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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: Curr Opin Chem Eng. 2020 Sep 12;30:86–95. doi: 10.1016/j.coche.2020.08.009

Caveolae-Mediated Transport at the Injured Blood-Brain Barrier as an Underexplored Pathway for Central Nervous System Drug Delivery

Alexander G Sorets 1, Jonah C Rosch 2, Craig L Duvall 1,3, Ethan S Lippmann 1,2,3,4,5
PMCID: PMC7497790  NIHMSID: NIHMS1627909  PMID: 32953427

Abstract

Drug delivery to the central nervous system (CNS) is generally hindered by the selectivity of the blood-brain barrier (BBB). However, there is strong evidence that the integrity of the BBB is compromised under certain pathological conditions, potentially providing a window to deliver drugs to injured brain regions. Recent studies suggest that caveolae-mediated transcytosis, a transport pathway suppressed in the healthy BBB, becomes elevated as an immediate response to ischemic stroke and at early stages of aging, where it may precede irreversible neurological damage. This article reviews early-stage caveolar transcytosis as a novel and promising drug delivery opportunity. We propose that albumin-binding and nanoparticle approaches have the potential to leverage this window of transcellular BBB disruption for trafficking therapeutic agents into the CNS.

Introduction

The blood-brain-barrier (BBB), which is effectively impermeable to all large molecule therapeutics and 98% of small molecules, presents a substantial challenge for the treatment of central nervous system (CNS) disorders [1]. Many engineering strategies have been explored to overcome this obstacle, particularly for the delivery of biologics. Some approaches to increase drug penetration include co-opting endogenous receptor-mediated processes that normally shuttle proteins and amino acids across the BBB, as well as temporarily opening the intact BBB using ultrasound or pharmacological agents [2]. However, using antibodies or peptides to exploit receptor-mediated transcytosis (RMT) is inefficient with only a small fraction of injected dose reaching the brain. Furthermore, drug delivery through RMT is usually not targeted to a focal area of the CNS, as the receptors are ubiquitously expressed in the cerebrovasculature. The alternate strategy of opening the BBB introduces the potential for toxicity from entry of blood-borne proteins, and its relative invasiveness is not well-suited for delivering drugs on a frequent schedule.

Recent work suggests that a promising but underappreciated approach for CNS drug delivery may be selective targeting of receptor-independent pathways that involve trafficking of caveolae, which are small lipid rafts that non-specifically shuttle proteins such as albumin and immunoglobulins across the brain endothelium. Although suppressed at the healthy BBB, robust upregulation of caveolar-dependent transcytosis occurs in early stages of a number of CNS diseases [3–5]. Enhanced caveolae-mediated trafficking of albumin has also been identified as an early hallmark of aging [6]. While sustained upregulation of caveolae-mediated transcytosis may lead to long-term detrimental neurological consequences, the onset of caveolae upregulation in aging and disease likely precedes irreversible neurological damage and may provide a gateway for therapeutic agents to reach brain targets [7]. Here, we review and provide a prospectus on the potential exploitation of early-stage caveolar transcytosis as a novel and promising approach for delivering therapeutic compounds to the CNS.

Neurovascular Unit Structure and Regulation of Transport

The restrictive properties of the BBB arise from the unique molecular signature of brain microvascular endothelial cells (BMECs) and their bidirectional communication with neighboring cells. These polarized BMECs, together with pericytes, glia (astrocytes and microglia), neurons, and the basement membrane, collectively form the neurovascular unit (NVU), which maintains cerebral homeostasis by regulating the transport of compounds between the blood circulation and brain parenchyma [8]. Unlike most peripheral endothelial cells, BMECs are connected by a continuous network of epithelial-like tight junctions that prevent paracellular diffusion of water, ions, and organic molecules. As a result, the NVU effectively controls material exchange between the blood and CNS through selective transporters expressed on the endothelium [9].

The transport pathways at the BBB include passive diffusion, carrier-mediated transport, and vesicular transcytosis (Figure 1). Free diffusion across the endothelium is dictated by the physicochemical properties of the compounds, favoring small (<450 Da) lipophilic molecules with fewer than 6 hydrogen bonds, though these factors are not an absolute prediction of BBB permeability [10]. Most nutrients including glucose, amino acids, and vitamins are too polar to diffuse across the lipid membrane and are therefore transported through specific solute carriers present at both the apical and basolateral membranes of BMECs.

