Colloids for drug delivery to the brain

Accepted Manuscript Colloids for drug delivery to the brain M.J. Santander-Ortega, M. Plaza-Oliver, V. Rodríguez-Robledo, L. Castro-Vázquez, N. Villaseca-González, J. González-Fuentes, P. Marcos, M.M. Arroyo-Jiménez, M.V. Lozano PII:

S1773-2247(17)30167-3

DOI:

10.1016/j.jddst.2017.07.012

Reference:

JDDST 431

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 25 February 2017 Revised Date:

30 May 2017

Accepted Date: 11 July 2017

Please cite this article as: M.J. Santander-Ortega, M. Plaza-Oliver, V. Rodríguez-Robledo, L. CastroVázquez, N. Villaseca-González, J. González-Fuentes, P. Marcos, M.M. Arroyo-Jiménez, M.V. Lozano, Colloids for drug delivery to the brain, Journal of Drug Delivery Science and Technology (2017), doi: 10.1016/j.jddst.2017.07.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Colloids for drug delivery to the brain.

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M.J. Santander-Ortega, M. Plaza-Oliver, V. Rodríguez-Robledo, L. Castro-Vázquez, N. VillasecaGonzález, J. González-Fuentes, P. Marcos, M.M. Arroyo-Jiménez, M.V. Lozano.*

Cellular neurobiology and molecular chemistry of the central nervous system group. Faculty of

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Pharmacy, University of Castilla-La Mancha, Albacete (Spain).

Regional Centre of Biomedical Research (CRIB)- University of Castilla-La Mancha, Albacete

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(Spain).

M.V. Lozano

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*Corresponding Author

email: [email protected]

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Tel: 967.59.92.00 ext 8238

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1. Abstract The field of neurodegenerative diseases have lately experienced significant advances. Despite that, we are still far from achieving the recovery of the patients. Nowadays there is more profound knowledge about the origins of the disorders and experimental therapies have become available. These attainments are encouraging, as the incidence of neurodegenerative

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diseases will notably increase in the coming years. This increases the demand for treatments that are more efficient. Up to now, the molecules that have proven to be effective cannot access the brain due to the restrictions posed by the blood-brain barrier (BBB). This review focuses on the most significant physiological approaches that used colloids as carriers to

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improve the delivery of therapeutics to the brain. In that sense, the general design considerations of colloids will be discussed with regard to the importance of size, surface properties, core composition, targeting and interaction with mucosal tissues. The

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improvement of the interaction with the BBB, the increase of drug bioavailability by the oral route and the nose-to-brain pathway illustrate the physiological efforts to circumvent the

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transport limitation of the BBB, and therefore they will be specially analysed.

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2. Introduction The world population is increasingly getting older. The World Health Organization (WHO) stated that by 2050 one in five people will be older than 60 years. The process of ageing is

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multifactorial and influenced by genetic, environmental, behavioural and epigenetic factors ultimately leading to deleterious consequences in the physiological functions of the body. Additionally it will determine the quality of life in old people and will require patient long-term care. Ageing is closely related to the development of neurodegenerative diseases such as

increasing

population-ageing

rate

will

end

in

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Alzheimer disease (AD) or Parkinson´s disease (PD), so it is reasonable to think that the higher

incidence

of

age-related

neurodegenerative diseases in the future [1]. Neurodegenerative disorders are described as

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the progressive and selective loss of neurons with impairment of different functional systems [2]. The decline in neurological functions is commonly represented by motor impairment, cognitive deterioration and loss of sensory and emotional functions [3]. The current therapeutic arsenal is able to slow down the progress of the disease to some extent but even not in all of the treated patients [4]. In addition, there are diseases that do not

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have any treatment options; this is partially due to the lack of understanding of the pathological basis of the diseases. Therefore, there are strong research efforts ongoing focusing on obtaining new pharmacological active molecules that improve the prospects of these patients. These molecules include as diverse classes as microRNAs (miRNA), intrabodies,

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stem cells or selective growth factors which try to balance neurotransmitters, chelate ions or confer neuroprotection [5]. For exerting their therapeutic action, these drugs need to access the brain, but this is hampered by the presence of different barriers. Amongst these, the blood

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brain barrier (BBB) is the most thoroughly known and more extended of barriers [6]. Some regions of the brain, such as choroid plexus or circumventricular organs, have a more permeable barrier [7]. These structures could be considered as an alternative opportunity for reaching the brain, but it should be taken into account that drugs would encountered increased enzymatic activity as well as the presence of the cerebrospinal fluid (CSF), which is endlessly being renewed [8]. The knowledge in the existence of the BBB started with the first observations of Paul Ehrlich about the exclusions of injected dyes from the brain. From these early experiments, much knowledge has been gained and nowadays it can be more clearly elucidated how the BBB participate in the brain-periphery crosstalk by the expression of mediators, transporters and

ACCEPTED MANUSCRIPT control of its permeability. The BBB is highly restrictive in the access of molecules to the brain, allowing only the entrance of those nutrients and mediators required to maintain brain homeostasis. Due to its restrictiveness most of the xenobiotics are excluded from the brain. Generally it has been considered that drugs with molecular weight (Mw) of about 400-600Da and hydrophobic character can cross the BBB by passive diffusion [9]. Nevertheless, not all of

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the small molecules freely crosses the BBB, indeed it is estimated that only 2% of them are able to reach the brain; meanwhile nearly all of the large ones (Mw>1 kDa) are blocked [10]. The physiological characteristics of the BBB are the basis of this restrictive behaviour. Unlike endothelial cells from other tissues of the body, brain endothelial cells are strongly packed by tight junctions so there are no fenestrations between them, hampering the paracellular

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transport of molecules to the brain. This structure is supported by the presence of astrocytes, neuronal endings, microglia, pericytes and circulating immune cells, all of them constitute the

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neurovascular unit. This complex structure is reinforced by the presence of efflux mechanisms at the brain endothelial cells, such as P-glycoprotein, that decrease the concentration of some xenobiotics by expelling them out of the brain [6]. The BBB is not just a static and complex barrier to overcome; its restriction is also due to its dynamic capacity to adjust to the

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physiological needs of the brain and to pathological conditions. This is attained by the

Figure 1: Schematic diagram showing the endothelial cells strongly packed by the tight junctions, the astrocytes and pericytes among other components of the neurovascular unit. interaction of the brain endothelial cells with the surrounding cells that constitutes the neurovascular unit [11] (Figure 1).

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The demand for more effective therapeutics has led to the development of multiple strategies to reach the brain tissue. Some of them rely on the transient disruption of the BBB by using osmotic pressure, ultrasound or pharmacological entities, meanwhile others are based on intra-cerebro-ventricular (ICV) infusion, convection-enhanced delivery, intra-cerebral injection

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or the use of implants [12]. These techniques have shown benefit in the treatment of diseases such as neuroblastoma, but are considered invasive approaches that may alter the homeostasis of the brain tissue and in addition requires the hospitalization of the patient to carry out the procedure. Besides there are various physiological strategies such as adsorptive

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mediated endocytosis or transport mediated transcytosis, which are based on the same mechanisms that hydrophilic high Mw molecules use for reaching the brain. Other approaches that are worth to be mentioned are the increase of the bioavailability of pharmacological

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active molecules to increase their brain levels by the oral route or the nose-to-brain transport. In this sense, the use of colloidal carriers can enlarge the scope of these strategies. Colloids are structures at the nanometer range that can associate bioactive molecules for improving their

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stability, transport and interaction with biological barriers (Lockman 2002). Their composition

Figure 2: Illustration of the physiological approaches for the treatment of neurodegenerative diseases. and properties can be rationally designed to the specific application (Pasha 2009). More specifically, surface and core composition, size, targeting properties and interaction with mucosae are key characteristics of colloids of special consideration when designing colloids for drug delivery. Then, the aim of this review is to present updated knowledge about the

ACCEPTED MANUSCRIPT physicochemical properties of colloidal carriers designed for brain delivery and to analyse the progress of those systems that follow the physiological approaches for the treatment of neurodegenerative diseases (Figure 2).

