The role of non-endothelial cells on the penetration of nanoparticles through the blood brain barrier

Progress in Neurobiology 159 (2017) 39–49

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Progress in Neurobiology journal homepage: www.elsevier.com/locate/pneurobio

Review article

The role of non-endothelial cells on the penetration of nanoparticles through the blood brain barrier Rui Pedro Mouraa , Andreia Almeidab,c,d , Bruno Sarmentoa,b,c,* a

CESPU – Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Rua Central de Gandra 1317, 4585-116 Gandra, Portugal INEB – Instituto Nacional de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 208, 4200-180 Porto, Portugal i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen 208, 4200-180 Porto, Portugal d ICBAS – Instituto Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal b c

A R T I C L E I N F O

Article history: Received 18 July 2017 Received in revised form 30 August 2017 Accepted 8 September 2017 Available online 9 September 2017 Keywords: Blood brain barrier Astrocytes Pericytes Macrophage Nanoparticle Permeability

A B S T R A C T

The blood brain barrier (BBB) is a well-established cell-based membrane that circumvents the central nervous system (CNS), protecting it from harmful substances. Due to its robustness and cell integrity, it is also an outstanding opponent when it comes to the delivery of several therapeutic agents to the brain, which requires the crossing through its highly-organized structure. This regulation and cell-cell communications occur mostly between astrocytes, pericytes and endothelial cells. Therefore, alternative ways to deliver drugs to the CNS, overcoming the BBB are required, to improve the efficacy of brain target drugs. Nanoparticles emerge here as a promising drug delivery strategy, due to their ability of high drug loading and the capability to exploit specific delivery pathways that most drugs are unable to when administered freely, increasing their bioavailability in the CNS. Thus, further attempts to assess the possible influence of non-endothelial may have on the BBB translocation of nanoparticles are here revised. Furthermore, the use of macrophages and/or monocytes as nanoparticle delivery cells are also approached. Lastly, the temporarily disruption of the overall organization and normal structure of the BBB to promote the penetration of nanoparticles aimed at the CNS is described, as a synergistic path. © 2017 Elsevier Ltd. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General overview of the blood-brain barrier structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nanoparticles as a drug delivery system to the central nervous system . . . . . . . . . . . . . . . . . . . . . . . . Blood-brain barrier cells as modulators of the blood-brain barrier permeability . . . . . . . . . . . . . . . . . The role of astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. 4.2. The role of pericytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Macrophages/Monocytes as a potential nanoparticle central nervous delivery system . . . . . . . . . . . . . Disrupting the blood-brain barrier as an adjuvant to improve nanoparticle delivery . . . . . . . . . . . . . . 6.1. The challenges of blood-brain barrier modulation to increase the penetration of nanoparticles Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: ALA, 5-aminolevulinic acid; AMT, Adsorptive-mediated transcytosis; BBB, Blood-brain barrier; B-CSF, Blood-cerebrospinal fluid barrier; CNS, Central nervous system; CSF, Cerebrospinal fluid; CYPs, Cytochromes P450; FUS, Focused ultrasound; GLUT-1, Glucose transporter 1; LDLR, Low-density lipoprotein receptor; PEG, Poly(ethyleneglycol); PEG-PLA, Polyethylene glycol-polylactic acid; PCI, Photochemical internalization; PTD, Photodynamic therapy; RM, Receptor-mediated transcytosis; TAT, Trans-activating transcriptor; TfR, Transferrin receptor. * Corresponding author at: i3S, Instituto de Investigação e Inovação em Saúde, Rua Alfredo Allen, 208, 4200-18035 Porto, Portugal. E-mail address: [email protected] (B. Sarmento). http://dx.doi.org/10.1016/j.pneurobio.2017.09.001 0301-0082/© 2017 Elsevier Ltd. All rights reserved.

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1. Introduction The blood-brain barrier (BBB), referred for the first time by Paul Ehrlich in 1885, is an extremely limiting barrier that isolates the central nervous system (CNS) from the rest of the body (Rubin and Staddon, 1999). It is mainly composed of highly-specialized brain endothelial cells, which form complex and compact tight and adherents junctions, characterized also by the lack of fenestrations, high expression of efflux transporters and minimal pinocytic activity (Almutairi et al., 2016; Neuwelt et al., 2008; Tietz and Engelhardt, 2015). Joining these endothelial cells are the astrocytes and their respective end-feet, pericytes, macrophages, neurons and a basement membrane, constituted mainly by structural proteins. The basement membrane can be split up into two portions, the vascular basement membrane secreted mainly by endothelial cells and pericytes, and the glial basement membrane, secreted by astrocytes (Blanchette and Daneman, 2015). All the previously mentioned components, together with the endothelial cells, make up the neurovascular unit, a definition proposed to elucidate how the different interactions between these components ultimately regulates the BBB properties (Bradbury, 1985; Weiss et al., 2009). Astrocytes have shown the ability to modulate several features of the BBB, such as promoting tighter tight junctions between the endothelial cells, regulating the number of receptors expressed on the surface of endothelial cells, managing the expression of certain transporters/efflux pumps, controlling the balance of fluid and electrolytes at the BBB, and also the ability to directly express efflux pumps to promote the removal of foreign compounds from the BBB (Abbott et al., 2006; Dombrowski et al., 2001; Rubin et al., 1991). Pericytes have shown to be able to control the development of endothelial cells, and to have an high influence on the regulation of the rate of transcytosis occurring at the BBB and to regulate the vascular permeability and immune cell trafficking of the BBB (Dohgu et al., 2005). Taking this into account, the possible disruption of the interactions between the endothelial cells and astrocytes/pericytes may prove as a valuable tool in further increasing the permeability of molecules and other structures across the BBB as it may result in a periodical opening of this barrier (Berezowski et al., 2004). Nanoparticles emerge as platforms to deliver drugs to the CNS due to their ability to cross the BBB without compromising its integrity and without exhibiting notable central or peripheral toxicity (Jain, 2012). Additionally, nanoparticles can increase the residence time of the drug in the body by circumventing certain physiological excretion mechanisms. Furthermore, nanoparticles load drugs and protect them from efflux pumps present in the endothelial cells, such as the P-glycoprotein, multidrug resistance proteins and breast cancer resistance proteins, overexpressed in the BBB endothelial cells (Gomes et al., 2014; Grabrucker et al., 2014). Nanoparticles may be prepared by a wide variety of materials, from polymers, lipids to inorganic materials. Based on their constitution and possible coating agents that can be added to ensure interaction with specific transport pathway, nanoparticles will cross the BBB mainly through receptor mediated transcytosis and adsorptive-mediated transcytosis (Saraiva et al., 2016). Therefore, the mechanisms of nanoparticle BBB-crossing may be intimately associated with the regulation of both astrocytes and pericytes exert on the BBB; either through chemical segregations or by direct cell-cell signalling, increasing or decreasing the amount of receptors and/or transporters expressed or the rates of transcytosis occurring in the endothelial cells (Abbott, 2013; Prat et al., 2001). Furthermore, nanoparticles can also be develop to target non-endothelial cells in certain situations to achieve BBB penetration and a therapeutical action in certain CNS disorders (Gu

