Cano et al. J Nanobiotechnol (2021) 19:122 https://doi.org/10.1186/s12951-021-00864-x Journal of Nanobiotechnology

REVIEW Open Access Nanomedicine‑based technologies and novel biomarkers for the diagnosis and treatment of Alzheimer’s disease: from current to future challenges Amanda Cano1,2,3,4*, Patric Turowski5, Miren Ettcheto2,6, Jason Thomas Duskey7,8, Giovanni Tosi7, Elena Sánchez‑López2,3,4, Maria Luisa García2,3,4, Antonio Camins2,6, Eliana B. Souto9,10, Agustín Ruiz1,2, Marta Marquié1,2† and Mercè Boada1,2†

Abstract Increasing life expectancy has led to an aging population, which has consequently increased the prevalence of dementia. Alzheimer’s disease (AD), the most common form of dementia worldwide, is estimated to make up 50–80% of all cases. AD cases are expected to reach 131 million by 2050, and this increasing prevalence will critically burden economies and health systems in the next decades. There is currently no treatment that can stop or reverse disease progression. In addition, the late diagnosis of AD constitutes a major obstacle to efective disease management. Therefore, improved diagnostic tools and new treatments for AD are urgently needed. In this review, we investigate and describe both well-established and recently discovered AD biomarkers that could potentially be used to detect AD at early stages and allow the monitoring of disease progression. Proteins such as NfL, MMPs, p-tau217, YKL-40, SNAP-25, VCAM-1, and Ng / BACE are some of the most promising biomarkers because of their successful use as diagnostic tools. In addition, we explore the most recent molecular strategies for an AD therapeutic approach and nanomedicine-based technologies, used to both target drugs to the brain and serve as devices for tracking disease progression diagnostic biomarkers. State-of-the-art nanoparticles, such as polymeric, lipid, and metal-based, are being widely investigated for their potential to improve the efectiveness of both conventional drugs and novel compounds for treating AD. The most recent studies on these nanodevices are deeply explained and discussed in this review. Keywords: Alzheimer’s disease, Biomarkers, Polymeric nanoparticles, Lipid nanoparticles, Metal nanoparticles, Nanotechnology

Highlights

• Alzheimer’s disease is the most common form of dementia worldwide, estimated to be responsible for 50–80% of all cases. *Correspondence: [email protected]; [email protected] • No currently available treatment can stop the disease † Marta Marquié and Mercè Boada—senior co-authors progression, and it is expected that AD cases will 1 Research Center and Memory Clinic, Fundació ACE. Institut Català de Neurociències Aplicades, International University of Catalunya (UIC), C/ reach 131 million by 2050. Marquès de Sentmenat, 57, 08029 Barcelona, Spain • Te late diagnosis of AD will have a serious impact Full list of author information is available at the end of the article on health and socio-economic systems worldwide.

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• Novel biomarkers, such as NfL, SNAP-25, Clusterin, Alzheimer’s disease (AD) is the most common form MMPs, and YKL-40, among others, are being studied of dementia worldwide, estimated to constitute up to alongside commonly used biomarkers as potential 50–80% of cases [1]. Undoubtedly, AD will critically new diagnostic tools. burden economies and health systems since cases are • Nanomedicine has aroused much interest in the last expected to reach 131 million by 2050 [2]. AD is com- decade and has shown promising results for improv- monly diagnosed symptomatically through the occur- ing drug targeting and delivery to the brain. rence of signifcant memory loss, global cognitive • Polymeric-, lipid-, and metal-based nanoparticles are decline, and the impairment of daily life activities. Later the most commonly investigated nanodevices in the in the course of the disease, the breakdown of physi- feld of dementia. cal functions, such as walking, swallowing, and general • State-of-the-art nanoparticles are emerging as prom- movement, ultimately leads to death [3]. Dementia was ising tools to improve the diagnosis and treatment of the ffth-leading cause of death in 2016 [1]. AD. AD can be classifed depending on the onset of the frst symptoms. Approximately 1–6% of all cases are cat- egorized as early-onset AD (EOAD), which manifests Introduction before the age of 65. By contrast, late-onset AD (LOAD) In the past few decades, the number of people living is characterized by the occurrence of symptoms at an with dementia has increased exponentially all around age greater than 65 years and accounts for around 90% the world, mainly due to aging populations and improve- of cases [4]. EOAD difers from LOAD in many aspects. ments in quality of life. Te latest Global Burden Disease Prominently, EOAD has a more aggressive course of Study estimated that the number of cases of demen- disease progression, greater delay to diagnosis, lower tia more than doubled from 1990 to 2016 and indicated cognitive reserves, lower incidence of diabetes, obesity, population growth and aging as the main drivers of this and circulatory disorders, relatively greater defcits in increase (Fig. 1) [1]. attention, executive functions, praxis, and visuospatial

D: 84 368 D: 260 080 P: 1 554 081 D: 12 669 P: 4 347 849 P: 247 867 D: 59 119 P: 1 033 615

D: 452 515 P: 6 586 827 D: 262 278 D: 120 035 P: 4 216 158 D: 67 443 P: 2 613 225 P: 1 268 251 D: 495 128 D: 10 351 P: 10 767 181 D: 13 624 P: 210 389 P: 244 819 D: 168 498 D: 10 893 D: 178 664 P: 3 337 721 D: 11 513 P: 205 047 P: 3 747 443 P: 201 807 D: 82 383 D: 17 668 P: 1 725 736 P: 251 413 D: 29 557 D: 23 831 D: 10 893 P: 508 756 P: 375 984 P: 205 047 High-incomeNorth America Central Lan America North Africa& MiddleEast Central Asia Caribbean Australasia Central sub-SaharanAfrica East Asia Andean Lan America Western Europe Eastern sub-SaharanAfrica SoutheastAsia Tropical LanAmerica Eastern Europe Western sub-SaharanAfrica South Asia Southern Lan America Central Europe Southern sub-SaharanAfrica High-incomeAsia Pacific Fig. 1 Global deaths (D) and prevalence (P) for Alzheimer’s disease and other dementias in 2016. Data are n (95% UI). UI uncertainty interval. Data = extracted from the Global Burden of Disease Study of Alzheimer’s disease and other dementias 2016 [1] Cano et al. J Nanobiotechnol (2021) 19:122 Page 3 of 30

functions, lower frequency of the APOE ε4 allele, greater for the development of novel cures. In addition, ef- white matter changes, and a higher burden of neuritic ciently and specifcally delivering the diagnostic and plaques and neurofbrillary tangles [5]. therapeutic molecules to the sites of interest in these pro- In general, AD is defned as a multifactorial disease in cesses is critical. Nanoparticles (NPs) have enabled great which genetic, environmental, behavioral, and develop- strides towards the delivery, treatment and diagnostics of mental components critically infuence its pathogenesis, diseases, mainly due to their various chemical character- with age being the most important risk factor. Although istics and their propensity for chemical modifcation to most cases of AD are sporadic, rare cases (< 1%) have modulate and refne required properties [11, 12]. NPs’ a genetic component (“Familial AD”, FAD) with a few core constituents comprise a wide variety of materials, hundred families identifed worldwide. Tese cases usu- such as lipids, polymers, and metals, that can encapsu- ally have an early onset at young ages (40–50). Te three late molecules with diferent chemical natures. In addi- known genes that cause FAD, with an autosomal domi- tion, these carriers promote the protection and delivery nant inheritance, are the amyloid precursor protein of bioactive molecules, which can reduce their potential (APP), presenilin-1 (PS1), and presenilin-2 (PS2) genes, toxicity and, in turn, enhance their solubility, stability, all of which are involved in the processing or production biodistribution, and pharmacokinetics. Molecules encap- of Aβ [6]. Our understanding of the pathophysiology of sulated in NPs range from small molecules, peptides, and AD is constantly changing [4]. Te overproduction and proteins to genetic material [13]. Crucially for chronic accumulation of amyloid-β (Aβ) peptide and hyperphos- CNS diseases, NPs have the demonstrated capacity to phorylated tau (p-tau) protein are the two most com- deliver molecules to hard-to-reach tissues, such as the mon hypotheses for AD pathogenesis, but recent fndings CNS where the crossing of the BBB and the release of have demonstrated that chronic oxidative stress, hor- drugs with controlled kinetics for long-term treatments mone imbalance, mitochondrial dysfunction, infamma- are required [11, 14]. tion, calcium mishandling, mitotic dysfunction, genetic In this review, we investigate and describe well-estab- components or blood–brain barrier (BBB) dysfunction lished AD biomarkers and highlight recently discovered also likely play key roles in the disease process [7, 8]. ones that could be potential diagnosis tools. In addition, Similarly, emerging studies have also reported that oli- we explore the most recent nanomedicine-based tech- godendrocytes act as antigen-presenting cells and pro- nologies used to target drugs across the BBB and increase duce immune molecules. Likewise, the activation of CNS delivery of these active molecules. both astroglia and oligodendrocytes, mainly due to BBB dysfunction and general toxic species produced in AD, Novel biomarkers in the diagnosis of Alzheimer’s occurs widely in AD [9]. In neuroinfammation processes, Disease oligodendrocytes express many factors known to activate AD was frst described in 1906 [15]. For a long time after astrocytes. In response to these factors, astrocytes and that, frm diagnosis was made when signs of memory loss oligodendrocytes secrete immune factors, underscoring and cognitive decline were already signifcantly advanced. the possible immune function of these cells. IL-1β, IL17 Nowadays, many observations indicate that the patho- tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), physiological alterations of AD in the brain begin dec- and fbroblast growth factor-2 (FGF-2) are some of these ades before the onset of clinical symptoms of dementia. secreted cytokines that induce pro-infammatory efects Historically, AD patients were classifed into three clini- [9]. Regardless of all these changes, neuronal death is the cal categories: cognitively unimpaired (CU), mild cogni- ultimate invariable outcome, which then drives the typi- tive impairment (MCI), and dementia patients [16]. AD cal neurodegeneration of AD. is now recognized as a continuum of neurological decline Currently there are only four FDA approved treatments that can be identifed and staged through a combination for AD, and these are linked mainly to the two molecular of neuropathological fndings and in vivo biomarkers pathways involving the accumulation of Aβ peptide and [17, 18]. Tis has led to a paradigm shift in the diagnosis neurofbrillary tangles (NFT) of p-tau protein [10]. How- of AD, opening new windows of opportunities for early ever, none of these drugs stops disease progression or treatment in the preclinical stages. In that context, the cures AD, highlighting the need for additional treatment National Institute on Aging and the Alzheimer’s Asso- approaches. Te discovery of novel biomarkers is hoped ciation have recently updated diagnostic criteria, with a to deliver earlier AD diagnosis and could also support the clear shift from a clinical to a biological defnition of AD identifcation of additional molecular targets, potentially [19]. leading to new treatments. Biomarkers now have key importance in the robust Identifying the pathophysiological processes involved diagnosis of AD. Biomarkers are quantifable mol- in AD and the best biomarkers to detect them is critical ecules or processes that can be related to the biological Cano et al. 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alterations and/or pharmacologic responses to a thera- development of AD, are being identifed as potential new peutic intervention for a specifc disease [18, 20]. An biomarkers for the early diagnosis of this disorder (Fig. 2) ideal biomarker should be specifc, sensitive, predictive, [24–27]. accurate, robust, inexpensive, and ideally non-invasive and measurable in common biological fuids such as Conventional AD biomarkers in CSF/plasma serum, saliva and/or urine [21, 22]. For AD, cerebro- Aβ‑related biomarkers spinal fuid (CSF) is considered the optimal biological In AD pathogenesis, the production of Aβ peptides is source for biomarker assessment, since its direct contact increased, while their clearance is reduced. Aβ1-40 and with the interstitial fuid where the brain is immersed Aβ1-42 fragments predominantly form, promoting the refects the pathophysiological changes of AD progres- aggregation of these peptides and thus leading to the for- sion in real time [18, 23]. For AD biomarkers, specifcity mation of senile plaques [28]. Aβ42 is one of the most and sensitivity of more than 80% are needed to be con- toxic isoforms of the Aβ peptide and is also one of the sidered a reliable biomarker [18]. Notably, not all patients most widely accepted biomarkers of AD diagnosis [29, with cognitive impairment develop Aβ plaques or tau 30]. Aβ42 quantifcation in CSF allows the identifcation neurofbrillary tangles (NFTs), and vice versa. Further- of AD in its preclinical stage, and possesses high diagnos- more, biomarkers have diferent uses and interpretations tic value with clear specifcity for AD over other neurode- depending on the degree of the cognitive deterioration generative diseases. However, absolute Aβ42 levels vary observed. Tus, the use of biomarkers has allowed the due to a variety of factors, particularly interindividual identifcation of prodromal AD in MCI subjects and the diferences [29]. To account for these, the ratio between confrmation of AD in individuals with dementia [18]. Aβ42/Aβ40 is also commonly used. Aβ40 concentration Several biomarkers are currently being used in a com- is 10-times higher than Aβ42 in CSF and its level does plementary way with conventional neuropsychological not usually vary in AD [31]. Similarly, the Aβ42/Aβ38 tests and routine neurological exams in the diagnosis of ratio is emerging as a potential predictive tool with com- AD. Te parameters and diagnostic implications of these parable power to the Aβ42/Aβ40 ratio [32–34]. conventional biomarkers have recently been reviewed Likewise, as with other disease biomarkers, current and updated. Furthermore, new molecules, related to eforts are also focused on improving Aβ42 detection in diferent pathophysiological pathways involved in the blood samples [28, 29]. CSF extraction through lumbar