Figure 1. Transport pathways across the BBB.

Figure 1.

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Passive diffusion through the brain endothelium is restricted to small (<450 Daltons), lipophilic compounds including but not limited to caffeine, alcohol, and a limited subset of small molecule drugs that are not efflux transporter substrates. Transport of small and large molecules between cells (paracellular route) is effectively non-present due to a dense network of specialized tight junctions. Carrier-mediated transport of solutes involves binding of compounds to specific proteins expressed on the brain endothelium. Importantly, this pathway does not involve vesicles, and notable molecules that are transported through this route include glucose, amino acids, and vitamins. In terms of vesicular transport mechanisms, receptor-mediated transport involves specific receptor-ligand interactions that trigger clathrin-dependent endocytosis. After the vesicle is pinched off by dynamin, the clathrin coat is shed and the vesicle fuses with a sorting endosome. Depending on a number of factors, including the affinity of the ligand of the receptor, the vesicle will either be directed to a late endosome and lysosomal degradation pathway or be passed to a transcytotic vesicle. In adsorptive mediated transcytosis, a macromolecule needs to be positively charged, in which binding of the cation to the plasma membrane leads to endocytosis through caveolae. These uncoated vesicles are lipid rafts stabilized by caveolin-1. While many studies suggest that caveolae are directly passed across brain endothelial cells, it is not well understood if there are alternative routes involving degradation pathways.

The transport of large molecules such as proteins and peptides is restricted predominantly to endocytotic transcellular pathways. Vesicular transport can occur via RMT or non-specific adsorptive-mediated transcytosis (AMT) [10]. The former requires specific protein-receptor interactions to trigger endocytosis, and examples of proteins transported by RMT include transferrin, insulin, and leptin. The less studied AMT pathway involves positively charged proteins accumulating on the negatively charged glycocalyx, triggering caveolar uptake and transport across the BBB. Although the hallmark of the BBB is low baseline rates of AMT from the luminal side of the endothelium into the brain [3], current advances in understanding AMT function and regulation suggest the potential to ‘backpack’ on the AMT pathway to deliver molecules across the injured BBB.

Structure, Transport, and Regulation of Caveolar Transcytosis at the Healthy BBB

Caveolae are flask-shaped plasma membrane invaginations that are vital for the endocytosis and transcytosis of plasma macromolecules [11]. First discovered in the 1950s, caveolae are highly abundant in adipocytes, smooth-muscle cells, fibroblasts, epithelial cells, and most importantly for this review, endothelial cells [11,12]. Brain endothelial caveolae are 50-80 nm in diameter and are highly enriched with cholesterol and glycosphingolipids, forming a lipid raft that is stabilized by caveolin-1 (cav-1) at the plasma membrane [11]. Relative to other endothelia, brain endothelial cells have the lowest frequency of caveolae (<100/μm3) with only a small fraction being internalized [13]. When transcytosis is activated, the vesicle is pinched off from the membrane and subsequently migrates across the brain endothelium to the basolateral membrane (Figure 1). Both activation and fission of caveolae are mediated by Src kinase tyrosine phosphorylation; phosphorylation of cav-1 induces internalization of bound and fluid-phase macromolecules within caveolae [14] and Src phosphorylation of dynamin mediates release of caveolae from the plasma membrane as intracellular vesicles [15]. Upon reaching the basolateral membrane, caveolae fuse and release their contents into the interstitial space [12].

While albumin is one of the most prominent proteins trafficked across the endothelium through caveolae, albumin transport mechanisms are tissue-specific and may be mediated by receptor-dependent or - independent pathways. Albumin binding to albondin (gp60), an endothelial sialoglycoprotein residing in caveolae, initiates non-degradative transport by triggering Src kinase-mediated phosphorylation of gp60 and cav-1[16]. However, despite facilitating over 50% of albumin transport across peripheral endothelia, gp60 is not expressed in healthy brain endothelial cells, further contributing to the inability of albumin to cross the intact BBB [17,18].