3. Colloids: general considerations

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Colloidal carriers are structures with a size between 10 and several hundred nanometers. They have been used as delivery systems of low soluble or unstable drugs, thus improving their solubility and avoiding their rapid degradation rates. These carriers are able to modify the biodistribution patterns of the drugs associated and prolong short circulation half-life values

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[13, 14]. Those therapeutic molecules can be either entrapped in the matrix of the carrier or attached to its surface. The versatility offered by colloids enables the association of molecules with a wide range of characteristics.

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Biodegradability and biocompatibility are common characteristics of natural and synthetic polymer-based colloids so they can exhibit a favourable toxicity profile. Inorganic nanoparticles are important in diagnosis and theragnosis because of excellent imaging properties, but mostly have degradability concerns for long chronic treatments. Conversely, natural and synthetic polymer-based colloids have the disadvantage of poor recognition by microscopy techniques

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[8]. This is a clear drawback in terms of understanding the mechanism of interaction with the BBB and for regulatory issues. The interaction of nanoparticles with the BBB is a complex interplay between those properties of particles such as size, surface charge of hydrophilicity and those many variable biological properties [15]. Hence, determining the mechanism of

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transport of nanoparticles across the BBB is quite a difficult task [7]. Multidisciplinary knowledge is the basis for the different drug delivery strategies developed

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and this is crucial for improving the future clinical outcome of neurodegenerative diseases. The following sections will remark the rational design of colloids for adjusting them to the requirements of their administration route.

3.1.

Size

The size of the colloidal systems has an effect on their interaction with the biological media. For instance, nanoparticles with dimensions lower than 50 nm administered by the systemic route have more chances to overcome the BBB that nanoparticles of 200 nm or higher [16, 17]. However, small nanoparticles (around 10 nm) are prone to be cleared from the blood stream by the hepatic and glomerular filtration. Researchers have also studied the influence of the size for colloids aimed for brain delivery by the olfactory route. Some recent studies analysed

ACCEPTED MANUSCRIPT the transport of polystyrene nanoparticles in the 20-200 nm range coated with polysorbate 80. For that purpose, nanoparticles were placed into the donor chamber of a Franz diffusion cell loaded with excised olfactory porcine epithelium. Although none of the systems were efficiently transported across the epithelial tissue, the systems of 200 nm entered more deeply through the tissue [18]. The same polysorbate 80 coated systems were tested by intranasal

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administration to mice. Particles with a size of 100 nm could penetrate more easily into the olfactory tissue compared to the 200 nm carriers [19]. Authors suggest that this can be due to the faster diffusion of smaller nanoparticles in mucus [20, 21].

There have been many studies trying to optimize the absorption of neuroprotective molecules

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through the oral route and some of them are focused on the effect that the size of the carriers have on the bioavailability. The in vitro studies carried out by Salvia et al. with the neuroprotective antioxidant β-carotene [22], have shown that it was possible to optimize both

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the digestion extent as well as the digestion time of β-carotene by reducing the size of corn oil/polysorbate 20 carriers from 23 to 0.21 µm, improving its bioavailability after oral administration. Analogously, Dey et al. formulated emulsions (1.58 ± 0.31 µm) and nanoemulsions (78-95 nm) with EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) rich fish lipid oil using polysorbate 20 and Span 80 as emulsifier and co-emulsifier respectively [23]. The administration of both formulations to Sprague-Dawley rats resulted in a significant

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2.5 fold increase in the lipid absorption. Additionally, the formulation of the ω-3 rich oil as nanoemulsion avoided the not always welcome fishy smell and unpleasant reflux associated to conventional fish oil emulsions [23]. In the same line, Lane et al. studied the effect of the fortification of yogurt (used as food vehicle) either with emulsions of ω -3 algal oil or with the

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bulk oil, in the intestinal absorption of DHA in human volunteers [24]. Interestingly, supplementation of yogurt with emulsions led to a significant increase of plasma levels of DHA

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in comparison with the bulk oil up to 8 hours after ingestion [24, 25]. It is known that the size of the nanostructure will affect the rate, the extension of the lipid digestion and the absorption of these neuroprotective molecules [26]. However, despite the efforts of scientist to formulate nanometric platforms to improve the bioavailability of the cargo, crossing the gastrointestinal tract (GIT) may provoke the aggregation of the particles and the formation of 10-20 µm droplets in the stomach irrespectively of the initial size [27-29]. As shown by Li et al. using Sprague-Dawley rats, premature aggregation of the nanostructures can led to a clear reduction of the serum levels of the neuroprotective molecule after oral intake [30].

ACCEPTED MANUSCRIPT This example clearly illustrates that the efforts of scientists should be focused not only on the formulation of novel nanometric platforms, but also in conferring them with the appropriate superficial properties to survive to the harsh physiological environment of the GIT [31].

3.2.

Surface Surface properties

The control of the surface properties of colloidal systems is a key parameter to understand the

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behaviour of the formulation under physiological conditions. The elevated surface to mass ratio of colloids make them thermodynamically unstable and highly reactive [32, 33]. Classical DLVO theory, developed by Derjaguin and Landau, Verwey and Overbeek, describes the stability of colloidal systems in aqueous medium [32, 33]. This theory states that the

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interaction between two approaching nanoparticles depends on an attractive potential (VA), result of the attractive hydrophobic interactions, and a repulsive potential created by the

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repulsive electrostatic interactions (VR). The total potential of interaction (VT) can be shown as follows: =

+

The control of the surface characteristics of the colloids could modulate the particle-particle interaction and, more interestingly, the particle-physiological medium interactions. Highly

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charged colloids will present a remarkable stability after their formulation, while highly hydrophobic colloids, in absence of a high enough repulsive VR, will tend to aggregate. The magnitude of VA and VR will affect the behaviour of the colloid not only in the formulation medium, but also in the physiological media. While high VR can be desirable to formulate

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colloidally stable systems, this aspect may be detrimental under physiological conditions. For example, the administration of highly cationic particles will lead to strong interactions with the negatively charged cellular membranes and macromolecules present in the physiological

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fluids, which can result in acute toxicity [34-36]. However, the use of highly negative colloids can result in low uptake of the nanoparticles by the cells due to the repulsion between the cell membrane and the nanoparticles. VA also presents a relevant effect on the performance of the formulation after administration. High VA values will promote strong hydrophobic interactions with macromolecules and mucosal tissues [32, 33, 36, 37] this may promote fast clearing of colloids by the reticulo-endothelial system (RES) after the adsorption of opsonins to the carriers [38, 39]. The interaction with plasma proteins and other macromolecules is one of the fastest events that take place once colloidal systems enter the blood stream [34, 36, 37]. Consequently, the adsorption of macromolecules will modify the surface properties of the nanoparticle [34, 40,

ACCEPTED MANUSCRIPT 41]. This physiological coating will affect to both their physicochemical stability and their biological interactions, controlling the final fate of the particle. Heterogeneous coating of positively charged nanoparticles with negative plasma proteins will lead to nanoparticles with positive (non-coated) and negative (coated) surface areas. This heterogeneous surface charge distribution will induce bridging flocculation mechanisms which end up in the particle

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aggregation [42] and the potential retention of the aggregates in the narrow lung capillaries [43-45].

Fortunately, these interactions between hydrophobic nanoparticles and plasma components may not necessary result in particle aggregation. This protein layer created on the surface of

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the particles, known as the protein corona [34-36, 42, 46], may actually be favourable. The improvement of the colloidal stability of the particles under physiological conditions [47], or advances on their biological performance are determined by the specific composition of the

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protein corona. On one side, the composition of the protein corona depends on the physicochemical properties of the particles, such as size, surface charge and hydrophobicity. On the other side, plasma properties, such as the specific concentration and affinity of the plasma proteins at the specific time and place of injection will affect to the protein corona composition.