Astrocytebiomarkers Tau biomarkers YKL-40 tTau pTau

Clearance biomarkers Clusterin, Orexin, Aβ biomarkers Transthyretin, Ubiquitin, Vascular Biomarkers Aβ42 LAMP-1, cystatin C… Albumin, VCAM1, Aβ42 / Aβ40 ratio ICAM1, VEGF, MMPs, Aβ42 / Aβ38 ratio sPDGFR-β… BACE1 Neuronal biomarkers Nfl VILIP-1 Microglia biomarkers tTau sTREM2 Progranulin Synaptic Biomarkers Synaptotagmin-1 Lipidbiomarkers Neurogranin ApoE,ApoH, SNAP-25 FABP3, VDBP… GAP-43

Fig. 2 Conventional and novel biomarkers related to molecular alterations and physiopathological changes of AD Cano et al. J Nanobiotechnol (2021) 19:122 Page 5 of 30

function is an invasive procedure that is only permit- However, longitudinal studies have revealed that CSF tau ted for certain patients. It is often not feasible, e.g., for levels vary depending on the diferent stages of the dis- patients with low back injuries, increased intracranial ease [49, 50]. Several recent studies have tried to eluci- pressure due to a space-occupying lesion, coagulopathy, date the prognostic value of tau in early-onset AD and or infected skin over the puncture site [35, 36]. Immuno- as a biomarker to monitor drug responses, but further precipitation coupled with mass spectrometry, ELISA, investigation is required [29]. and protein misfolding cyclic amplifcation assays are As with Aβ, progress is also being made regarding the possible techniques for detecting blood Aβ42 [29, 37]. predictive value of tau in blood samples thanks to ultra- Recently, a blood analytical test for the detection of sensitive immunoassays techniques. Some studies have plasma Aβ by mass-spectrometry gained approval by reg- already identifed higher plasma and/or serum p-tau181 ulators at the Centers for Medicare & Medicaid Services levels in AD patients versus controls using innovative under the Clinical Laboratory Improvement Amend- techniques such as Simoa®, immunomagnetic reduc- ments protocol [38]. Researchers predict that the next tion, and label-free real-time surface plasmon resonance tests to be approved are plasma p-tau181 and p-tau217, [51–53]. Interestingly, recent fndings have shown the as these tests will be more robust since they possess a predictive value of plasma p-tau217 for discriminating larger dynamic range. Indeed, phosphorylated fragments AD from other neurodegenerative diseases, specifcally of tau increase by as much as tenfold in the blood as AD as an accurate predictor of β-amyloidosis at asympto- progresses, whereas the Aβ42/40 ratio falls by about 15 matic and symptomatic stages [54–57]. In these stud- percent at most [38]. ies, p-tau-217 and p-tau-181 were highly specifc for BACE1, the key enzyme that initiates the formation of amyloid plaque pathology, as p-tau-217 measurement Aβ peptide [39], has increasingly been studied as a poten- was still more specifc to amyloid status than p-tau-181 tial AD biomarker. BACE1 can be measured in CSF, but [54–57]. Collectively, these fndings raise the possibility there is controversy regarding its predictive value. Some for a novel, highly sensitive, and specifc biomarker based studies have shown increased levels and protein activity on circulating tau, with improved diagnostic accuracy. of BACE1 in AD patients and that this is a good predic- Further studies are needed to validate these fndings in tor of the progression of MCI patients [40, 41]. However, unselected and diverse populations and determine its more recent studies have reported no changes in CSF potential role in clinical practice [54–57]. BACE1 levels among controls, MCI and AD patients [42–44]. Novel AD biomarkers in CSF/plasma Neuronal damage‑related biomarkers Tau‑related biomarkers Other molecules and cellular structures have been sug- It is widely accepted that hyperphosphorylated tau gested as potential biomarkers of neuronal damage. (p-tau), accumulating as intracellular NFTs, is the other Among these, neuroflaments, specifcally neuroflament main pathological hallmark of AD, together with Aβ light chain (NfL), have emerged as promising biomarkers. plaques [29, 45]. However, in many neurodegenerative After axonal and/or neuronal damage, NfL leaks into CSF diseases, collectively known as "tauopathies", p-tau NFTs and increases to detectable levels [58]. are found that difer in their phosphorylated residues Although NfL is not specifc to AD, it has been shown [29, 46]. Te diagnostic value of total tau (t-tau) levels for to be intimately linked to neurodegenerative diseases, diferentiating AD from normal aging is well described. and its predictive value increases in combination with T-tau has been considered a relevant biomarker of neu- other biomarkers [59]. However, one of the benefts of ronal injury and thus is not a specifc marker of AD; it NfL compared to other biomarkers is the high correla- is also found to be elevated in other neurological condi- tion of its levels in CSF and blood [60]. Moreover, simi- tions such as Creutzfeldt-Jakob’s disease, frontotemporal larly to Aβ, increased levels of NfL can be detected in dementia with parkinsonism, and Pick’s disease, among the early stages of autosomal dominant AD, even before others [47]. P-tau (specifcally tau phosphorylated on the onset of frst symptoms [61]. In addition, a recent threonine 181, p-tau181) has been described as a more 18-month trial with mild AD patients showed that NfL, specifc biomarker for AD since p-tau is present at nor- as a Tau-independent marker of axonal degeneration, had mal levels in most other neurodegenerative disorders, a stronger association with clinical scales than did t-tau but not in AD [29]. In clinical practice, the correlation [62]. Tus, the ability of NfL to detect changes before between both t-tau and p-tau in CSF is important for dif- clinical manifestations occur reveals its promise as a ferentiating among several dementia types [48]. diagnostic biomarker. Levels of tau deposition display a stronger correlation Visinin-like protein 1 (VILIP-1), a calcium sensor pro- to cognitive decline than does Aβ in AD patients [49]. tein highly expressed in neurons, is another protein that Cano et al. J Nanobiotechnol (2021) 19:122 Page 6 of 30

