siRNA as a tool to improve the treatment of brain diseases: Mechanism, targets and delivery

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Contents lists available at ScienceDirect

Ageing Research Reviews journal homepage: www.elsevier.com/locate/arr

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siRNA as a tool to improve the treatment of brain diseases: Mechanism, targets and delivery

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Maria João Gomes a , Susana Martins b , Bruno Sarmento a,c,∗ a b c

INEB – Instituto de Engenharia Biomédica, Biocarrier Group, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark CESPU, Instituto de Investigac¸ão e Formac¸ão Avanc¸ada em Ciências e Tecnologias da Saúde, Rua Central de Gandra, 1317, 4585-116 Gandra PRD, Portugal

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Article history: Received 28 November 2014 Received in revised form 10 March 2015 Accepted 16 March 2015 Available online xxx

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Keywords: Brain diseases Blood–brain barrier Efflux transporters Nanoparticles siRNA

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Contents

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As the population ages, brain pathologies such as neurodegenerative diseases and brain cancer increase their incidence, being the need to find successful treatments of upmost importance. Drug delivery to the central nervous system (CNS) is required in order to reach diseases causes and treat them. However, biological barriers, mainly blood–brain barrier (BBB), are the key obstacles that prevent the effectiveness of possible treatments due to their ability to strongly limit the perfusion of compounds into the brain. Over the past decades, new approaches towards overcoming BBB and its efflux transporters had been proposed. One of these approaches here reviewed is through small interfering RNA (siRNA), which is capable to specifically target one gene and silence it in a post-transcriptional way. There are different possible functional proteins at the BBB, as the ones responsible for transport or just for its tightness, which could be a siRNA target. As important as the effective silence is the way to delivery siRNA to its anatomical site of action. This is where nanotechnology-based systems may help, by protecting siRNA circulation and providing cell/tissue-targeting and intracellular siRNA delivery. After an initial overview on incidence of brain diseases and basic features of the CNS, BBB and its efflux pumps, this review focuses on recent strategies to reach brain based on siRNA, and how to specifically target these approaches in order to treat brain diseases. © 2015 Published by Elsevier B.V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Obstacles to brain diseases treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BBB and drug efflux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. BBB as a protective membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Drug efflux transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. How to overcome BBB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Common strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. siRNA – mechanism and targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. siRNA delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. siRNA/drug co-delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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∗ Corresponding author at: INEB – Instituto de Engenharia Biomédica, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. Tel.: +351 226 074 900; fax: +351 226 094 567. E-mail address: [email protected] (B. Sarmento). http://dx.doi.org/10.1016/j.arr.2015.03.001 1568-1637/© 2015 Published by Elsevier B.V.

Please cite this article in press as: Gomes, M.J., et al., siRNA as a tool to improve the treatment of brain diseases: Mechanism, targets and delivery. Ageing Res. Rev. (2015), http://dx.doi.org/10.1016/j.arr.2015.03.001

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1. Introduction The World Health Organization has indicated that central nervous system (CNS) disorders are the major medical challenge of the 21st Century (ResearchAndMarkets, 2007). Disorders of the CNS are numerous, diverse, frequently severe, and affect a large portion of the world population. These diseases can debilitate conditions that significantly affect the morbidity and mortality of modern society. Neurodegenerative diseases including Alzheimer’s diseases (AD), Parkinson’s diseases (PD) and amyotrophic lateral sclerosis – which symptoms are related to loss of movement, memory, and dementia due to the gradual loss of neurons – are constantly and rapidly increasing as population ages. As well, brain tumours constitute a severe and unsolved clinical condition and are a common cause of cancer-related death. Longer life expectancy should be followed by better quality of life, however, current therapies to CNS disorders (which are mainly incident on old-age population) do not positively correspond to their expectations (Bhaskar et al., 2010). Neurodegenerative diseases, deeply associated with ageing, are usually linked to a loss of brain and spinal cord cells. As examples, in AD and PD the neuronal damage occur due to abnormal protein processing and accumulation, which results in gradual cognitive and motor deterioration (Gilmore et al., 2008). According to Brain Tumour Research website statistics (Farm, 2013), brain tumours kill more children and adults under the age of 40 than any other cancer, and only 18.8% of those diagnosed with brain tumour survive beyond five years, compared to 50% average survival prognostic for all cancers. Moreover, the incidence of brain metastases has increased over the last decade mainly due to improved treatment of primary peripheral cancers resulting in increased patient survival, as well as due to the development of newer tools to image and diagnostic tumours of the CNS (Agarwal et al., 2011). Among the different ways to treat cancer, such as surgery and radiotherapy (which uses high-energy particles or waves to destroy or damage cancer cells, that arise the possibility to damage also normal cells, reason why this treatment must be carefully planned to minimize side effects), tumour therapy is usually based on the interplay between chemotherapeutic and antiangiogenic agents (Murthy, 2007). In general, treatment of many ageing disorders and tumours require drugs acting on the CNS, highlighting the need and importance to reach CNS on a therapeutic concentration. Simultaneously, the field of nanomedicine is rapidly expanding and promises revolutionary advances to the diagnosis and treatment of devastating human diseases (Gilmore et al., 2008).

2. Obstacles to brain diseases treatment Drug delivery to the CNS represents a challenge in developing effective treatments for neurodegenerative diseases and brain tumours due to the unique and complicated environment imposed by the CNS itself. There are protective barriers which restrict the passage of foreign substances into the brain, namely the blood–brain barrier (BBB), the blood cerebrospinal fluid barrier (BCSF), and other specialized CNS barriers as the arachnoid barrier (Abbott et al., 2010; Bhaskar et al., 2010). Therefore, an important part of this CNS challenge is overcoming the natural tendency of the BBB to block drug transport. This barrier, a tightly packed layer of endothelial cells surrounding the brain (Bhaskar et al., 2010), is designed to protect and prevent high-molecular weight molecules in blood from entering the brain by filter harmful compounds from the brain back to the bloodstream. As the BBB cannot recognize many therapeutic compounds, high doses must be administered to have a drug therapeutic concentration at the brain, with increased risks of adverse side effects (Murthy, 2007).

Fig. 1. BBB cellular structure (Wilhelm et al., 2011).

Due to the difficulty of physically active molecules overcome BBB and reach CNS, it becomes crucial to understand the structural composition as well as how the factors that regulate permeability of the substances across the BBB act. BBB is constituted by the brain endothelial cells which form the cerebral microvascular endothelium. The cerebral microvascular endothelium, together with astrocytes, pericytes, neurons, and the extracellular matrix, constitute a “neurovascular unit” that is essential for the health and function of the CNS (see Fig. 1). Pericytes play an important role on the integration of endothelial cells and astrocytes functions at the neurovascular unit (Armulik et al., 2010; Fisher, 2009); astrocytes are crucial on the induction of BBB functions; and neurons on BBB cerebral flow and vessel dynamics (Cardoso et al., 2010; Choi and Kim, 2008; Weiss et al., 2009). Several molecular and receptor structures are present on the surface of the endothelial cells, able to mediate the transport of solutes and other substances including drugs in and out of the brain. BBB is also responsible for leucocyte migration and maintenance of brain microenvironment homeostasis, which is crucial for neuronal activity and proper functioning of CNS. The transport of solutes and other substances across BBB is also dependent on tight junctions (TJs) between adjacent endothelial cells, adherent junctions (AJs) and metabolic barriers (enzymes, diverse transport systems). Besides TJs, as special characteristics of BBB that limit drug uptake, there is also a lack of fenestrations and a low endogenous pinocytotic activity. Next to the BBB, BCSF is the second important feature of the CNS, formed by the epithelial cells of the choroid plexus. BCSF mainly regulates the exchange of molecules between the blood and CSF, controlling the penetration within the interstitial fluid of the brain parenchyma (Bhaskar et al., 2010; Gilmore et al., 2008; Mahringer et al., 2011). Moreover, another interface, the avascular arachnoid epithelium, has a relatively small surface that is the main reason why it is not a significant surface for exchange between blood and CNS (Abbott et al., 2010). Some other CNS barriers, like blood tumour barrier and blood retina barrier, may also play a role in drug transport (Bhaskar et al., 2010). Concerning these limitations, some current strategies used for drug delivery to the brain include invasive delivery, temporary disruption of the BBB, as well as the use of specific drug delivery systems. While direct injection can be an effective invasive modality for local delivery in some cases (e.g., in some tumours), it is not efficient for brain metastasis or neurodegenerative diseases, which require therapeutic agents to be widely spread in the brain. Reversible opening of the BBB, by an osmotic or chemical method as well as ultrasound techniques, allows therapeutic agents to enter the brain. However, this approach can also result in significant damage to the brain (Gilmore et al., 2008), with partial irreversibility of

