Melissa J. Simon

Permeability of Endothelial and Astrocyte Cocultures: In Vitro Blood–Brain Barrier Models for Drug Delivery Studies

The blood–brain barrier (BBB) is a major obstacle for drug delivery to the brain. To seek for in vitro BBB models that are more accessible than animals for investigating drug transport across the BBB, we compared four in vitro cultured cell models: endothelial monoculture (bEnd3 cell line), coculture of bEnd3 and primary rat astrocytes (coculture), coculture with collagen type I and IV mixture, and coculture with Matrigel. The expression of the BBB tight junction proteins in these in vitro models was assessed using RT-PCR and immunofluorescence. We also quantified the hydraulic conductivity (Lp), transendothelial electrical resistance (TER) and diffusive solute permeability (P) of these models to three solutes: TAMRA, Dextran 10K and Dextran 70K. Our results show that Lp and P of the endothelial monoculture and coculture models are not different from each other. Compared with in vivo permeability data from rat pial microvessels, P of the endothelial monoculture and coculture models are not significantly different from in vivo data for Dextran 70K, but they are 2–4 times higher for TAMRA and Dextran 10K. This suggests that the endothelial monoculture and all of the coculture models are fairly good models for studying the transport of relatively large solutes across the BBB.

TAT Is Not Capable of Transcellular Delivery Across an Intact Endothelial Monolayer In Vitro

Developing delivery vehicles capable of penetrating cell barriers is critical for drug delivery to the brain due to the presence of the blood–brain barrier (BBB). Cell-penetrating peptides (CPPs) are one potential solution since they can enter cells; however, it is unclear whether CPPs can pass through cell barriers. In this study, the ability of the TAT CPP to cross an endothelial barrier without disrupting the integrity of its tight junctions was investigated. Endothelial cell monolayers (bEnd.3) were exposed to the TAT peptide, and cell integrity was quantified by zona occludens-1 immunofluorescence, trans-endothelial electrical resistance, and hydraulic conductivity. None of these parameters were significantly altered following exposure to TAT. To evaluate the passage of TAT through the monolayer, the permeability of a green fluorescent protein (GFP)–TAT fusion protein was not significantly different from the permeability of GFP or fluorescent dextrans of similar sizes. Furthermore, GFP–TAT was unable to significantly transduce astrocytes on the opposite side of the bEnd.3 monolayer. We conclude, therefore, that although TAT may not be an efficient delivery vehicle for trans-BBB delivery, our TAT construct may have utility in delivering therapeutic cargos to endothelial cells or to the brain parenchyma after BBB disruption.

TAT-MEDIATED INTRACELLULAR PROTEIN DELIVERY TO PRIMARY BRAIN CELLS IS DEPENDENT ON GLYCOSAMINOGLYCAN EXPRESSION

Although some studies have shown that the cell penetrating peptide (CPP) TAT can enter a variety of cell lines with high efficiency, others have observed little or no transduction in vivo or in vitro under conditions mimicking the in vivo environment. The mechanisms underlying TAT‐mediated transduction have been investigated in cell lines, but not in primary brain cells. In this study we demonstrate that transduction of a green fluorescent protein (GFP)‐TAT fusion protein is dependent on glycosaminoglycan (GAG) expression in both the PC12 cell line and primary astrocytes. GFP‐TAT transduced PC12 cells and did so with even higher efficiency following NGF differentiation. In cultures of primary brain cells, TAT significantly enhanced GFP delivery into astrocytes grown under different conditions: (1) monocultures grown in serum‐containing medium; (2) monocultures grown in serum‐free medium; (3) cocultures with neurons in serum‐free medium. The efficiency of GFP‐TAT transduction was significantly higher in the monocultures than in the cocultures. The GFP‐TAT construct did not significantly enter neurons. Experimental modulation of GAG content correlated with alterations in TAT transduction in PC12 cells and astrocyte monocultures grown in the presence of serum. In addition, this correlation was predictive of TAT‐mediated transduction in astrocyte monocultures grown in serum free medium and in coculture. We conclude that culture conditions affect cellular GAG expression, which in turn dictates TAT‐mediated transduction efficiency, extending previous results from cell lines to primary cells. These results highlight the cell‐type and phenotype‐dependence of TAT‐mediated transduction, and underscore the necessity of controlling the phenotype of the target cell in future protein engineering efforts aimed at creating more efficacious CPPs. Biotechnol. Bioeng. 2009; 104: 10–19

Increased delivery of TAT across an endothelial monolayer following ischemic injury.