et al., 2017). Macrophages and monocytes have also been explored as drug delivery candidates to the CNS, due to their ability to incorporate nanoparticles, and to cross the BBB as local immune cell (Klyachko et al., 2014). This review aims to summarize the role that non-endothelial cells of the BBB hold, and their possible interaction and regulation of the penetration of nanoparticles through the BBB. Moreover, methods that use a temporary disruption of the BBB, either physically or through chemical segregations affecting astrocytes and/or pericytes will also be presented. 2. General overview of the blood-brain barrier structure The BBB is the main physiological barrier that isolates the CNS, holding the ability to perform physiological actions to ensure the homeostasis of CNS. The main roles of the BBB are to regulate the passage of substances to the brain, to maintain a correct environment for all neuronal activities, to block the passage of potentially toxic compounds and pathogens and to allow a correct and fluid communication between the brain and the rest of the body (Campos-Bedolla et al., 2014). To complete its functions, a very delicate balance is maintained by the endothelial cells which, with assistance of all the components of the neurovascular unit, act as a team to correctly safeguard the CNS (Abbott et al., 2006; Balabanov and Dore-Duffy, 1998). The biggest feature that allows the well-known selective behaviour in the BBB is mainly the existence of highly specialized tight junctions between endothelial cells, that are responsible for the intrinsic high BBB trans-endothelial electrical resistance, and contrary to the lower values reported to other vascular endothelial cells (Stamatovic et al., 2008). Considering all the limiting factors of the BBB presented, only very specific substances and/or endogenous compounds, or highly lipophilic, small-sized drugs can cross the BBB through specific pathways. Those include a paracellular pathway, directly through the tight junctions between the endothelial cells (mainly for small and water soluble agents); a direct pathway in which compounds can directly cross the cell, through diffusion (applicable only by very small and lipophilic molecules, or certain gaseous molecules such as O2 and CO2; through transport proteins that exist on endothelial cells to transport certain nutrients/compounds required in the CNS; cell surface receptor mediated transcytosis, in which a compound interacts with a receptor expressed on the surface of the endothelial cell, and is interiorized; non-cell surface-receptor mediated transcytosis, or adsorptive transcytosis, in which a compound does not require a receptor or a transporter to be internalized by the endothelial cell (Pinto et al., 2017). Out of the pathways presented, certain drugs exploit them to adequately cross the BBB. Some of these pathways are also effectively used by nanoparticles to cross the BBB with relative efficiency and limited side effects (Li et al., 2017). After crossing the BBB, drugs face a second barrier, the blood-cerebrospinal fluid barrier (B-CSF). Unlike its other barrier counterpart, the B-CSF is relatively permeable to most drugs. After crossing to the cerebrospinal fluid (CSF), drugs will cross into the brain parenchyma, through diffusion. However, the diffusion process is founded to be very slow, usually resulting in much higher concentrations of free drug in the CSF than in the brain parenchyma. Therefore, due to the accumulation at the CSF, toxicity can occur (Pardrige, 2011). Nanoparticles hold an additional advantage, due to the ability to hold within numerous molecules. Also, the CSF can promote drug movement to the brain parenchyma through Virchow-Robin (perivascular) spaces. However, as this pathway has low rates of fluid flow, it is usually not considered important for drug transport to the brain parenchyma (Pardrige, 2011; Rennels et al., 1985). Nanoparticles can also enter the brain parenchyma through the

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CSF, but preferably exploit the paravascular pial-glial basement membranes pathway (Morris et al., 2016). Although this review will not present in detail the glymphatic system, readers are pointed to this eloquently written review by Jessen et al. to clear any questions regarding the glymphatic system (Jessen et al., 2015). Another naturally occurring feature at the BBB is the presence of macrophages. Macrophages mainly play a regulatory role at the BBB, in part regulating the transient immune cell trafficking at the BBB. Macrophages/monocytes hold the ability to innately cross the BBB, without resistance, something that is highly desirable when planning a delivery system to the CNS (Obermeier et al., 2016). However, the BBB can also limit the passage of therapeutic drugs to the CNS through the expression of efflux pumps, such as glycoprotein-P, multidrug resistance proteins or other ATP binding cassettes, which have not only an high expression on the BBB endothelial cells, but are also thoroughly expressed on pericytes and astrocytes (Abbott et al., 2002; Mahringer and Frickler, 2016). This adds even more efficiency to the BBB highly selective behaviour, further limiting what drugs can cross the barrier for the treatment of neurological disorders or degenerative CNS disorders. When these efflux pumps are physiological impaired, either due to a pathological condition or due to the natural efficiency decrease when aging, the BBB starts to allow higher quantities of substrates through, with potential CNS toxicity (Bauer et al., 2017). Joining efflux pumps are cytochromes P450 (CYPs), such as CYP1B1 or CYP2E1, that can act as a metabolic defence, promoting the metabolism of certain drugs as they reach the BBB (Dauchy et al., 2008). Like efflux pumps, recent research has shown that CYPs can have a role in brain disorders. A decrease in expression of certain CYPs can lead to physiological imbalances at the brain, and can promote a pathological state within the CNS (Ghosh et al., 2010). An increase of the expression of efflux pumps

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and/or CYPs may be seen as an added defence to the BBB, but it will also hinder the ability to deliver drugs to the CNS (Tang et al., 2017). Thus, nanoparticles can promote here another advantage towards CNS delivery, shielding of the drug loaded within nanoparticles from these metabolic defences, promoting higher penetration to the CNS. To add to the very resistant phenotype of the BBB, it also has significantly lower rates of transcytosis/endocytosis than other endothelial tissues of the body expressed within the body (Abbott et al., 2006) (Fig. 1). 3. Nanoparticles as a drug delivery system to the central nervous system With all the restrictive factors and the current limitations of the therapeutic arsenal currently available, it is necessary to come up with different pathways to promote adequate drug delivery to the CNS without causing problems or harm to the patient (Jain, 2012). Here, nanoparticles seem to be a great candidate as a drug delivery system across the BBB, due to their ability to circumvent some of the BBB extremely limiting defences and, the fact that most nanoparticles are generally well-tolerated by the organism. Nanoparticles for brain delivery can be made from different materials, such as polymers, metallic compounds or lipids (Kim et al., 2010). These systems are characterized due to their submicron dimensions, although those ranging between 10 and 300 nm have higher potential to cross the BBB (Shilo et al., 2015; Zhang et al., 2015). Nanoparticles have the ability to encapsulate or adsorb a widespread number of different drugs. It brings advantages to what is carried, mainly based on the ability of control the delivery of the drug, the possibility of targeting specific organs or locations, as well as an increased ability to cross biological barriers exploring pathways not available to the free

Fig. 1. illustrating the main transport pathways that occur at the BBB. Pathway a. is seldom exploited as it requires the transient disruption of the tight junctions previously to delivery, a relatively dangerous procedure with regards to the integrity of the BBB. Pathway b. represents the pathway most free drugs use to permeate the BBB. Pathways c., d. and e. are the ones exploited to deliver nanoparticles across the BBB, after respective surface modifications of the nanoparticles, adding a specific peptide or antibody to adequately target the transporter or receptor previously decided upon. Some of the main receptors and transporters used are also expressed on the figure. Reproduced with permission from Nature Publishing Group© (2005).