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the disruption of BBB, compromising its protective role to the CNS. These restrictions are the main reasons for lack of successful strategies that can allow localized and controlled delivery of drugs across the BBB to the desired site of injury (Bhaskar et al., 2010). Thus, the existence of few safe and effective therapeutic options for many devastating and pervasive disorders encourages the raising of drug delivery systems of colloidal dimensions, as described in following sections. 3. BBB and drug efflux 3.1. BBB as a protective membrane As already mentioned, BBB is an endothelial dynamic interface that shields the CNS from exposure to circulating toxins and potentially harmful chemicals (Hawkins and Davis, 2005). This protective barrier controls the influx and efflux of a wide variety of substances, excluding therapeutic drugs from entering the brain, and thus, at the same time, maintains a favourable environment for the CNS and becomes an obstacle for drugs intended to treat CNS diseases (Agarwal et al., 2011; Bhaskar et al., 2010). The ability of a substance to penetrate the BBB or be transported across BBB is dependent on its physiochemical properties, as small size, liposolubility and customizable surface are examples of favourable properties. The cerebral pharmacological efficacy of any drug depends on its CNS uptake which, in turn, depends on a combination of factors, including CNS physical barriers and the affinity of the substrate for specific transport systems located at both sides of these interfaces. In this context, efflux transporters present at the BBB are one of the main limitations of brain penetration as well as the intra- and extracellular distribution of a variety of endogenous and exogenous compounds. Active efflux transport or carrier mediated efflux involve extrusion of drugs from the brain in the presence of efflux transporters. This type of transport causes the active efflux of drugs from brain back to blood (Bhaskar et al., 2010). Therefore, one strategy to improve the efficacy of CNS drugs would be the modulation of transport proteins mainly responsible for the drug efflux, being responsible for their passage across the BBB (Mahringer et al., 2011). 3.2. Drug efflux transporters A key element of the BBB function is the expression of ATPbinding cassette (ABC) drug efflux transporters in the luminal membrane of brain microvessel endothelial cells, which besides restrict the entry of many compounds into the brain, also plays a major role for the maintenance of brain homeostasis and detoxification. Among these ABC transporters, are the P-glycoprotein (Mdr1 gene product, P-gp; ABCB1), breast cancer resistance protein (BCRP, encoded by the ABCG2 gene), and members of the multidrug resistance related proteins (Mrp1, 2, 4 and 5, encoded by ABCC1, ABCC2, ABCC4 and ABCC5 genes, respectively). These proteins collectively hamper brain uptake of a huge variety of lipophilic xenobiotics, potentially toxic metabolites and also drugs. Several other transport proteins and receptors are expressed at the BBB, such as the non-ABC transporters Oat3 (organic anion transporter) and Oatp2 (organic anion transporting polypeptide that handles steroid and drug conjugates, certain opioid peptides, and the cardiac glycoside, digoxin) that, when coupled to the appropriate ion gradients, are capable of driving organic anions into the endothelial cells (Agarwal et al., 2011; Bauer et al., 2005; Hermann and Patak, 2011; Mahringer et al., 2011). A deeply studied export pump is P-gp, a 170 kDa phosphorylated glycoprotein expressed in multiple cell types within the brain parenchyma, including astrocytes and microglia. Its

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highest activities seem to be in the luminal plasma membrane of the capillary endothelium (Mahringer et al., 2011). Mdr1 is often overexpressed in tumour cells which contributes to the multi-drug resistant phenotype commonly seen in cancer (Fisher et al., 2007). The fact that Mdr1 mRNA and P-gp are highly expressed in multidrug resistant cells, while P-gp has a long turnover time, make this target very attractive to inhibition with antisense or siRNA oligonucleotides (Fisher et al., 2007). Moreover, in vivo dosing studies in Mdr1-knockout mice show greatly increased plasma-to-brain ratios (5–50-fold) for a large number of drugs that are P-gp substrates (Schinkel et al., 1996), which indicates the relevance of P-gp efflux system (Bauer et al., 2005). Another example that highlights P-gp efflux mechanism, by decreasing the effect of drugs which are its substrates, was studied by using morphine on rats-induced peripheral inflammatory pain (PIP) (Sanchez-Covarrubias et al., 2014). PIP results in increased expression and activity of P-gp and, consequently, there is a significant reduction in CNS uptake of morphine (since morphine is a P-gp substrate) and reduced morphine analgesic efficacy. Considering this, they induced PIP and examined the administration of diclofenac, a non-steroidal anti-inflammatory drug (NSAID) that is commonly administered in conjunction with opioids (i.e., morphine) during pain therapy and that have been reported to modulate P-gp (Akanuma et al., 2010), on BBB transport of morphine via P-gp, as well as on its analgesic and anti-inflammatory efficacy. Authors observed a significant decrease in brain morphine uptake in PIP animals. Moreover, in situ brain perfusion studies showed that not only PIP induction but also diclofenac treatment alone increased P-gp efflux activity which results in decreased morphine brain uptake (Sanchez-Covarrubias et al., 2014). Robillard and colleagues investigated in vivo the tissue distribution of the HIV protease inhibitor atazanavir in wild-type (WT) mice and Pgp/breast cancer resistance protein (Bcrp)-knockout mice. In this study, some WT mice were pre-treated with a P-gp/BCRP inhibitor, elacridar. In P-gp and Bcrp KO mice, authors demonstrated a significant increase in atazanavir brain concentration of 5.4-fold compared to those in WT mice (P < 0.05). Moreover, elacridartreated WT mice showed a significant increase in atazanavir brain concentration of 12.3-fold compared to those in vehicle-treated WT mice. These in vivo results show how P-gp (as well as BCRP) is involved in limiting the ability of atazanavir to permeate mice brain (Robillard et al., 2014). Also Liu and co-workers studied P-gp consequences as an efflux transporter. A novel anti-Parkinson’s disease (PD) candidate drug, which is a synthetic squamosamide derivative (FLZ), has shown poor BBB penetration, but the main reason for that (if P-gp and/or BCRP) was still unclear. Therefore, in vitro permeability experiments of FLZ were carried out on BBB models (one mimicking physiological, and other PD pathological-related BBB properties). In PD models, both expressions of P-gp and BCRP were significantly greater, which is associated with the lower BBB permeability of FLZ in pathological model compared with physiological model. Transport studies were also performed, where only P-gp blocker effectively inhibited the efflux of FLZ. Thus, from this study it is possible to conclude that P-gp is the main responsible for poor brain penetration of FLZ and low BBB permeability (Liu et al., 2014). BCRP (72 kDa, Paturi et al., 2010) mainly transports sulphated conjugates of drugs and sterols (Hori et al., 2005), which largely corresponds to cationic and uncharged substrates (Bauer et al., 2005). Its function is regulated by steroid hormones, particularly estrogens. In brain capillaries from male and female rats and mice BCRP-mediated transport was rapidly and reversibly reduced after short term exposure to nanomolar concentrations of 17␤-estradiol (Mahringer et al., 2011). Multidrug resistance-associated protein 4 (Mrp4, ABCC4; 150 kDa (Sauna et al., 2004)) shares many features with P-gp

