the coupling reaction. The Bradford method was adopted to determine the protein content and revealed ~14.4 Lf molecules per Fe3O4 nanoparticle on average for the Fe3O4-Lf. The chemical yield and purity of Fe3O4-Lf were not reported.

In Vitro Studies: Testing in Cells and Tissues


Qiao et al. first tested the permeability of Fe3O4-Lf using an in vitro model of the BBB (1). The model was established with primary porcine BCECs that were cultured on microporous filter membrane inserts within a chamber (9, 10). Transendothelial electrical resistance (TEER) was measured after incubation of BCECs with Fe3O4-Lf or PEG-coated Fe3O4 at concentrations of 0.04, 0.1, and 0.3 mg Fe/mL, respectively (1). Water was used as a reference for the changes of TEER after nanoparticle exposure. TEER is an indicator of BBB integrity and is usually expressed as measured resistance multiplied by the area of endothelial monolayer (Ω cm2). TEER level is mainly determined by the tight junctions between endothelial cells. For the model used by Qiao et al., the TEER was 1,500–2,000 Ω cm2, typically >700 Ω cm2 after 7 days of culture (1).

At the concentration of 0.04 mg Fe/mL, both Fe3O4-Lf and PEG-coated Fe3O4 decreased the TEER level during the initial several hours of exposure. The TEER level recovered over time. Water resulted in similar changes of TEER. These results suggested that the initial decrease of TEER was mainly due to the cell response to the environmental change. However, compared to water, Fe3O4-Lf led to a lower degree of TEER reduction, while PEG-coated Fe3O4 resulted in a higher degree of TEER reduction. This trend was more evident at the concentration of 0.1 mg Fe/mL, indicating that the BBB remained more intact in the presence of Fe3O4-Lf than PEG-coated Fe3O4. At the high concentration of 0.3 mg Fe/mL, both Fe3O4-Lf and PEG-coated Fe3O4 led to much weaker TEER signals than water, suggesting considerable damage to the tight junctions by both Fe3O4-Lf and PEG-coated Fe3O4 at this concentration. Overall, the results indicated that Lf could protect the tight junctions from being damaged by the Fe3O4 nanoparticles at low concentrations. Experiments performed by incubating Lf alone with the BCECs further confirmed that Lf could increase the TEER value.

Qiao et al. then evaluated the efficiency of particle transport by measuring the iron content in the media of the basolateral side after ~18 h incubation of the BCECs with either Fe3O4-Lf or PEG-coated Fe3O4 particles (1). At concentrations of 0.04 and 0.1 mg Fe/mL of the agents at the apical side, the transport efficiency of Fe3O4 particles was strongly enhanced by the Lf conjugation (Table 1). The transport of Fe3O4-Lf (0.1 mg Fe/mL) could be blocked with Lf, showing that the iron content dropped from 22.0 ± 2.9% to 1.0 ± 0.6% in the presence of 16 times the amount of Lf in the apical media. Consistent with other data, these results also indicate that Lf could facilitate the cross of Fe3O4 particles through the BBB.

Table 1 Transport efficiency of Fe3O4-Lf or PEG-coated Fe3O4 particles in in vitro BBB model.

Animal Studies



Qiao et al. evaluated the BBB-penetrating efficiency of Fe3O4-Lf in vivo with T2*-weighted MRI after intravenous injection of PEG-coated Fe3O4 or Fe3O4-Lf (10 mg Fe/kg) into Sprague-Dawley rats (n = 2/group) (1). At 15 min after injection, stronger contrast-enhanced vascular images of the brain were obtained with Fe3O4-Lf than with PEG-coated Fe3O4. Similarly, a stronger effect on reducing T2* value in the thalamus, brain stem, and frontal cortex was observed at 24 h with Fe3O4-Lf than with PEG-coated Fe3O4. These results supported the conclusion that the BBB-penetrating efficiency of Fe3O4 particles could be enhanced through Lf-receptor-mediated transport in vivo.

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.


Qiao R., Jia Q., Huwel S., Xia R., Liu T., Gao F., Galla H.J., Gao M. Receptor-mediated delivery of magnetic nanoparticles across the blood-brain barrier. ACS Nano. 2012;6(4):3304–10. [PubMed: 22443607]
Fortin D. The blood-brain barrier: its influence in the treatment of brain tumors metastases. Curr Cancer Drug Targets. 2012;12(3):247–59. [PubMed: 22229251]
Fu B.M. Experimental methods and transport models for drug delivery across the blood-brain barrier. Curr Pharm Biotechnol. 2012;13(7):1346–59. [PMC free article: PMC5349708] [PubMed: 22201587]
Huang R., Ke W., Han L., Liu Y., Shao K., Jiang C., Pei Y. Lactoferrin-modified nanoparticles could mediate efficient gene delivery to the brain in vivo. Brain Res Bull. 2010;81(6):600–4. [PubMed: 20026388]
Huang R.Q., Ke W.L., Qu Y.H., Zhu J.H., Pei Y.Y., Jiang C. Characterization of lactoferrin receptor in brain endothelial capillary cells and mouse brain. J Biomed Sci. 2007;14(1):121–8. [PubMed: 17048089]
Suzuki Y.A., Lopez V., Lonnerdal B. Mammalian lactoferrin receptors: structure and function. Cell Mol Life Sci. 2005;62(22):2560–75. [PubMed: 16261254]
Fillebeen C., Descamps L., Dehouck M.P., Fenart L., Benaissa M., Spik G., Cecchelli R., Pierce A. Receptor-mediated transcytosis of lactoferrin through the blood-brain barrier. J Biol Chem. 1999;274(11):7011–7. [PubMed: 10066755]
Xie H., Zhu Y., Jiang W., Zhou Q., Yang H., Gu N., Zhang Y., Xu H., Yang X. Lactoferrin-conjugated superparamagnetic iron oxide nanoparticles as a specific MRI contrast agent for detection of brain glioma in vivo. Biomaterials. 2011;32(2):495–502. [PubMed: 20970851]
Garcia-Garcia E., Gil S., Andrieux K., Desmaele D., Nicolas V., Taran F., Georgin D., Andreux J.P., Roux F., Couvreur P. A relevant in vitro rat model for the evaluation of blood-brain barrier translocation of nanoparticles. Cell Mol Life Sci. 2005;62(12):1400–8. [PMC free article: PMC2773840] [PubMed: 15905957]
Roux F. andCouraud, P.O. Rat brain endothelial cell lines for the study of blood-brain barrier permeability and transport functions. Cell Mol Neurobiol. 2005;25(1):41–58. [PubMed: 15962508]