Targeted Delivery of GDNF through the Blood–Brain Barrier by MRI-Guided Focused Ultrasound

Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), are promising therapeutic agents for neurodegenerative diseases. However, the application of GDNF to treat these diseases effectively is limited because the blood–brain barrier (BBB) prevents the local delivery of macromolecular therapeutic agents from entering the central nervous system (CNS). Focused ultrasound combined with microbubbles (MBs) using appropriate parameters has been previously demonstrated to be able to open the BBB locally and noninvasively. This study investigated the targeted delivery of GDNF MBs through the BBB by magnetic resonance imaging (MRI)-guided focused ultrasound. Evans Blue extravasation and histological examination were used to determine the optimum focused ultrasound parameters. Enzyme-linked immunosorbent assay was performed to verify the effects of GDNF bound on MBs using a biotin–avidin bridging chemistry method to promote GDNF delivery into the brain. The results showed that GDNF can be delivered locally and noninvasively into the CNS through the BBB using MRI-guided focused ultrasound combined with MBs under optimum parameters. MBs that bind GDNF combined with MRI-guided focused ultrasound may be an effective way of delivering neurotrophic factors directly into the CNS. The method described herein provides a potential means of treating patients with CNS diseases.     

Strategies for enhancing antibody delivery to the brain

Antibodies and antibody conjugates have emerged as important tools for cancer therapy. However, a major therapeutic challenge for the use of antibodies is their inability to cross the blood–brain barrier (BBB) to reach tumors localized in the central nervous system (CNS). Multiple methods have been developed to enhance antibody delivery to the CNS, including direct injection, mechanical or biochemical disruption of the BBB, conjugation to a ‘molecular Trojan horse’, cationization, encapsulation in nanoparticles and liposomes, and more recently, stem cell-mediated antibody delivery. In this review, we discuss each of these approaches, highlighting their successes and the obstacles that remain to be overcome.     

Ultrasound, microbubbles and the blood-brain barrier

The blood–brain barrier (BBB) is a specialized system of capillary endothelial cells that protects the brain from harmful substances in the blood stream, while supplying the brain with the required nutrients for proper function. The BBB controls transport through both tight junctions and metabolic barriers and is often a rate-limiting factor in determining permeation of therapeutic drugs into the brain. It is a significant obstacle for delivery of both small molecules and macromolecular agents. Although many drugs could be potentially used to treat brain disease, there has been no method that allows non-invasive-targeted delivery through the BBB. Recently, promising studies indicate that ultrasound can be used to locally deliver a drug or gene to a specific region of interest in the brain. If microbubbles are combined with ultrasound exposure, the effects of ultrasound can be focused upon the vasculature to reduce the acoustic intensity needed to produce BBB opening. Several avenues of transcapillary passage after ultrasound sonication have been identified including transcytosis, passage through endothelial cell cytoplasmic openings, opening of tight junctions and free passage through injured endothelium. This article reviews the topic of transient disruption of the BBB with ultrasound and microbubbles and addresses related safety issues. It also discusses possible roles of the BBB in brain disease and potential interactions with ultrasound and microbubbles in such disease states.     

Peripheral delivery of a CNS targeted, metalo-protease reduces aβ toxicity in a mouse model of Alzheimer's disease

Alzheimer's disease (AD), an incurable, progressive neurodegenerative disorder, is the most common form of dementia. Therapeutic options have been elusive due to the inability to deliver proteins across the blood-brain barrier (BBB). In order to improve the therapeutic potential for AD, we utilized a promising new approach for delivery of proteins across the BBB. We generated a lentivirus vector expressing the amyloid β-degrading enzyme, neprilysin, fused to the ApoB transport domain and delivered this by intra-peritoneal injection to amyloid protein precursor (APP) transgenic model of AD. Treated mice had reduced levels of Aβ, reduced plaques and increased synaptic density in the CNS. Furthermore, mice treated with the neprilysin targeting the CNS had a reversal of memory deficits. Thus, the addition of the ApoB transport domain to the secreted neprilysin generated a non-invasive therapeutic approach that may be a potential treatment in patients with AD.     