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(ABCB1) and BCRP (ABCG2), including broad substrate affinity and expression at the BBB (Lin et al., 2013). Regarding the difficulty to evaluate the role of ABCC4 at the BBB (since most drugs are also substrates of ABCB1 and/or ABCG2), Lin and co-workers created a mouse strain in which all these alleles are inactivated to assess their impact on brain delivery of camptothecin analogues (as gimatecan and itinotecan active metabolite SN-38), an important class of antineoplastic agents with antitumor activity and substrates of these transporters. They were able to observe that additional deficiency of Abcc4 in Abcb1; Abcg2− /− mice significantly increased the brain concentration of all camptothecin analogues by 1.2-fold (gimatecan) and 5.8-fold (SN-38). The presence of Abcb1 or Abcc4 alone was sufficient to reduce the brain concentration of SN-38 to the level in WT mice. From this study, it is possible to conclude that ABCC4 limits the brain penetration of camptothecin analogues and teams up with ABCB1 and ABCG2 to form a robust cooperative drug efflux system. This intensive action limits the usefulness of selective ABC transport inhibitors to enhance drug entry for treatment of intracranial diseases (Lin et al., 2013). ABCA1, a 254 kDa protein, is another protein responsible for the efflux transport of drugs and endogenous compounds at the BBB and it is involved in the complex process of brain cholesterol homeostasis (Farke et al., 2006). ABCA1-inhibition decreases cholesterol efflux to apoA-I, apoE, and HDL particles. Since ABCA1 is also a key player in amyloid accumulation and deposition, it represents a promising therapeutic target in AD (Saint-Pol et al., 2014). Regarding BBB composition features, namely these drug efflux transporters, only a small class of drugs or small molecules (molecular mass of <400–500 Da), electrically neutral and with high lipid solubility actually goes across the BBB (Bhaskar et al., 2010). Thus, conventional pharmacological drugs or chemotherapeutic agents are unable to pass through the barrier. Consequently, the future of CNS diseases treatment depends on drug delivery systems based on novel strategies able to modulate these key efflux transporters. Here we discuss different strategies, highlighting the possibility to temporarily silence efflux transporters, in a controlled way.

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To overcome the BBB, without compromising its integrity, four main strategies have been applied, namely direct transporter inhibition, targeting signalling pathways that control transporter regulation (function and/or expression), targeting inflammatory and stress pathways in order to modify the transporter synthesis, and direct down-regulation of efflux transporters expression and/or transport activity (Agarwal et al., 2011). Of all the xenobiotic efflux pumps highly expressed in brain capillary endothelial cells, P-gp handles the largest fraction of commonly prescribed drugs and thus is an obvious target for manipulation. Some mechanisms by which P-gp activity in the BBB can be modulated are, regarding the strategies mentioned: (i) direct inhibition by specific competitors, (ii) functional modulation, and (iii) transcriptional modulation. All have the potential to specifically reduce P-gp function and thus selectively increase brain permeability of its substrates. The first approach is related to the modification of pump function by inhibitors and intracellular signals. Several relatively specific P-gp inhibitors have been developed and tested (Kemper et al., 2003, 2004). This option depends on several issues, as the inhibitor affinity and reversibility, which could become serious problems (Bauer et al., 2005). The first P-gp inhibitor was found

by chance in 1981 by Tsuruo et al., who showed that verapamil, a calcium channel blocker, inhibits P-gp-mediated drug efflux in resistant tumour cells. Then, a variety of inhibitors were developed that differ in potency, selectivity, and side effects (Avendano and Menendez, 2002; Pleban and Ecker, 2005). However, until now, only a few compounds have been tested for their potential to enhance drug delivery to the brain (Agarwal et al., 2011). Due to their similarities, P-gp and BCRP have an overlap in substrate specificity. Concerning this, there are some evidences that demonstrate, for some compounds, inhibition of either BCRP or P-gp alone is not sufficient to increase delivery into the brain (Agarwal et al., 2011). Thus, it became also important to study the combined impact of these two efflux transporters on the delivery of drugs across the BBB. The first proof-of-principle that P-gp inhibition can be used to treat brain cancer came from a study in nude mice with intracerebrally implanted human U-118 MG glioblastoma. In this study, Fellner et al. identified P-gp as the major factor in limiting the anticancer therapeutic paclitaxel from crossing the BBB and permeating into the CNS. Consistent with this, treating glioblastoma-bearing mice with paclitaxel had no effect on tumour size but pre-treating mice with the P-gp inhibitor PSC833 (valspodar) increased paclitaxel brain levels and reduced tumour size by 90% (Fellner et al., 2002). Some studies already showed that absence of both P-gp and BCRP resulted in an effect that was significantly larger than the combined effects from the single transporter knockout mice. These findings suggest that inhibition of either P-gp or BCRP can be compensated by the respective other transporter, and that both transporters “cooperate” with each other in preventing chemotherapeutic drugs from entering the brain. Furthermore, for the majority of drugs, P-gp is the dominant transporter, rather than BCRP. Given the synergic effect of P-gp and BCRP at the BBB, developing compounds that are potent inhibitors of both transporters may prove beneficial. As so, elacridar (GF120918) is a dual P-gp/BCRP inhibitor that has undergone extensive preclinical and clinical evaluation (Agarwal et al., 2011; Hyafil et al., 1993) – a phase I study was performed where the lowest effective dose of elacridar to obtain maximum oral bioavailability of topotecan was determined, as well as the optimal schedule of coadministration of both drugs (Kuppens et al., 2007). The second approach, ideally, is the transiently and specific decrease of efflux to the blood through rapid regulation of transporter function. Two signalling pathways have been identified that could potentially be used to down-regulate P-gp transport activity at the BBB – one involves signalling of inflammatory mediators through PKC beta(I), the other one involves vascular endothelial growth factor (VEGF) signalling though flk-1 and Src (Agarwal et al., 2011). Several signalling pathways, which contribute for a rapid and reversible functional regulation, are also related to P-gp function. One of them is triggered by VEGF and results in a reversible loss of P-gp function without a decrease in export protein expression. Depression of P-gp activity by VEGF is associated with phosphorylation of caveolin-1, a signal known to trigger caveolin-dependent endocytosis. Another example is the endothelin-1 (ET-1), acting through an ET(B) receptor, NO synthase, and protein kinase C, which rapidly and reversibly reduces P-gp transport function (Mahringer et al., 2011). As well, various signalling pathways have been shown to down-regulate BCRP. As an example, it was demonstrated that estrogens play a role in BCRP regulation– estrone and 17␤-estradiol (E2) reverse BCRP-mediated drug resistance (Imai et al., 2002), and E2 triggers post-transcriptional down-regulation of BCRP in human breast cancer cell lines. Also the PTEN/PI3K/Akt signalling pathway regulates the BCRP activity (Agarwal et al., 2011).

Please cite this article in press as: Gomes, M.J., et al., siRNA as a tool to improve the treatment of brain diseases: Mechanism, targets and delivery. Ageing Res. Rev. (2015), http://dx.doi.org/10.1016/j.arr.2015.03.001

