Research reportLactoferrin-modified nanoparticles could mediate efficient gene delivery to the brain in vivo
Introduction
The blood–brain barrier (BBB), due to the presence of tight junctions and lack of fenestrae, is usually the rate-limiting factor for the penetration of proteins, peptides or genes into the central nervous system (CNS) [2]. Fortunately, the BBB possesses specific receptor-mediated transport mechanisms that potentially can be exploited as a means to target drugs to the brain [19], [16]. Till date, receptors discovered on the BBB mainly include transferrin (Tf) receptors, insulin receptors, epidermal growth factor receptors, insulin-like growth factor receptors, and so on [10], [11], [23]. Ligand-mediated brain-targeting drug delivery is one of the focuses at present. The natural ligand, Tf, and the synthetic monoclonal antibody to Tf receptors, for example, have been extensively used as brain-targeting ligands for constructing drug delivery systems to the brain [10], [23], [15], [1]. Some other ligands such as Angiopep-2 to low-density lipoprotein receptor-related protein-1 (LRP1) and RVG29 to GABA (B) receptor have also been exploited for conjugating brain delivery systems [18], [13], [22], [14].
Recently, lactoferrin (Lf) has been exploited as a novel brain-targeting ligand in our lab. Lf is a single-chain iron-binding glycoprotein that belongs to the Tf family. One of the advantages of Lf to be used as a brain-targeting ligand is the low plasma concentration of endogenous Lf, approximately 5 nM [21]. The plasma concentration of Lf is much lower than the Kd of Lf receptors in the BBB [9], efficiently avoiding the competitive inhibition of endogenous Lf to Lf-conjugated exogenous drug delivery systems. Furthermore, the transport of Lf across the BBB monolayer model was reported to be unidirectional, from the apical to the basolateral side [4]. This unidirectional transport might result in higher accumulation of Lf-conjugated drug delivery systems in the neuron, compared to Tf-conjugated counterparts. These advantages were partly reflected in a recent study, the results of which showed that exogenous gene expression of Lf-modified NPs in brains was about 2.3-fold of that of Tf-modified NPs [8]. Similar results were obtained in the biodistribution study of Lf-modified vectors and unmodified counterparts [8]. Hu et al. also reported that an approximate 3-fold of coumarin-6 was found in mice brains carried by Lf-modified NPs compared to that carried by unmodified counterparts [6].
In this study, the brain-targeting properties of Lf-modified NPs were characterized in vivo. The brain accumulation of different NPs was evaluated in macro-level using an in vivo imaging system and in microscopic level via an analytical transmission electron microscope. The expression of exogenous genes in the brain was also studied by staining Von Willebrand in the BBB.
Section snippets
Materials
Polyamidoamine (PAMAM) dendrimer [generation = 5, 21.43%, w/w solution in methanol, containing 128 surface primary amino groups (MW 28,826)], was purchased from Dentritech (Midland, MI, U.S.A.). α-Malemidyl-ω-N-hydroxysuccinimidyl polyethyleneglycol (NHS-PEG-MAL, MW 3400) was obtained from Nektar Therapeutics (Huntsville, AL, U.S.A.). Ethidium monoazide bromide (EMA) was purchased from Molecular Probes (Eugene, OR, U.S.A.). Copper chlorophyll was supplied by Qingdao Green Source Bioengineering
In vivo imaging of mice administrated with different NPs
Nude mice were administered with EMA-labeled Lf-modified NPs, and unmodified NPs as control. In vivo fluorescent images were taken at 4 h after injection. As shown in Fig. 1, EMA-labeled DNA was obviously accumulated in the brain of the mouse treated with Lf-modified NPs (Fig. 1B and F), the fluorescence of which was much stronger than that of unmodified NPs (Fig. 1A and E). Furthermore, the fluorescence relatively concentrated in the central region of the brain, which might be mainly dependent
Discussion
The brain-targeting delivery of Lf-modified NPs was verified in this study. The results fully demonstrated the brain-targeting potential of Lf-modified NPs via several separate means.
Several lines of evidence demonstrated the presence of specific Lf receptors in the brain [21], [3], [20]. Talukder et al. carried out a binding assay for Lf receptors with 125I-Lf and revealed a Kd of 0.11 μM of epithelial membranes of the choroid plexus in young calves [21]. Our previous studies also provided
Acknowledgments
This work was supported by the grants from National Basic Research Program (2007CB935802) of China (973 Program), and National Natural Science Foundation of China (30901861 and 30973652).
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