There is a great need for the development of vehicles capable of delivering therapeutic cargoes across the blood-brain barrier (BBB) and into brain cells. Cell-penetrating peptides (CPPs), such as TAT, present one such solution, and have been used successfully in vivo to deliver neuroprotective cargoes to the brain in models of stroke and seizure. However, a significant discrepancy exists in the literature, as other groups have not had the same success. One commonality between the successful studies is a compromised BBB. In this study, we hypothesized that ischemic injury increases the transport of TAT across an endothelial monolayer (comprised of bEnd.3 cells) in vitro and, consequently, increases TAT-mediated delivery into astrocytes on the other side. In the 24h following in vitro ischemia (oxygen-glucose deprivation), transendothelial electrical resistance (TEER) significantly decreased, indicating disruption of BBB integrity. Concomitantly, the transport of a green fluorescent protein (GFP)-TAT fusion protein significantly increased, and the transduction of GFP-TAT into astrocytes cultured on the other side of the endothelial monolayer significantly increased. These results explain why TAT-mediated delivery of therapeutic cargoes is successful in the ischemic brain but not in the uninjured brain with an intact BBB, highlighting the necessity for continued development of delivery vehicles. We conclude that although TAT may not be an efficient vehicle for trans-BBB delivery across an intact BBB, it may have utility in clinical situations when the BBB is disrupted.

Bifunctional chimeric fusion proteins engineered for DNA delivery: Optimization of the protein to DNA ratio ☆

BACKGROUND Cell penetrating peptides (CPPs) have been used to deliver nucleotide-based therapeutics to cells, but this approach has produced mixed results. Ionic interactions and covalent bonds between the CPPs and the cargos may inhibit the effectiveness of the CPPs or interfere with the bioactivity of the cargos. METHODS We have created a bifunctional chimeric protein that binds DNA using the p50 domain of the NF-kappaB transcription factor and is functionalized for delivery with the TAT CPP. The green fluorescent protein (GFP) has been incorporated for tracking delivery. The new chimeric protein, p50-GFP-TAT, was compared to p50-GFP, GFP-TAT and GFP as controls for the ability to transduce PC12 cells with and without oligonucleotide cargos. RESULTS The p50-GFP-TAT construct can deliver 30 bp and 293 bp oligonucleotides to PC12 cells with an optimal ratio of 1.89 protein molecules per base pair of DNA length. This correlation was validated through the delivery of a fluorescent protein transgene encoded in a plasmid to PC12 cells. Thus, self-assembling CPP-based bifunctional fusion proteins can be engineered for the non-viral delivery of nucleotide-based cargos to mammalian cells. GENERAL SIGNIFICANCE This work represents an important step forward in the rational design of protein-based systems for the delivery of macromolecular cargos.

An unusual cell penetrating peptide identified using a plasmid display-based functional selection platform

Cell penetrating peptides (CPPs) have tremendous potential for use in gene and drug delivery applications. The selection of new CPPs with desired capabilities from randomized peptide libraries is challenging, since the CPP phenotype is a complex selection target. Here we report the discovery of an unusual new CPP from a randomized peptide library using a functional selection system based on plasmid display (PD). After four rounds of screening of a 14-mer peptide library over PC12 cells, several peptides were identified and tested for their ability to deliver the green fluorescent protein (GFP). One peptide (SG3) exhibited a cell penetrating phenotype; however, unlike other well-known CPPs such as TAT or Penetratin, the newly identified peptide was not highly cationic. The PD protocol necessitated the addition of a cationic lipid (Lipofectamine2000), and in the presence of this compound, the SG3 peptide significantly outperformed the well-known TAT CPP in the delivery of GFP to PC12 cells and primary astrocytes. When the SG3 peptide was fused to the pro-apoptotic BH3 peptide from the Bak protein, significant cell death was induced in cultured primary astrocytes, indicating relevant, intracellular delivery of a functional cargo. The PD platform is a useful method for identifying functional new CPPs from randomized libraries with unique delivery capabilities.