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The third regards the alteration of the transporter synthesis due to enhanced or inhibited transcription and translation. This topic is related with the possibility of use inflammatory and stress pathways to lower P-gp expression at the BBB. Several promoter elements have been found so far, as pregnane X receptor (PXR) ligands, but it is not clear to what extent P-gp expression can be down-regulated by removing PXR ligands from the diet. In contrast to inflammation and inflammatory mediators (as interleukin-6 (IL6), IL-1b, and tumour necrosis factor-␣ (TNF-␣)) which reduces transporter expression (Hartmann et al., 2002; McRae et al., 2003), cellular stress (e.g., exposure to heavy metals, reactive oxygen species, and some chemotherapeutics as well as heat stress) upregulates expression of P-gp (Bauer et al., 2005). The forth strategy related to directly down-regulate efflux transporters expression and/or transport activity constitute a new and promising approach which takes advantage of siRNA and is here further discussed. To overcome BBB, several administration strategies may be used, which can be divided into two categories: local delivery (such as intraparenchymal and intraventricular) (Alam et al., 2010) and systemic delivery (such as intravenous and intranasal). The local route enables drug delivery directly into the parenchymal space of the brain (intraparenchymal), however a large dose is required since access to the parenchyma is minimal (De Boer and Gaillard, 2007). For the intraventricular route, therapeutic agents are administrated directly into cerebral ventricle (Alam et al., 2010), thus will only be distributed to the ependymal surface of the ipsilateral which means that just a limited amount will be available (Pardridge, 2003). Moreover, BBB disruption could also be used by directly delivering substances to the brain (Pardridge, 2005), using certain chemicals or applying energy (ultrasonic waves or electromagnetic radiations), which exposes brain to infection and damage (Alam et al., 2010). On the other hand, intravenous delivery is the most commonly used route to administrate large doses of drugs (Huynh et al., 2006) since, although drug availability is affected by its exposure to peripheral organs and rapid clearance, it avoids first-pass metabolism and has the great potential to deliver drugs to almost all neurons in the brain (almost all neurons have their own brain capillary for oxygen and nutrient supply) (Alam et al., 2010; Pardridge, 2003). The intranasal route is based on the principle that drugs exit nose sub-mucosa space into the brain compartment. Advantages of this route came from nasal epithelium high permeability, avoidance of first-pass metabolism and selfadministration. However, this administration damages the nasal mucosa and decreases the quantity of drug available (Alam et al., 2010; Nagpal et al., 2013).

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There is a potential therapeutic exploitation of gene regulation based on RNA interference. Its endogenous regulatory mechanisms rely on the sequence specific interaction between small interfering RNA (siRNA) and mRNA. Therefore, these mechanisms can be activated via the delivery of siRNA to the cell interior, a short double stranded RNA (dsRNA) that guides sequence specific mRNA degradation and highly result in specific inhibition of gene expression through degradation of target mRNA or inhibit translation (Dorsett and Tuschl, 2004). RNA interference was first discovered in Caenorhabditis elegans when it was observed that injected dsRNA was far more potent at limiting gene expression than either sense or antisense strands alone (Fire et al., 1998). The RNA interference mechanism of action starts by cleavage of the dsRNA into short ∼21 nucleotide RNA duplexes by an endonuclease called Dicer (Zamore et al., 2000). These short RNA sequences, the siRNAs, are rapidly taken up into an enzyme complex, RNA induced silencing complex (RISC), that

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Fig. 2. P-gp-labelled area of the hilus and the parietal cortex of control mice and mice treated with siRNA targeting Mdr1 for four consecutive days. Significant differences between groups are indicated by an asterisk (P < 0.05, Student’s t-test, one sided) (Fuest et al., 2009).

degrades the mRNA through guidance to a specific target mRNA resulting in specific gene silencing (Fuest et al., 2009; Hammond et al., 2000). At RISC (Nascimento et al., 2014), one siRNA strand is taken into the effector complex, the catalytic subunit Argonaute2, and then serves as a template, guiding the hydrolysis of complementary or near complementary mRNA sequences (Fountaine et al., 2005; Martinez and Tuschl, 2004). Moreover, since it is very specific, siRNA technology is considered to be a useful approach to study the role of various proteins in neuronal physiology. Even better than the old methods of generating knock-out mice who lack the target protein (Morrison et al., 1996) or generating conditional knock down or knock-in mice that only remove or express the protein following a specific treatment (Christophorou et al., 2005; Perez-Martinez et al., 2012). However, as all existing strategies, the use of siRNA could also have some drawbacks important to mention. Among them is poor cellular uptake and instability under physiological conditions, therefore successful siRNA therapy needs the development of drug delivery systems. Besides the saturation of the endogenous RNA interference pathway, silencing of unwanted genes known as off-target effects and the activation of innate immunity may also arise from siRNA approach (Sioud, 2015; Xu and Wang, 2015). Regarding siRNA mechanism, it becomes clear how this molecule can be used as a unique strategy to directly down-regulate efflux transporters expression and increase the chances to overcome BBB and reach brain treatments. On a small study, siRNA was used to target P-gp in mouse brain capillary endothelial cells. siRNA was administrated intravenously (once/day during 4 days) and it resulted in a significant reduction of the P-gp-labelled area in the hippocampal hilus and parietal cortex. P-gp expression proved to be down-regulated in these brain regions by 31 and 16%, respectively (Fig. 2). This was the first preliminary evidence that a downregulation of P-gp can be achieved in brain capillary endothelial cells by administration of siRNA in vivo. Furthermore, since just 2 day-treatment was not able to down-regulate BBB P-gp in a significant manner, authors conclude that this down regulation could only be achieved with a prolonged treatment (Fuest et al., 2009). Hori and colleagues specifically silence the rat ATP-binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of siRNA. They demonstrated that three of the four different siRNAs tested were able to reduce rABCG2 mRNA between 54.7 and 78.8% (analyzed by quantitative real-time PCR), as well as protein levels in HEK293 cells (examined by western blot analysis, using an anti-c-myc antibody). Moreover, rABCG2mediated mitoxantrone efflux transport was suppressed by the introduction of siRNAs into HEK293 cells, detected through the mean fluorescence intensity of mitoxantrone. In addition, these siRNAs did not affect the mRNA levels of other ABC transporters, suggesting that it can selectively silence rABCG2 at the BBB (Hori

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et al., 2005).siRNA had already proven to be a promising strategy for the treatment of Huntington’s disease (HD), a progressive neurodegenerative disease caused by an expansion of a CAG repeat in the Huntingtin (Htt) gene, as it can specifically decrease the expression of the toxic mutant Huntingtin protein (Htt). In order to locally and transiently disrupt the BBB to improve siRNA efficiency, focused ultrasound (FUS) combined with intravascular delivery of microbubble contrast agent was used in the right striatum brain of adult rats. This technique is based on the transcranially application of concentrated acoustic energy to the target location and simultaneously systemic administration of a microbubble contrast agent. Then circulating microbubbles begin to oscillate, which leads to mechanical changes in the blood vessel wall and a transient increase in the permeability of the BBB (Hynynen et al., 2001). 48 h following treatment with siRNA, the right (treated) and the left (control) striatum were dissected and analyzed for Htt mRNA levels. The authors shown that FUS can non-invasively deliver siRNA-Htt directly to the striatum, which leads to a significant 32% reduction of Htt expression (which is similar to decreases observed in other studies (Rodriguez-Lebron et al., 2005)) in a dose dependent manner. Furthermore, they show that reduction of Htt with siRNAHtt was greater when the extent of BBB disruption was increased, measuring the intensity of the enhancement in the right striatum and the left striatum in post-treatment MR images, the relative enhancement ranged from 12 to 58% (Burgess et al., 2012). As an important BBB feature, TJs reduce the space between the plasma membranes of contacting endothelial cells, forming this selective and regulatable barrier (Miller, 2002), and representing a key factor related to the low permeability properties of BBB (Matter and Balda, 2003). TJs are composed of a complex of intracellular and transmembrane proteins including occludin and claudin-5 (Fanning and Anderson, 1998; Zahraoui et al., 2000). Therefore, another approach to overcome BBB and reach the brain could be the delivery of siRNA targeting the TJ protein claudin-5 to the endothelial cells of the BBB, as Campbell and co-workers evaluated in mice (Campbell et al., 2008). From this study it is possible to see a significant decrease in claudin-5 mRNA levels, by RT-PCR analysis, 24 h (up to almost 80%) and 48 h post-delivery of siRNA (with levels returning to normal between 72 h and 1 week after injection). As well, levels of protein expression evaluated through immunohistochemical analysis decrease up to 48 h post-injection compared to uninjected, phosphate-buffered saline (PBS)-injected and nontargeting siRNA-injected mice, which was similar to previously reported (Nitta et al., 2003). Consequently, using a tracer molecule perfusion and MRI analysis, they also observed increased permeability at the BBB to molecules up to 740 Da (molecular weights which normally do not cross the BBB if the TJs are intact), but not 4400 Da. Furthermore, to understand if these results really have functional efficacy, authors tested the efficiency of delivery of a small neuropeptide thyrotropin-releasing hormone (TRH), which stimulates the release of thyroid-stimulating hormone from the anterior pituitary and crosses BBB via passive paracellular diffusion. They have shown an enhanced delivery of TRH (MW 360 Da) to the brain of mice 48 h post-injection of siRNA targeting claudin5, through significant increase in the period of time C57/Bl6 mice remained immobile after systemic injection of 20 mg/kg TRH (up to 4.5 min) (Fig. 3). Therefore, by silencing a protein responsible for TJs at the BBB, it is possible to transiently and size-selectively open the BBB in mice which promotes TRH passage (Campbell et al., 2008). One of the first studies in this area used siRNA to suppress gene expression of organic anion transporter 3 (OAT3) in brain microvascular endothelial cells (BMECs) (Hino et al., 2006). After intravenous injection of a large siRNA dose to mice, suppression of endogenous protein and the BBB function was investigated regarding the brain-to-blood transport function. The authors saw that siRNA against OAT3 was delivered to BMECs and efficiently reduced the