could act as a biomarker of neuronal-injury in AD [18, Progranulin, a glycoprotein mainly secreted by acti- 29]. Tis is supported by the strong correlation between vated microglia, is involved in the modulation of neuroin- increased levels of both VILIP-1 and tau in the CSF of fammation [73]. Tis protein has aroused much interest AD patients [63, 64]. Importantly, some studies have since its loss-of-function has been closely correlated with reported the predictive value of VILIP-1 for atrophy some types of frontotemporal lobar degeneration [29, rates and cognitive decline, indicating that this protein 74]. However, although a cross-sectional study reported could help identify MCI patients that will progress to AD increased CSF progranulin levels in autosomal-dominant [64]. In blood samples however, the predictive value of AD and late-onset AD, progranulin from CSF or blood is VILIP-1 remains uncertain [27, 29]. currently not validated as diagnostic markers [75, 76]. Another neuroinfammation-related biomarker that has attracted attention is the triggering receptor Neuroinfammation‑related biomarkers expressed on myeloid cells 2 (TREM2), which has an Infammatory processes play important roles in the arguably controversial role in AD development. Tis pathogenesis of AD. Te activation of glial cells, the resi- transmembrane protein belongs to the immunoglobulin dent immune cells of the CNS, is well described for dif- family and is expressed in microglial cells [77]. TREM2 ferent neurodegenerative diseases, especially AD [65, 66]. is involved in many biological processes, such as migra- Historically, it was accepted that neuroinfammation, ini- tion, proliferation, cytokine release, APOE binding, or tiated primarily as a reaction to Aβ and p-tau neurotoxic- sealing of Aβ plaques [29, 68]. Moreover, a rare missense ity, triggered the production of neurotoxic molecules (e.g. mutation, predicted to lead to an R47H substitution in ROS, glutamate or infammatory cytokines). Tis pat- TREM2, has been linked to a signifcant risk of AD in tern is reproduced in a continuous molecular feedback Iceland [78]. It was also reported that the soluble ecto- loop, which has been described to be more pronounced domain of TREM2 (sTREM2) is released into the extra- in late stages of AD [67]. However, recent evidence also cellular space and may serve as a CSF biomarker [29]. reveals the neuroprotective role of microglia and astro- However, to the best of our knowledge, the biological glia in earlier stages of AD development [67, 68]. When role of sTREM2 is unclear, particularly in neuroinfam- the deposition of Aβ plaques appears, glial cells create an mation. Some studies have shown that hyperstimulation immune barrier that surrounds and isolates these senile of the TREM2 pathway correlates to the attenuation of plaques. Tis process protects the axons adjacent to Aβ microglial activation [79–81]. However, further studies plaques from Aβ neurotoxicity [50, 60]. Recent genetic are needed to provide frm mechanistic evidence. studies have found correlations between the defciency Taken together, most of these infammation-related of microglia encapsulation toward Aβ deposits and an biomarkers are not specifc to AD and have thus been increased risk of late-onset AD [53, 58]. In addition, an proposed as “neuroinfammation-tracking assistants” inverse correlation between neuroinfammation in the that can provide a more solid diagnosis together with brain cortex and plasmatic NfL levels has been reported specifc AD biomarkers. Additionally, they could be used [69, 70]. In normal aging, serum levels of NfL show a to identify AD patients who would beneft from novel nonlinear increase from 60 years old in both males and microglia-targeted treatments. females [71]. However, low plasma NfL levels are associ- ated with raised cortical microglial activation, suggesting Synaptic dysfunction‑related biomarkers that infammation acts to protect prodromal AD [70]. Synaptic degeneration is another hallmark of early AD Consequently, neuroinfammation biomarkers may be pathology and appears to closely correlate with cognitive of great interest for the early diagnosis of preclinical AD. decline [82]. Tis event is associated, in turn, with the YKL-40, also known as human cartilage glycoprotein 39 neurotoxic efects of Aβ and tau species and glutamater- and predominantly related to astroglial proteins, is one gic excitotoxicity, which lead to alterations in axonal of the most studied neuroinfammation-related biomark- transport that later promote dendritic alterations and ers [72]. YKL-40 is not only upregulated in AD, but in eventually neuronal loss [83]. Many molecules involved other diseases in which infammation plays an important in synaptic degeneration have been identifed as poten- role [72]. Longitudinal studies and recent meta-analyses tial biomarkers and have been divided into pre- and post- have reported that CSF YKL-40 levels are higher in AD synaptic biomarkers. patients compared with controls, increase throughout Growth-associated protein 43 (GAP-43) belongs to the disease progression and are positively correlated the group of potential presynaptic biomarkers [27, 29]. with neuronal-injury biomarkers, mainly in the preclini- Tis protein is involved in synaptogenesis and neuronal cal stages of AD [30, 64]. However, YKL-40 has not yet development and is mainly expressed in the cortex, hip- shown consistent results in blood samples. pocampus, olfactory bulb, and cerebellum. In fact, three Cano et al. J Nanobiotechnol (2021) 19:122 Page 7 of 30

decades ago, the correlation between an increased den- dysfunction in AD. Te scientifc evidence to date has sity of neurons containing NFTs and decreased levels of not clarifed the predictive value of blood Ng as an AD GAP-43 in the brain and cerebellar cortex of AD patients synaptic-damage biomarker. In addition, further studies had already been reported [84]. More recently, the signif- are needed to defne the specifc range of Ng values for icant elevation of CSF GAP-43 in AD patients has been diagnoses of diferent stages of AD. reported [85]. Interestingly, this increase also correlates with the amount of Aβ and p-tau found in the cortex, BBB dysfunction‑related biomarkers amygdala and hippocampal structures [85]. Tese fnd- ings indicate that GAP-43 is a promising biomarker for Cerebrovascular disease and AD share multiple risk fac- AD in CSF. However, its usefulness in blood samples has tors. Substantial evidence suggests that vascular dysfunc- not yet been well described. tion is the earliest event in the pathogenic development Synaptosomal-associated-protein-25 (SNAP-25) is a of late-onset AD [94]. Vascular dysfunction, associated newly discovered potential biomarker related to pre-syn- with aging, causes reduced oxygen, glucose, and nutri- aptic damage. Recent studies reported that CSF SNAP-25 ent supply to the brain, which directly damages not only increases in MCI and AD, which is associated with the the parenchymal cells, but also the BBB structure. Tis in rate of hippocampal atrophy and cognitive decline [86]. turn promotes the overproduction of ROS, nitric oxide, Moreover, CSF SNAP-25 levels are also substantially and infammatory cytokines in response to the neuro- higher in MCI patients who are APOE ε4 carriers, com- toxic efects of vascular dysfunction, which contributes to pared to non-carriers. Tese fndings suggest the poten- a vicious circle of global neurotoxicity afecting both BBB tial relevance of SNAP-25 to predict the conversion from dysfunction and AD pathogenesis [94]. Te standard bio- MCI to AD [87]. SNAP-25 may even have relevance as marker of BBB dysfunction measured in clinical practice a blood biomarker. Agliardi et al. showed that SNAP-25 is the CSF/serum ratio of albumin [95]. Although the role can be detected in serum, where it appears in neuron- of albumin and its predictive value are still controversial, derived plasma exosomes, which are extracellular vesicles many eforts have been made to exploit the usefulness involved in intercellular communication. Serum levels of of this biomarker. Other molecules have also emerged SNAP-25 are lower in AD patients compared to healthy as potential biomarkers of BBB-dysfunction, such as controls [88]. Although further studies are needed, these VCAM-1, ICAM-1, MMPs, VEGF, PIGF, sPDGFR-β, and fndings indicate the potential use of SNAP-25 as a pre- tight junction proteins (such as claudins and occludin) synaptic injury-related biomarker. [25, 29]. However, further studies are needed to deter- Synaptotagmin-1 was one of the frst proteins detected mine their predictive value in CSF, as well as their cor- in the CSF of early-onset AD patients [89]. Synaptotag- relation in peripheral blood. min-1 is a pre-synaptic vesicle protein involved in the maintenance of correct synaptic transmission and cog- Lipid metabolism‑related biomarkers nitive function. Recent studies reported increased CSF Brain tissue is highly enriched in lipids. Lipid metabolism Synaptotagmin-1 levels in both MCI and AD patients is decisive for the synaptic activity, neuronal survival, and compared to healthy controls [90], indicating that Synap- immune responses of glial cells, and AD pathogenesis is totagmin-1 is a potential CSF biomarker of both AD and accompanied by continuous changes in brain lipid pat- conversion from MCI to AD. terns [96]. Tus, proteins related to lipid transport and Neurogranin (Ng) is a post-synaptic protein that regu- metabolism in the CNS have been proposed as poten- lates calcium signaling and synaptic plasticity mainly tial biomarkers of AD pathogenesis. Undoubtedly, ApoE found in the dendritic spines of the amygdala, hippocam- is the prototypical protein involved in lipid homeostasis pus, and caudoputamen [29, 82]. High CSF Ng levels have and AD development. Te expression of the ApoE ɛ4 been reported to predict future cognitive decline specifc allele is well known as the strongest genetic risk factor to AD pathogenesis, with better specifcity than p-tau for AD [27, 97]. Terefore, CSF ApoE levels have been [91]. Furthermore, several studies have shown elevated investigated as AD progression biomarkers and for the CSF Ng levels in MCI and prodromal AD patients [91, diferential diagnosis of AD and other neurodegenera- 92]. Interestingly, a recent study carried out by Kirsebom tive disorders [27]. However, further studies are needed et al. identifed an elevated CSF Ng/BACE1 ratio as a pre- to validate its potential predictive value in blood sam- dictor of very early cognitive decline in AD. Specifcally, ples. Similarly, heart fatty acid-binding protein (FABP3), elevated Ng/BACE1 is associated with lower hippocam- ApoH, and vitamin D-binding protein are related to pal and amygdala volumes of MCI patients compared to lipid metabolism and increased levels of them have been healthy controls [93]. All these fndings highlight Ng as found in the CSF of AD patients. Moreover, these have one of the most relevant biomarkers related for synaptic been proposed for the diferential diagnosis of AD from Cano et al. J Nanobiotechnol (2021) 19:122 Page 8 of 30