The rostral migratory stream plays a key role in intranasal delivery of drugs into the CNS

Background

The blood brain barrier (BBB) is impermeable to most drugs, impeding the establishment of novel neuroprotective therapies and strategies for many neurological diseases. Intranasal administration offers an alternative path for efficient drug delivery into the CNS. So far, the anatomical structures discussed to be involved in the transport of intranasally administered drugs into the CNS include the trigeminal nerve, olfactory nerve and the rostral migratory stream (RMS), but the relative contributions are debated.

Methods and Findings

In the present study we demonstrate that surgical transection, and the resulting structural disruption of the RMS, in mice effectively obstructs the uptake of intranasally administered radioligands into the CNS. Furthermore, using a fluorescent cell tracer, we demonstrate that intranasal administration in mice allows agents to be distributed throughout the entire brain, including olfactory bulb, hippocampus, cortex and cerebellum.

Conclusions

This study provides evidence of the vital role the RMS has in the CNS delivery of intranasally administered agents. The identification of the RMS as the major access path for intranasally administered drugs into the CNS may contribute to the development of treatments that are tailored for efficient transport within this structure. Research into the RMS needs to continue to elucidate its limitations, capabilities, mechanisms of transport and potential hazards before we are able to advance this technique into human research.     

Delivery of peptides to the brain: emphasis on therapeutic development

Peptides and regulatory proteins hold great promise as therapeutic agents for the central nervous system (CNS). However, the blood-brain barrier (BBB) is a major obstacle to the delivery of these potential therapeutics to their site of action. We concentrate here on the vascular BBB, which is comprised of the capillary bed of the brain specially modified to prevent the production of a plasma ultrafiltrate. For many peptides and proteins, this physical barrier is reinforced by enzymatic activities at the BBB, CNS, and peripheral tissues, short half-lives and large volumes of distribution in the blood, binding proteins in blood, and brain-to-blood efflux systems. Nevertheless, there are pathways through which substances can cross. Small, lipid soluble substances cross by the nonsaturable mechanism of transmembrane diffusion, but even water-soluble peptides can cross to some degree. Many endogenous peptides and regulatory proteins cross the BBB by way of selective, saturable transport systems. For enzymatically resistant substances with long circulating half-lives and small volumes of distribution, such as antibodies, erythropoietin, and enzymes, substances can enter the CNS in therapeutic amounts through the residual leak of the BBB, termed the extracellular pathways. Recent examples show that the BBB transporters for peptides and regulatory substances are modifiable. This provides both a therapeutic opportunity and the potential for disease to arise from BBB dysfunctions. In the last case, the BBB itself is a therapeutic target.     

Noninvasive, transient and selective blood-brain barrier opening in non-human primates in vivo

The blood-brain barrier (BBB) is a specialized vascular system that impedes entry of all large and the vast majority of small molecules including the most potent central nervous system (CNS) disease therapeutic agents from entering from the lumen into the brain parenchyma. Microbubble-enhanced, focused ultrasound (ME-FUS) has been previously shown to disrupt noninvasively, selectively, and transiently the BBB in small animals in vivo. For the first time, the feasibility of transcranial ME-FUS BBB opening in non-human primates is demonstrated with subsequent BBB recovery. Sonications were combined with two different types of microbubbles (customized 4–5 µm and Definity®). 3T MRI was used to confirm the BBB disruption and to assess brain damage.     

A review of multifunctional nanoemulsion systems to overcome oral and CNS drug delivery barriers

The oral and central nervous systems (CNS) present a unique set of barriers to the delivery of important diagnostic and therapeutic agents. Extensive research over the past few years has enabled a better understanding of these physical and biological barriers based on tight cellular junctions and expression of active transporters and metabolizing enzymes at the luminal surfaces of the gastrointestinal (GI) tract and the blood-brain barrier (BBB). This review focuses on the recent understanding of transport across the GI tract and BBB and the development of nanotechnology-based delivery strategies that can enhance bioavailability of drugs. Multifunctional lipid nanosystems, such as oil-in-water nanoemulsions, that integrate enhancement in permeability, tissue and cell targeting, imaging, and therapeutic functions are especially promising. Based on strategic choice of edible oils, surfactants and additional surface modifiers, and different types of payloads, rationale design of multifunctional nanoemulsions can serve as a safe and effective delivery vehicle across oral and CNS barriers.     