Fig. 3. Changes in period of immobility of C57/BI6 mice observed after administration of 20 mg/kg TRH, 48 h upon tail vein injection of a non-targeting siRNA or siRNA targeting claudin-5. (**P = 0.0041). Adapted from Campbell et al. (2008).

expression of OAT3 in HEK293 cells by 86.2% compared with control siRNA. This suppression effect was enough to reduce the in vitro uptake of OAT3 substrate, benzylpenicillin, as well as its brainto-blood transport at BBB. After 36 h of the siRNA injection, the percentage of benzylpenicillin remaining in the brain was significantly higher than that of control siRNA by 26.4% (Hino et al., 2006). This promising siRNA strategy has already attempted to be improved by siRNA modification. In some cases, these efforts resulted in significant improvement in the biological effectiveness of siRNA. One example is to create siRNA mimics by using altritol- Q3 modified nucleic acids-modified siRNAs (ANA), where a 6-carbon sugar, altritol, is used in place of the usual pentose ring (Fisher et al., 2007). In fact, ANA-modified siRNAs targeting the Mdr1 gene can exhibit improved efficacy as compared to unmodified control. These observations suggest that altritol modifications may be helpful in developing siRNA with enhanced pharmacological effectiveness. Treatment of drug resistant cells with Mdr1-targeted siRNAs resulted in reduction of P-gp expression, reduction in Mdr1 mRNA levels, increased intracellular accumulation of the P-gp substrate rhodamine 123, and reduced resistance to anti-tumour drugs (Fisher et al., 2007). 4.3. siRNA delivery Delivery is considered a hurdle for the development of siRNAbased therapeutics (Aagaard and Rossi, 2007). Therefore, several strategies had been proposed, including delivery of the siRNA precursor, short hairpin RNA, (shRNA), which is introduced in cells encoded on viral vectors, thus promoter choice is essential to regulate its expression. Although it has a low rate of degradation and turnover, compared to siRNA it has some disadvantages as safety concerns (some viral vectors are related to putative insertional mutagenesis) and saturation of the nuclear exportin-5 responsible for export shRNA from the nucleus (Grimm et al., 2006). Although siRNA delivery through viral vectors as lentivirus and adenovirus is highly efficient, this type of carriers are not desired due to production issues, such as limited loading capacities and high production costs, and most essentially potential for mutagenicity and safety risks related to their inflammatory and immunogenic effects. Therefore, non-viral vectors arise as promising alternatives able to deliver siRNA and other nucleic acids into cells, potentially resulting in their functional expression (Gao et al., 2011). Chemical stabilization and conjugation with functional molecules can be used to improve the stability and permeability of siRNA (Lorenz et al., 2004; Muratovska and Eccles, 2004;

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Table 1 Enhanced siRNA delivery through used nanosystems. Nanosystem

Target

Main results

References

Peptide RVG

FITC GFP

siRNA delivered specifically to neuronal cells

Chitosan NPs

P-gp

P-gp mRNA levels reduced ∼20% compared to the untreated cells

Amphiphilic ␤-cyclodextrins

HTT

Htt expression reduced by 85%, 4 h post injection

EGFP-EGF1-PLGA NPs (ENPs)

TF

NPs complexes PEG–PEI TAT-Pegylated chitosan NPs

ROCK-II Ataxin-1

Down-regulation efficiency was 1.4-fold higher for TF-siRNA-loaded ENPs, compared with TF-siRNA-loaded NPs Expression of ROCK-II mRNA inhibited by 20% Suppression of the target protein, after 48 h of transfection

Lipid NPs

PTENGRIN1

Pathogen-mimicking microparticles Copolymer PPAA-g-PAO and DOTAP liposome Cationic lipoplex D2CH

IL10

Reduction of 72% and 51% expression, compared with noninjected control Significantly enhances DCs Th1/Th2 cytokine ratio

Bcl-2

Gene expression reduction to 40% of the untreated control

RRM1

Increased cellular uptake (∼84%, 6× higher than with naked siRNA); reduced gene expression (∼27%, ∼84% with naked siRNA) Reduction in GFP expression by 62% for NP-siRNA-CTX, compared to 35% for NP-siRNA

Kumar et al. (2007) Malmo et al. (2013) Godinho et al. (2013) Chen et al. (2013) Liu et al. (2013) Malhotra et al. (2013) Rungta et al. (2013) Pradhan et al. (2014) Peddada et al. (2014) Khatri et al. (2014) Veiseh et al. (2010)

CTX-superparamagnetic iron oxide NP with PEG-grafted chitosan and PEI

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GFP

Turner et al., 2007). Another issue is the lack of cellular specificity, since P-gp is not only localized in brain capillary endothelial cells, but also in many other tissues including the gut (Watkins, 1997) and mediates protection at hematopoetic cells (Fromm, 2004). Non-selective modulation of P-gp expression by siRNA will enhance exposure of sensitive tissues to harmful xenobiotics (Fuest et al., 2009; Loscher and Potschka, 2005). Regarding these topics, among the different approaches explored in recent years to overcome this limitation are nanoparticle-based systems ranging from polymer particles to liposomes and inorganic systems as gold nanoparticles. In addition, different examples and types of nanoparticles were studied and it was observed that they facilitate drug transport across the BBB (Murthy, 2007). NPs have long been noticed to pass across the BBB, which is related to their advantageous properties as small size, customizable surface, improved solubility, targeted drug delivery and multifunctionality. These provide a substantial advantage for drug and gene delivery systems across the BBB, which is the first step for effective treatments of many CNS disorders (Bhaskar et al., 2010). To improve the nanosystem ability to reach BBB, the incorporation of PEG on its surface is common and useful because it is not recognized as a foreign material by macrophages in blood and can therefore increase the half-life of carriers (Murthy, 2007; Perez-Martinez et al., 2012), also enhancing the ability to cross the BBB due to the possibility to conjugate BBB targets to PEG tips promoting their exposition to receptors (Schlachetzki et al., 2004). Regarding toxicity, polymeric and liposomal NPs are probably the least problematic since typically they are made from or covered with natural or highly biocompatible components (as PEG or chitosan) (Murthy, 2007). NPs can also be functionalized to a specific target with, for example, transferrin (Huang et al., 2007; Lee et al., 2000), to favour the uptake mechanism (with the cell penetrating peptide TAT or lowdensity lipoproteins LDL, Kleemann et al., 2005; Sethuraman and Bae, 2007), or simply to improve their capacities of prolonging the pharmacokinetics of drugs through PEGylation of the surface (Jokerst et al., 2011). Another ligand used to target systems to BBB is glutathione, which receptor is abundantly expressed at the BBB and thus is able to mediate safe targeting and enhanced delivery of drugs to the brain (Georgieva et al., 2014). As a result, active targeting of NPs, a non invasive way to transport drug/gene to target organs using site-specific ligands, could highly promote BBB permeability. After surpass the BBB and be internalized to the cell,