Attenuation of Astrocyte Activation by TAT-Mediated Delivery of a Peptide JNK Inhibitor

Astrocyte activation contributes to the brains response to disease and injury. Activated astrocytes generate harmful radicals that exacerbate brain damage including nitric oxide, peroxides and superoxides. Furthermore, reactive astrocytes hinder regeneration of damaged neural circuits by secreting neuro-developmental inhibitors and glycosaminoglycans (GAGs), which physically block growth cone extension. Therefore, targeted therapeutic strategies to limit astrocyte activation may enhance recovery from many neurodegenerative states. Previously, we demonstrated that the HIV-1 TAT cell-penetrating peptide, a short non-toxic peptide from the full-length TAT protein, delivered a protein cargo to astrocytes in a process dependent on cell-surface GAG. Since activated astrocytes produce GAG, in this study we tested whether TAT could transduce activated astrocytes, deliver a biologically active cargo, and produce a physiological effect. Astrocyte activation was induced by IL-1β, lipopolysaccharide (LPS), or mechanical stretch injury, and quantified by increased GAG and nitrite content. TAT-mediated delivery of a mock therapeutic protein, GFP, increased significantly after activation. Nitrite production, GAG expression, and GFP-TAT transduction were significantly attenuated by inhibitors of JNK, p38, or ERK. TAT fused to a peptide JNK inhibitor delivered the peptide inhibitor to activated astrocytes and significantly reduced activation. Our study is the first to report significant and direct modulation of astrocyte activation with a peptide JNK inhibitor. Our promising in vitro results warrant in vivo follow-up, as TAT-mediated protein delivery may have broad therapeutic potential for preventing astrocyte activation with the possibility of limiting off-target, negative side effects.

A plasmid display platform for the selection of peptides exhibiting a functional cell-penetrating phenotype.

Cell‐penetrating peptides (CPPs) represent a promising nonviral platform for the delivery of therapeutic cargos to cells and tissues. However, these peptides are often nonspecific, and their mechanism of action is still a subject of debate, which hinders the design of new CPPs. The alternative to rational protein design is the combinatorial approach to protein engineering, whereby large libraries of peptides are created and a screening or selection procedure is used to identify members with the desired phenotype(s). Here we describe a novel procedure for selecting peptides with a CPP phenotype using a plasmid display (PD) platform to link the peptides to their encoding DNA sequences. The PD system is based on genetic fusions to a DNA binding domain. The plasmid was designed to concomitantly express a fluorescent reporter protein to serve as a mock therapeutic cargo indicating its functional delivery into a cell. We have demonstrated this selection strategy using a control CPP (the TAT peptide) in the PC12 neuronal‐like cell line. In the absence of transfection reagents, TAT was unable to deliver the protein/DNA complexes. The inclusion of the HA2 peptide from the hemagglutinin protein and the addition of polyethylenimine (PEI) were similarly ineffective. The addition of Lipofectamine, however, enabled the TAT‐mediated delivery of the protein/DNA complexes, which was significantly better than control experiments without a CPP. This new PD selection platform will be a valuable new approach for use in identifying unique CPPs from randomized libraries with novel abilities and specificities.

Evaluation of the cell-penetrating peptide TAT as a trans-blood-brain barrier delivery vehicle

Therapeutic delivery into the brain parenchyma is difficult due to the presence of the blood-brain barrier (BBB). One potential solution is to use cell-penetrating peptides (CPPs). CPPs are short peptides made up of primarily positively charged amino acids that have the ability to transduce cell membranes and deliver cargoes to cells. It is unclear, however, whether CPPs can penetrate cell barriers, such as the BBB, and deliver cargoes into cells on the other side. In this study, we examined the ability of the CPP TAT to deliver a green fluorescent protein (GFP) cargo across an endothelial monolayer. GFP-TAT had only minimal permeability across the monolayer, and the rate was no different from GFP alone or other similarly sized dextrans. Furthermore, this permeability was too small to detect transduction of GFP-TAT into astrocytes on the other side of the monolayer. We conclude that TAT does not have the ability to deliver cargoes across an intact BBB, and therefore may not be a suitable vehicle for therapeutic delivery to the brain.

Permeability of in vitro blood-brain barrier models

The blood-brain barrier (BBB) is a major obstacle for drug delivery to the brain for treatment of CNS disorders. To search for more convenient models in studying the transport across the BBB, we compared four in vitro models: endothelial monoculture (bEnd3 cell line), coculture of bEnd3 and astrocytes (coculture), coculture with collagen type I and IV mixture, and coculture with Matrigel. We also quantified the hydraulic conductivity (Lp), trans-electrical resistance (TER) and diffusive permeability (P) of these models to three solutes: TAMRA, Dextran 10K and Dextran70K. Our results showed that Lp and P of the endothelial monolayer and coculture models are not different from each other. Compared with in vivo permeability data from rat pial microvessels [1], P of the endothelial monolayer and coculture models are not significantly different from in vivo data for Dextran 70K while they are 2–4 times higher for TAMRA and Dextran 10K. The results suggested that endothelial monolayer and all the coculture models are fairly good models for studying transport of relatively large solutes across the BBB.