Strategies to overcome the barrier: use of nanoparticles as carriers and modulators of barrier properties

The blood–brain barrier (BBB) protects the brain from toxic substances within the bloodstream and keeps the brain’s homeostasis stable. On the other hand, it also represents the main obstacle in the treatment of many CNS diseases. Among different techniques, nanoparticles have emerged as promising tools to enhance brain drug delivery of therapeutic molecules. For successful drug delivery, nanoparticles may either modulate BBB integrity or exploit transport systems present on the endothelium. In this review, we present two different nanoparticles to enhance brain drug delivery. Poly(butyl cyanoacrylate) nanoparticles were shown to induce a reversible disruption of the BBB in vitro which may be exploited by simultaneous injection of the drug in question. By coating the poly(butyl cyanoacrylate) nanoparticles with, e.g., ApoE, it is also possible to circumvent the BBB via the LDL-receptor. Another example of the use of receptor-mediated endocytosis to enhance brain uptake of nanoparticles are poly(ethylene glycol)-coated Fe3O4 nanoparticles which are covalently attached to lactoferrin. These nanoparticles have been shown to facilitate the transport via the lactoferrin receptor, and so could then be used for magnetic resonance imaging.     

A review of multifunctional nanoemulsion systems to overcome oral and CNS drug delivery barriers

Abstract

The oral and central nervous systems (CNS) present a unique set of barriers to the delivery of important diagnostic and therapeutic agents. Extensive research over the past few years has enabled a better understanding of these physical and biological barriers based on tight cellular junctions and expression of active transporters and metabolizing enzymes at the luminal surfaces of the gastrointestinal (GI) tract and the blood-brain barrier (BBB). This review focuses on the recent understanding of transport across the GI tract and BBB and the development of nanotechnology-based delivery strategies that can enhance bioavailability of drugs. Multifunctional lipid nanosystems, such as oil-in-water nanoemulsions, that integrate enhancement in permeability, tissue and cell targeting, imaging, and therapeutic functions are especially promising. Based on strategic choice of edible oils, surfactants and additional surface modifiers, and different types of payloads, rationale design of multifunctional nanoemulsions can serve as a safe and effective delivery vehicle across oral and CNS barriers.     

Perforin Competent CD8 T Cells Are Sufficient to Cause Immune-Mediated Blood-Brain Barrier Disruption

Numerous neurological disorders are characterized by central nervous system (CNS) vascular permeability. However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood. In order to address this, we developed an inducible model of BBB disruption using a variation of the Theiler''s murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide induced fatal syndrome (PIFS) model is initiated by virus-specific CD8 T cells and results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. While perforin is required for BBB disruption, the cellular source of perforin has remained unidentified. In addition to CD8 T cells, various innate immune cells also express perforin and therefore could also contribute to BBB disruption. To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques. We determined that C57BL/6 perforin^−/− mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited: 1) heightened CNS vascular permeability, 2) increased astrocyte activation as measured by GFAP expression, and 3) loss of linear organization of BBB tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability when compared to mock-treated controls. These results are consistent with the characteristics associated with PIFS in perforin competent mice. Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.     