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drugs. Overall, nanoparticles improve the stability of the formulated drug and prolong the residence time in the body due to circumventing certain excretion mechanisms (Zhang et al., 2016). Their surface can be modified with certain ligand moieties that selectively target specific receptors or transporters to the BBB (De Jong and Borm, 2008). Indeed, these modifications show a lack of toxic responses and a high tolerability to their administration as compared to the same dosage of the drug supplied without nanoparticles. These features make them a very tempting drug delivery system for the CNS, and as possible candidates to avoid the BBB filtration (Updahyay, 2014). Lastly, nanoparticles can be adsorbed by some previously established BBB disrupting agents and, these can transiently disrupt the BBB to promote higher nanoparticle BBB crossing and delivery efficiency (Kuo and Lee, 2012; Toman et al., 2015). Different variables will influence the transport of nanoparticles, as the mean particle size, the type of materials used, the drug encapsulated and their global structure (Jain, 2012). Also, the penetration of nanoparticles through the BBB can be achieved through a direct disruption of the tight junctions in the BBB, diminishing the tight junction effectiveness and opening tight junctions to the point that these can be freely permeated. Cellsurface receptor-mediated transcytosis (RMT), where the nanoparticle adheres to a specific receptor/transporter, through a certain antibody or peptide on its external coating, and is subsequently interiorized by the endothelial cell. Lastly, adsorptive-mediated transcytosis (AMT), where the nanoparticle interacts with the membrane of the endothelial cell and is subsequently interiorized without the necessity or previous interaction with a specific receptor; or a certain combination of these mechanisms (Gomes et al., 2014; Kreuter, 2014; Saraiva et al., 2016). The main mechanisms used by nanoparticles to cross the BBB are receptor mediated transcytosis and adsorptive-mediated transcytosis as these offer the most satisfactory results. Bearing this in mind, the main approaches to target the BBB involve designing nanoparticles that target specific receptors or transporters, that exist in moderate expression on the surface of endothelial cells, in higher quantities than in other locations, through surface modifications by promoting the adsorption of some antibodies or peptides to their surface (Gutkin et al., 2016). The materials used to make the nanoparticle are also taken in account as some can be used to harness special delivery methods, such as the use of magnetic nanoparticles with an external magnetic stimulus to promote higher CNS concentrations (Jain et al., 2003). The first nanoparticles created with this goal, were coated with Polysorbate 80, where levels of BBB penetration were adequately achieved. However, researchers found that without Polysorbate 80 the nanoparticles would not cross the BBB satisfactorily, suggesting that Polysorbate 80 had a key role in BBB crossing. This realization led to the exploration of other coating agents and targets at the BBB to realize which ones offered the most advantages target as a drug delivery system (Kreuter et al., 1995; Patel et al., 2012). Thus, Jiang et al., developed poly(ethylene glycol)-co-poly(trimethylene carbonate) nanoparticles for the treatment of glioma. This included 2-deoxy-D-glucose as a surface modifier, to allow targeting of the glucose transporter 1 (GLUT1 transporter), responsible for the transport of glucose to the brain. These nanoparticles achieved higher therapeutic efficacy when compared to nanoparticles without the surface coating agent. The nanoparticles also showed a lack of significant toxicity at the proposed dosage by the authors (Jiang et al., 2014). Ulbrich et al., developed human serum albumin nanoparticles, combined with both transferrin and transferrin receptor antibody to target the transferrin receptor (TfR), showing that these nanoparticles achieved adequate CNS penetration when compared to a similar variation of the nanoparticles, but with a different surface-coating

agent (Ulbrich et al., 2009). Zhang et al., engineered polyethylene glycol-polylactic acid (PEG-PLA) nanoparticles, surface coated with peptide-22, a peptide with high affinity for the low-density lipoprotein receptor (LDLR), and showed that these nanoparticles not only had great brain penetration values, but also promising glioma targeting effects and chemotherapeutic efficiency (Zhang et al., 2013). Geldenhuys et al., made poly(lactic-co-glycolic acid) (PLGA) nanoparticles, externally coated with glutathione, and also showed that these nanoparticles were able to moderately circumvent the P-glycoprotein extrusion and their penetration across the BBB was substantially higher than nanoparticles without glutathione surface-coating. Furthermore, the nanoparticles also showed higher cytotoxic activity than previously mentioned non surface-coated nanoparticles, showing that their therapeutic potential was enhanced (Geldenhuys et al., 2011). Nanoparticles can also be used to circumvent efflux pumps, such as P-glycoprotein. This has been attempted in relation to the BBB (Abbott et al., 2002; Dagenais et al., 2009) as a method to avoid the expulsion of the drug once inside the cell. An example of this was described by the use of nanoparticles as delivery systems of siRNA to silence the P-glycoprotein, a technique that greatly increases the permeability of drugs targeted to the CNS, as reported by Gomes et al. (Gomes et al., 2017). Also, using nanoparticles surface-coated with cell-penetrating peptides such as the HIV-1 trans-activating transcriptor (TAT), can allow not only the effective delivery across the BBB of drugs but also the bypass of excretion mechanisms by P-glycoprotein (Rao et al., 2008). Also, nanoparticles can be used to target the non-endothelial cells at the BBB. Gu et al. demonstrated the use of chitosan nanoparticles, surface coated with either transferrin or bradykinin antibodies to target astrocytes, carrying siRNA for the treatment of HIV. (Gu et al., 2017). HIV can replicate inside astrocytes, and due to astrocytes being the most numerous cell at the BBB, barring endothelial cells, this study highlights how non-endothelial cells can also be considered therapeutic targets for CNS disorders. Thus, targeting non-endothelial cells to achieve BBB penetration by nanoparticles could one day be considered a viable strategy, but further research is still required. Lastly, nanoparticles can also be internalized by macrophages or monocytes and delivered to the CNS. These cells can cross the BBB completely undetected and, after having successfully crossed the BBB, the macrophages/monocytes are able to release the nanoparticles within them, resulting in a higher amount of nanoparticles delivered to the CNS, compared to delivering them without these cell carriers (Klyachko et al., 2014). This chapter however, will be evaluated later in further detail this review. 4. Blood-brain barrier cells as modulators of the blood-brain barrier permeability The endothelial cells of the BBB are transiently regulated by most of the components of the neurovascular unit. However, these cells are constantly being modulated by different signals produced by both astrocytes and pericytes that induce, maintain, up-regulate or down-regulate some features of the BBB and hold plenty of other regulatory functions within the BBB contributing to its function (Abbott et al., 2006; Dohgu et al., 2005). This led to the proposed theory that strategies targeting these cells can open the BBB, allowing a more effective delivery of nanoparticles to the CNS. The main adversity to the targeting approach of these cells could possibly be due to the experienced toxicity of a non-selective and possibly widespread opening of the BBB, as will be presented. The basement membrane, although not considered properly a cellular component of the BBB, allows the anchoring of BBB cells in place (pericytes and perivascular macrophages), contributing to the correct structural organization of the BBB and, subsequently,