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Interestingly, non-specific plasma protein interactions may also induce the targeting of the nanoparticles themselves [48]. In this sense, the research group of Prof. Kreuter formulated a polysorbate 80 coated colloidal system able to promote the formation of a protein corona enriched in apolipoprotein E (ApoE), a protein involved in the transport across the BBB due to

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receptor mediated transport [49-51]. The analysis of this protein corona led to the identification of ApoE targeting moieties and its use for covalent modification of the particles

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for brain delivery [52].

Non-specific interactions with proteins can be reduced by modulating the hydrophobic character of the nanoparticles. This can be achieved by using hydrophilic raw materials for the preparation of the nanoparticles or by the coating of the already formed nanoparticles with hydrophilic polymers [53-57]. In this sense, the use of polymers such as chitosan or hyaluronic acid can result in the formation of low protein affinity hydrophilic colloids [54, 55]. As demonstrated by Lalatsa et al. [58, 59], the use of hydrophilic polymers, such as chitosan may also promote the interaction of the colloidal system with the intestinal mucosa after oral intake and thus facilitate the transport of the associated peptide to the brain. In the same line, the use of HA has the added value to promote the mucosal interaction of the formulation and

ACCEPTED MANUSCRIPT also the targeting of the nanoparticles towards CD44 receptor positive cells [53, 54]. This aspect could be potentially exploited for dual brain targeting, as CD44 is usually expressed by the astrocytes of the glia [60]. In addition, it has been described for primates that the agerelated astrogliosis of the CNS in non-pathological ageing process leading to the overexpression of CD44 receptors [61], and a constant upregulation in the expression of CD44

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receptors in the brain astrocytes was observed in AD patients [62]. The arrangement of these polymers, used as coatings or structural components, on the colloidal surface can be studied through the stability of the formulation as a function of the medium salinity [54, 56]. According to the DLVO theory, the increase of the medium salinity

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does not affect VA, but may reduce the repulsive VR, leading to a reduction on the colloidal stability. However, it is well described for hydrophilic colloids that the increase of the medium salinity can lead to the creation of a repulsive structural force known as hydration forces (VH)

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that can stabilize the formulation under physiological conditions [33, 54]. The intensity of hydration forces depends on the surface hydrophilicity and the electrolyte concentration. Under this scenario, the quantification of VH at a given electrolyte concentration is a helpful tool to compare the hydrophilic character of different colloidal systems. In this sense, the studies carried out with colloidal systems formulated either with chitosan or hyaluronic acid revealed that the Mw plays a key role on the arrangement of the polymer in the colloidal

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surface, where low Mw allowed a better adjustment of the polymer chains and then produced a more homogeneous coating [54, 56].

Another well-known strategy to enhance the hydrophilic character of the colloidal system is

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the use of poly(ethylene glycol), PEG, derivatives as coating agents [39, 63-66]. In addition to the reduction of the hydrophobic character, this type of coatings can result in the creation of

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repulsive osmotic (VOSM) and elastic (VEL) potentials of interaction. This potentials may sterically stabilize the formulation [32, 33]; where VOSM depends on the volume fraction (φ) and the length (δ) of the PEG segments and (VEL) on the coating density (ρ) and the PEG Mw [32, 33]. As a function of ρ, the formulation will present different colloidal behaviour. For low ρ values, the presence of the non-ionic PEG derivative on the colloidal surface will screen the surface charge of the formulation resulting in the destabilization of the system [63]. From certain ρ value, VOSM and VEL may create an intense potential barrier enough to ensure the stabilization of the system [63]. Contrary to VR or VH, neither VOSM nor VEL depend on the electrolytes present in the medium, which guarantees the colloidal stability of the formulation independently of the physiological medium salinity [32, 63]. Apart from the protection of the formulation from the salinity of the medium, PEG coatings can isolate the nanoparticles from

ACCEPTED MANUSCRIPT the unspecific interaction with macromolecules present in the physiological medium. Stealth particles have been widely reported in literature for their capacity to avoid the capture from the RES and then increase their systemic circulation time, obviously enhancing the efficiency of the system [39, 67]. PEG derivatives can be anchored to the nanoparticles surface mainly by covalent binding or by

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adsorption. Covalent binding implicates the presence of active groups in both the nanoparticle and the PEG derivative. This aspect can limit the application of this technique to nanoparticles without appropriate reactive groups on the surface. Additionally, covalent binding involves the formation of a new chemical entity (NCE), which can present some issues from a regulatory

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point of view. Adsorption is a versatile and accessible approach for obtaining PEG coated colloidal systems [63, 67]; however, the main drawback of adsorption is the relative weakness of the particle-PEG interaction. In this sense, molecules with higher affinity (proteins, enzymes

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and co-enzymes) than PEG derivatives for the nanoparticle surface can displace the surfactant and modify the final fate of the formulation after administration [31, 68].

3.3.

Core composition

Core composition is another aspect that has to be carefully designed for drug delivery, because along with the surface properties, colloidal core composition can also affect to the

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performance of the formulation. The delivery to the central nervous system has been faced using colloids with a wide variety of cores, e.g. purely hydrophobic systems (such as solid lipid nanoparticles), liposomes (aqueous core surrounded by a hydrophobic lipid bilayer), and purely hydrophilic materials such as chitosan [12]. In all these cases, the inner core has been

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thoughtfully engineered in order to facilitate the interaction of the nanoparticle with the BBB and other biological barriers, i.e. intestinal or nasal mucosa, or for an optimal accommodation

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of the therapeutic cargo. Poly(lactic-co-glycolic) acid (PLGA) is a good example of the use of synthetic polymers for brain drug delivery [12]. In comparison to natural materials, the use of synthetic polymers allows the design of the nanostructure from the unitary blocs that will form the colloid matrix. In this sense, PLGA can be synthetized with different lactic/glycolic ratios to modulate the degradation kinetic of the matrix and then the release of the therapeutic cargo [69]. Focused on the bioavailability improvement of neuroprotective molecules after oral intake, McClements et al. have demonstrated that the core composition (more exactly the use of medium chain triglycerides, MCT, or long chain triglycerides, LCT) can also control both the digestion of the nanostructure and the bioaccessibility of neuroprotective molecules after oral

ACCEPTED MANUSCRIPT intake [70-72]. Bile salts, phospholipids and enzymes secreted by the pancreas in the small intestine can digest the lipid matrix of these nanostructures leading to the formation of vesicles and mixed-micelles enriched in the neuroprotective molecule that are ready to be absorbed [73, 74]. The capacity of these vesicles and mixed micelles to solubilize the bioactive molecule and promote the absorption will depend on the nature of the lipid matrix of the

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nanostructure. It has been recently described that although MCT matrices present faster digestion kinetics, LCT matrices (such as corn oil) promote the formation of vesicles and mixedmicelles with higher solubilisation capacity of the bioactive molecule, and then higher absorption potential [71, 72].

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Another aspect to consider is the physical state in the nanostructure core. Studies carried out with the neuroprotector β-carotene have shown that liquid lipid nanoparticles (LLN) confer better protection to the antioxidant than solid lipid nanoparticles (SLN) [75]. The lower

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stability of β-carotene in SLN seems to be related to the displacement of the antioxidant towards the nanoparticle surface during the core crystallization, and then to its exposition to external oxidants [75].

Interestingly, the nanostructure core composition can also affect the conformation of its surface, and in the end determine the colloidal behaviour of the formulation. Lipid

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nanocapsules are core-shell nanostructures, which electrostatic and hydrophobic interactions between the lipid core and the outer polymer will clearly affect the polymer arrangement on the surface. These results show that the colloidal behaviour of the formulation under physiological conditions can be modulated by selecting the adequate core composition [31,

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41, 68, 76].

The inclusion of different specific additives such as secondary antioxidants or chelating agents

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to improve the stability of the main bioactive molecule or permeation enhancers to facilitate its absorption are alternatives routinely used to improve the performance of colloids [25, 30, 77]. Additionally, new advances in nanotechnology are allowing the engineering of novel nanostructures made of stimuli-response materials able to modulate their behaviour as a function of the external physiological conditions [78]. In this sense, it would be possible to design nanoparticles with a matrix composition able to selectively protect or release the cargo depending on the physiological conditions of the selected administration route.