Isolation of blood-brain barrier-crossing antibodies from a phage display library by competitive elution and their ability to penetrate the central nervous system

The blood-brain barrier (BBB) is a formidable obstacle for delivery of biologic therapeutics to central nervous system (CNS) targets. Whilst the BBB prevents passage of the vast majority of molecules, it also selectively transports a wide variety of molecules required to maintain brain homeostasis. Receptor-mediated transcytosis is one example of a macromolecule transport system that is employed by cells of the BBB to supply essential proteins to the brain and which can be utilized to deliver biologic payloads, such as antibodies, across the BBB. In this study, we performed phage display selections on the mouse brain endothelial cell line, bEND.3, to enrich for antibody single-chain variable fragments (scFvs) that could compete for binding with a known BBB-crossing antibody fragment, FC5. A number of these scFvs were converted to IgGs and characterized for their ability to bind to mouse, rat and human brain endothelial cells, and subsequent ability to transport across the BBB. We demonstrated that these newly identified BBB-targeting IgGs had increased brain exposure when delivered peripherally in mice and were also able to transport a biologically active molecule, interleukin-1 receptor antagonist (IL-1RA), into the CNS. The antagonism of the interleukin-1 system within the CNS can result in the relief of neuropathic pain. We demonstrated that the BBB-targeting IgGs were able to elicit an analgesic response in a mouse model of nerve ligation-induced hypersensitivity when fused to IL-1RA.     

Sufficient virus-neutralizing antibody in the central nerve system improves the survival of rabid rats

Background

Rabies is known to be lethal in human. Treatment with passive immunity for the rabies is effective only when the patients have not shown the central nerve system (CNS) signs. The blood–brain barrier (BBB) is a complex functional barrier that may compromise the therapeutic development in neurological diseases. The goal of this study is to determine the change of BBB integrity and to assess the therapeutic possibility of enhancing BBB permeability combined with passive immunity in the late stage of rabies virus infection.

Methods

The integrity of BBB permeability in rats was measured by quantitative ELISA for total IgG and albumin levels in the cerebrospinal fluid (CSF) and by exogenously applying Evans blue as a tracer. Western blotting of occludin and ZO-1, two tight junction proteins, was used to assess the molecular change of BBB structure.The breakdown of BBB with hypertonic arabinose, recombinant tumor necrosis factor-alpha (rTNF-γ), and focused ultrasound (FUS) were used to compare the extent of BBB disruption with rabies virus infection. Specific humoral immunity was analyzed by immunofluorescent assay and rapid fluorescent focus inhibition test. Virus-neutralizing monoclonal antibody (mAb) 8-10E was administered to rats with hypertonic breakdown of BBB as a passive immunotherapy to prevent the death from rabies.

Results

The BBB permeability was altered on day 7 post-infection. Increased BBB permeability induced by rabies virus infection was observed primarily in the cerebellum and spinal cord. Occludin was significantly decreased in both the cerebral cortex and cerebellum. The rabies virus-specific antibody was not strongly elicited even in the presence of clinical signs. Disruption of BBB had no direct association with the lethal outcome of rabies. Passive immunotherapy with virus-neutralizing mAb 8-10E with the hypertonic breakdown of BBB prolonged the survival of rabies virus-infected rats.

Conclusions

We demonstrated that the BBB permeability was altered in a rat model with rabies virus inoculation. Delivery of neutralizing mAb to the infected site in brain combined with effective breakdown of BBB could be an aggressive but feasible therapeutic mode in rabies when the CNS infection has been established.     

How do immune cells overcome the blood–brain barrier in multiple sclerosis?

The presence of the blood-brain barrier (BBB) restricts the movement of soluble mediators and leukocytes from the periphery to the central nervous system (CNS). Leukocyte entry into the CNS is nonetheless an early event in multiple sclerosis (MS), an inflammatory disorder of the CNS. Whether BBB dysfunction precedes immune cell infiltration or is the consequence of perivascular leukocyte accumulation remains enigmatic, but leukocyte migration modifies BBB permeability. Immune cells of MS subjects express inflammatory cytokines, reactive oxygen species (ROS) and enzymes that can facilitate their migration to the CNS by influencing BBB function, either directly or indirectly. In this review, we describe how immune cells from the peripheral blood overcome the BBB and promote CNS inflammation in MS through BBB disruption.     