Lewy body dementia, Parkinson’s disease, and other disease [28, 101]. α-secretase is the main enzyme in amy- dementias [27]. loid-precursor protein (APP) metabolism and its action is followed by that of γ-secretase in physiological condi- Neurotoxins clearance‑related biomarkers tions. Amyloidogenic processing occurs when an alter- Te clearance of toxic metabolites from the brain is nate enzyme, β-secretase, acts instead of α-secretase essential for healthy function. Since the dysregula- [102]. Generally, Aβ-based therapies target several tion of clearance mechanisms has been identifed as a aspects of APP metabolism [101]. Likewise, Aβ-binding direct cause of AD development, proteins related to the to several receptors has been related with some of its removal of cerebral metabolic waste, Aβ and p-tau pep- neurotoxic mechanisms (Table 1) [103]. Tus, thera- tides, and reactive oxidative species have been suggested peutic targeting to these proteins has been proposed to as candidate biomarkers [27, 98]. Clusterin, Orexin and decrease amyloidogenic APP processing and Aβ-related Transthyretin, which are involved in the clearance of par- toxicity. tially unfolded proteins and Aβ peptides, have thus been In this context, recent pre-clinical studies have sug- described as potential biomarkers [27]. Interestingly, gested that epigallocatechin-gallate (EGCG), the most clusterin, also called APOJ, is also related to lipid trans- abundant polyphenol in green tea, induces α-secretase port, infammation, and chaperone activities. Similarly, activity and improves non-amyloidogenic APP pro- LAMP-1, carboxypeptidase E, cystatin C, and ubiqui- cessing [104]. Likewise, the efect of a one-year treat- tin CSF levels are increased in patients with AD and are ment with EGCG on cognitive Aβ biomarkers, as well as emerging as potential neurotoxin clearance-related bio- metabolomics, microbiota, saliva, plasma, and urine, is markers [27, 99]. being evaluated in a randomized, double-blind clinical trial in 200 subjects (NCT03978052) Tis study aims to Metal ion homeostasis‑related biomarkers demonstrate the potential prevention of cognitive decline Metal ions have been widely described as potential tar- in patients with positive ApoEε4 with subjective cogni- gets for the diagnosis and treatment of AD. Te abnormal tive decline after a multimodal intervention with EGCG accumulation of metal ions, like zinc, copper, and iron, [105]. Te estimated study completion date is September in the brain has been closely related to the overproduc- 2021, when the frst results will reveal the potential of tion of Aβ peptide and p-tau, and the accumulation of this drug for AD. senile plaques and NFTs [100]. Likewise, abnormalities in Etazolate is another compound that has been shown to metal-binding proteins are a key factor in promoting the possess neuroprotective efects in AD. It is a pyrazolopyr- erroneous distribution and deposition of metal ions in idine with anxiolytic-like properties that selectively mod- the brain [100]. Some of the mechanisms by which metal ulates the ­GABAA receptor. Marcade et al. demonstrated ions promote these abnormalities are the induction of that Etazolate also promotes neuroprotection via the oxidative stress, autophagy dysfunctions, the disruption α-secretase pathway, leading to an induction of sAPPα, of endoplasmic reticulum and mitochondria structures, which is a neurotrophic and precognitive molecule [106]. activation of β- or γ-secretases, inhibition of α-secretase, Tus, modulators of the ­GABAA receptors could ofer a and activation of protein kinases such as cyclin- promising opportunity for the treatment of AD. dependent protein kinase-5 (CDK5), glycogen synthase Te active enantiomer of phenserine, which directly kinase-3β (GSK-3β), mitogen-activated protein kinases decreases levels of APP by interacting with APP (MAPKs), etc. [100]. Moreover, all these alterations mRNA, has also been found to be efective in enhanc- enhance the abnormal brain deposition of metal ions and ing α-secretase and AChE activity [4]. However, other further dysregulate their homeostasis. Terefore, these secretase-targeting drugs have not succeeded. For metal ions have been proposed as potential biomarkers instance, tarenfurbil, the active enantiomer of furbi- for tracking AD progression. Likewise, adjusting metal profen, ELND006 and Semagacestat are all γ-secretase balance may be a potential treatment for AD pathologies inhibitors developed to reduce Aβ levels that failed in and is a promising path for future research. clinical trials because of their signifcant adverse efects, and the absence of results extrapolatable from pre-clini- Molecular strategies for the therapeutic approach cal models to patients [4]. to Alzheimer’s disease Modulating Aβ transport is another therapeutic Amyloid‑β strategies approach to AD. Apolipoproteins have an important As explained above, the overproduction and accumula- role in Aβ transport and metabolism since they regu- tion of Aβ lead to several neuropathological processes late the movement of Aβ peptides between the periph- that trigger neuronal death, which translates into the ery and CNS. ApoEε4 increases the passage of Aβ from memory loss and cognitive disorders typical of this blood to the brain through the low-density lipoprotein Cano et al. J Nanobiotechnol (2021) 19:122 Page 9 of 30

Table 1 Main receptors and their implications in Aβ binding-mediated neurotoxic efects [103] Receptor Localization Proposed mechanisms

NMDAR Postsynaptically located on dendrites and dendritic spines Impairment of NMDAR activity: removal from the cell surface and triggering of synaptic depression signalling pathways Increase of NMDAR function: AβOs induce neuronal oxidative stress through an NMDAR-dependent mechanism AβOs bind to NMDAR excessive activation of NMDAR infow of 2 → → Ca­ + to neurons excitotoxicity → AMPAR Hippocampal pyramidal neurons and dendritic spines AβOs synaptic dysfunction by inducing calcineurin-dependent internalization→ of AMPAR PrPC Brain neurons and spinal cord Initial interaction of AβOs with PrPC on the neuronal surface which leads to: Disturbed regulation of BACE1 activity Inhibition of elongation of Aβ fbrils 2 Intracellular ­Ca + increase in neurons via the complex PrPC- mGluR5 impairment of synaptic plasticity → mGluR5 Hypothalamus and cortex Complexes of AβOs with PrPC generate mGluR5-mediated infux of 2 Ca­ + in neurons excitotoxicity AβO-PrPC-mGluR5→ complexes signalling pathway involved in den‑ dritic spine loss β2ARs Locus coeruleus, hippocampus and cortex AβOs induce the degradation of β2ARs, which leads to: Enhanced γ-secretase activity Aβ plaque formation Reduction of neurogenesis → Reduction of the levels of synapse-associated proteins such as synap‑ tophysin, synapsin 1, and PSD-95 α7nAChR Septo-hippocampal region and cortical neurons Aβ42 binds to α7nAChR loss of cholinergic neurons in the brain receptor internalization→ and intracellular accumulation of Aβ → IR Choroid plexus, olfactory bulb and regions of the striatum and AβOs bind to neuronal IR impaired insulin signalling and brain cerebral cortex insulin resistance elevated→ Aβ production and reduced AβO clearance Aβ deposits→ in the brain neuronal damage → → p75NTR White matter brain regions and spinal cord AβOs bind to membrane p75NTR formation of annular amyloid pores and ion channels induction→ of aberrant cytoskeletal changes in dendritic spines→ AβOs bind to IGF-1R phosphorylation of IGF-1R induced p75NTR expression→ cell death by fbrillary form→ of Aβ → ILR Hippocampus and surface of B lymphocytes, dendritic cells, natural AβOs bind to PirB impartment of synaptic plasticity disruption killer cells, macrophages, granulocytes, mast cells, etc of hippocampal →long-term potentiation Aβ-induced→ defcits of memory → AβOs bind to FcγRIIb AβO-induced inhibition of long-term poten‑ tiation Aβ-mediated→ neuronal dysfunction → TREM2 Surface of immune cells of myeloid origin AD-associated TREM2 mutations reduction of AβOs binding and → degradation of Aβ microglial depolarization, induction of ­K+ current into cells as→ well as increased cytokine expression and secretion, cells migration, proliferation, apoptosis, and morphologi‑ cal changes of microglia Eph4A Hippocampal neurons AβOs reduce Eph receptor expression, promote its endocytosis and EphB2 its degradation in the proteasome, which leads to: Loss of dendritic spine Synaptic dysfunction Increase of synaptoneurosomes Impairment of NMDAR functioning and cognitive defcits RAGE Blood–brain barrier Expression of RAGE is increased in the AD brain RAGE is responsi‑ ble of Aβ infux from plasma to BBB increase→ of free Aβ fraction in plasma → LPR2 Choroid plexus epithelium and ependymal cells covering the brain Aβ alone did not bind directly to LRP-2, whereas complexes of Aβ-40 ventricles with ApoJ are able to react with LRP-2 clearance of Aβ → VDR Broadly expressed in all brain regions 1,25-(OH)2D3 binds to VDR increase the expression of amyloid transporters (i.e. LRP-1) → increase of transport of Aβ across the BBB Aβ clearance → → Cano et al. J Nanobiotechnol (2021) 19:122 Page 10 of 30

Table 1 (continued) Receptor Localization Proposed mechanisms SIRT1 Predominantly located in the nucleus, but also in the cytosol of SIRT1 defciency has been described to be responsible for the neurons of the hippocampus and hypothalamus increased risk of insulin resistance, obesity and diabetes, which in turn are risk factors of AD SIRT1 defciency has been described to be involve in the reduction of normal cognitive function and synaptic plasticity Reduction of SIRT1 reduction of α-secretase activity enhance‑ ment of amyloidogenic→ processing of APP → AβOs, amyloid-β oligopeptides; α7nAChR, Acetylcholine Receptor; AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; β2ARs, β2-Adrenergic Receptors; LPR2, lipoprotein-related protein 2; mGluR5, Metabotropic Glutamate Receptor 5; EphA4, EphB2, Tyrosine Kinase Ephrin Receptors; FcγRIIb, Fragment crystallizable gamma receptor II b; IGF-1R, insulin-like growth factor 1 receptor; ILR, Immunoglobulin-Like Receptors; IR, Insulin Receptor; NMDAR, N-methyl-D- aspartate receptor; p75NTR, p75 Neurotrophin Receptor; PirB, paired immunoglobulin-like receptor B: PrP­ C, Cellular Prion Protein; RAGE, Receptor for Advanced Glycation Endproducts; SIRT 1, Sirtuin 1; TREM2, Triggering Receptor Expressed on Myeloid Cells 2; VDR, vitamin D receptor receptor-related protein (LRP) [107, 108]. Te periph- of disease progression in a multicenter, randomized, eral administration of soluble LRP has been proposed blinded and placebo-controlled, parallel-group, phase as a promising treatment for AD by increasing Aβ efux IIb/III clinical trial (AMBAR) of mild-to-moderate AD from the brain to peripheral blood [109]. Similarly, patients (NCT01561053) [112, 113]. Te molecular basis increasing the Aβ clearance has been studied as a thera- of this innovative therapeutic approach was the uptake peutic option for AD. Some proteases, such as metallo- and clearance of blood Aβ by replaced albumin, thereby proteinase 9, neprilysin, and insulin-degrading enzyme, promoting the transport of Aβ peptides from CSF to can degrade Aβ plaques. Te levels of these enzymes plasma, which in turn reduces the Aβ burden by restor- decline in AD and this could contribute to Aβ accumula- ing the normal balance of Aβ between brain and blood. tion and senile plaque formation. Te activation of these Te trial obtained promising results in terms of disease types of enzymes has also been proposed as a therapeutic progression, indicating that this approach is an important approach to AD [109]. development in AD therapeutics [114]. Terapeutically decreasing Aβ aggregation is one of the most explored routes to interfere clinically with AD Tau‑based strategies progression. Drugs like tramiprosate, EGCG, ELND005, Targeting AD as a tauopathy is another main therapeutic and melatonin have been studied in pre-clinical and clini- approach. Soluble tau is found in neuronal cells and plays cal trials because of their demonstrated inhibition of the a dominant role in axonal growth and neuronal develop- aggregation of Aβ peptides and dissolution of pre-formed ment [115]. Its importance lies in its role in regulating fbrils [4]. Likewise, the use of monoclonal antibodies and stabilizing microtubules, essential structures of the against Aβ aggregation has been explored. For instance, cell cytoskeleton. While under physiological conditions, aducanumab has been shown to enter the brain, bind the phosphorylation and de-phosphorylation of tau are parenchymal Aβ, and reduce soluble and insoluble Aβ in equilibrium, thus maintaining its ability to bind to in a dose-dependent manner in both transgenic mouse microtubules; in pathological situations, its hyperphos- models and patients with prodromal or mild AD [110]. phorylation generates insoluble flaments in the form of Aducanumab is currently being investigated in a phase III tangles, which leads to synaptic dysfunction and neu- clinical trial (NCT01677572) that is expected to provide ronal degeneration [115]. Several studies have also shown compelling support for the amyloid hypothesis. How- a relationship between the amyloidogenic pathway and ever, the FDA’s advisory committee recently expressed tau, demonstrating that the acceleration of tau hyper- concern about the biostatistical and neurologic results of phosphorylation is promoted by soluble Aβ oligomers this trial. Moreover, experts agreed that the aducanumab and, in turn, p-tau enhances the formation and aggrega- efcacy data were weak and pointed to inconsistencies in tion of Aβ plaques [116]. the data that came from two futility-stopped Phase 3 tri- Terefore, targeting tau phosphorylation is another als and one Phase 1b trial. Tus, the committee deemed important strategy in AD therapeutics, in particular the the evidence premature for approval and recommended a inhibition of tau protein kinases to prevent tau phos- confrmatory trial. Te decision now rests with the FDA, phorylation and the concomitant microtubule instabil- which is predicted to decide by June 2021 [111]. ity. Of the tau protein kinases, glycogen synthase kinase Boada et al. investigated the efects of plasmapher- 3 (GSK3) has possibly aroused the most interest [117]. esis with albumin replacement, plus intravenous immu- Specifcally, valproate, lithium, tideglusib, cafeine, noglobulin and obtained promising results in terms and several other GSK3 inhibitors have been studied in Cano et al. J Nanobiotechnol (2021) 19:122 Page 11 of 30