NPs are enclosed in vesicles from which they must escape, to find their target sites, before they enter the lysosomes where the low pH and the high concentration of degrading enzymes can inactivate them. Regarding this, positively charged NPs and dendrimers are more efficient transfection agents, since they are able to escape from endosomes (Perez-Carrion et al., 2012; Perez-Martinez et al., 2012; Zhu and Mahato, 2010). To improve safe siRNA NPs delivery to the CNS, the proper design of nanoparticles (Table 1) needs to be studied to enhance BBB crossing and endosomal escape (O’Mahony et al., 2013; PerezMartinez et al., 2012). As an example, pluronic block copolymers contain two hydrophilic PEG and one hydrophobic PPG blocks (PEG–PPG–PEG) were shown to bind to the membranes of brain microvessel endothelial cells (Gilmore et al., 2008). Kumar and colleagues have shown that a short peptide derived from the rabies virus glycoprotein (RVG) enables transvascular delivery of siRNA to the brain. The 29-amino-acid peptide described specifically binds to the acetylcholine receptor expressed by BBB endothelial cells and neuronal cells, and, by modifying the carboxy terminus of the peptide (by adding arginine residues), they were able to conjugate siRNAs to the RVG, allowing for delivery across the BBB and subsequent transduction of neuronal cells and gene silencing. Authors saw this when, after intravenous injection into mice, modified RVG delivered siRNA specifically to the neuronal cells, resulting in specific gene silencing within the brain. Furthermore, intravenous treatment with modified RVG-bound antiviral siRNA afforded robust protection against fatal viral encephalitis in mice. Thus, modified RVG provides a safe and non-invasive approach for the delivery of siRNA and potentially other therapeutic molecules across the BBB (Kumar et al., 2007). In another study, chitosan nanoparticles were assessed as a delivery system to silence P-gp in an in vitro BBB model. siRNA loaded in chitosan NPs transfection of rat brain endothelial cells mediated effective knockdown of P-gp, since P-gp mRNA levels reduced to approximately 20% compared to the untreated cells. As well, a subsequent decrease in P-gp substrate efflux was detected, resulting in increased cellular delivery, which doubles, and increased efficacy of the model drug doxorubicin (Malmo et al., 2013). Another strategy to silence Htt gene is by using modified amphiphilic ␤-cyclodextrins (CDs), based on naturally occurring oligosaccharide molecules, as siRNA neuronal carriers (Godinho

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Fig. 4. Htt gene expression in the R6/2 mouse brain mediated through CD-HttsiRNA. Mice were injected with vehicle (white), naked siRNA (light grey), CD-HttsiRNA (grey), and CD-NSsiRNA (dark grey) nanoparticles. RNA was extracted from tissue and reverse transcribed to cDNA, at different time points. Relative expression of Htt mRNA was assessed by quantitative PCR and Htt gene expression was normalized against the expression of ␤-actin. All results are expressed in mean ± SEM, n = 3–8 per group, ***P < 0.001 compared to vehicle treated animals (Godinho et al., 2013).

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et al., 2013). CDs could form particles with a diameter between 100 and 350 nm, which were stable up to 6 h in artificial cerebrospinal fluid. After 24 h transfection, these complexes were able to reduce the expression of the Htt gene by ∼51% in rat striatal cells (ST14A-HTT120Q) and ∼78% in human HD primary fibroblasts. Additionally, Htt protein levels were found to be reduced by ∼35% after 72 h transfection in rat model. Only limited toxicity (after 48 h transfection with these siRNA-NPs complexes, cell viability was maintained above 78%) was observed with CD-siRNA nanoparticles in any of the in vitro models used. In order to investigate knockdown efficiency in vivo, R6/2 mice were treated with Htt naked siRNA, CD-HttsiRNA (CD with Htt target siRNA), or CD-NSsiRNA (CD with non-silencing siRNA). A total of 2.5 ␮g of siRNA was injected bilaterally into the striatum of R6/2 males and females. 4 h post injection, Htt gene expression was reduced by 85% and maintained up to seven days with Htt gene expression still reduced by 66% (Fig. 4). CD-HttsiRNA nanoparticles were able to significantly reduce the expression of the Htt gene in vivo (Godinho et al., 2013). The potential application of these modified ␤-CDs as siRNA carriers for CNS delivery is not restricted to HD but applicable to other neurodegenerative diseases, such as Alzheimer’s and Parkinson (Godinho et al., 2013). It is known that injured BMECs overexpress tissue factor (TF), a plasma membrane glycoprotein and the primary initiator of blood coagulation, which is involved in the thrombosis and inflammation that are associated with sepsis, atherosclerosis, and cancer (Mackman, 2009). The fusion protein EGFP-EGF1, derived from coagulation factor VII, could be targeted to TF. In order to study the silencing of TF, the inflammatory mediator TNF-␣ was chosen as a stimulus for primary BMECs to produce injured endothelium in vitro (Kim et al., 2002; Steffel et al., 2006), and then EGFP-EGF1PLGA nanoparticles (ENPs) with loaded siRNA against TF were used as a new carrier for targeted delivery to the injured BMECs. These studies show that the ENP-based transfections result in a more efficient down-regulation of TF, when compared to NP-mediated transfections. In fact, ENPs could efficiently bind to the TFexpressing BMECs in a higher extension than non-functionalized NPs. These findings also show that the TF-siRNA-loaded ENPs had minimal toxicity, with almost 96% of the cells viable 24 h after transfection. Moreover, the real-time PCR results showed that the TF mRNA level of the injured BMECs exhibited a decrease following transfection with TF-siRNA-loaded NPs, which was even higher for TF-siRNA-loaded ENPs. TF protein expression indicates that the down-regulation efficiency exhibited a 1.4-fold increase for TF-siRNA-loaded ENPs when compared with TF-siRNA-loaded NPs

transfection. Therefore, ENP-based transfection could be used for efficient siRNA transfection to injured BMECs and consequent associated down-regulation. This transfection could serve as a potential treatment for diseases, such as stroke, atherosclerosis and cancer (Chen et al., 2013). Rho-associated kinase (ROCK) is a serine/threonine kinase and one of the major downstream effectors of the small GTPase RhoA, which is involved in many parts of neuronal function, including axon regeneration (Tan et al., 2011). Several growth inhibitory molecules limited the ability for axons to regenerate–this limitation can be done by activating the Rho/ROCK pathway (Gopalakrishnan et al., 2008). Recent studies suggest ROCK as a potential target for the treatment of AD, since it is a major effector in the Rho/ROCK pathway and by blocking this pathway, protection of neuronal cells is promoted and axonal regeneration occurs (Fujimura et al., 2011; Yang et al., 2010). Thus, and as ROCK-II mRNA (one of the two types of ROCK isomers) is expressed in brain in large quantities and its knockdown leads to a decrease of Amyloid-␤ (A␤) production (Herskowitz et al., 2011), suppress its expression with a siRNA approach was what Liu and colleagues studied. They synthesized complexes of nanoparticles made of polyethylene glycol-polyethyleneimine (PEG–PEI) loaded with siRNA against ROCK-II and transfected to C17.2 neural stem cells in vitro. PEI is one of the most successful polycationic carriers used for non-viral gene delivery (Huang et al., 2011), and hydrophilic PEG moieties were engrafted onto the PEI core to decrease its cytotoxicity and improve resistance to enzymatic degradation (Weber et al., 2012). From their results, they conclude that the complexes with the best characteristics (high transfection efficiency of 70% and low cytotoxicity – cell viability was 80%) were achieved with a charge ratio between the amino groups of PEG–PEI and phosphate groups of the siRNA (N/P ratio) of 50. Laser confocal microscopy, used to investigate the cell uptake of the complexes, showed that ROCKII-siRNA was mainly distributed in the cytoplasm and synapses. RT-PCR and western blot demonstrated effective gene silencing for mRNA (expression of ROCK-II mRNA inhibited by 20%) and protein level, respectively. These results indicated that PEG–PEI/ROCK-IIsiRNA complexes effectively suppressed ROCK-II mRNA expression, which could promote AD treatment (Liu et al., 2013). In order to delivery siRNA for use in neurodegenerative diseases, Malhotra and co-workers synthesized pegylated chitosan with further conjugation of PEG to a cell-penetrating peptide, transactivator of transcription (TAT), to develop nanoparticles able to carry siRNA to be delivered in neuronal cells. The nanoparticles were tested to deliver a functional siRNA against the Ataxin-1 gene in an in vitro established model of a neurodegenerative disease – Spino Cerebellar Ataxia (SCA1). On SCA1 there is an over-expression of this ataxin protein, and it is characterized by a loss of cells of the cerebellum and spinal cord affecting motor coordination, posture and balance. The results indicate successful suppression of the SCA1 protein following 48 h of transfection as siRNA loaded on these NPs was able to down-regulate its target compared to bare siRNA (Malhotra et al., 2013). Rungta and colleagues develop a method for using siRNA in lipid nanoparticles (LNPs) to efficiently silence neuronal gene expression in cell culture and in brain, in vivo, through intracranial injection. Previous work has shown that similar LNP systems were able to silence target genes in hepatocytes following IV injection at low siRNA dose levels (Belliveau et al., 2012; Jayaraman et al., 2012) in an apolipoprotein E (ApoE)-dependent way (Akinc et al., 2010). With this study, authors also show that neurons accumulate these NPs in an ApoE-dependent way, resulting in very efficient uptake in cell culture (100%) with little apparent toxicity. In vivo, both intracortical or intracerebroventricular (ICV) siRNA-LNP injections resulted in knockdown of target genes (PTEN, which is a protein highly expressed in pyramidal neurons, Kwon et al., 2006) either