3.4.

Targeted colloidal systems

The surface properties of colloidal systems can be adapted not only to fit the colloidal stability of the formulation according to the administration route, but also to promote the active

ACCEPTED MANUSCRIPT targeting of the nanoparticles towards the desired tissue by the incorporation of antibodies, peptides, and others cell receptor-recognizing molecules [12, 54, 60, 79-84]. It is important to highlight that, from a physicochemical point of view, these modifications may affect the colloidal behaviour of the formulation. It is well known that the use of antibodies may lead to the destabilization of the colloidal formulation under physiological conditions [33, 40]. The

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incorporation of antibodies, with an isoelectric point in the 6-8 pH range will result in the reduction of VR. Then, in the absence of other stabilizing mechanisms, like steric stabilization, the colloidal stability of the targeted nanoparticles can be diminished under physiological conditions.

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Another important issue related to the incorporation of antibodies is the modification of the hydrophobic character of colloids. Anchoring of antibodies, either by covalent binding or adsorption [85], to the colloidal surface takes place through the hydrophobic Fc fragment of

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the antibody. In this way, the hydrophilic and active Fab fragments are exposed towards the external medium increasing the hydrophilicity of the colloidal system. This modification can contribute to the colloidal stabilization of the formulation under physiological conditions, thanks to the repulsive structural force known as hydration forces [40]. With respect to the anchoring strategy followed, covalent binding through the Fc fragment is preferred to increase the number of antibodies with an adequate orientation of the active Fab epitopes for targeting

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purposes [85]. However, direct binding of the targeting moieties to the colloidal system may actually result in a loss of binding affinity with the targeted tissue due to the steric hindrance of the surface-immobilized targeting molecules [86, 87]. This issue was solved by the inclusion

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of spacer-like molecules between the nanoparticle and the targeting molecule [82]. In 1983 Couvreur et al. used protein A as spacer [88]. Once adsorbed onto the preformed colloidal system, protein A could be covalently bound to IgGs resulting in more than 95% of the

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antibodies efficiently coupled to the nanoparticles. Despite the better results obtained with this strategy, in comparison with the direct anchoring of the antibody to the nanoparticle, in vivo studies led to controversial results [89-91]. Most probably this is due to the shedding of the protein A-IgG complex from the colloidal system surface by the competitive adsorption of other plasma components after administration [89, 90]. Based on this knowledge, the next generation of spacers was focused on the use of chemically activated PEG molecules that could be covalently bind by one reactive terminal group to the colloidal surface and by the other to the targeting molecule [86, 87]. In this sense, the length of the spacer is a key parameter in order to facilitate the adequate interaction of the anchored targeting molecule with the desired cell receptor [82].

ACCEPTED MANUSCRIPT The potential degradation of the targeting moiety is another aspect to be considered when designing targeted colloidal systems. Antibodies, peptides and aptamers are prone to be degraded by the plasma enzymes [92]. Bearing this in mind Wei et al. have tested the potential of D-Angiopep-modified nanoparticles for active brain targeting [93]. Angiopep is selectively recognized by the LRP-1 receptors overexpressed on the endothelial cells of the BBB; however,

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this peptide can be easily metabolized in the systemic circulation [94] [83]. The results showed that D-Angiopep was much more stable in plasma than the natural L-analogue and that the functionalized colloidal system displayed faster internalization across the BBB.

The studies carried out by Gao et al. revealed that the coating density can also play a role in

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the active targeting of the formulation towards the brain [95]. These authors studied the effect of Angiopep coating density on the brain targeting capacity of dendritic nanoparticles. As expected, the incorporation of this peptide clearly enhanced the brain accumulation of the

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colloidal system after injection to mice. However, the increase of the Angiopep coating density resulted in an accumulation of the nanoparticles in the cortex, hippocampus, striatum and cerebellum quite similar to that of the non-targeted nanoparticles [95]. Authors suggest that the reduction of the efficacy was due to the RES response that rapidly remove the colloidal system from the systemic circulation. Pang et al. observed a similar effect with OX26, a monoclonal antibody able to interact selectively with the transferrin receptor expressed in the

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BBB [84]. The pharmacokinetic studies in rats clearly showed that the increase of the OX26 coating density led to a faster clearance of the associated polymersomes from the systemic

3.5.

Interaction with mucosal tissues

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circulation.

Oral and nasal routes are considered suitable gateways for brain drug delivery, so it is

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reasonable to evaluate the interaction of colloids with these mucosal tissues [58, 59, 96]. The fundamental approach for nose-to-brain has been the formulation of colloidal systems able to increase their retention time in the nasal mucosa, including both the respiratory and the olfactory mucosae [18, 19]. As it will be described in more detail in the following section, two of the main raw materials used for nasal administration to the brain are chitosan and PLGA. The interest of these polymers relies on their capacity to interact with the nasal mucosa through a variety of mechanisms that include hydrogen bonds, attractive electrostatic or unspecific hydrophobic interactions [[97]. The main objective in oral drug delivery is to enhance the bioavailability of neuroprotective molecules to facilitate their uptake by the central nervous system. Muco-adhesive

ACCEPTED MANUSCRIPT nanoparticles made of chitosan, poly(acrylic), poly(lactic) (PLA), PLGA or hyaluronic acid have been widely reported as structural materials that confer bio-adhesive properties to the corresponding nanostructure and then prolong the intestinal retention of the nanostructure [21, 98]. Irrespective of the specific mucosal route, it is important to highlight that the use of muco-

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adhesive colloidal systems may compromise the protective role of the mucosa [21, 99]. Mucins, as glycosylated proteins, present the ability to form the well-known gel-like structure of the mucus [21]. Mucin mesh is the responsible to filter out those pathogens and foreign particles that can potentially damage the organism. Under this scenario, muco-adhesive

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colloids can cross-link the mucin fibres, which result in an increase of the mean pore size of the mucin mesh and subsequently loss of the mucus protection capacity. This is of special concern in the case of chronic diseases [21, 99]. Wang et al. recently demonstrated that the incubation

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of muco-adhesive nanoparticles with mucus can lead to a mean mesh pore size increase of up to ∼160% [99]. Additionally, it was observed an increase of the fraction of ≥500nm pores from 11% to 19%, meanwhile the pores with a mean size of ≥1 µm increased from 3% to 9%. Another issue related to the use of muco-adhesive colloidal systems is the mucus clearance time of the specific mucosal tissue. It is well described that muco-adhesive nanoparticles are

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efficiently retained by the mucus blanket that cover the epithelial cells. However, the mucus clearance process can slough off more than the 90% on the initially retained particles in a period of time that can range from minutes to hours, which can compromise the efficacy of the formulation [100, 101].

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As alternative approach, other authors have proposed the use of mucolytic enzymesdecorated colloidal systems to overcome the mucus barrier of the intestinal mucosa and

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promote the intimate contact between the particle and the epithelial cells of the intestine [102]. The studies carried out in porcine intestinal mucus with papain or bromelain embedded poly(acrylic) acid nanoparticles showed that the incorporation of the enzymes result in a at least 2.5-fold increase of the mucus diffusion rate in comparison to the bare poly(acrylic) acid nanoparticles [102, 103].

Others authors have focused on the design of virus-like colloidal systems able to efficiently diffuse across the mucus and release its cargo in the proximity of the epithelial cells [20]. These authors have shown that it is possible to protect the colloids from the interaction with the mucins of the mucus mesh by playing with the Mw (≤10KDa) and the coating density of PEG as coating agent [101, 104]. These colloidal systems, formulated either by covalent

ACCEPTED MANUSCRIPT binding or by the adsorption of the PEG derivatives have shown a similar diffusiveness in both saline and mucus [20, 104-106]. Maisel et al. have shown that after the oral administration of muco-adhesive colloids barely 40% of the intestinal epithelial cells was covered, meanwhile mucodiffusive colloidal systems achieved a remarkable 80% of coverage [107]. In addition, once they have diffuse across the mucus blanket, mucodiffusive colloidal systems can prolong

for at least 24h [101].