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aid it in its functions (Obermeier et al., 2016). It consists of compounds that promote its structure (i.e., collagen, elastin), proteins (i.e., laminin, fibronectin) and proteoglycans (i.e., perlecan, agrin). Its constitution will also contribute to the transendothelial electrical resistance of the BBB, it will affect pericyte migration and differentiation (Maherally et al., 2015) and furthermore it can affect the BBB permeability, as a lack of certain components such as laminin can be directly associated with lower astrocyte end-feet association with endothelial cells, a lower number of pericytes and an overall decrease of certain tight junctions proteins (Menezes et al., 2014). Lastly, some components of the basement membrane can also regulate certain processes and signals between astrocytes/pericytes and the endothelial cells of the BBB (Baeten and Akassoglou, 2011). The main roles that astrocytes and pericytes hold within the BBB is summarised briefly in Table 1. Nonetheless, the role of laminar blood flow through the BBB, which generates a certain force at the apical surface of the endothelium, denominated shear stress, has shown to have a role in both BBB-like phenotype induction and in the maintenance of a healthy and correctly functioning BBB. A study by Cucullo et al., demonstrated that an exposure to blood flow promoted increases in RNA levels for genes encoding for tight junctional proteins and cell-adhesion proteins, compared to a similar model that was not exposed to blood flow and failed to develop barrier-like properties (Cucullo et al., 2011). The same study also remarked that shear stress promoted the increase of the expression of genes encoding several efflux pumps. Furthermore, it was also presented that exposure to shear stress also positively modulated the expression of ionic transporters (Ca2+ and K+) and solute transporters (such as the various glucose transporters). Thus, this breakthrough study highlighted that importance of shear stress, showing that it positively affects various features for which the BBB is known, and necessarily has to execute with perfection. 4.1. The role of astrocytes Astrocytes are one of the main types of glial cells, making up the overwhelming majority of the cells that exists in the brain (Cabezas et al., 2014; Kimelberg and Nedergaard, 2010). Astrocytes were seen as supportive glial cells at the BBB, but recent research has

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shown that their role is undeniably vital to a correct function of the neurovascular unit (Abbott et al., 2006). These cells are the main inductors of the special phenotype endothelial cells of the BBB acquire, and envelop almost the totality of the BBB endothelium through their end-portions known as their end-feet (Persidsky et al., 2006). Thus, astrocytes act as the main regulator of the barrier functions of the BBB, mostly through their secretion of soluble factors, due to the lack of direct physical contact with endothelial cells (Abbott et al., 2006). However, astrocytes by themselves do not induct all these features in endothelial cells during its development, as an early-BBB seems to appear before astrocytes are fully differentiated in some species (Daneman et al., 2010). Subsequently, their development and differentiation is triggered by the endothelial cells of the BBB (Garg et al., 2015). Without directly considering endothelial cells, but no-less important, astrocytes are also responsible for the development of neurons, controlling neurotransmitter levels and managing immune reactions within the CNS (Obermeier et al., 2013). Taking this in account, their correct interaction with the endothelial cells is unequivocally essential for the global function of the neurovascular unit and the integrity of the BBB (Abbott et al., 2006). In vivo studies comparing endothelial cells cultured with astrocytes and without astrocytes showed a direct decrease in the permeability of these cells, highlighting that astrocytes have the ability to tighten the tight junctions between endothelial cells and to reduce the area of gap junctions (Janzer and Raff, 1987; Sobue et al., 1999; Tao-Cheng and Brightman, 1988). Additionally, astrocytes also have the ability to up-regulate several transporters expressed by brain endothelial cells, where some of which hold a key role in nanoparticle targeting, such as the GLUT-1 expression and glutathione transporters (Abbott, 2002; Omidi et al., 2008). Similarly, the expression of receptors thoroughly used in receptormediated transcytosis, such as LDLR and TfR is also tightly regulated by astrocytes. On the other hand, astrocytes are also able to induce the expression of P-glycoprotein multidrug resistance proteins expressed at the BBB (Dehouck et al., 1994; Hayashi et al., 1997). Considering the use of receptors or transporters is currently the main and most efficient pathway for nanoparticle BBB penetration (Gomes et al., 2014; Kreuter, 2014). This highlights

Table 1 The main roles of non-endothelial cells of the BBB on nanoparticles penetration to the CNS. Non-endothelial cells

Main functions

Astrocytes

Regulation of tight junctions and tight junction proteins expression

Broux et al. (2015), Sobue et al. (1999) and Tao-Cheng and Brightman (1988) Control of the expression of receptors and/or transporters of endothelial cells Abbott (2002), Dehouck et al. (1994) and Omidi et al. (2008) Up-regulation of efflux pumps such as P-glycoprotein and direct expression of efflux pumps (Hayashi et al., 1997)) Regulation of fluid and electrolyte levels which can ultimately impact certain transporters/ Simard and Nedergaard (2004), Zlokovic (2008) receptors expression Yao et al. (2014) Control of pericytes differentiation, preventing their alteration to a more disruptive one Production and regulation of basal membrane compounds Kose et al. (2007), Shimizu et al. (2013) and Wang and Bordey (2008)

Pericytes

Increase of the trans-endothelial electrical resistance of BBB endothelial cells Inhibition of immune cell trafficking at the BBB and the permeation of certain agents that promote vascular permeability Direct expression of efflux pumps Regulation of the rates of transcytosis of the endothelial cells Synthesis of basal membrane compounds and maintenance of their correct association

Macrophages

References

Nakagawa et al. (2007) Liu et al., (2012) Shimizu et al. (2008) Armulik et al. (2010) Brachvogel et al. (2007) and Engelhardt and Sorokin (2009)

BBB crossing without impairment due to their normal presence at the BBB Ali and Chen (2015) In increased quantities at the BBB in certain diseases Tong et al. (2016b) Ability to hold nanoparticles within them and to release the nanoparticles in a controlled Klyachko et al. (2014) manner after crossing the BBB