While caveolae-mediated transcytosis constitutes a primary albumin transport mechanism in peripheral organs, caveolae vesicle formation is heavily suppressed at the BBB. In 2014, Ben-Zvi et al. discovered that a BBB-specific transporter, Major facilitator super family domain containing 2a (Mfsd2a), is responsible for regulating vesicular transcytosis [19]. Using transgenic Mfsd2a−/− mice, they observed elevated vesicular transport despite the maintenance of tight junctions. Recently, Andreone et al. demonstrated that Mfsd2a−/− mice displayed enhanced caveolar transcytosis without changes to paracellular permeability [3]. Cav1 knockout in the Mfsd2a−/− mice prevented the increase in transcytosis, suggesting that caveolae-mediated transcytosis is the pathway suppressed by Mfsd2a [3]. These researchers proposed that Mfsd2a functions as a lipid flippase that transports docosahexaenoic acid (DHA) onto the inner plasma membrane leaflet. DHA is an omega-3 fatty acid important for neural and cognitive function, but its presence on the plasma membrane displaces cholesterol and cav-1, thereby preventing the formation of caveolae [3,20]. This recent insight into transcytosis mechanisms at the BBB highlights a critical role for the lipid composition of the plasma membrane in regulating caveolae formation in BMECs.

Previous Strategies for CNS Drug Delivery through Transcytotic Routes

In contrast to carrier-mediated transport which is restricted by size limitations, RMT and AMT pathways are well-suited for delivery of proteins, drugs, and nanoparticles across the BBB [2]. BMECs are highly enriched in clathrin-coated vesicles, which mediate RMT. Thus, strategies to deliver biologics across the BBB have primarily exploited the mechanisms of RMT pathways [2]. The general principle of delivering drugs through RMT is to conjugate the therapeutic agent to a vector that selectively binds the target receptor and triggers transcytosis. The most prominent proteins that undergo RMT and have been used to guide drug delivery are transferrin, which shuttles iron into brain, and insulin, which is not produced in the CNS [1]. For example, targeting the transferrin receptor with OX26 (a mouse monoclonal antibody against the rat receptor) successfully enables transcytosis of pharmaceuticals without impeding natural transferrin transport [2]. Fusion of OX26 with proteins such as brain-derived neurotrophic factor [21] and basic fibroblast growth factor [22] has been explored as a possible neuroprotective approach following ischemic stroke. However, while these RMT-leveraging strategies do achieve brain targeting, they are limited in the requirement for substantial and frequent dosing to achieve high extent of uptake into the brain parenchyma. The absolute amount of OX26 transported into the CNS is typically far below 1% of the injected dose per gram, with an upper limit of ~3-4% for bi-specific antibody designs [23,24].

In contrast, AMT has a far greater binding capacity than RMT because AMT is not limited by the expression of specific receptors and is therefore saturated at much higher concentrations [25]. Intriguingly, some approaches to brain drug delivery have targeted AMT pathways by cationizing serum proteins such as albumin and immunoglobulin. The negative charge arising from sialoglycoproteins and heparin sulfate proteoglycans in the endothelial glycocalyx preclude the transport of native albumin, which is negatively charged at neutral pH due to an abundance of acidic amino acid residues [26]. For delivering chimeric peptides through the healthy brain endothelium, Kang and Partridge originally used cationized human serum albumin (cHSA) bound to neutral avidin and demonstrated increased brain delivery of H3-biotin conjugated to the cHSA-avidin vector [27]. Overall, cationized albumin improved pharmacokinetic properties by greatly enhancing half-life and brain targeting compared to other organs [26]. However, these studies were published in 1994 when the transport of albumin and regulation of AMT pathways at the BBB were poorly understand, and few efforts have built upon this nascent work. Below, we delve into the mechanisms by which caveolar transport pathways become elevated during disease and aging as a potential motivation for revisiting caveolar-based drug delivery strategies.

Changes to BBB Permeability Following Ischemic Stroke

Caused by thrombosis or embolism, ischemic stroke accounts for over 80% of strokes and is characterized by reduced cerebral blood supply resulting in poor delivery of oxygen and glucose [28]. Nutrient inaccessibility causes rapid neuronal death in the ischemic core. However, salvageable neurons in the surrounding penumbra slowly die due to a complex secondary injury cascade involving glutamate-mediated excitotoxicity, neuroinflammation, and oxidative stress [28]. Clinical and experimental findings indicate that sustained BBB breakdown is a hallmark of ischemic stroke, and that greater BBB disruption exacerbates secondary injuries, thereby contributing to adverse long-term prognoses [29,30]. BBB integrity is compromised within several hours of ischemic injury permitting the uncontrolled leakage of blood-borne molecules into the brain parenchyma, which increases the risk of hemorrhage and induces vasogenic edema through disruption of osmotic gradients, leading to elevated intracranial pressure [31,32]. While BBB breakdown as a cause or consequence of secondary injury is debated, recent studies demonstrated that an increased number of endothelial caveolae after medial cerebral artery occlusion (MCAO) correlated with the extent of BBB damage and widespread extravasation of large plasma proteins such as albumin and IgG [33,34].