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the

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4. Physiological approaches neurodegenerative diseases

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their contact with the epithelial cells facilitating the localized release of the encapsulated drug

4.1. Promote interaction with the BBB

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As already mentioned, the BBB is an extraordinarily complex barrier that efficiently maintains the homeostasis of the brain. Despite its restrictive nature, it still offers opportunities for those colloids administered by the parenteral route that aim to deliver their cargo in the brain. Indeed, double resveratrol brain concentrations can be reached by the encapsulation of the antioxidant in nanocapsules [108]. Curcumin encapsulation resulted in neuroprotection at doses that were 20 times lower than those for the free molecule [109]. As commented above,

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the blood circulation time of colloids depends on their ability to avoid opsonisation and recognition by the mononuclear-phagocyte system. Longer circulation times will confer better chances for particle accumulation or interaction with the BBB that may consequently improve drug payload to the brain. The interaction with the BBB is achieved by colloids through

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unspecific mechanisms like adsorptive-mediated endocytosis or taking advantage of transporters and receptors expressed at the BBB. These transport routes are in charge of the

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access of highly polar or macromolecular entities that control brain homeostasis [12], but they can be used as gateways for targeting colloids to the brain. Careful design of colloids is paramount for following each different route, as it will be disclosed in the following sections.

4.1.1. Adsorptive mediated interactions

Adsorptive mediated interactions involve mechanisms for overcoming the BBB that consists on the electrostatic interactions between positively charged colloids and the negatively-charged membranes of endothelial cells. The interaction of colloids with the luminal membrane of the cells may trigger the endocytosis of the constructs and their internalization or maintain the colloid at the BBB increasing its residence time [10]. Colloids do not need to be modified in their surface with any targeting moiety to benefit from this mechanism. Consequently,

ACCEPTED MANUSCRIPT adsorptive mediated interactions are considered as non-specific mechanisms that do not differentiate among the different regions of the brain and the endothelial cells of other tissues of the body. Chitosan is one of the polymers used to this aim. More specifically, quaternary ammonium palmitoyl glycol chitosan interacts with the lumen of blood capillaries increasing the retention in the brain tissue while scarcely being observed at the brain parenchyma [58].

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The interesting properties of this naturally occurring polysaccharide were the reason for its incorporation in formulations for the improvement of dopamine delivery to the brain. Trapani et al. observed that there was an increase in dopamine levels at striatum after the intraperitoneal administration of the nanoparticles, considering adsorptive mediated

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processes as the mechanism underlying this effect [110].

4.1.2. Brain targeted delivery delivery

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The lipidic nature of the BBB allows the free diffusion of hydrophobic low Mw to the brain. Nevertheless, those water-soluble molecules, most commonly charged, need the expression of transporters at the BBB for gaining access to the brain. The homeostasis of the brain and its crosstalk with the body is achieved by the action of some molecules that are transported after interacting with their receptors expressed at the luminal side of the endothelial cells of the BBB [10]. This is the mechanism of access for essential molecules needed for the well-

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functioning of the brain, such as electrolytes, glucose or regulatory proteins. Some therapeutics benefit from these transporters, like L-dopa, baclofen, valproic acid or verapamil, but they must closely resemble those substrates in order to be transported by the carrier [111, 112]. Transporters at the BBB have also been used to direct a delivery system and boost drug

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delivery into the brain. The Dutch company To-BBB has developed the G-Technology that consist of (PEG)-ylated liposomes modified at the distal PEG ends with glutathione molecules.

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Glutathione is an endogenous tripeptide that naturally interacts with its transporters at the BBB. The liposomal product, 2B3-201, containing methylprednisolone hemisuccinate has completed phase I clinical trials with positive data about improved pharmacokinetics and safety of the product. In this product, glutathione is present at micromolar concentrations on the liposomes, which are values much lower than the millimolar endogenous levels and the saturable transport capacity. This modification effectively balance the targeting capacity and the average plasma half-life of the formulation, without affecting the physiological glutathione transport systems of the body [113, 114]. Those drugs that access the brain by diffusion will indiscriminately reach the tissue, display their therapeutic effect in its target area but also producing side effects through the brain. On

ACCEPTED MANUSCRIPT the contrary, those drugs that enter the brain by specific interaction with a receptor will reach the tissue in a more selective pattern. Then, one strategy to delivery therapeutics to the brain is to use those endogenous systems to trigger a specific recognition of the targeting moieties located at the surface of the nanoparticles by the BBB receptors, which shuttle them across the barrier. Different receptors have been identified to be used as gateways to the brain,

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among them transferrin, insulin or LDL receptor-related protein (LRP) are the most relevant [115]. One physiological aspect to highlight is that the inherent polarisation of the endothelial cells of the BBB allows the unidirectional transport of the systems from blood to brain after their interaction with the receptors. Despite the fact that many receptors have been identified at the BBB, it is estimated that most of them are yet to be discovered. In line with this,

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Daneman et al. studied the transcriptome of the BBB in mice, which would help to better understand the barrier and the possibilities that it offers for enhancing drug transport [116].

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The expression distribution is different among brain regions and the localization of the receptors appears to be determined by the area over the peptide exerts its biological action [117]. This heterogeneous distribution could be useful for targeting specific areas of the brain, so the selection of the targeting molecule should be in accordance to the targeted area. For instance, areas such as hypothalamus and occipital cortex preferentially transport tumour necrosis factor (TNF) [7]. This fact becomes even more important if we take into account the

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restricted diffusion in brain parenchyma and the rich capillary irrigation of the brain. Receptor mediated strategies entails various ligands of different sources like transferrin, insulin, lipoproteins, diphtheria toxin or peptides obtained by phage display. This section will

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highlight the most relevant advances in colloids for receptor mediated targeting to the brain in neurodegenerative diseases, but further information can be found in excellent reviews of the

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field [12, 118].

Circulating transferrin provides iron to the brain after its interaction with the transferrin receptor located at the BBB. Antibodies that specifically interact with the transferrin receptor are effective targeting tools [119, 120] compared to transferrin- anchored colloids that might suffer from competitive inhibition towards transferrin receptor by the physiological high blood transferrin concentration [12]. Some limitations such as size of antibodies and safety concerns [121] have stimulated the development of peptides with affinity towards the transferrin receptor as alternative targeting molecules. In this perspective, new peptides that maintain specificity towards transferrin receptor without inhibiting the interaction with circulating transferrin have been designed by phage display. For instance the peptide denoted as B6 was obtained using this technique. Its further conjugation to PEG-PLA nanoparticles that

ACCEPTED MANUSCRIPT encapsulated the neuroprotective peptide NAP led to significant accumulation at the brain even 12 hours after the administration of the nanoparticles. Neuroprotective effects were also observed on an AD murine model by biochemical indicators and behavioural tests [81]. Another iron-binding protein involved in the targeting of colloids is lactoferrin. This protein is translocated to the brain by the LRP, an endocytic receptor expressed at the BBB [122].

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Therefore, the functionalization of colloids with lactoferrin promotes the accumulation of the systems in the brain. Great efforts have been put into improving the brain biodistribution of therapeutics that do not naturally cross the BBB. One of these molecules is urocortin, which is related to the corticotropin releasing factor and is able to reverse the nigrostriatal

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degeneration in the long term. The intravenous administration of urocortin loaded PEG-PLGA nanoparticles functionalized with lactoferrin on their surface attenuated the lesion produced by 6-OHDA in rat striatum, a model of PD. Nevertheless, acute toxicity was observed in liver,

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spleen and kidney that diminished at 48 hours post treatment. This fact should be taken as a word of caution for the development of colloids, as toxicity issues may arise not only by the components of the formulation by themselves, but also from the new entity that is formed when a colloid is formulated. Changes in the interaction with the biological media or different biodistribution may give rise to new toxicity concerns. Another approach for treating neurodegenerative diseases is through gene therapy. The human gene encoding glial cell line-

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derived neurotrophic factor (GDNF) was selected as therapeutic gene for its association to polyamidoamine (PAMAM) conjugated to lactoferrin. Multiple intravenous administrations of the system ensured the expression of the gene in the brain. The behavioural tests in the

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rotenone-induced PD model led to improved locomotor activity. In addition, enhanced monoamine transmitter levels in striatum and reduced dopaminergic neuronal loss confirmed the neuroprotective effect of the colloid [123]. There is evidence that pathological processes

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may induce changes in the integrity of the BBB, the blood flow or the transport of insulin [7]. More specifically, it has been observed an overexpression of lactoferrin receptors in the dopaminergic neurons of the substantia nigra as well as in the BBB in PD [124, 125]. Then, lactoferrin could be considered for the design of colloids for PD therapy to improve their access to the brain.

