The blood-brain barrier (BBB) poses great difficulties for gene delivery to the brain. To circumvent the BBB, we investigated a novel brain-targeting gene vector based on the nanoscopic high-branching dendrimer, polyamidoamine (PAMAM), in vitro and in vivo. Transferrin (Tf) was selected as a brain-targeting ligand conjugated to PAMAM via bifunctional polyethyleneglycol (PEG), yielding PAMAM-PEG-Tf. UV and nuclear magnetic resonance (NMR) spectroscopy were used to evaluate the synthesis of vectors. The characteristics and biodistribution of gene vectors were evaluated by fluorescent microscopy, flow cytometry, and a radiolabeling method. The transfection efficiency of vector/DNA complexes in brain capillary endothelial cells (BCECs) was evaluated by fluorescent microscopy and determination of luciferase activity. The potency of vector/DNA complexes was evaluated by using frozen sections and measuring tissue luciferase activity in Balb/c mice after i.v. administration. UV and NMR results demonstrated the successful synthesis of PAMAM-PEG-Tf. This vector showed a concentration-dependent manner in cellular uptake study and a 2.25-fold brain uptake compared with PAMAM and PAMAM-PEG in vivo. Transfection efficiency of PAMAM-PEG-Tf/DNA complex was much higher than PAMAM/DNA and PAMAM-PEG/DNA complexes in BCECs. Results of tissue expression experiments indicated the widespread expression of an exogenous gene in mouse brain after i.v. administration. With a PAMAM/DNA weight ratio of 10:1, the brain gene expression of the PAMAM-PEG-Tf/DNA complex was approximately 2-fold higher than that of the PAMAM/DNA and PAMAM-PEG/DNA complexes. These results suggested that PAMAM-PEG-Tf can be exploited as a potential nonviral gene vector targeting to brain via noninvasive administration.

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We present, herein, the evidence for lactoferrin (Lf) binding sites in brain endothelial capillary cells (BCECs) and mouse brain. The results from confocal microscopy showed the presence of Lf receptors on the surface of BCECs and the receptor-mediated endocytosis for Lf to enter the cells. Saturation binding analyses revealed that Lf receptors exhibited two classes of binding sites in BCECs (high affinity: dissociation constant (K (d)) = 6.77 nM, binding site density (B (max)) = 10.3 fmol bound/mug protein; low affinity: K (d) = 4815 nM, B (max) = 1190 fmol bound/mug protein) and membrane preparations of mouse brain (high affinity: K (d) = 10.61 nM, B (max) = 410 fmol bound/mug protein; low affinity: K (d) = 2228 nM, B (max) = 51641 fmol bound/mug protein). The distribution study indicated the effective uptake of (125)I-Lf in brain after intravenous administration. The present study provides experimental evidence for the application of Lf as a novel ligand for brain targeting.

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