pre-clinical and clinical trials as therapeutic candidates In terms of neurotransmission, other routes related to for AD tauopathy [4]. Similarly, the reduction of tau oli- the development of AD have recently come into thera- gomerization, prevention of microtubule stabilization, peutic focus, particularly the GABAergic system, the and enhancement of tau degradation are other strategies serotonin receptor, and the modulation of histaminergic currently being studied to improve AD therapy manage- and adenosine receptors [4]. However, further research is ment [4]. needed to clarify the roles of these systems in AD.

Oxidative stress Neurotransmission Oxidative stress crucially drives many disorders, espe- Te cholinergic system may be strongly afected in AD. cially those inherent to the CNS, where there is very high Specifcally, the degeneration of cholinergic neurons in oxygen consumption but normally low concentrations the basal anterior brain has been reported to play a pre- of antioxidant enzymes [124]. In 2001, Nunomura et al. dominant role in the progression of the disease [118]. reported the level of oxidative stress in the early stages Much like tau hyperphosphorylation, cholinergic defcit of AD [125]. Unexpectedly, oxidative damage decreases is also related to Aβ. More than two decades ago, Pit- with disease progression and the formation of senile tel et al. showed the potentiation of the non-amyloido- plaque and tau tangles, suggesting the existence of com- genic pathway by activating the cholinergic receptors of pensatory mechanisms. Some researchers have suggested the cerebral cortex and cerebellum and the consequent that the appearance of both senile plaque and tau tangles decrease in Aβ formation [119]. is enhanced with increasing oxidative stress. However, Te inhibition of acetylcholinesterase (AChE) causes an other authors have claimed that the participation of Aβ increase in acetylcholine in the synaptic space and, there- in the deregulation of the intracellular homeostasis of fore, an increase in cholinergic activity. Indeed, three Ca­ 2+ and diferent mitochondrial mechanisms instead of the four marketed AD drugs are inhibitors of AChE, lead to the appearance of oxidative processes [124]. namely Donepezil, Rivastigmine and Galantamine. Tey Tere is inconsistent evidence about the efect of exog- were approved by the FDA between 1998 and 2001, and enous antioxidants such as vitamins, carotenoids, phy- since their inception have been used in mild or moder- tochemicals, and synthetic compounds against already ate phases of the disease [120]. However, these drugs established oxidative stress damage. However, rutin, cur- have poor efectiveness, with patients continuing to show cumin, and melatonin, potent antioxidant compounds, progressive cognitive impairment, suggesting that these have been shown to possess several benefcial roles apart compounds have only palliative efects. In contrast, their from their powerful antioxidant efects, such as amyloid- adverse efects, due to peripheral cholinergic hyperac- disaggregating properties and anti-infammatory activity tivity, reduce patients’ adherence to the treatments and [126]. Likewise, boosting endogenous antioxidant activ- therefore limit their success [120, 121]. ity is another strategy for the oxidative stress approach AD neuronal pathogenesis also extends to the gluta- to AD. Te nuclear factor 2 (Nrf2) /antioxidant response matergic system, especially in later stages of the disease. element (ARE) cascade is the primary endogenous anti- At the central level, glutamate intervenes in most excita- oxidant pathway. Te translocation of Nrf2 from the tory signals and participates in several physiological pro- cytosol into the nucleus is blocked in AD. Tus, drugs cesses, such as neurogenesis, synaptic plasticity, memory, inducing the Nrf2/ARE pathway could be an interesting and learning [122]. Glutamatergic excitotoxicity is due approach for the treatment of AD [4]. to a massive infux of Ca­ 2+ ions. In AD, this toxicity is mainly due to the overstimulation of NMDARs caused Neuroinfammation by deposits of Aβ and tau tangles. Likewise, the increase Neuroinfammation is the physiological response of the in Aβ levels induces a decrease in glutamate reuptake by CNS immune system against molecular alterations in glial cells, which translates into an increase in neuronal the brain tissue. Growing evidence suggests that the death [123]. Memantine, a non-competitive NMDAR pathogenesis of AD is not restricted to neurons, but antagonist, was approved for AD treatment in 2003 by also involves immunity in the brain [66]. Tus, the acti- the FDA and is used in moderate to severe phases. Like vation of astrocytes and microglia, resident immune AChE inhibitors, it acts at a symptomatic level, reducing cells of the CNS, is a hallmark of neuroinfammation the neurotoxic efects caused by glutamatergic excito- that is observed in most neurodegenerative conditions, toxicity and improving memory and learning processes. including AD [127]. However, the underlying molecular Although its efectiveness is superior to that of AChE mechanisms remain unclear. In general, the activation inhibitors, it does not stop the disease progress or resolve of glial cells induces many biochemical and cytological the pathogenesis of AD [4]. changes, such as the production of ROS, the secretion of Cano et al. J Nanobiotechnol (2021) 19:122 Page 12 of 30

proinfammatory cytokines, and the degradation of neu- Metabolic alterations roprotective retinoids, thus endangering the surround- In recent years, several studies have focused on clarifying ing healthy neurons. Similarly, external factors, such as the relationship between dementia and metabolic disor- systemic infammation and obesity, likely interfere with ders such as diabetes, obesity, dyslipidemia, and hyper- immune processes in the brain and further promote dis- tension. Te alteration of the insulin pathway, a reduction ease progression [65]. Glial cells, mainly composed of in the expression of its receptors, an increase in free fatty astroglia, microglia, and oligodendroglia, become acti- acids and cholesterol, an increase in lipogenesis, and vated and adopt diferent gene expression profles in altered vascular function are some comorbidities related pathological conditions, which leads to a neuroinfam- to the appearance of AD [7]. All these processes alter matory response [128]. Tis response causes an overpro- the clearance of Aβ in brain tissue, stimulate the hyper- duction of pro-infammatory cytokines, which gives rise phosphorylation of tau, promote astrocyte and micro- to an increase of amyloidosis state, looser maintenance glia activation and the secretion of ROS, and, ultimately, of the myelin that surrounds CNS axons, an increase of exacerbate the signs and symptoms of AD [136]. Tis ROS, mitochondrial damage, and ER stress, among other highlights the importance of addressing both disorders efects. Neuroinfammation fnally contributes to the as a single entity and the need to elucidate the underly- neurotoxic processes that produce neuronal death in AD ing molecular connections to clarify the relationship [128]. between them and, therefore, their etiology. Tus, statins Epidemiological data show the protective efect of or antidiabetic drugs are emerging as potential, promis- anti-infammatory agents in neurodegenerative diseases. ing therapeutic candidates in AD management [137, 138]. Tus, the hypothesis linking neuroinfammation to the pathogenesis of AD has gained strength in recent years, Controlled drug delivery systems even suggesting that its onset occurs long before memory Until the mid-twentieth century, the most common impairment becomes clinically evident [129]. Tis has pharmaceutical formulations were tablets, capsules, and also promoted studies of NSAIDs in AD pre-clinical and syrups. Tese types of formulations meant an average clinical trials. In the context of AD NSAIDs may not only of 3–4 administrations per day for patients. Likewise, inhibit cyclooxygenase, but also target α-secretase or many drugs with limited solubility and bioavailability had maintain ­Ca2+ homeostasis [4]. However, although epi- restricted use in the clinic, regardless of the therapeutic demiological and observational studies have highlighted potential of the active molecule. Controlled drug delivery the benefcial efect of NSAIDs in reducing the symptoms systems address many of these obstacles. and progression of AD, randomized clinical trials and In 1952, Smith Klein Beecham designed the frst con- meta-analyses have failed to corroborate this signifcantly trolled release system (CRS) called “Spansule® technol- [130]. ogy” [139]. Tis resulted in the frst generation (1G) of CRS, which focused on the control of release kinetics. Mitochondria damage approach Until the 1980s, 1G CRS were most productive, with a Te implications of mitochondrial dysfunction, mainly large number of new formulations with easy adminis- caused by ROS overproduction have also recently been tration (mainly oral and transdermal routes) [140]. Te described in the pathogenesis of AD [131]. Controlling second generation (2G) focused on developing more ROS production is considered even more promising than advanced systems, such as CRS of intelligent poly- traditional antioxidant strategies, which only act against mers sensitive to the environment, zero-order releases, accumulated ROS. Some molecules that have been stud- depot formulations aimed at very long administrations ied in terms of mitochondrial anti-oxidants are coenzyme (months) or hydrogels. Tis generation was not as pro- Q10, L-carnitine, triphenylphosphonium, and lipoic acid ductive as the 1G in terms of the number of formulations [132]. Te latter also increases acetylcholine production that entered into clinical practice, due to the complexity and down-regulates the expression of pro-infammatory of development presented by these new systems [139]. cytokines [133]. Dimebon, an anti-histaminic drug, was It was not until 2010 that the third generation of CRS also evaluated for its activity towards mitochondria and (3G) emerged. 3G CRS comprise present and future shown to block mitochondrial permeability transition developments to improve the clinical problems noted pore opening, which translates to protection against with 2G CRS. Eforts have focused on developing CRS cellular dysfunction and apoptosis processes [134]. with more predictable kinetics through in vitro methods, However, the clinical translation of mitochondrial thera- selective releases, such as longer-lasting systems and easy peutics has not yet occurred [135] and further studies are administration, and, overcoming the physicochemical needed to elucidate the specifc role of mitochondria in and biological barriers that were not breached by 2G CRS AD development. [140]. Currently, the use of diferent nanocarriers is one Cano et al. J Nanobiotechnol (2021) 19:122 Page 13 of 30