Apolipoproteins are natural ligands that interact with the LRP at the BBB for their internalization. Because of that, they have been studied as targeting molecules for the transport of colloids to the brain. Apolipoproteins can be adsorbed to the particle surface by its recruitment from plasma components when the tensioactive polysorbate 80 coats the surface of the particle. Among the adsorbed proteins ApoB and ApoE are the predominant

ACCEPTED MANUSCRIPT ones [126]. In this sense, the work published by da Rocha et al. showed that resveratrol-loaded poly(lactide) nanoparticles, coated with polysorbate 80, was superior over free resveratrol after intraperitoneal injection to 3-4 months old mice. Their performance was evaluated by monitoring, through behavioral tests and biochemical analysis, the symptoms associated to the previous intranasal administration of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-

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tetrahydropyridine (MPTP), which are analogous to early preclinical effects of PD. The encapsulation of resveratrol helped to mitigate the effects of the neurotoxin in the brains to the values of non neurotoxin treated mice [127]. Poly(butyl) cyanoacrylate nanoparticles were also loaded with the nerve growth factor and coated with polysorbate 80 for promoting apolipoprotein adsorption to the nanoparticles. The intravenous administration of the colloid

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improved the cognitive condition and the locomotor performance of the animals pretreated with scopolamine for inducing amnesia in rats [80]. Recently, Song et al. have reported a novel

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nanocarrier formed by the auto-assembly of recombinant ApoE and synthetic lipids. The intravenous administration of the formulation led to a remarkable decrease of amyloid deposits in addition to the rescue of the memory of SAMP8 mice. The target delivery of the therapeutic molecule encapsulated, α-mangostin, to the fibrils by the biomimetic nanocarrier formed the basis for the successful results obtained [128]. Among the variety of ligands that interact with LRP it is worth to highlight Angiopep, a family of peptides derived from the kunitz

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domain that experience a fast tranlocation to the brain parenchyma [129]. Hence, Angiopep was selected as the ligand to promote the transport of poly-L-lysine dendrigraft by brain capillary endothelial cells, as another targeting approach for non-viral gene therapy treatment of PD. Multiple intravenous administrations of the dendrigraft led to the expression of GDNF

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that improved the locomotor activity of the animals and to apparent recovery of dopaminergic neurons after rotenone lesion [130]. Angiopep and other peptides used to shuttle colloids

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across the BBB offer high selectivity and easier characterization, apart of avoiding the immunogenicity, compared to proteins and antibodies. Much effort has been put into obtaining new peptides for carrier functionalization, an updated review of the field can be attained in the recent work of Oller-Salvia et al. [131]. In that sense, phage display have contributed with peptides such as NAP that helped PEG-PLGA nanoparticles to accumulate in brain [132]. Further research with this system has yielded the first evidence of NAP-mediated delivery leading to neuroprotection by its display on nanoparticles effectuating targeting to the brain by this phage-display peptide [133]. Dual targeting is an interesting approach for improving colloids translocation through the BBB and distribution to their target in the brain parenchyma. This was the rationale for selecting TGN and QSH, two peptides obtained by phage display. TGN is an effective brain targeting peptide, meanwhile QSH shows high affinity

ACCEPTED MANUSCRIPT for Aβ42, whose brain accumulation is related to cognitive impairment in AD. PEG-PLA nanoparticles loaded with the β-sheet breaker peptide H102 were modified on their surface with both targeting peptides. The administration of the construct helped to ameliorate the synaptic degeneration induced by Aβ42 and effectively increased their accumulation in hippocampus in an AD model mice [134]. The same dual strategy was followed in the recent

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work of Zheng et al., where they modified PEGylated Poly(2-(N,N-dimethylamino) ethyl methacrylate) nanoparticles this time with the peptide CGN for brain penetration and QSH for β-amyloid binding. These particles were used as non-viral vectors for the delivery of siRNA against BACE1 as new gene target for AD. This enzyme, BACE1, cleaves the amyloid precursor protein into the peptide β-amyloid, which is the main component of amyloid plaques. The

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results of the studies showed that BACE-1 expression was silenced in hippocampus after the intravenous administration of the complexes in every 5 days dose regimen to an AD model

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mice. This was related to the higher accumulation in that area of the brain most probably due to the dual targeting approach followed [135].

4.2. Increase drug bioavailability

The advances performed in the basis of neurodegenerative diseases have led to new hopes for achieving effective therapeutic options. As mentioned previously, the current therapies are

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based on alleviating symptoms and their success is somehow limited in many cases. Hence, prevention of the diseases arises as an interesting therapeutic approach by tackling the prolonged silent stage of the disease before the onset of clinical symptoms in the patient. In other words, it helps to preserve brain function before needing the reversal of stablished

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pathologies. Oxidative stress is a common process of non-pathological ageing and age-related neurodegenerative diseases such as AD or PD. The process consists on high amount of free

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radicals that outgrow the natural body defences, subsequently leading to mitochondrial dysfunction, energy impairment or protein damage to end up in the death of the cell [136]. There are scientific evidences about the protection role of antioxidants and ω-3 fatty acids by providing cognitive stabilization to AD patients [137]. Indeed there are studies that correlate the concentration of carotenoids in plasma and CSF with the concentration of nicotinamide adenine dinucleotide NAD(H), a coenzyme related to redox reactions, in both fluids. These antioxidant molecules are characterized by their compromised stability after intake due to the physiological conditions that they encountered in the gastrointestinal tract [138, 139]. In addition, this instability might get worse by the oxidation and temperature modifications typical of the manufacturing processes when they are included in functional food. Apart from

ACCEPTED MANUSCRIPT their chemical degradation, neuroprotective molecules need to circumvent the intestinal barrier and the BBB for exerting their protecting action, which are two of the most challenging barriers of the body. Illustrative examples of both barriers are curcumin, that achieves only 0.4 µg/g brain after the oral administration of 100 mg/kg to mice [140] or the flavonoid quercetin that apart from its low absorption it poorly crosses the BBB [141]. Therefore, both of them

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account for the low bioavailability and poor performance of neuroprotective molecules. The encapsulation of neuroprotective molecules in colloids for oral intake is a promising strategy that can improve their scope. On one side, the oral route is the major route for drug delivery due to the acceptance by the patient and subsequent high compliance. On the other

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side, the physicochemical characteristics of colloids promote higher bioavailability of neuroprotectants and subsequently increased levels at the central nervous systems [31, 142]. From a technological perspective, colloids can improve the solubility of the bioactive molecules

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and protect them from the harsh conditions of the gastrointestinal tract. The use of polyethylene oxide derivatives can avoid the interaction of colloids with gastrointestinal enzymes, avoiding the premature degradation of the system.

Due to their huge surface area and surface properties, colloids can promote the interaction with biological barriers such as the intestinal mucosa. According to their behaviour, colloidal

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carriers for the oral route can be classified into i) lipid-based digestible systems when they are degraded by the natural enzymes of the gastrointestinal tract for yielding mixed micelles which can be readily absorbed, ii) mucoadhesive, when they strongly interact with the mucus layer or iii) mucodiffusive if colloids can get across the mucus blanket. Therefore, the proper selection

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of the components and subsequently the physicochemical properties of the colloids obtained are paramount for improving the oral absorption of neuroprotectants. In this way, lecithin-

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stabilized nanoemulsions are excellent delivery systems that improve the interaction with enzymes increasing the bioaccessibility values of the antioxidant α-tocopherol [31]. The selection of the polysaccharide chitosan would yield mucoadhesive carriers that would principally attach to the mucus blanket releasing the cargo molecules close to the enterocytes or promoting the uptake of the carrier itself. The use of PEG derivatives that confer colloids with a hydrophilic shell is the basis of mucodiffusive carriers. This approach requires both adequate Mw and surface density of the coating to avoid the interaction with the enzymes of the gastrointestinal tract. Recently, we have observed that the surface properties of the systems are closely related to the composition of the core of nanoemulsions [68].