Re-engineering biopharmaceuticals for delivery to brain with molecular Trojan horses

Biopharmaceuticals, including recombinant proteins, monoclonal antibody therapeutics, and antisense or RNA interference drugs, cannot be developed as drugs for the brain, because these large molecules do not cross the blood-brain barrier (BBB). Biopharmaceuticals must be re-engineered to cross the BBB, and this is possible with genetically engineered molecular Trojan horses. A molecular Trojan horse is an endogenous peptide, or peptidomimetic monoclonal antibody (mAb), which enters brain from blood via receptor-mediated transport on endogenous BBB transporters. Recombinant neurotrophins, single chain Fv antibodies, or therapeutic enzymes may be re-engineered as IgG fusion proteins. The engineering of IgG-avidin fusion proteins enables the BBB delivery of biotinylated drugs. The IgG fusion proteins are new chemical entities that are dual or triple function molecules that bind multiple receptors. The fusion proteins are able both to enter the brain, by binding an endogenous BBB receptor, and to induce the desired pharmacologic effect in brain, by binding target receptors in the brain behind the BBB. The development of molecular Trojan horses for BBB drug delivery allows the re-engineering of biopharmaceuticals that, owing to the BBB problem, could not otherwise be developed as new drugs for the human brain.     

Noninvasive and Targeted Gene Delivery into the Brain Using Microbubble-Facilitated Focused Ultrasound

Recombinant adeno-associated viral (rAAV) vectors are potentially powerful tools for gene therapy of CNS diseases, but their penetration into brain parenchyma is severely limited by the blood-brain barrier (BBB) and current delivery relies on invasive stereotactic injection. Here we evaluate the local, targeted delivery of rAAV vectors into the brains of mice by noninvasive, reversible, microbubble-facilitated focused ultrasound (FUS), resulting in BBB opening that can be monitored and controlled by magnetic resonance imaging (MRI). Using this method, we found that IV-administered AAV2-GFP (green fluorescence protein) with a low viral vector titer (1×10⁹ vg/g) can successfully penetrate the BBB-opened brain regions to express GFP. We show that MRI monitoring of BBB-opening could serve as an indicator of the scale and distribution of AAV transduction. Transduction peaked at 3 weeks and neurons and astrocytes were affected. This novel, noninvasive delivery approach could significantly broaden the application of AAV-viral-vector-based genes for treatment of CNS diseases.     

CD8 T cell-initiated blood-brain barrier disruption is independent of neutrophil support

Blood-brain barrier (BBB) disruption is a common feature of numerous neurologic disorders. A fundamental question in these diseases is the extent inflammatory immune cells contribute to CNS vascular permeability. We have previously shown that CD8 T cells play a critical role in initiating BBB disruption in the peptide-induced fatal syndrome model developed by our laboratory. However, myelomonocytic cells such as neutrophils have also been implicated in promoting CNS vascular permeability and functional deficit in murine models of neuroinflammatory disease. For this reason, we evaluated neutrophil depletion in a murine model of CD8 T cell-initiated BBB disruption by employing traditionally used anti-granulocyte receptor-1 mAb RB6-8C5 and Ly-6G-specific mAb 1A8. We report that CNS-infiltrating antiviral CD8 T cells express high levels of granulocyte receptor-1 protein and are depleted by treatment with RB6-8C5. Mice treated with RB6-8C5, but not 1A8, display: 1) intact BBB tight junction proteins; 2) reduced CNS vascular permeability visible by gadolinium-enhanced T1-weighted magnetic resonance imaging; and 3) preservation of motor function. These studies demonstrate that traditional methods of neutrophil depletion with RB6-8C5 are broadly immune ablating. Our data also provide evidence that CD8 T cells initiate disruption of BBB tight junction proteins and CNS vascular permeability in the absence of neutrophil support.     

Improved delivery of broadly neutralizing antibodies by nanocapsules suppresses SHIV infection in the CNS of infant rhesus macaques

Broadly neutralizing antibodies (bNAbs) directed to HIV-1 have shown promise at suppressing viremia in animal models. However, the use of bNAbs for the central nervous system (CNS) infection is confounded by poor penetration of the blood brain barrier (BBB). Typically, antibody concentrations in the CNS are extremely low; with levels in cerebrospinal fluid (CSF) only 0.1% of blood concentrations. Using a novel nanotechnology platform, which we term nanocapsules, we show effective transportation of the human bNAb PGT121 across the BBB in infant rhesus macaques upon systemic administration up to 1.6% of plasma concentration. We demonstrate that a single dose of PGT121 encased in nanocapsules when delivered at 48h post-infection delays early acute infection with SHIV_SF162P3 in infants, with one of four animals demonstrating viral clearance. Importantly, the nanocapsule delivery of PGT121 improves suppression of SHIV infection in the CNS relative to controls.     