of the most widely used state-of-the-art technologies to (MRI) were the frst attempt to use this technology for overcome the disadvantages of available drugs and ofer AD diagnosis. Tis type of nanovehicle, which could be new theragnostic approaches, especially in those diseases surface modifed with specifc antibodies to recognize whose afected organs are difcult to access, such as AD. AD biomarkers, has shown elevated targeting in in vitro, Te main properties that a device should possess to be in vivo, and ex vivo AD models. Tus, SPIONs coated a CRS are: (i) release the drug at a predetermined rate; (ii) with ganglioside carbohydrate (sialic acid), Aβ1-42 anti- can be administered locally or systemically; (iii) remain in body, and curcumin are MRI agents used to identify Aβ the body for a specifed period of time; (iv) target the car- plaques [144, 145]. Similarly, enhanced diagnosis was ried drug to the specifc site of action [141]. In addition, provided by liposomes modifed with gadolinium and due to Fick’s law, difusion is the mechanism that governs ET6-21, an amyloid-targeting ligand, compared to con- the controlled release of a drug and depends on the aque- ventional MRI in an in vivo model of AD [146]. Te pre- ous solubility, ionization, PKa, stability, partition coef- ferred administration route for these kinds of diagnostic cient, and molecular weight of the drug [141]. nanocarriers is intranasal since it is non-invasive, allows Te general classifcation of CRS is based on the mech- penetration across the BBB, and avoids systemic adverse anisms that govern the release process and the entrap- efects. A hybrid surface-modifed graphene oxide with ment of the drug [142]. Tus, the main types of CRS are: both tau and Aβ antibodies linked to magnetic core– (i) difusion-controlled CRS, which act as reservoirs and plasmonic coat nanomaterials was also developed. Tis monolithic systems; (ii) water penetration-controlled nanodevice readily detected Aβ and tau proteins in an CRS, where osmotic and swelling processes control the in vitro model of AD [143]. release of the drug; (iii) chemically-controlled CRS, Another diagnostic application of nanocarriers is the which can be designed as biodegradable reservoirs and specifc binding of disease-specifc proteins. As explained monolithic systems or biodegradable polymer backbones above, CFS and plasma show some of the molecular with pendant drugs, where the release ratio is man- alterations that occur in the brain parenchyma of AD aged by chemical reactions between the device and the patients, with levels of Aβ peptides, tau, NfL, and SNAP- medium; (iv) responsive CRS, which act as physically or 25, among others, increased in both. Nanocarriers could chemically responsive systems, mechanical-, magnetic-, act as uptake tools to monitor these biomarkers, thus or ultrasound-responsive systems, and biochemically- contributing to a solid diagnosis of the stage and severity responsive self-regulated systems; (v) particulate CRS, of AD pathogenesis [143]. which can be designed as microparticles, liposome sys- tems, and polymer-drug conjugates, among others [142]. Te most important CRS developed for nanomedicine Nanocarriers as therapeutic tools in AD applications are liposomes, micelles, nanoparticles, car- Te complexity of the therapeutic approach to AD lies bon nanotubes, graphene sheets, hydrogels, dendrimers, not only in its unknown etiology and the lack of available polyelectrolyte complex, and quantum dots. Table 2 sum- efective treatments, but also in the restricted access to marizes the main characteristics of all CRS developed for the afected organ. Tree biological barriers control the biomedical applications [141, 142]. passage of most substances into the brain and thus sig- nifcantly limit drug access: the BBB, blood-CSF barrier Nanotechnologic strategies for Alzheimer’s disease (BCSFB), and ependymal barrier [143]. For orally admin- Nanocarriers as diagnostic tools in AD istered drugs, the gastrointestinal barrier and hepatic One of the main problems of AD is its late diagnosis due frst-pass efect must also be added [147]. Likewise, clear- to the delayed manifestation of frst clinical symptoms ance mechanisms and efux pumps signifcantly reduce compared to the onset of its molecular and cellular mani- the half-life of drugs in the body, thereby contributing to festation in the brain. When the frst symptoms appear, the reduction of pharmacological efectiveness [148]. the neuronal damage is already established and irreversi- Apart from accessibility problems, drugs administered ble. Terefore, devices that can recognize AD biomarkers to the CNS, as well as other organs, must fulfll specifc in the early stages preceding memory loss and cognitive biopharmaceutical characteristics that confer high bio- decline have attracted much interest in clinical research. availability, but these are not always present. Although Historically, the frst clinical biomarker of AD was the Aβ their pharmacological activity might be relevant, many peptide of senile plaques, which accumulates in the gray drugs have physicochemical disadvantages, such as low matter of AD brains. Tus, nanodevices specifc for Aβ solubility, short stability, and high molecular weight, that have been designed for diagnostic purposes [143]. signifcantly reduce their bioavailability and, therefore, In this context, superparamagnetic iron oxide nanopar- their fnal therapeutic efectiveness [149]. Additional ticles (SPIONs) used with magnetic resonance imaging challenges that CNS therapies face include the presence Cano et al. J Nanobiotechnol (2021) 19:122 Page 14 of 30

Morphology dimensional printing reduction - 4 Layer fabrication - Layer - by tion Solvent evaporationSolvent dispersion Solvent Reverse phase evaporation Solvent evaporationSolvent Direct dilution Salting out Dialysis nanoprecipitation Flash Divergent synthesis Divergent synthesis Convergent Self - assembly synthesis Solvent evaporationSolvent Double emulsion Homogenization Salting out NaBH Laser vaporization Chemical vapor deposition discharge Arc Colloidal synthesis Colloidal synthesis Plasma Self - assembled synthesis dual injectionHigh temperature Solution spontaneous associa ‑ Simple PECs titration technique Three Layer Microfuidic - based fabrication Fabrication methods Fabrication Sol–Gel synthesis ‑ - soluble responsive hydro - responsive Metallo dendrimers Sensor applications QDs PECs, gels, Multilamellar Single compartment Macrovesicles Polymeric micelles Polymeric Lipid micelles Supermicelles Simple, Chiral, Micellar, Hybrid, Micellar, Chiral, Simple, Polymeric Lipid Metal Gold/Silver Magnetic Silica Imaging QDs, Drug deliveryImaging QDs, QDs, SWCNTs, MWCNTs SWCNTs, Nonstoichiometric water Stoichiometric insoluble PECs, colloidal stable PECs, Turbid coacervatesComplex PECs Temperature hydrogels, pH - responsive Electric hydrogels, - sensitive hydrogels - sensitive Ionic strength Types ‑ - like structure vesicular systems vesicular phobic shell architecture branched tree porus speheres cles with optical and electronic properties built from sp2 hybridized sp2 hybridized built from carbon atoms charged polyelectrolytes with polyelectrolytes charged but reversible strong polymers containing a large polymers containing a large amount of water Lipid based spherical shaped Hydrophilic inner core–HydroHydrophilic Macromolecules with a regularly with a regularly Macromolecules Nanocapsules, nanospheres, nanospheres, Nanocapsules, Nanosized semiconductor partiNanosized ‑ Rolled sheets of graphene ringsRolled sheets of graphene Polymeric matrix with oppositely Polymeric links electrostatic - linked Natural/ synthetic cross Structure 20 nm–10 µm 10–200 nm 20–400 nm 10–300 nm 5–50 nm 0.5–50 nm Ø 20–300 nm – Size ‑ Phosphatidyl choline, PEG, PVP, PEG, PVP, choline, Phosphatidyl PCL, PLA… PEOz, PEO, lesterol, Palmitate, Au, Si, Ag, Fe, Fe, Si, Ag, Au, Palmitate, lesterol, Cu, Pb… fbroin, SCMC… fbroin, PEG, PVA, PAAM… PEG, PVA, Cholesterol Phospholipids Phosphatidyl ethanolamine, ethanolamine, Phosphatidyl PAMAM, PPI PAMAM, CdSe, CdS, ZnS, PbS… CdS, CdSe, PLGA, PEG, PCL, Chitosan, Cho PEG, PCL, Chitosan, PLGA, Carbon atoms Chitosan, PGA, SDS, CMCG, PGA, SDS, CMCG, Chitosan, Fibrin, collagen, hyaluronic acid, acid, collagen, hyaluronic Fibrin, Material Diferent types and main characteristics drug delivery biomedical applications [ 141 , 142 ] of controlled Diferent for systems Liposomes Micelles Dendrimers Quantum dots Nanoparticles Carbon nanotubes Polyelectrolyte complexes Polyelectrolyte Hydrogels 2 Table drug delivery Controlled system oxide); poly (propylene PEO, glycol); (ethylene (ε-caprolactone); PEG, Poly poly (amido amine); PCL, Poly poly(acrylamide); PAMAM, Multiple-walled carbon nanotubes; PAAM, cashew gum; MWCNTs, carboxymethyl CMCG, SDS, cellulose; SCMC, sodium carboxymethyl pyrrolidone); poly (N-vinyl poly (lactic-co-glycolic); PVP, acid; PLA, poly (lactide); poly (2-ethyl-2-oxazoline); PGA, polygalacturonic PEOz, PLGA, PPI, poly(propylenemine); Single-walled carbon nanotubes SWCNTs, sodium dextran sulfate; Cano et al. J Nanobiotechnol (2021) 19:122 Page 15 of 30