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in discrete regions around the injection site or in more widespread areas following ICV injections, with no apparent toxicity or immune reactions from the LNPs. This knockdown corresponded to a reduction of 72% compared with noninjected control. Effective targeted knockdown was demonstrated by showing that intracortical delivery of siRNA against GRIN1 (which encods the GluN1 subunit of the NMDA receptor (NMDAR)) resulted in significant knockdown of GluN1 expression by 51% compared with noninjected control. Consequent reduction synaptic NMDAR currents occurred in vivo, as compared with synaptic AMPA receptor currents. Therefore, LNP delivery of siRNA through in vivo intracranial injection manipulates expression of proteins involved in primary neuronal culture, possibly enabling the development of gene therapies for neurological disorders (Rungta et al., 2013). Microparticles (1.18 ± 0.28 ␮m) could also be used to transport siRNA as Pradhan and colleagues assessed through an immunomodulatory strategy regarding the critical balance of T helper 1 (Th1; key to eliminating tumour cells) and T helper 2 (Th2) responses driven by antigen presenting cells (as dendritic cells, DCs). The basis of this work is by reducing IL10 production while maintaining IL12 levels during CpG (Toll-like receptor 9 (TLR9) agonist, used to enhance Th1 response, and also induces high levels of the anti-inflammatory, Th2-promoting cytokine IL10, which could decrease the resulting Th1 response) delivery, it is possible to enhance the Th1/Th2 cytokine balance and therefore improve anti-tumour immune response. In this study the delivery of IL10silencing siRNA and CpG oligonucleotides (ODN) to DCs using pathogen-mimicking microparticles (PMPs) was performed. PMPs were PLGA nanoparticles functionalized with polyethyleneimine (PEI) to become cationic, with further surface loading of different nucleic acid based immunomodulatory molecules (as CpG and IL10 siRNA). This system significantly enhances DCs Th1/Th2 cytokine ratio, which suggest an efficient inhibition of IL10 production due to siRNA delivery to DCs. Thus, PMPs are nanosystems able to precisely modulate TLR ligand-mediated immune-stimulation in DCs, promoting siRNA to reach its target (Pradhan et al., 2014). The combination of two systems could be advantageous, like Peddada and co-workers tested. A complex consisting of an anionic copolymer (based on backbone poly(propylacrylic acid) (PPAA), a pH-sensitive hydrophobic polymer, with grafted poly(alkylene oxides) (PAOs)), and a cationic DOTAP liposome was the authors idea to carrier an antisense oligonucleotide (AON). The addition of PPAA to DOTAP/AON complexes improved the antisense gene silencing effect in A2780 human ovarian cancer cells, reducing the gene expression to 40% of the untreated control. In vivo studies showed that, after intraperitoneal injection in mice, a greater amount of AON is delivered to ovarian tumour xenografts using the ternary copolymer-stabilized delivery system, compared to a binary DOTAP/AON complex. These results highlight the importance of the hydrophilic–lipophilic balance of the transport system in achieving stability and cellular uptake (Peddada et al., 2014). In order to achieve stable and non toxic lipoplexes able to carrier siRNA, Khatri and colleagues evaluated them as a transport system for siRNA targeted to RRM1 (that is responsible for development of resistance to gemcitabine in cancer cells). A cationic lipoplex (D2CH) composed of dioleoyl-trimethylammoniumpropane (DOTAP), dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), hydrogenated soya phosphocholine (HSPC), cholesterol, and methoxy(polyethyleneglycol)2000-1,2-distearoyl-sn-glycero-3phosphoethanolamine (mPEG2000-DSPE) was tested. Cell uptake studies and gene expression studies have confirmed intracellular availability of siRNA. D2CH lipoplexes showed high cellular uptake of 83.83 ± 1.01% in A549 cells and 67.07 ± 0.86% in HT1299 cells which was five to six fold higher than that obtained with naked siRNA. As well, at 5 nM concentration D2CH lipoplexes showed 27.15 ± 1.85% gene expression while naked siRNA has 83.50 ± 2.5%

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gene expression in A549 cells (similar values were obtained in H1299 cells) – results that emphasize siRNA carriers importance (Khatri et al., 2014). Another system used to transport siRNA to brain is magnetic nanoparticles. Regarding this field, Veiseh and colleagues studied a cancer-cell specific magnetic nanosystem for siRNA delivery and simultaneous non-invasive monitoring through magnetic resonance imaging (MRI) (Veiseh et al., 2010). Their nanosystem was composed by superparamagnetic iron oxide nanoparticle core coated with PEG-grafted chitosan and polyethylenimine (PEI) and further functionalized with siRNA (against GFP) and a tumourtargeting peptide (chlorotoxin, CTX). When C6 cells (rat glioma cells) were treated with targeted and non-targeted nanovectors, NP-siRNA-CTX was internalized by cells 2-fold more than NPsiRNA, which highlights how NP-siRNA-CTX readily binds to glioma cells through the affinity of CTX to its receptor. These results were further confirmed by MRI imaging through nanovectors magnetic properties. Moreover, the knockdown transgene expression of GFP in C6/GFP+ cells were evaluated and found to be enhanced when cells were treated with NP-siRNA-CTX (62% reduction in GFP expression) by comparison with NP-CTX (no appreciable change in GFP expression) and NP-siRNA (35% reduction in GFP expression). These results suggest how receptor-mediated cellular internalization affects an enhanced knockdown (Veiseh et al., 2010). Before siRNA technique can be used as effective treatment in the brain, efficient methods of in vivo delivery must be devised. For that, a careful understanding of biological barriers and their interaction with nanocarriers is the key for effective nucleic acid therapy. 4.4. siRNA/drug co-delivery In order to create an even more efficient system, the combination of siRNA and drugs in the same formulation (loaded in functionalized NPs, for example) could be really advantageous, since it would have distinct ways to act. Previously it was shown that TAT, a cell-penetrating peptide, and the modified poly(ethyleneglycol) and poly(␧-caprolactone) block copolymers (MPEG–PCL–TAT) can form stable complexes with siRNA, or can be loaded with an anticancer drug and efficiently deliver the drug to the brain tissue via intranasal delivery (Kanazawa et al., 2013; Nori et al., 2003). Therefore, another study was performed to develop a novel, efficient, and safe therapeutic strategy for managing brain disorders, especially malignant glioma. By using MPEG–PCL–TAT micelles with a nose-to-brain delivery system, authors investigated therapeutic effects on a rat model of malignant glioma using siRNA against Raf-1 (which plays a role in cell proliferation and apoptosis; siRaf-1) and camptothecin (CPT) co-delivery system. MPEG–PCL–TAT and CPT-loaded MPEG–PCL–TAT can form a stable complex with siRNA. Additionally, MPEG–PCL–TAT/siRaf-1 and CPT-loaded MPEG–PCL–TAT/siRaf-1 have promoted cell death in rat glioma cells after the high cellular uptake of siRaf-1/drug by the MPEG–PCL–TAT carrier. Furthermore, the mean survival period of the untreated rats was 16.6 days, and all rats died within 20 days of tumour inoculation. The mean survival period of rats treated with naked siRaf-1 solution (18.4 days) was not significantly different from that of untreated rats. The mean survival period of rats treated with MPEG–PCL–TAT/siRaf-1 was 20.4 days, CPT-loaded MPEG–PCL–TAT/siControl 20.6 days, and CPT-loaded MPEG–PCL–TAT/siRaf-1 was 28.4 days, this last was significantly greater than any other option (Fig. 5). So it can be concluded that a CPT-loaded MPEG–PCL–TAT/siRaf-1 complex achieved the highest therapeutic effect because of the additive effects of CPT and siRaf-1. These results indicate that drug/siRNA co-delivery using MPEG–PCL–TAT nanomicelles with nose-to-brain delivery

