Abstract
BackgroundTherapeutic intervention of numerous brain-associated disorders currently remains unrealized due to serious limitations imposed by the blood-brain-barrier (BBB). The BBB generally allows transport of small molecules, typically <600 daltons with high octanol/water partition coefficients, but denies passage to most larger molecules. However, some receptors present on the BBB allow passage of cognate proteins to the brain. Utilizing such receptor-ligand systems, several investigators have developed methods for delivering proteins to the brain, a critical requirement of which involves covalent linking of the target protein to a carrier entity. Such covalent modifications involve extensive preparative and post-preparative chemistry that poses daunting limitations in the context of delivery to any organ. Here, we report creation of a 36-amino acid peptide transporter, which can transport a protein to the brain after routine intravenous injection of the transporter-protein mixture. No covalent linkage of the protein with the transporter is necessary.ApproachA peptide transporter comprising sixteen lysine residues and 20 amino acids corresponding to the LDLR-binding domain of apolipoprotein E (ApoE) was synthesized. Transport of beta-galactosidase, IgG, IgM, and antibodies against amyloid plques to the brain upon iv injection of the protein-transporter mixture was evaluated through staining for enzyme activity or micro single photon emission tomography (micro-SPECT) or immunostaining. Effect of the transporter on the integrity of the BBB was also investigated.Principal FindingsThe transporter enabled delivery to the mouse brain of functional beta-galactosidase, human IgG and IgM, and two antibodies that labeled brain-associated amyloid beta plaques in a mouse model of Alzheimer's disease.SignificanceThe results suggest the transporter is able to transport most or all proteins to the brain without the need for chemically linking the transporter to a protein. Thus, the approach offers an avenue for rapid clinical evaluation of numerous candidate drugs against neurological diseases including cancer. (299 words).
Highlights
Numerous potential drug candidates for treating brain-associated disorders involving mood, behavior, addiction, aging, infection, cancer and neurodegenerative disease exist but therapeutic use of these candidate drugs currently remains unrealized due to serious impediment imposed by the blood-brain-barrier (BBB) [1,2,3,4,5,6,7,8,9]
Intense beta-galactosidase activity was observed in the mouse brain when the enzyme-peptide mix was injected intravenously and brain slices were prepared for enzyme activity staining 6 h after injection,{data presented in Figure S1 demonstrate that 6 h of time is sufficient for visual evaluation of intracerebral beta-galactosidase activity after K16ApoE-mediated iv delivery in brain}whereas no activity was seen when the enzyme was injected alone (Figure 1)
High-magnification scan of several regions of brain-sections stained for K16ApoE-delivered betagalactosidase activity show that the enzyme was delivered in nearly every area of the brain, and appeared to have stained all cells when compared to cells stained with hematoxylin
Summary
Numerous potential drug candidates for treating brain-associated disorders involving mood, behavior, addiction, aging, infection, cancer and neurodegenerative disease exist but therapeutic use of these candidate drugs currently remains unrealized due to serious impediment imposed by the blood-brain-barrier (BBB) [1,2,3,4,5,6,7,8,9]. Some receptors present on the BBB allow passage of cognate proteins to the brain Utilizing such receptor-ligand systems, several investigators have developed methods for delivering proteins to the brain, a critical requirement of which involves covalent linking of the target protein to a carrier entity. Such covalent modifications involve extensive preparative and postpreparative chemistry that poses daunting limitations in the context of delivery to any organ. No covalent linkage of the protein with the transporter is necessary
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