Annexin A1 restores Aβ1‐42‐induced blood–brain barrier disruption through the inhibition of RhoA‐ROCK signaling pathway

The blood–brain barrier (BBB) is composed of brain capillary endothelial cells and has an important role in maintaining homeostasis of the brain separating the blood from the parenchyma of the central nervous system (CNS). It is widely known that disruption of the BBB occurs in various neurodegenerative diseases, including Alzheimer's disease (AD). Annexin A1 (ANXA1), an anti‐inflammatory messenger, is expressed in brain endothelial cells and regulates the BBB integrity. However, its role and mechanism for protecting BBB in AD have not been identified. We found that β‐Amyloid 1‐42 (Aβ42)‐induced BBB disruption was rescued by human recombinant ANXA1 (hrANXA1) in the murine brain endothelial cell line bEnd.3. Also, ANXA1 was decreased in the bEnd.3 cells, the capillaries of 5XFAD mice, and the human serum of patients with AD. To find out the mechanism by which ANXA1 recovers the BBB integrity in AD, the RhoA‐ROCK signaling pathway was examined in both Aβ42‐treated bEnd.3 cells and the capillaries of 5XFAD mice as RhoA was activated in both cases. RhoA inhibitors alleviated Aβ42‐induced BBB disruption and constitutively overexpressed RhoA‐GTP (active form of RhoA) attenuated the protective effect of ANXA1. When pericytes were cocultured with bEnd.3 cells, Aβ42‐induced RhoA activation of bEnd.3 cells was inhibited by the secretion of ANXA1 from pericytes. Taken together, our results suggest that ANXA1 restores Aβ42‐induced BBB disruption through inhibition of RhoA‐ROCK signaling pathway and we propose ANXA1 as a therapeutic reagent, protecting against the breakdown of the BBB in AD.     

Brain uptake of multivalent and multi-specific DVD-Ig proteins after systemic administration

Therapeutic monoclonal antibodies and endogenous IgG antibodies show limited uptake into the central nervous system (CNS) due to the blood-brain barrier (BBB), which regulates and controls the selective and specific transport of both exogenous and endogenous materials to the brain. The use of natural transport mechanisms, such as receptor-mediated transcytosis (RMT), to deliver antibody therapeutics into the brain have been studied in rodents and monkeys. Recent successful examples include monovalent bispecific antibodies and mono- or bivalent fusion proteins; however, these formats do not have the capability to bind to both the CNS target and the BBB transport receptor in a bivalent fashion as a canonical antibody would. Dual-variable-domain immunoglobulin (DVD-Ig) proteins offer a bispecific format where monoclonal antibody-like bivalency to both the BBB receptor and the therapeutic target is preserved, enabling independent engineering of binding affinity, potency, valency, epitope and conformation, essential for successful generation of clinical candidates for CNS applications with desired drug-like properties. Each of these parameters can affect the binding and transcytosis ability mediated by different receptors on the brain endothelium differentially, allowing exploration of diverse properties. Here, we describe generation and characterization of several different DVD-Ig proteins, specific for four different CNS targets, capable of crossing the BBB through transcytosis mediated by the transferrin receptor 1 (TfR1). After systemic administration of each DVD-Ig, we used two independent methods in parallel to observe specific uptake into the brain. An electrochemiluminescent-based sensitive quantitative assay and a semi-quantitative immunohistochemistry technique were used for brain concentration determination and biodistribution/localization in brain, respectively. Significantly enhanced brain uptake and retention was observed for all TfR1 DVD-Ig proteins regardless of the CNS target or the systemic administration route selected.