Please cite this article in press as: Gomes, M.J., et al., siRNA as a tool to improve the treatment of brain diseases: Mechanism, targets and delivery. Ageing Res. Rev. (2015), http://dx.doi.org/10.1016/j.arr.2015.03.001

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inhibit cell proliferation (A549 and H226) in a time-dependent manner, and induced a remarkable apoptosis of cancer cells with elevated levels of caspase 3/7 activity (apoptosis markers). Moreover, lipo-DTX/siRNA exhibited greater apoptotic populations and a superior caspase activity than comparing to lipo-DTX throughout the study period. In vivo antitumor study exhibited a significant tumour regression profile for lipo-DTX/siRNA with 100% survival rate, which never occurred with any other treatment option (Qu et al., 2014). All these experiments and results highlight the synergistic antitumour activity of co-loaded siRNA and drug. 5. Conclusions

Fig. 5. Effect on survival of intranasal administration of siRaf-1 complexed with camptothecin-loaded micelles on intracranial C6 glioma-bearing rats. The test substances administered to the rats were 0.1 mg/kg siRNA and 0.36 mg/kg CPT (n = 5) (Kanazawa et al., 2014).

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is an excellent therapeutic approach for brain and CNS diseases (Kanazawa et al., 2014). Zhu and colleagues also evaluated the co-delivery of hydrophilic siRNA (anti-survivin siRNA, survivin is an inhibitor protein of apoptosis found up-regulated in malignant tumours, especially in drug resistant cells) and hydrophobic drugs (PTX, paclitaxel) through micelles constructed by a matrix metalloproteinase 2 (MMP2)-sensitive copolymer (PEG–pp-PEI–PE) via self-assembly. These micelles act as transporters by siRNA condensation by PEI and hydrophobic drug solubilization in the lipid “core”. To study the synergistic effect, PTX-sensitive (A549; mimic normal cells) and – resistant (A549 T24; mimic drug resistant cancer cells) cells were used. Compared to A549 cells with IC50 of about 5.2 nM PTX, the resistant cells A549 T24 cells have a higher IC50 of about 96 nM PTX. Incubation of PEG–pp-PEI–PE/PTX with A549 or A549 T24 cells increased drug solubility and enhanced cellular uptake which leads to significantly increase of PTX cytotoxicity, compared to those of free PTX or its nonsensitive micelles. The simultaneous delivery of anti-survivin siRNA and PTX, made in order to kill tumour cells, significantly brought down the IC50 of PTX to about 15 nM (Zhu et al., 2014). Also Li and co-workers assessed layered double hydroxide nanoparticles (LDHs) to simultaneously deliver an anticancer drug 5-fluorouracil (5-FU) and Allstars Cell Death siRNA (CD-siRNA, siRNA targeting ubiquitously expressed human genes that are essential for cell survival) for effective cancer treatment. These LDHs have an anion exchange ability that promotes 5-FU to be intercalated into their interlayer spacing, and load siRNA on their surface. By testing the combination of CD-siRNA and 5-FU within LDH particles in three cancer cell lines (MCF-7, U2OS and HCT-116), a significantly enhanced cytotoxicity was observed compared to the single treatment with either CD-siRNA or 5-FU. For example, treatment with CD-siRNA-5-FU/LDH caused 70% cell death, while treatment with either 5-FU/LDH or siRNA/LDH at the same concentration resulted in only 46% or 34% cell death. Authors relate these results to synergistic effect of two therapeutics on cancer cells by effectively inducing cell death in complementary pathways, namely mitochondrial damage process (Li et al., 2014). The co-delivery promising strategy was also evaluated by Qu and colleagues, who incorporate in a PEGylated liposome docetaxel (DTX), a chemotherapeutic drug, and BCL-2 siRNA (BCL-2 gene is a key apoptosis inhibitor that is overexpressed in many tumours; silencing BCL-2 could enhance the chemotherapeutic effect in cancer), to systemically deliver in a lung cancer model (A549) and suppress the tumour growth. NPs lipo-DTX/siRNA shown to

Due to an increased importance of brain diseases, effective treatments are needed. siRNA, as a key to several approaches that silence specific genes associated with BBB protective functions, already overcome difficulties from existence alternatives which do not express a protein (as knock-out mice who lack the target protein or conditional knock down mice that only remove the protein following a specific treatment). When compared to these alternatives, siRNA has many advantages, since, in contrast to knock-out animals which usually have problems due to a permanent protein lack, siRNA related silence is reversible. Furthermore, as opposed to the combination of inhibitory drugs for efflux transporters, siRNA do not promote side effects that commonly arise from simultaneous administration of several drugs. Gene and drug delivery had been studied for several years, and their associated promising nanotechnology methods could as well be used to transport and target siRNA to its final goal. Many studies were performed assessing also the advantages of nanotechnology systems to transport siRNA which, as expected, become even more evident. Nevertheless, the main and innovative approach related to siRNA is the ability to promote the BBB surpass. As it is reviewed here, several studies assessed siRNA effectiveness and specificity against different proteins, especially drug efflux systems as P-gp, which are fundamental to maintain xenobiotics out of the brain and the main cause for many brain diseases treatment fail. As these studies showed, siRNA was able to decrease mRNA levels as well as protein expression of target genes. Therefore, it was proven to have an enormous potential in this field as a promising way to the treatment of neurodegenerative diseases, which incidence numbers have been increasing and have no treatment so far. Conflicts of interest The authors report that they have no conflicts of interest in this work. Acknowledgments This work was financed by European Regional Development Q4 Fund (ERDF) through the Programa Operacional Factores de Competitividade – COMPETE, by Portuguese funds through FCT – Fundac¸ão para a Ciência e a Tecnologia in the framework of the project PEst-C/SAU/LA0002/2013, and co-financed by North Portugal Regional Operational Programme (ON.2 – O Novo Norte) in the framework of project SAESCTN-PIIC&DT/2011, under the National Strategic Reference Framework (NSRF). Maria João Gomes gratefully acknowledges Fundac¸ão para a Ciência e a Tecnologia (FCT), Portugal for financial support (grant SFRH/BD/90404/2012). References Aagaard, L., Rossi, J.J., 2007. RNAi therapeutics: principles, prospects and challenges. Adv. Drug Deliv. Rev. 59, 75–86.

Please cite this article in press as: Gomes, M.J., et al., siRNA as a tool to improve the treatment of brain diseases: Mechanism, targets and delivery. Ageing Res. Rev. (2015), http://dx.doi.org/10.1016/j.arr.2015.03.001

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