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the importance that astrocytes may hold when it comes to nanoparticle BBB penetration. Some chemical segregations mediated by astrocytes, such as glutamate, aspartate, nitric oxide and serotonin can cause an opening of the tight junctions between the endothelial cells (Abbott, 2000, 2002; Abbott et al., 2006; Broux et al., 2015). However, some chemical or enzymatic signals from astrocytes can greatly diminish the BBB permeability, such as the production of angiotensin-converting enzyme-1, which ultimately allows angiotensin-II binding to AT1 receptors on endothelial cell allowing for the stabilization of these cells, or angiopoietin-1 that increases tight-junction effectiveness through an up-regulation of junctional proteins (Broux et al., 2015). The maintenance of a correct brain fluid and electrolyte balance has also been attributed to astrocytes, through the expression of water channels in their end-feet (aquaporin-4) (Simard and Nedergaard, 2004; Zlokovic, 2008). This, in turn, can affect the expression of transporters and receptors on endothelial cells, as in certain electrolyte or fluid imbalances can influence either the upregulation or down-regulation of certain receptor and transport pathways (Abbott et al., 2006). Not only is the interaction with endothelial cells vital, but astrocytes have also shown the ability to regulate the differentiation of the conformation of pericytes, preventing them from altering their natural configuration to a configuration that promotes BBB opening and subsequently leakage (Yao et al., 2014). Finally, astrocytes are also responsible for the production and the correct association of basement membrane compounds, such as laminin, N-cadherin, neural cell adhesion molecule and fibronectin or some catabolic proteins such as metalloproteinases. This is a function that astrocytes share with pericytes, although pericytes seem to have a higher importance in this matter (Kose et al., 2007; Shimizu et al., 2013; Wang and Bordey, 2008). 4.2. The role of pericytes Pericytes are located on the smaller calibre blood vessels, in close association to endothelial cells throughout the whole body, but with a particularly high expression on the CNS. Due to this, their expression across the BBB has been subject to a lot of research as result of their role regulating the maintenance and permeability of the BBB, and the stability of the blood vessels at the BBB. Their presence is not required for the endothelial cells to develop BBBspecific features, but their absence will result in an abnormally leaky BBB, unable to filter all compounds as it normally does and ultimately lead to CNS disorders that can impair a normal life (Hill et al., 2014). Pericytes can also limit BBB transport through their ability to phagocytose certain compounds after the compounds have successfully crossed the endothelial barrier, ultimately preventing these compounds from disseminating through the CNS (Broadwell and Salcman, 1981). Unlike astrocytes, there is not a consensus on the surface of the BBB that pericytes are in contact with and subsequently cover, with reports varying from 22% to 99% (Engelhardt and Sorokin, 2009; Frank et al., 1987). As astrocytes, pericytes play a role in the regulation of the tight junctions between endothelial cells of the BBB, because of their close association with them (Sá-Pereira et al., 2012). When co-cultured with astrocytes and endothelial cells, in a triple culture exam, pericytes offer a substantive increase in the trans-endothelial electrical resistance of the endothelial cells, further suggesting that a three-way interaction between astrocytes and pericytes is vital for an adequate BBB function (Nakagawa et al., 2007). Pericytes can also inhibit both immune cells to cross the BBB and certain molecules that increase vascular permeability, which is a negative point for the delivery of nanoparticles concealed within macrophages or monocytes (Liu et al., 2012).

Furthermore, pericytes can express efflux pumps like Pglycoprotein and multidrug resistance proteins, acting themselves as key team-players to remove foreign compounds from the BBB area, assisting endothelial cells and astrocytes (Shimizu et al., 2008). Also, a study by Armulik et al., showed that lack of pericytes increases BBB permeability, due to higher transcytosis, which suggests that pericytes have a role in the low transcytosis activity that is observed in the brain-endothelial cells (Armulik et al., 2010). Lastly, pericytes have also been reported to synthesize components present in the basement membrane such as collagen, fibronectin, glycosaminoglycans, elastin and laminin. Pericytes also have the ability to directly stimulate the endothelial cells of the BBB to produce these components themselves, further showing that these cells hold a direct role on the regulation of the neurovascular unit (Brachvogel et al., 2007; Engelhardt and Sorokin, 2009; Kose et al., 2007). 5. Macrophages/Monocytes as a potential nanoparticle central nervous delivery system The macrophages at the BBB transiently reside mainly in the perivascular space, often being referred to as perivascular macrophages. Although there are other cells at the BBB, such as microglia and “surveillance” macrophages (Guillemin and Brew, 2004), perivascular macrophages are the ones to hold interest relating to nanoparticle delivery systems. These cells play a regulatory function in the BBB, controlling immune cells that can permeate the BBB and, regularly recruiting other macrophages to travel to the BBB as a support in certain neurological conditions (Obermeier et al., 2016). In normal conditions, macrophage and monocytes, which subsequently transform themselves into macrophages, routinely transverse across the BBB (Ali and Chen, 2015). However, in certain pathological conditions of the CNS, this macrophage/ monocyte trafficking is heavily increased (Tong et al., 2016b), a process that is highly dependent on cell-cell interactions between endothelial cells and astrocytes (Madsen et al., 2012). This is a feature that is of the utmost interest when considering macrophages and/or monocytes as delivery cells of nanoparticles for CNS disorders. Also, macrophages/monocytes possess high phagocytic activity, and are able to interiorize most types of nanoparticles (Lameijer et al., 2013; Madsen et al., 2012). Due to their presence in the BBB, an opportunity opens to exploit monocytes or macrophages as delivery agents to the CNS, traversing the BBB completely undetected, containing nanoparticles inside, simulating the fabled “Trojan horse” (Klyachko et al., 2014). This technique would require nanoparticles to be directly inserted into a macrophage and/or monocyte, to later administer these cells harbouring the nanoparticles, and aim for them to cross the BBB and, subsequently, discharge their content, after crossing it. These cells can permeate the BBB through their innate ability to alter their shape, a process called diapedesis (Pawlowski et al., 1988). In some cases, BBB disruption can promote increased penetration by macrophages/monocytes through the BBB, although it is highly dependable on the conditions allowed. Usually, when coupled with BBB disruption, cell based carriers can cross the disrupted barrier, but will usually be confined to the perivascular spaces, not quite reaching the brain parenchyma (Marchi et al., 2010). Some limiting conditions exist that must be taken in account for the nanoparticles to be internalized by the macrophages, such as their composition, dimensions, surface charge and shape (Oh and Park, 2014; Sagar et al., 2014). Special attention should be payed to the possibility of premature degradation of the nanoparticles by the lysosomes within macrophages/monocytes, requiring certain types of coating agents to resist this (Hersh et al., 2016). Choi et al., showed the ability to use monocytes/macrophages to deliver gold nanoparticles to treat brain metastases. It was shown