Further, while the prevailing belief over the last few decades was that BBB breakdown in ischemic injury is caused by destabilization of endothelial tight junction proteins, recent studies instead support a biphasic BBB breakdown profile with an initial early period of increased caveolar transcytosis and therefore elevated transcellular permeability, followed by a later period of tight junction remodeling and elevated paracellular permeability (Figure 2) [33]. Early evidence for this novel mechanism came from ultrastructural studies that revealed minimal damage to tight junctions in areas of massive albumin accumulation following embolic stroke in rats [35]. A further report from Knowland et al. demonstrated that caveolae-mediated transcytosis precedes tight junction structural defects in a transient-MCAO (t-MCAO) stroke model [4]. By expressing green fluorescent protein fused with claudin-5 in a transgenic mouse and visualizing junctions with in vivo time lapse two-photon microscopy, the authors delineated the contribution of caveolar transport of fluorescent albumin from tight junction breakdown-induced transport [4]. They further found that caveolar vesicle number and transcytosis rate increase within 6 hours of t-MCAO without tight junction disruption, which becomes evident after 48-58 hours, concurrent with a second phase of albumin hyperpermeability, where both the paracellular and transcellular routes contribute to albumin transport. Mechanistically, others have shown that an ischemic stroke induces charge neutralization of the endothelial glycocalyx, potentially enabling caveolar transport of native (anionic) albumin [36,37]. Overall, microvascular pathology following ischemic stroke appears to be characterized by an early window of caveolae-mediated transport that precedes extensive neuronal damage.

Figure 2. Overview of temporal changes in BBB integrity following an ischemic stroke.

Figure 2.

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The healthy BBB is characterized by low rates of transcytosis, an intact negatively charged glycocalyx, and a stringent paracellular barrier that collectively prevent non-specific transport of material into the brain parenchyma. At early stages of injury post-stroke (<6 hours), the dissolution of the glycocalyx coupled with activation of caveolae contribute to a robust transcytosis of plasma proteins across the brain endothelium without evidence of disruption to tight junctions. At late stages after injury (48-72 hours), breakdown of tight junctions allows paracellular transport of proteins in addition to caveolar transport. At this stage, the secondary injury cascade has developed to include neuroinflammation, excitotoxicity, and oxidative stress that all contribute to neuronal cell death.

Age-associated Changes in BBB Integrity

Structural and biochemical changes to the cerebrovasculature have long been observed in aging, which is the greatest risk factor for many neurodegenerative diseases [38]. A meta-analysis of 1,953 individuals demonstrated that BBB permeability increases both in normal aging and in patients with vascular or Alzheimer’s disease, whereas the age-dependent BBB disruption was greater in the diseased groups [39]. It is clear that aging causes pathological changes to vascular structure and function and that in many cases BBB dysfunction precedes neurodegeneration and cognitive impairment [40–42]. Furthermore, while the mechanisms and timing of aging-induced BBB dysfunction are not fully understood, recent studies suggest a prominent role for elevated endothelial caveolae-mediated transcytosis as an early BBB disruption event that contributes to neurodegeneration [6].

As the most prominent protein in the blood that is trafficked intracellularly by caveolae, albumin extravasation may be one of the earliest hallmarks of the aging brain [7]. A recent study by Senatorov et al. demonstrated that in naturally aged mice (12-15 months), there is marked increase of albumin extravasation into the CNS where it binds to the transforming growth factor beta receptor (TGFβR) on astrocytes and triggers hyperexcitability in the hippocampus to yield a phenotype that is indicative of early-stage mild cognitive impairment [7]. Infusion of albumin into the brains of young mice was sufficient to mimic these age-related neurological deficits, while genetic knockdown of TGFβR or injection of a TGFβR small molecule inhibitor reversed these pathological effects. Thus, albumin infiltration through the BBB leading to TGFβ hyperactivation, as mediated by astrocytes, represents an early event in age-related cognitive decline.