ACCEPTED MANUSCRIPT Many antioxidants have benefited from their inclusion in colloids [143, 144], but among them it is worth to highlight β-carotene with a significant increase of its poor bioavailability, lower than 10%) after inclusion in lipid-based systems [138]. Another example is curcumin, a polyphenol that can inhibit Aβ formation reducing amyloid level. This compound, despite its activity, has poor aqueous solubility and oral bioavailability which hamper its therapeutic use.

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Curcumin encapsulation in PEG-PLA di-block polymer micelles provided a formulation that was able to improve working and cue memory after its chronic administration for 3 months to Tg2576 mice [145]. The reduction of the AD symptoms observed correlates to the significant increase of curcumin concentration both in plasma and brain, clearly showing that increasing the oral bioavailability of drugs can be turned into pharmacological improvement of the

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disease. Recently, Kundu et al. have reported lipid based nanoparticles loaded with curcumin and piperine that were able to improve PD pathogenesis after oral administration. The

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nanoparticles were able to increase curcumin bioavailability in plasma and brain which implications were motor coordination restoration and improvement of molecular features of the disease due to the neuroprotective effect of curcumin [146].

4.3. Nose to brain

Intranasal administration has arisen as an interesting strategy to circumvent the BBB and reach

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the brain tissue. Among the benefit of nasal route, it is worth to mention the local administration of the formulations with higher patient acceptance and lower degradation rates compared to oral or parenteral routes. In this respect, it should be taken into account that nasal delivery is mainly applicable to highly potent therapeutics because the brain

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accumulation is typically lower than 1% of the administered dose [79]. Indeed, the nasal cavity comprises a small volume and only 10% of the contact area constitutes the olfactory region,

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which is the gateway for the direct connection to the brain [147]. Drug Mw and hydrophobicity are parameters that may limit the nose-to-brain transport. In these cases, the association to colloidal carriers may strengthen the possibilities of these therapeutic molecules. Colloidal carriers increase the residence time in the nasal cavity and improve the interaction to the membranes, thus promoting the transport of the cargo molecules. They can even prolong the life span of the molecules [148] by protecting them against the enzymatic degradation of the nasal cavity. The most representative materials used for the formulation of nanoparticles for nasal delivery are chitosan, alginate, PLA or PLGA. As stated before, surface properties are decisive in the behaviour of the systems, so different coating agents and targeting moieties were applied to increase the interaction with the tissue [147].

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Figure 3: Illustration of the tentative routes for nose-to-brain drug delivery, showing the olfactory, trigeminal and systemic pathways. The nasal cavity is protected by the mucus that is complex secretion with a renewal of 15-20

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min, an obvious obstacle for the carriers to deliver their payload [149]. Mucoadhesion is the strategy followed to increase the residence time in the nasal cavity and delay mucociliary clearance, and this is achieved by using colloids constituted by polymers such as chitosan. A recent work reported chitosan nanoparticles formulated by the ionic gelation method for the

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encapsulation of bromocriptine, a dopamine receptor agonist and dopaminergic neurons protector currently used for the treatment of PD. The clinical evidence accounts oral administration of bromocriptine for low bioavailability (6%) and side effects related to its

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distribution to non-targeted tissues. In this regard, the nanoparticles formulated (less than 200 nm and highly charged) significantly improved the retention time of the drug in the nasal mucosa 4 h after their administration. In addition, they were able to raise bromocriptine levels in brain as shown by the pharmacokinetics parameters obtained by UHPLC/MS determination of the drug, this increase was observed just after 30 min post administration. These results correlated to the histopathological analysis of the brains and the dopamine concentration in striatum, which was increased by the treatment with bromocriptine loaded chitosan nanoparticles nasally given. Authors suggested that this effect could be due to the transport by the olfactory and trigeminal nerves pathways, although performing studies to understand the fate of the nanoparticles would be desirable to determine the potential of these promising

ACCEPTED MANUSCRIPT results for future treatments for PD [150]. As mentioned in the work of Md et al., the connection between the nasal cavity and the brain is achieved through the olfactory or trigeminal pathways [12]. Hence, it was proposed that therapeutics administered to the nasal epithelium could be transported directly to the brain without distributing in the bloodstream. This direct pathway has several implications like avoiding the restrictive BBB, minimizing the

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systemic exposure of the drug and reducing first-pass metabolism of the molecules. Some molecules are able to reach the brain by the nasal route such as growth factors [151] or insulin [152]. Insulin, among others gastrointestinal hormones, like leptin or vasoactive intestinal peptide, have shown neuroprotective effects by improving cognition [153]. These molecules are transported to the brain by specific transport mechanisms at the BBB but their effect in

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peripheral tissues impede their systemic administration for neurodegenerative therapies, so the nose-to-brain route might improve their scope [11]. The mechanism that governs the

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transport across the olfactory mucosa is still concealed. As mentioned before, the olfactory neural cells originate at the olfactory bulb and terminate at the olfactory neuroepithelium, located at the roof of the nasal cavity. There are different mechanisms of interaction with the olfactory epithelium such as paracellular transport through tight junctions of the olfactory mucosa, passive diffusion and endocytosis or pinocytosis by olfactory neurons. On one side, extracellular mechanisms are quick transport routes to the brain (few minutes) that follow

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most of the therapeutic molecules. On the other side, intracellular mechanisms involved in the intraneural pathway require from hours until days for drug transport to the olfactory bulbs along the axons of neurons [154]. The diameter of the neuronal axons is estimated to be about 200 nm in 2-month-old rabbits, but these values are larger in the case of humans ranging from

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100 to 700 nm [155]. This suggests that it might be limitation in the size of the colloids aimed to this direct nose-to-brain pathway and that this threshold might depend on the animal

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model used. This mechanism would transport drugs to the olfactory bulb and from there to the olfactory cortex, for subsequent caudal pole of the cerebral hemisphere, cerebrum and the cerebellum transport [147, 156]. Besides, the trigeminal pathway, that involves trigeminal nerve endings innervating the olfactory and respiratory mucosa, enters at two different locations of the brain, caudal and rostral areas. This fact and the possibility that other nerves and sensory structures of the nasal cavity may be involved in the direct nose-to-brain transport of therapeutics implies that the potential areas of the brain where those molecules might reach are numerous. In addition, there is evidence that nasal lymphatics may drain into the cerebral ventricles and subarachnoid space, which contain the CSF, leading to drug accumulation in distant regions of the brain.

ACCEPTED MANUSCRIPT A different mechanism of transport to the brain consists on the improvement of drug absorption by the highly vascularized nasal mucosa to reach the systemic circulation, mainly focused on the respiratory mucosa due to its higher blood vessel density compared to the olfactory one. This systemic pathway implies drug permeation across the nasal mucosa, whose fenestrations allow the crossing of even large molecules, and entering the carotid arteria that

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directly feed the brain and the spinal cord. Altogether, these examples illustrate the complications in the understanding of brain distribution and the pathways involved after nasal administration which are simplified in Figure 3. In that sense, it is highly recommendable to determine drug distribution in different brain areas instead of the whole brain, as it might help

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to understand the pathway followed [154].