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that these macrophages exploited the recruitment of monocytes and/or macrophages to the metastatic lesions and achieved satisfactory BBB penetration (Choi et al., 2012). Also, Dou et al., employed a similar method, using nanoparticles loaded into macrophages to treat HIV infections that had spread to the nervous system, showing that these macrophages achieved adequate concentrations within the CNS (Dou et al., 2009). Feng et al., also reported the use of macrophages as delivery systems of nanoparticles, using a surface-coating of the folate receptor, for the treatment of brain tumours. This specific receptor is adequately expressed in macrophages, which presents as an opportunity to exploit its expression to increase macrophage nanoparticle uptake. The study resulted in high adequate macrophage distribution and satisfying nanoparticle-macrophage uptake was achieved (Feng et al., 2014). Pang et al., likewise, reported the use of a mouse macrophage-like cell line to successfully deliver nanoparticles across the BBB for the treatment of glioma (Pang et al., 2016). Macrophages can also be loaded with magnetic nanostructures and their migration can be controlled by the presence of a magnet near the brain, attracting macrophages to this site, as reported by Jain et al. (Jain et al., 2003). Tong et al., proposed optimization parameters for the delivery of monocytes carrying nanoparticles to cross the BBB, with the added bonus of demonstrating that the synergistic use of BBB disruption using mannitol, bradykinin and serotonin promoted an increase in monocyte BBB crossing (Tong et al., 2016a). Lastly, it has been demonstrated that macrophages/monocytes can also be used as delivery cells of certain metallic nanoparticles and their ability to migrate through the BBB, carrying these previously impermeable nanoparticles, applied for the treatment of glioma coupled with photothermal therapy, as shown by Madsen et al. (Madsen et al., 2012). With all this, even though there is promise in using macrophages to cross the BBB for nanoparticle delivery, there is still some concerns. Certain chemical compounds and reactive oxygen species that macrophages and/or monocytes can produce, can potentially cause more damage to the CNS than the beneficial effect that the drug inside will promote (Klyachko et al., 2014; Pang et al., 2016). Therefore, further research is still required in this area to guarantee that these cell carriers can both travel in adequate quantities to the BBB and that macrophages/monocytes do not damage the central nervous system. For instance, an optimization on the quantity of nanoparticles that can be inserted into the macrophage whilst resisting degradation, while inside the macrophages, and the ability to release the drug inside in a sustained way after BBB crossing and not prematurely in an undesired location (Zhao et al., 2011). Furthermore, most of these techniques have currently limited or very little clinical data and so further research is still required before the possibility of these drug delivery systems start being implemented and used effectively in therapy for humans. 6. Disrupting the blood-brain barrier as an adjuvant to improve nanoparticle delivery Due to the immense resistance of the BBB to the passage of compounds through it, studying ways to disrupt it can offer valuable intel on different ways to increase the penetration of nanoparticles through the BBB. It is known that the BBB can suffer alterations on certain pathological states that can alter its highlylimiting characteristics. However, there are some methods that have been proposed that can either chemically or physically, temporarily disrupt the barrier to increase delivery efficiency across the BBB. Some of these methods can target the association between endothelial cells and astrocytes/pericytes or the stimulation of a certain function of astrocytes/pericytes to allow a

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transiently, and preferably temporary, and reversible BBB opening (Bidros and Vogelbaum, 2009). Physical methods use non-chemical substances to physically alter the structure of the BBB endothelial cells and their normal organization, to allow an higher permeability of compounds through the BBB (Burgess and Hynynen, 2014). Focused ultrasound (FUS) barrier disruption is a BBB disruption technique that is considered safer than osmotic disruption due to the ability to induce disruption in localized areas of the BBB. This technique consists in the use of microbubbles coupled with low energy bursts resulting in a periodical opening of the tight junctions between endothelial cells, and also an increase of endocytosis within these cells (Etame et al., 2012b; Sheikov et al., 2004). The work carried out by Nance et al., exemplifies this, where poly(ethyleneglycol) (PEG) coated nanoparticles were administered intravenously in rats, coupled with BBB disruption through FUS (Nance et al., 2014). Also, the use of FUS was observed to promote the penetration of gold nanoparticles through the BBB, promoting disruption in only one hemisphere of the brain, showing that this hemisphere obtained higher concentrations of gold nanoparticles, than the non-disrupted hemisphere (Etame et al., 2012a). Similarly, Díaz et al. demonstrated the use of FUS to promote BBB disruption for the delivery of gold nanoparticles, that had Surface Enhanced Raman Scattering tags with excitation wavelengths at near-infrared, to promote the uptake of the nanoparticles by glioma cells (Díaz et al., 2014). Lastly, FUS allows for an excellent coupling with magnetic metallic nanostructures and an external, adequately placed magnetic stimulus, resulting in satisfactory, relatively safe and directed nanoparticle delivery towards the CNS, (Liu et al., 2010; Price, 2015). Osmotic disruption of the BBB consists in the use of a hyperosmotic agent (usually mannitol) injected directly into the bloodstream that will cause a decrease in size of the endothelial cells of the BBB, altering their conformation, and subsequently, affecting the tight junctions between them. This can result in an increased permeability for a short amount of time (usually 20– 30 min). This, however, is not the only reported effect of osmotic disruption. It can also promote a water gradient, which in turn causes vasodilation leading to the membrane of endothelial cells to stretch and lastly, this physical alteration will also interfere with the association of endothelial cells with astrocyte end-feet due to the obvious shape alteration (Hersh et al., 2016; Rapoport and Robinson, 1986; Rousseau et al., 1997). However, considering this method affects the barrier as a whole, specifically the positional interaction between endothelial cells and the astrocytic end-feet, it limits the role that astrocytes have in regenerating the tight junctions. Subsequently, the permeability of the BBB is threatened and the possible permeation of previously limited compounds may occur, and certain neurological lesions or physiological imbalances can appear, harming the CNS more than the benefit the therapeutic plan could have possibly supplied (Rapoport and Robinson, 1986). Photodynamic therapy (PDT) is a technique that employs a photosensitizer agent coupled with activation by light at a specific wavelength to match the drug in question peak absorbance (Dougherty et al., 1998). Commonly, 5-aminolevulinic acid (ALA) is the drug usually employed, due to its usually rapid clearance and excellent tropism for tumours, especially glioblastomas (Madsen et al., 2003). Although this technique is commonly used in tumours, as it promotes irreversible damage to tissues (Madsen and Hirschberg, 2013), Hirschberg et al., reported the use of PDTALA on rats, showing that PDT-ALA was extremely efficient in opening the BBB at limited regions of the brain, with recuperation of normal function after 72 h (Hirschberg et al., 2008). Also, a study by Madsen et al., showed how PDT can be utilized to promote the delivery of macrophages, loaded with iron oxide nanoparticles, through the BBB. The previously filtered nanoparticles at the BBB,