The study by Senatorov et al. did not examine why albumin infiltration into the CNS occurred with aging, and indeed, the possibility of upregulated caveolae transport as an age-related BBB defect represents a relatively unexplored connection to neurodegeneration. Along these lines, a seminal paper by Park et al. causally linked acid sphingomyelinase (ASM), a sphingolipid metabolizing enzyme secreted by brain endothelial cells, with age-related neurological phenotypes [6]. The authors suggest that ASM becomes elevated with age and upregulates caveolae-mediated transcytosis via protein phosphatase-1 mediated ezrin/radixin/moesin dephosphorylation. Genetic inhibition of ASM reduced cav-1 phosphorylation, leading to improved BBB integrity and cognitive abilities compared to age-matched controls. Furthermore, conditional transgenic mice engineered to overexpress ASM at a young age demonstrated significant memory impairment and neurodegeneration [6]. These findings suggest that ASM triggers BBB disruption through upregulation of caveolae-mediated transcytosis, which enables albumin transport and accelerates aging-associated cognitive changes.

More broadly, these studies reveal that the loss of BBB integrity, particularly the ability to suppress caveolae-mediated transcytosis, is a hallmark of natural aging. These studies, along with recent revelations in mechanisms of BBB disruption during ischemia, highlight upregulation of caveolae transport as an early pathological event and reveal a possible opportunity to co-opt transport of albumin for brain drug delivery.

Drug Delivery Opportunities

Delivering efficacious concentrations of drug to diseased brain regions is a challenge for developing effective treatments to combat neurodegeneration. As detailed above, caveolae-mediated transcytosis is a transport pathway that is suppressed in healthy BMECs but becomes elevated as an immediate response to ischemic stroke and at early stages of aging [5,6], where it may precede irreversible neurological damage [7,43]. While blood-borne protein transport into the brain parenchyma may contribute to neurodegeneration, these extravasation events also may enable the selective delivery of normally excluded drugs to regions of CNS injury (Figure 3). Nanoparticles and albumin conjugates are promising drug delivery strategies that may be well suited to leverage this window of enhanced caveolae trafficking into the CNS.

Figure 3. Approaches for leveraging caveolae-mediated transcytosis as a drug delivery route to the CNS.

Figure 3.

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One possibility for targeting caveolae is to conjugate therapeutic compounds to endogenous albumin, which is highly abundant in blood and accumulates in brain regions where BBB integrity is compromised. As a natural lipid carrier, albumin contains binding pockets that interact with fatty acid moieties, which can be chemically engineered to carry a variety of payloads. Liposomal nanocarriers have also been shown to traffic through caveolae following stroke and can deliver both hydrophobic and hydrophilic compounds. In each case, delivery would be expected only at sites of injury where caveolae-mediated transcytosis is selectively upregulated.

One approach to improving the pharmacokinetic properties of rapidly metabolized or excreted compounds is encapsulation in nanoparticles. Nanoparticles are versatile drug carriers that can be functionalized with peptides, proteins, or antibodies to target regions of injury [44]. Liposomes are well-studied carriers for treating ischemic stroke as they can be engineered to enhance CNS bioavailability by appropriately tuning physical and chemical properties (size, charge, and surface properties) [45]. For example, to elucidate the window of maximum liposomal delivery following stroke, Al-Ahmedy et al. monitored fluorescently labeled liposome accumulation in the ischemic hemisphere of mice following t-MCAO; they identified an early window of accumulation (0.5 hours after reperfusion) due to transcellular transport and a delayed window (48 hours after t-MCAO) where both the paracellular and transcellular pathways contribute to liposome delivery [43]. Interestingly, in contrast to treatment at 48 hours, delivery of liposomes in the acute phase preceded histological evidence of neurological damage, suggesting that targeting the neurovascular unit itself to improve BBB integrity may improve outcomes. To investigate if liposome transport was mediated by caveolae, fluorescently labeled liposomes were intravenously administered and subsequently colocalized with brain regions of enhanced cav-1 expression and immunoglobulin penetration [43].