of peripheral adverse efects and the difculty of fnding extract is highly enriched in alkaloids and tannins, which the therapeutic threshold and maintaining it over time. have powerful antioxidant efects. Te results showed To overcome all these obstacles, the therapeutic poten- that, compared to untreated controls, animals treated tial of drug-loaded nanocarriers in diferent neurode- with such LNPs exhibited decreased NFTs and Aβ depos- generative diseases has been explored [150]. Te high its, improved cognitive test scores, and increased bio- surface-to-volume ratio of nanocarriers and the possibil- markers of antioxidant activity in the brain homogenates. ity of surface functionalization with desired ligands are Similarly, Giacomeli et al. investigated the neuroprotec- the two main exploited characteristics of these devices tive efects of curcumin-loaded LNPs in a mice model for drug delivery to CNS [150]. In addition, Aβ target- of AD [158]. Te therapeutic potential of curcumin has ing has been the main objective of nanomedicine to date. been widely recognized for many diseases, but its low Tree methods from nanotechnology have been used to solubility and, consequently, reduced bioavailability limit target and modify senile plaques: (i) genetic regulation its pharmacological efect [159]. In this study, the authors and/or inhibition of the synthesis of Aβ peptide; (ii) inhi- developed a hybrid nanocarrier of a lipidic core with bition and/or delay of the Aβ nucleation dependent pro- a PLC coat to confer better solubility. Animals treated cess; (iii) clearance of already-formed Aβ plaques [143]. with this nanocarrier showed improved spatial memory Álvarez-Erviti et al. employed naturally inert exosome and reduced neuroinfammation biomarker levels in nanocarriers to deliver BACE1-siRNA into the brain serum and the hippocampus and cortex homogenates. and silence BACE expression, thus inhibiting the cleav- Pinheiro et al. combined natural products and LNPs. age of APP and, in turn, the overproduction of Aβ pep- Te bioactive load was quercetin, a favonoid present tide [151]. Recent fndings related to the most common in many vegetables and fruits with strong antioxidant nanovehicles in preclinical assays of AD are described in activity. Te authors went one step further and coated depth below. the LNPs with transferrin to enhance transport across the BBB [160]. Tese LNPs were found to be non-toxic Lipid‑based nanoparticles for Alzheimer’s disease for hCMEC/D3 cells, a frequently used in vitro model of Lipid-based NPs (LNPs) are among the most used con- human BBB, and to enhance the penetration of quercetin trolled drug delivery systems to target and deliver drugs through the BBB. Moreover, these LNPs inhibited Aβ1- to the brain. Teir safety, biocompatibility, and biodeg- 42 fbril formation in an in vitro model [161]. Similarly, radability make these systems an interesting choice for α-bisabolol, a sesquiterpene alcohol found in Matricaria drug nanocarriers. Furthermore, since their inherent chamomilla essential oil with demonstrated anti-plasmo- lipidic structure is similar to the lipid composition of dial, anti-microbial, anti-infammatory, anti-cancer and the BBB, their penetration into the brain by the transcel- anti-cholinesterase properties, has signifcantly reduced lular pathway is favored [152]. Te core of LPNs is com- bioavailability due to low solubility [162]. Sathya et al. monly composed of monoglycerides, triglycerides, fatty developed α-bisabolol-loaded Cholesterol LNPs and acids, fatty alcohols, mixtures and waxes [153]. Te most explored their neuroprotective efects in an Aβ-induced common LNPs are liposomes, solid lipid NPs (SLN), in vitro model of Neuro-2a cells, a fast growing mouse and nanostructured lipid carriers (NLCs), which mainly neuroblastoma cell line [163, 164]. Tese studies show difer in the disposition of the lipid layers, morphology, that such LNPs possess antioxidant potential, signif- loading capacity, average particle size, and electrokinetic cantly reduce β-secretase, caspase-3, and cholinesterase behavior [154]. Tis type of nanocarrier can encapsu- activities, inhibit Aβ aggregation, and protect Neuro- late both hydrophilic and hydrophobic drugs, which sig- 2a cells from Aβ-induced neurotoxicity by reducing the nifcantly increases their versatility [155]. However, their expression of Bax and inducing the expression of Bcl-2 low stability, complex fabrication method, unexpected proteins [163, 164]. polymorphic transitions, and drug release during storage Finally, Dara et al. recently investigated the therapeutic are some disadvantages that complicate large-scale pro- potential of erythropoietin (EPO) in AD. EPO is neuro- duction and, therefore, widespread use [154]. However, protective in several diseases, such as spinal cord injury, many eforts are being made to improve these issues and cerebral ischemia, epilepsy, and diabetic neuropathy the potential of LNPs as diagnostic and therapeutic tools [165]. Moreover, EPO contributes to neuronal survival in AD and other CNS diseases [156]. Recent preclinical and the regulation of neurogenesis in both Parkinson’s studies have produced interesting results by combining disease and AD [166, 167]. However, EPO has restricted state-of-the-art LNPs and AD animal models (Table 3). penetration through the BBB due to its hydrophilicity, Almuhayawi et al. designed pomegranate extract- rapid clearance from the bloodstream, and high molec- loaded LNPs to evaluate their efects in an aluminum ular weight. Encapsulated in SLNs, EPO reduced oxida- chloride-induced rat model of AD [157]. Pomegranate tive stress and Aβ deposition in the hippocampus more Cano et al. J Nanobiotechnol (2021) 19:122 Page 16 of 30 Refs al. [ 57 ] et al. Almuhayawi al. [ 164 ] et al. Sathya 161 ] [ et al. Pinheiro al. [ 165 ] Dara et al. al. [ 158 ] Giacomedi et al. al. [ 163 ] et al. Sathya ‑ ‑ - induced mice 3, cholinesterase - 3, cholinesterase 2a cells from Aβ - 2a cells from NPs signifcantly restore restore signifcantly - NPs the oxidative reduce - NPs the AD animal model object in novel tiveness of increase test, recognition biomarkers of antioxidant lipid peroxidation activity, ‑ reduction, and neurofbril lary tangles and the senile plaques reduction the production of free of free the production β - secretase, reduce radicals, caspase of activities and expression Bax, and induce expression of Bcl - 2 proteins hCMEC/D3 cells and perme the BBB than free more ate inhibit NPs Moreover, drug. of Aβ peptide fbril formation memory rats in treated those treated to compared EPO with free and Aβ ratio, ADP/ATP stress, plaque deposition in the hip efectively pocampus more EPO than the free cant neuroprotection against cant neuroprotection Aβ42 - induced behavioral changes and neurochemical in the AD model Neuro and induced neurotoxicity Aβ aggregation inhibited Results PE shows a protective efect in efect a protective PE shows ‑ the PE efec improve NPs NPs signifcantly suppress suppress signifcantly NPs NPs are not toxic for for not toxic are - NPs Quercetin EPO EPO ‑ signif display - NPs Curcumin NPs protect the protect α - bisabolol NPs - 2a cells - 2a cells mice In vitro/In vivo model In vivo vitro/In induced Wistar rats Wistar AlCl 3 - induced Aβ25–35 Neuro hCMEC/D3 cells induced Wistar Aβ42 - induced Wistar rats Aβ42 - induced Swiss Albino Aβ25–35 Neuro Admin. route Admin. v.o incubation Incubation i.p i.c.v Incubation Dose 50 mg/kg 5–10 μg/ml 10–30 µM 1250–2500 IU/Kg 1–10 mg/kg 5–10 μg/ml Surface modifcations – – Transferrin – PCL – ‑ clinical studies based on recent LNPs for the diagnosis and treatment of AD and treatment the diagnosis for LNPs clinical studies based on recent - ides, sorbitan‑ monostea ides, rate Lipid matrix Lecithin Stearic acid Cholesterol Cholesterol - 812 miglyol Cetyl palmitate GMS Caprylic/capric triglycer Cholesterol Selected pre relevant 3 Table molecule Loaded extract PCL, poly( ɛ -caprolactone); endothelial cells; pomegranate PE, Immortalized microvascular hCMEC/D3 cells, human cerebral Erythropoietin; monostearate; GMS, Glycerin EPO, PE α - bisabolol Quercetin EPO Curcumin α - bisabolol Cano et al. J Nanobiotechnol (2021) 19:122 Page 17 of 30