The implications of the complexity and multiple pathways involved in nose-to-brain delivery are that a rational design of the carriers is quite challenging. In this sense, some authors have

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tried to elucidate which size and surface characteristics of colloids affect to the interaction with mucosa. The work of Mistry et al. in excised porcine olfactory nasal tissue, selected for its similarities with the human tissue, used fluorescent carboxylated polystyrene nanoparticles as basic model nanoparticles. The advantage of model nanoparticles are their controlled properties and narrow size distribution that the authors used for understanding the effect that particle size have on the transport across the olfactory epithelium selecting nanoparticles of

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20, 100 and 200 nm. In addition, they modified the negatively charged hydrophobic surface of the nanoparticles with chitosan for obtaining positively charged hydrophilic nanoparticles, and with polysorbate-80 for yielding almost neutral hydrophilic nanoparticles. Authors stated that

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none of the prototypes was able to cross the epithelium in the 4 h that lasted the experiment and suggested that this could be due to longer periods required for the transport across the olfactory neural pathway. Despite this absence of transport across the epithelium, it was

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observed that the polysorbate-coated nanoparticles diffused deeper compared to the chitosan-modified nanoparticles, which remained highly attached to the mucus layer probably due to their mucoadhesiveness [18]. These same authors have reported that the nasal administration of polystyrene coated polysorbate 80 nanoparticles (100 and 200 nm) to mice led to the transport of the 100 nm nanoparticles across the olfactory membrane without reaching the olfactory bulb or other brain areas. The divergence of the in vitro/in vivo results could rely on the different time frame of both studies, as the in vivo data were collected 4 days after the first administration [19]. The surface modification of colloids with targeting molecules is a mechanism used to increase brain accumulation after nasal administration of the carriers by preferentially promoting their

ACCEPTED MANUSCRIPT interaction with the olfactory epithelium with respect to the respiratory one. In this sense, lectins have been widely used as they bind to the glycosylated nasal mucosa by specific recognition of the epithelium sugar moieties [157]. As one drawback of lectins is their associated immunogenicity, researchers have worked to develop active targeting moieties like the peptide odorranalectin that binds to L-fucose, which displays less immunogenicity

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compared to wheat germ agglutinin [158]. Wen et al. used this peptide for the surface modification of urocortin-loaded-PLGA nanoparticles yielding a system of about 100 nm. The results showed that odorranalectin modification significantly improved the therapeutic effects of urocortin compared to the unmodified ones. The therapeutic efficacy of the nanoparticles was determined by the behaviour test of apomorphine-induced rotations in a hemiparkisonian

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rat model showing that the surface modified nanoparticles were able to reduce the number of rotations; this fact was supported by the recovery on dopaminergic cells observed by

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immunohistochemistry and the increased levels of monoamine neurotrasmitters in striatum [159].

Another strategy is to modify the surface of colloids with cell penetrating peptides, HIV-1 Tat peptide, oligoarginines or polyarginine [147] for increasing the transport of the system across epithelia. Kanazawa et al. have obtained complexes of less than 100 nm, with the amphiphilic block copolymer constituted by PEG and poly(ε-caprolactone) modified with the Tat peptide as

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a gene delivery platform. These authors have determined using fluorescence techniques that the nasal administration of the complexes led to their higher brain accumulation in the olfactory bulb and trigeminal nerves, suggesting that both pathways could be involved in the

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delivery of the complexes. These encouraging results could be the basis for non-invasive gene delivery treatments for central nervous disorders [160].

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Despite the advances achieved, there is still much research to be done for clarifying the pathways that colloids and their cargo drugs follow from their nasal administration towards the brain. The mechanistic routes underlying the pharmacodynamic effects that are frequently observed is paramount for understanding and properly designing future colloids for this application, because it is likely that a combination of pathways may be involved. In this regard, it is worth to remind that the transport of the drug and the carrier do not necessarily need to be by the same route. Determining whether active molecules are released in the olfactory mucosa or if they are truly transported in the colloids where they are included is of great importance, as the approach to be followed should adjust to these evidences. Further factors, inherent to the specific methodology used, such as head position or administration technique, have a considerable impact of the outcome and should not be overlooked [154].

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5. Concluding remarks and future perspectives The main challenge in brain delivery is to overcome the different barriers, mostly the BBB, that protect the central nervous system and selectively controls the access of the molecules to the tissue. It is important to bear in mind that the BBB is not just a physical barrier, it communicates brain with peripheral tissues and adapts to the needs of the brain tissue that it

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protects. This protection may be impaired in neurodegenerative diseases. AD patients have decreased cerebrovascular blood flow, impared CSF drainage, decreased Pg-p function or different pattern on the transport of compounds. Closely mimicking the disease characteristics is of importance for the model used to evaluate the formulations. This is the case of the

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chronic exposure to rotenone that adequately reproduce the features of PD in rats [161]. In addition, in vitro screening methods validated with in vivo studies are powerful tools for

replacement approach [6].

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understanding the mechanisms of interaction from a more economical and animal

It is obvious that colloid science is notably promoting the delivery of therapeutics for neurodegenerative diseases. Although it is necessary to understand better the correlation between the physicochemical properties of colloids and their interaction with the biological media. In this sense, predicting colloids in vivo behaviour and correlating it to the

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physicochemical properties is a complex process that could be supported by the development of new tools for properly evaluating the formulations and understanding the mechanisms for overcoming those highly specialized barriers. More concretely, scientists need to develop methods that determine the uptake of the particle itself, not only observe the behavioural

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effects by pharmacodynamics studies of the cargo molecule delivered. This is crucial for understanding the interaction and uptake mechanism of colloids, and for that purpose there

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are different techniques available like scintigraphy, near-infrared in vivo imaging, ex vivo optical imaging, confocal microscopy or chromatographic quantification [6]. The application of techniques such as coherent anti-Stokes Raman spectroscopy (CARS) to drug delivery have shown their potential as label-free technique to determine the interaction of colloids with the BBB [59].

It is important to highlight that there is not a unique type of requirement for brain delivery, indeed, the design and properties of the formulation should be adjusted to the drug and therapy needed. An illustrative example are neurodegenerative diseases that do not generally affect the brain in a global manner, like PD. In this context, additional formulation strategies will be required to improve the delivery efficiency and distribution of therapeutics to specific

ACCEPTED MANUSCRIPT brain areas, thus avoiding the accumulation in non-target regions of the brain [12]. The localization of drugs into the brain by dual targeting strategies using delivery systems anchored to BBB-targeting ligands and targeted cell-binding ligands could improve brain distribution [162]. Another aspect that deserves special attention is whether colloids diffuse in brain parenchyma and the characteristics of size and surface composition that determine their

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diffusion. Some works have stated that colloids may follow neural pathways, such as the olfactory nerve route. Of great concern is the fact that getting in a neural pathway does not necessarily mean that colloids might follow that route by moving from one neuron to other, they could diffuse in the presynaptic space without continuing forward. In addition, the multiple connection that interneurons afford can disperse colloids to different areas of the

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brain. So achieving deeper knowledge on BBB functionality and unveiling the multiple factors involved in the nose-to-brain delivery would help to clarify the interaction and transport to the

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brain.

Nowadays our knowledge in the mechanisms and regulatory pathways of the central nervous system is considerably broader, but the research in molecular neurobiology still needs to discover the molecular mechanisms that rule the brain, like the conditions that promote receptor expression, so as the active molecule will certainly interact with the receptor for activating or inhibiting a specific route. Other facts that need to be determined are in one side

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the kinetics and turnover of the receptors. On the other side, the desensitisation and other negative feedback mechanisms will undoubtedly affect the therapeutic efficacy of the delivery systems despite their ability to overcome the BBB and achieve significant therapeutic amounts

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of active molecules in the brain.

Transport of therapeutics to the brain is one of the greatest challenges for drug delivery.

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Although significant advances have been achieved, the progress and complexity of neurodegenerative diseases require that researchers blur disciplines boundaries and work together towards therapies that are more efficient.

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ACCEPTED MANUSCRIPT Gao, H., Z. Pang, and X. Jiang, Targeted delivery of nano-therapeutics for major disorders of the central nervous system. Pharmaceutical Research, 2013. 30(10): p. 2485-2498.

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Figure 1: Schematic diagram showing the endothelial cells strongly packed by the tight junctions, the astrocytes and pericytes among other components of the neurovascular unit.