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showed adequate iron oxide accumulation around brain capillaries, when preceded by PDT (Madsen et al., 2013). Although the exact mechanisms through which PDT can accomplish transient BBB opening are still relatively unknown. It is currently thought that PDT accomplishes the disruption of the BBB by promoting direct effects on the cytoskeleton of the endothelial cells of the BBB, causing configuration changes (Sporn and Foster, 1992). Lastly, photochemical internalization (PCI) is a novel BBB disrupting technology that also employs the use of photosensitizers that tend to distribute themselves along the membranes of endocytic vesicles. After activation with light, the photosensitizer interacts with oxygen, damaging the vesicular membrane, promoting the release of the target molecules from said vesicles, instead of potentially being degraded by lysosomes (Madsen and Hirschberg, 2013). Recent research using clostridium perfingens epsilon prototoxin (ETXp) has shown that the active toxin can promote reversible, although widespread, BBB disruption when coupled with photosensitizers to promote PCI (Hirschberg et al., 2009a, 2009b). Although there is still more to learn about the exact mechanism through which PCI accomplishes BBB disruption, the mechanism seems to be similar to that presented for PDT (Madsen and Hirschberg, 2013). Certain chemical methods have been shown to be linked to the reaction that astrocytes provide to the agent used. Therefore, certain chemical techniques can be highly associated with the role non-endothelial cells of the BBB have, as will be presented. To prove the importance and the astrocytes role in the regulation of the BBB functions, a-chlorohydrin was used to selectively destroy astrocytes at the BBB. This revealed an increase in the permeability of the BBB and a simultaneous decrease in both tight junction effectiveness and tight junction protein expression. Subsequently, after normal regeneration of astrocytes, the BBB functions were quickly restored, showing no long term damage to its function (Willis, 2011). This study highlighted the major role of astrocytes in the BBB, and led to the study of certain agents that can disrupt the BBB, either by supressing their productions in astrocytes, or mimicking their role. The use of bradykinin-agonists to target the b2 receptor expressed by endothelial cells can increase tight junction permeability, but will also stimulate astrocytes to produce Interleukin-6. The synergistic effect of these processes will further cause the opening of the BBB (Abbott et al., 2006; Azad et al., 2015; Bartus et al., 1996), increasing the probability of nanoparticles crossing it. A great example of this can be found on the work carried out by Kuo et al., which engineered methylmethacrylatesulfopropylmethacrylate nanoparticles, surface coated with RMP7, a synthetic bradykinin analogue. In this study, the nanoparticles that were surface-coated with RMP-7, when compared to a different variety without RMP-7 coating, had superior BBB crossing ability. Furthermore, It also showed that nanoparticles coated with RMP-7 could cause tight junction opening and a modulation of endocytosis, which ultimately aided in their improved BBB crossing capability (Kuo and Lee, 2012). Similarly to the use of a bradykinin analogue to disrupt the BBB, the use of alkylglycerols has been described. With a similar mode of action, Toman et al., reported the use of an alkyglycerol as a surface-coating agent on poly(lactic acid) nanoparticles. This, ultimately resulted in an increased penetration of these nanoparticles through the BBB, when compared to nanoparticles formulated without the alkylglycerol. Furthermore, these nanoparticles revealed little toxic effect over a short exposure to the nanoparticles, even on relatively high dosages, although when the incubation time was increased, toxic effects were noted on the integrity of the cell monolayer used to test (Toman et al., 2015). Endothelin-1, a peptide produced by astrocytes, has shown that it can increase the permeability of the BBB via protein kinase C

stimulation (Abbott et al., 2006; Rapoport, 1996). Also, in vitro studies supplying ATP and histamine to a co-culture of astrocytes and endothelial cells showed an increase of the GLUT-1. This was suggested to happen due to astrocytes requesting more glucose, and, consequently, causing an up-regulation of the GLUT-1 transporter in endothelial cells (Abbott et al., 2006; Leybaert, 2005). This can be considered a form of BBB opening to nanoparticles that are aimed at the GLUT-1 transporter since a higher number of transporters will result in higher quantities of nanoparticles interacting with it and, subsequently, using it to cross the BBB. Some inflammatory mediators can transiently increase the BBB permeability, but there is a distinct lack of studies regarding their use concomitantly with nanoparticles and their use in BBB permeating formulations (Abbott et al., 2006). Although some of these chemical agents are possible candidates to aid in BBB disruption, their side effects are still more damaging than the opportunity offered to open the BBB. Thus, further research is still required before being regularly employed in an effective manner. 6.1. The challenges of blood-brain barrier modulation to increase the penetration of nanoparticles Although the modulation of the BBB looks to be a great strategy to effectively open the BBB, it has its notable downsides that need to be optimized before barrier disruption can become a widespread method. Osmotic modulation has been reported to cause brain edema, an increase of the amount of water in the brain (Rapoport et al., 1980). It also has lacks localization, which causes a widespread disruption of the BBB. Subsequently, this allows certain endogenous components or pathogenic compounds, previously limited by the BBB to cross it, resulting in physiological imbalances and possibly severe toxic effects. The time window to when the barrier is transiently open to nanoparticles is also very short, which can result in a rather low efficiency when coupling it with nanoparticles(Hersh et al., 2016). Also, studies by both Angelov et al. and Marchi et al. have directly correlated an intrinsic risk of seizures after BBB disruption. (Angelov et al., 2009; Marchi et al., 2007). Marchi et al., reported focal motor seizures occurred in the hemisphere opposed to the location of BBB disruption, whilst the delivery of the same agent without BBB disruption resulted in no seizures. Angelov et al., reported similar findings, highlighting that the most common complication was the presence of seizures. It was also added that patients who had suffered a first seizure, were likely to experience another seizure when submitted to subsequent procedures. Although both studies reported that the seizures were manageable, special attention should be payed when considering BBB disruption. The risks caused by osmotic disruption has to be weighed against the benefit of a positive outcome, to decide whether osmotic disruption should be undertaken. FUS is a novel method for BBB modulation and it has minimal downsides. Reported downsides included a minimal passage of red blood cells in excess to the brain, without injury and, the possible inability of the BBB to recover its barrier properties in certain neurological diseases. This may be due to the disruption that astrocytes suffer in certain neurological diseases, resulting in an inability to correctly heal and restore the BBB function. This leads to the necessity of an optimization of FUS parameters for each pathological state of the brain in order to minimize the negative effects (Burgess et al., 2015). PDT also looks to be a promising and safe BBB disruption technique. The reported downsides to the use of PDT seem to revolve around the parameters set on the light source, thus requiring a tight control on the fluence and fluence rate of said light source to avoid tissue damage or brain edema, which can