An alternate approach to CNS drug delivery is to design nanoparticles that actively target caveolae as a transcytosis pathway. For example, to leverage the overexpression of gp60 on the glioma endothelium, Lin et al. intravenously administered chemotherapeutics encapsulated in albumin nanoparticles and observed enhanced BBB penetration and glioma targeting via gp60-mediated caveolar transport [46]. However, this approach has only been explored in glioblastoma and the large size (~150 nm) of these biomimetic albumin nanoparticles likely renders them ill-suited for uptake in caveolae. Voigt et al. developed polyanionic lipid nanoparticles that selectively undergo caveolae-mediated transcytosis in endothelial cells [47]. The principle of this technology is that high affinity hydrophobic interactions promotes association of the lipid nanoparticle with caveolar lipid rafts and the negatively charged sulfonate polymer on the nanoparticle surface prevents non-specific electrostatic interaction with the cell membrane [47]. While these nanoparticles were developed for cancer applications and not CNS delivery, this formulation may have potential for targeting the injured cerebrovasculature following stroke.

Despite substantial pre-clinical research on nanoparticle efficacy, a major obstacle to their widespread clinical translation is the possibility of cytotoxicity and preferential accumulation in the liver and spleen [48]. In contrast, albumin-mediated drug delivery can increase circulation half-life of therapeutic cargo and enhance target site accumulation without overt dose-limiting toxicity [49]. As a native carrier of fatty acids, albumin is well-suited for a variety of conjugation platforms, the majority of which have been developed for diabetes, vaccine, and cancer applications [50]. Commercially available Semaglutide leverages a fatty acid modification to promote in vivo association of glucagon-like peptide 1 (GLP-1) with native albumin to extend the peptide bioavailability in the treatment of type 2 diabetes [51]. In the context of vaccines, ‘albumin backpacking’ of antigen and adjuvants with lipophilic albumin binding domains improves lymph node targeting and T-cell priming [52]. Albumin is also an attractive drug carrier for cancer applications due to preferential accumulation in tissues with leaky vasculature [50]. For example, Sarett et al. developed a PEGylated diacyl lipid moiety to link endogenous albumin to siRNAs and achieved uptake in 99% of breast cancer cells with significant target protein knockdown in mice [53]. This approach could possibly be translated to treating CNS diseases, as the albumin-binding conjugate greatly enhances the pharmacokinetic properties of siRNA therapeutics, and the hyperpermeability of tumor vasculature is analogous in many ways to increased caveolin-mediated transport in BMECs under disease conditions. While the transport processes contain a number of differences as well, a notable similarity is that the transport of both albumin and nanoparticles across the leaky tumor endothelium is predicted to be predominantly mediated by active trans-endothelial processes that result in accumulation in the tumor interstitium [54,55].

Overall, while leveraging albumin as a drug carrier has been primarily investigated for cancer and diabetes therapeutics, we suggest this approach is a promising strategy for CNS delivery, as albumin is one of the most prominent proteins in the blood trafficked across the damaged BBB by caveolae. Furthermore, it is likely that the albumin-associated cargos would preferentially target cells in regions of BBB damage, as the healthy BMECs should not transport albumin. The proposed strategy for albumin backpacking involves conjugating drugs to fatty acid molecules that non-covalently interact with endogenous albumin after intravenous injection (Figure 3). An advantage of this approach is that albumin is readily passed through the brain endothelium and is not associated with early endosomes or lysosomal pathways (Figure 1) [4]. After leaking through the BBB, evidence suggests albumin is endocytosed to some degree by astrocytes, neurons, and microglia [56], where it could theoretically deliver a drug payload. To our knowledge, albumin-drug conjugates have not yet been translated to the treatment of CNS disorders in this manner, but they may represent a versatile avenue for localized drug delivery in the early stages of neurodegeneration.

Conclusion

In summary, the restrictive properties of the BBB hinder drug transport into the CNS, spurring research into approaches that exploit endogenous transcytosis pathways to enhance drug delivery. While extensive research has focused on RMT, we posit that emerging data suggests that leveraging caveolae-mediated transport pathways for CNS delivery of biologics is a promising strategy for treating neurodegenerative diseases or combating age-associated cognitive decline. Recent advances highlight robust albumin transport through caveolae as an early pathological response to stroke and natural aging. As such, backpacking of drugs on albumin or other serum proteins could be an unrecognized approach for localized drug delivery during early stages of BBB dysfunction prior to widespread CNS disease.

Acknowledgments

Funding in our laboratories related to the topic of this review is graciously provided by a Chan Zuckerberg Initiative Ben Barres Early Career Acceleration Award (grant 2018-191850 to ESL) and NIH grant R01 HL122347 (to CLD).

Footnotes

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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