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ultimately result in drastic complications, including death (Hirschberg et al., 2008). Similarly, PCI holds promise as well as a BBB disruption technique, but still has room to improvements. The transient opening of the BBB for long periods of time (can be up to 14 days) can promote unwanted passage of compounds through the BBB. Also, ETXp has shown to be toxic in animal models, casting doubts on whether this technology could translate to human trials (Madsen and Hirschberg, 2013). The use of chemical techniques such as the use of inflammatory mediators has the same downside osmotic regulation has: a widespread disruption of the BBB and some have a rather longlasting effect. This can allow the passage of compounds that were previously limited by the BBB, creating physiological imbalances and, possibly causing damage within the CNS. Also, on the usage of some inflammatory mediators, although able to increase the permeability of the BBB, there is a distinct lack of studies made correlating their possible BBB-disruption features and nanoparticles. Lastly, it can cause adverse side effects due to the stimulation caused on astrocytes, and the potential immune response that can be generated, as some of them are in fact, chemical agents that are produced while in stress by astrocytes (Abbott et al., 2006; Etame et al., 2012b). 7. Conclusions and future perspectives The BBB is a notable adversary when it comes to the necessity of adequate drug delivery for the CNS, always aided by the whole neurovascular unit. Therefore, there is a necessity to come up with more efficient ways to deliver drugs into the CNS, ways that can cross the BBB without notable downsides. Nanoparticles can provide an effective alternative delivery system to the CNS, successfully bypassing the BBB, without showing any severe side effects or toxicity. The role astrocytes, pericytes and macrophages hold within the BBB is well established. However, regarding their interactions with nanoparticles, and the possible benefits of enhancing or inhibiting a certain function that these cells have, to increase the permeability of the BBB to nanoparticles, the existing information is still sparse. Further research is still required with the goal of a better understanding on how the penetration of nanoparticles aimed at the CNS can be moderately increased and higher therapeutic effectiveness achieved. Meanwhile, more nanoparticles are aimed at BBB receptors or transporters and higher efficacy and efficiency is obtained. In the future, coupling nanoparticles targeted to these receptors with a reversal disruption of the BBB (the ones that have been attempted with surface-coated bradykinin analogues or alkylglycerols) or, with a chemical factor, able to target both the endothelial cells and either astrocytes or pericytes causing a periodical weakness in the BBB function may prove valuable as auxiliaries in therapy and increase the effectiveness of CNS disorders treatment. The use of macrophages/monocytes as drug delivery vehicles to allow nanoparticles to cross BBB concealed within these cells shows to be a promise, with perspectives of exploiting the macrophages present at the BBB to segregate factors that will attract these nanoparticle-loaded macrophages to them. The main obstacles to these techniques will always be the adverse side effects an influx of immune cells to the BBB might have, and finding a way to either “call” macrophages to the BBB or to correctly target astrocytes without irreversibly damaging the BBB. Until then, targeting some over-expressed receptors or transporters on the endothelial cells of the BBB seems to be the most advanced and efficient pathway for nanoparticles to delivery drugs in the CNS, although very little clinical progress has yet been achieved in humans.

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Acknowledgements This article is a result of the project NORTE-01-0145-FEDER000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was financed by FEDER – Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI01-0145-FEDER-007274). This research was also partially supported by CESPU/IINFACTS under the project MicelCampt-CESPU2017. Andreia Almeida would like to thank Fundação para a Ciência e a Tecnologia (FCT), Portugal for financial support (Grant SFRH/ BD/118721/2016). References Abbott, N., Khan, E., Rollinson, C., Reichel, A., Janigro, D., Dombrowski, S., Dobbie, M., Begley, D., 2002. Drug resistance in epilepsy: the role of the blood brain barrier. Novartis Found. Symp. 243, 38–47. Abbott, N., Rönnbäck, L., Hansson, E., 2006. Astrocyte-endothelial interactions at the blood-brain barrier. Nat. Rev. Neurosci. 7, 41–53. Abbott, N., 2000. Inflammatory mediators and modulation of blood-brain barrier permeability. Cellular Mol. Neurobiol. 20, 131–147. Abbott, N., 2002. Astrocyte–endothelial interactions and blood?brain barrier permeability. J. Anat. 200, 629–638. Abbott, N., 2013. Blood-brain barrier structure and function and the challenges for CNS drug delivery. J. Inherit. Metab. Dis. 36, 437–449. Ali, I., Chen, X., 2015. Penetrating the blood–brain barrier: promise of novel nanoplatforms and delivery vehicles. ACS Nano 9, 9470–9474. Almutairi, M., Gong, C., Xu, Y., Chang, Y., Shi, H., 2016. Factors controlling permeability of the blood-brain barrier. Cell. Mol. Life Sci. 73, 57–77. Angelov, L., Doolittle, N., Kraemer, D., Siegal, T., Barnett, G., Peereboom, D., Stevens, G., McGregor, J., Jahnke, K., Lacy, C., Hedrick, N., Shalom, E., Ference, S., Bell, S., Sorenson, L., Tyson, R., Haluska, M., Neuwelt, E., 2009. Blood-brain barrier disruption and intra-Arterial methotrexate-Based therapy for newly diagnosed primary CNS lymphoma: a multi-Institutional experience. J. Clin. Oncol. 27, 3503–3509. Armulik, A., Genové, G., Mae, N., Nisancioglu, M., Wallgard, E., Niaudet, C., He, L., Norlin, J., Strittmatter, K., Lindblom, P., Johansson, B., Betsholtz, C., 2010. Pericytes regulate the blood–brain barrier. Nature 468, 557–561. Azad, T., Pan, J., Connolly, I., Remington, A., Wilson, C., Grant, G., 2015. Therapeutic strategies to improve drug delivery across the blood-brain barrier. Neurosurg. Focus 38. Baeten, K., Akassoglou, K., 2011. Extracellular matrix and matrix receptors in bloodBrain barrier formation and stroke. Dev. Neurobiol. 71, 1018–1039. Balabanov, R., Dore-Duffy, P., 1998. Role of the CNS microvascular pericyte in the blood-brain barrier. J. Neurosci. Res. 53, 637–644. Bartus, R., Elliott, P., Dean, R., Hayward, N., Nagle, T., Huff, M., Snodgrass, P., Blunt, D., 1996. Controlled modulation of BBB permeability using the bradykinin agonist, RMP-7. Exp. Neurol. 142, 14–28. Bauer, M., Wulkersdorfer, B., Karch, R., Philippe, C., Jäger, W., Stanek, J., Wadsak, W., Hacker, M., Zeitlinger, M., Langer, O., 2017. Effect of P-glycoprotein inhibition at the blood-brain barrier on brain distribution of (R)-[11C]verapamil in elderly vs. young subjects. Br. J. Clin. Pharmacol. 83, 1991–1999. Berezowski, V., Landry, C., Dehouck, M., Cecchelli, R., Fenart, L., 2004. Contribution of glial cells and pericytes to the mRNA profiles of P-glycoprotein and multidrug resistance-associated proteins in an in vitro model of the blood-brain barrier. Brain Res. 1018, 1–9. Bidros, D., Vogelbaum, M., 2009. Novel drug delivery strategies in neuro-oncology. Neurotherapeutics 6, 539–546. Blanchette, M., Daneman, R., 2015. Formation and maintenance of the BBB. Mech. Dev. 138, 8–16. Brachvogel, B., Pausch, F., Farlie, P., Gaipl, U., Etich, J., Zhou, Z., Cameron, T., von der Mark, K., Bateman, J., Poschl, E., 2007. Isolated Anxa5+/Sca-1 + perivascular cells from mouse meningeal vasculature retain their perivascular phenotype in vitro and in vivo. Exp. Cell Res. 313, 2730–2743. Bradbury, M., 1985. The blood-brain barrier: transport across the cerebral endothelium. Circ. Res. 57, 213–222. Broadwell, R., Salcman, M., 1981. Expanding the definition of the blood-brain barrier to protein. Proc. Natl. Acad. Sci. U. S. A. 78, 7820–7824. Broux, B., Gowing, E., Prat, A., 2015. Glial regulation of the blood-brain barrier in health and disease. Smin. Immunopathol. 37, 577–590. Burgess, A., Hynynen, K., 2014. Drug delivery across the blood-brain barrier using focused ultrasound. Expert Opin. Drug Deliv. 11, 711–721.

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