efciently in an Aβ42-induced Wistar rat model of AD and bioavailability have restricted its use in the food and [165]. Moreover, animals treated with EPO-LNPs exhib- pharmaceutical industries [175]. In this study, the authors ited increased spatial memory compared to free EPO- demonstrated that the developed nanocarrier possesses treated littermates. the optimal physicochemical characteristics for suitable in vivo administration. Te PNPs have good entrapment Polymeric‑based nanoparticles for Alzheimer’s efciency, sustained LT release, protection of LT integ- disease rity, biocompatibility with brain cellular models, and ef- Due to their versatility and ease of fabrication by difer- cient passage through an in vitro BBB [174]. Moreover, ent methods, polymeric NPs (PNPs) are among the most after intranasal administration in rats, LT-loaded PNPs used carriers in nanomedicine applications [168, 169]. accumulated in the brain and exhibited reduced toxicity Teir mean average size is between 10 and 1000 nm and and signifcant ROS scavenging activity [174]. Similarly, they can load high amounts of both hydrosoluble and Sathya et al. evaluated the therapeutic potential of phytol- hydrophobic drugs [170]. Depending on the polymer loaded PNPs in the regulation of the expression of AD- composition, which can be natural or synthetic, PNPs related genes and neuronal degeneration in both in vitro can have both positive and negative surface charges. Tis and in vivo models of AD [176]. As with LT, phytol is a characteristic signifcantly conditions their biological natural compound with several pharmacological proper- behavior, muco-adhesiveness, and penetration [171]. ties but poor solubility and low absorption, resulting in PNPs can be formulated as nanocapsules and nano- reduced bioavailability that limits its clinical use [177]. spheres. Nanocapsules possess a vesicular structure in In the in vitro Neuro-2a cell model, phytol-loaded PNPs which drugs are dissolved in a liquid core surrounded inhibit apoptosis-mediated cell death and cholinester- by the polymeric capsule. In contrast, nanospheres are ase activity. In a transgenic nematode AD model, these composed of a polymeric matrix in which drugs are dis- PNPs were found to increase chemotaxis and lifespan persed in the matrix gaps or adsorbed onto the sphere and reduce ROS production and Aβ deposition. Further- surface [172]. Te polymers most used to manufacture more, PNP treatment upregulated a gene involved in the PNPs are polylactide (PLA), poly(lactide-co-glycolide) longevity of nematodes, downregulated the expression of (PLGA), chitosan, polyethyleneimine (PEI), and poly-ε- several AD-associated genes, and reduced the expression caprolactone (PCL), all of which were approved by the of Aβ peptide at the protein level [176]. FDA for biomedical applications twenty years ago [173]. Cano et al. also studied natural-compound-loaded Tese kinds of vehicles have many important advan- PNPs for AD [178]. In this study, the selected molecule tages, such as their high loading capacity, controlled drug was EGCG (see also above), which has shown therapeutic release kinetics, surface modifcations for brain target- activity in many diseases, such aas breast cancer, diabe- ing, safety, biodegradability, biocompatibility, and easy tes mellitus, Down syndrome, and diferent neurodegen- elimination [168]. However, these systems also have some erative diseases, but its instability in water solutions and disadvantages that limit their use, especially the need for in vivo administrations reduce its efectiveness [179]. organic solvents during their fabrication. Nevertheless, Co-encapsulating EGCG into PEGylated PLGA NPs with their pharmacological potential has encouraged many ascorbic acid (AA) to prevent auto-oxidation produces a researchers to explore the potential of PNPs for the diag- nanosystem with signifcantly enhanced EGCG integrity, nosis and therapeutic management of diferent neuro- which is correlated with improved EGCG efectiveness in degenerative diseases [150]. Table 4 summarizes recent a variety of biological assays relevant to AD [178]. EGCG/ advances on the preclinical evaluation of state-of-the-art AA PNPs readily cross the BBB both in vitro and in vivo. PNPs in AD. Moreover, compared to free EGCG, EGCG/AA NPs Te lack of efective treatments for AD has led to an exhibit improved bioavailability and pharmacokinetic incessant search for new pharmacological alternatives. profle and, in an AD mouse model, improved memory Among these alternatives, natural compounds are emerg- learning processes, reduced cognitive decline, neuroin- ing as potential therapeutic drugs for several diseases, fammation, and Aβ plaque burden, and increased synap- such as cancer, diabetes, and cardiovascular and neuro- tic expression. degenerative diseases, including AD. Tus, Dhas et al. Taken together, these fndings highlight the relevance developed cationic biopolymer core/shell NPs loaded of natural compounds as promising therapeutic strate- with lutein (LT), a natural dietary carotenoid mainly gies in AD management. However, PNPs loaded with obtained from food such as green vegetables, eggs, and natural compounds have not only been studied as thera- corn [174]. Although this molecule has shown promis- peutics, but also for diagnostic purposes. In this regard, ing benefcial efects, such as anti-infammatory, anti- Ahlschwede et al. developed curcumin-loaded PLGA oxidant, and anti-cancer activity, its reduced solubility NPs functionalized with K16ApoE, a BBB penetration Cano et al. J Nanobiotechnol (2021) 19:122 Page 18 of 30 176 ] [ et al. Sathya al. [ 174 ] Dhas et al. 178 ] [ et al. Cano al. [ 185 ] Guo et al. Refs al. [ 180 ] et al. Ahlschwede al. [ 182 ] Jeon et al. al. [ 181 ] Liu et al. ‑ ‑ ‑ - 1 and hsp 4 upregu PNPs show higher binding afnity show either GT1b to - PNPs ‑ Aβ deposition and ROS produc and decrease PNPs Moreover, models of AD. tion in the vivo of AD the expression downregulate treatment ace genes viz Aβ, associated in the longevity the gene dnj - 14, involved late of Aβ the expression and reduce of nematodes, level peptide at the protein and demonstrated to possess signifcant ROS possess signifcant to and demonstrated activityscavenging without damaging the BBB integrity. NPs treat NPs the BBB integrity. without damaging Aβ plaque neuroinfammation, ment reduce soluble and insoluble Aβ42 peptide levels burden, and memory processes ganglioside receptor or brain capillaryganglioside receptor endothe the BBB - penetration increase cells, lial bEnd.3 enhance ROS efcacy, - targeting and neuron from ability microtubule and protect scavenging inhibit okadaic Aβ25 ‐ 35 - induced neurotoxicity, and neuronal acid - induced tau aggregation performance the cognitive of improve apoptosis, the tau phosphoryla- regulate down ‑ mice, treated transport axonal and attenuate promote tion level, microgliosis K16ApoE accumulation, neuroinfammation, neuronal loss neuronal accumulation, neuroinfammation, dysfunction and cognitive probe for the detection of Aβ monomers. In the detection of Aβ monomers. CSF for probe are NPs developed and various brain tissues of rats, the Aβ peptide and fuoresce, recognize able to its detection and quantifcation thus leading to PNPs increase the lifespan, chemotaxis behaviour chemotaxis behaviour the lifespan, increase PNPs NPs are highly deposited in brain following i.n. route i.n. route in brain following highly deposited are NPs vitro BBB the in vitro through penetrate efectively NPs spatial learning and enhance synapsis expression, TPL Results NPs improve BBB transcytosis coating with a by improve NPs detect Aβ plaques enhance MRI contrast to NPs vitro. Attenuation of Aβ Attenuation in vitro. Inhibition Aβ aggregation Developed NPs act as a ratiometric fuorescent Developed NPs - 2a cells - 5Y cells TNA2 cells - (CL2006, CL4176) (CL2006, SHSY 2650 cells RPMI BMVECs In vitro/in vivo model vivo In vitro/in Neuro Caenorhabditis elegans Caenorhabditis bEnd.3 cells bEnd.3 PC12 cells HT22 cells CTX 5XFAD mice 5XFAD Sprague Dawley rats Sprague Dawley AD rats APP/PS1 mice ICR mice Tg2576 mice Tg2576 CSF sample Incubation Incubation Admin. route Admin. Incubation Exposition Incubation i.v i.n incubation - NPs v.o N.A i.v 0–20 mM 15–500 μg/ml Dose 5–10 µg/ml 25, 50 and 100 μg/ml 10 μM 2.5 mg/kg 4 mg LT/kg 0.67 mg/ml 40 mg/kg/day 6–24 μg NPs/kg/day 100 µCi/100 µL Chitosan PEG Surface modifcations – TPL PEG – Eu(NO3)3 CuCl2 [Gd]DTPA Chitosan IgG4.1 K16ApoE clinical studies based on recent PNPs for the diagnosis and treatment of AD and treatment the diagnosis for PNPs clinical studies based on recent - PLGA PLGA Polymeric matrix Polymeric PLGA PLA PLGA Chitosan PLGA Selected pre relevant 4 Table disodium; guanosine monophosphate GMP, Epigallocatechin-3-gallate; EGCG; D-binding protein; Vitamin fuid; DBP, cerebrospinal carbon dots; CSF, CDs, endothelial cells; microvascular brain acid; BMVECs, AA, ascorbic Tet1 binding peptide and a neuron TGN peptide a BBB-penetrating TPL, fusion peptide comprising peptide; ROS, species; Reactive oxygen neuroprotective NAP, Lutein EGCG/AA Loaded molecule Loaded Phytol NAP DBP CDs GMP Curcumin Cano et al. J Nanobiotechnol (2021) 19:122 Page 19 of 30

peptide. Te authors aimed to create an efective tool for loss, and signifcantly reduced the cognitive impairment detecting cerebrovascular Aβ and treating cerebral amy- of treated transgenic mice. loid angiopathy, which is widely observed in AD devel- Finally, Guo et al. recently developed a PNP coated opment [180]. Using a quartz crystal microbalance with with both a BBB-penetrating ligand and a neuron-target- dissipation monitoring technology, the authors found ing ligand for carrying the neuroprotective peptide NAP. that developed PNPs efectively migrated from the blood NAP has been shown to provide neuroprotective activity fow to the vascular endothelium. As expected, K16ApoE against NMDA receptors and Aβ-mediated excitotoxic- coating signifcantly improved BBB transcytosis and pro- ity by interacting with glial and neuronal tubulin, thereby vided specifc MRI contrast to detect brain Aβ plaques. promoting microtubule assembly and the protection Moreover, K16ApoE-PNPs also showed specifc targeting of the neuronal cytoskeleton [185]. As explained above, of vasculotropic DutchAβ40 peptide that accumulated in the clinical application of NAP is restricted due to its the cerebral vasculature. Despite this study’s innovations, enzymatic degradation, inefective neuron targeting and in vitro and in vivo assays are still required to describe reduced half-life in the bloodstream [185]. Te encap- the therapeutic efects of curcumin-loaded K16ApoE- sulation of NAP in the nanocarrier improved its stabil- PNPs on memory impairment, cognitive decline, and ity in vivo. Likewise, coating the PNP’s surface efectively molecular alterations related to AD development. Liu increased the accumulation of developed NPs in brain et al. used nanotechnology for diagnosis. Te authors neurons. Te evaluation of its pharmacological proper- designed carbon dots (CDs) sensitized lanthanide inf- ties highlighted that NAP-PNPs promote the alleviation nite coordination polymer (ICP) NPs and a PNPs-based of oxidative stress, microtubule disruption, the attenu- ratiometric fuorescent probe mainly composed of CDs, ation of neuroinfammation, and the inhibition of tau 2+ Europium Nitrate (Eu(NO3)3), and ­Cu [181]. Te detec- aggregation and apoptosis processes. Likewise, a rescue tion technique was based on the competitive coordina- of the memory defcits and spatial learning in AD mice tion interaction of Cu­ 2+ between the guest CDs and Aβ was observed after PNP treatment. Moreover, treatment monomer. Briefy, in the absence of Aβ, the coordination with these PNPs also prevented tau hyper-phosphoryla- interaction between CDs and ­Cu2+ disrupts the antenna tion and restored axonal transport. efect, leading to the fuorescence quenching of Eu­ 3+. When developed PNPs contact an Aβ-enriched sample, Metal‑based nanoparticles for Alzheimer’s disease the stronger coordination between Aβ monomer and Metal-based NPs (MNPs) are the most relevant inorganic Cu­ 2+ restores the red fuorescence of ­Eu3+, leading to nanocarriers because of their widespread use in nano- the detection and quantifcation of Aβ peptide by fuo- medicine applications [186]. Tese vehicles usually have rescence. In this study, developed PNPs were exposed to an average size of 10 to 100 nm. Moreover, their high CSF/brain tissue samples of AD rats. Te authors showed surface area allows for coating with diferent molecules that their method is highly sensitive for the in vivo analy- (e.g., antibodies, genes, peptides), which lets them act sis of Aβ monomer, thereby demonstrating the utility of as biosensors and targeting tools. MNPs are commonly this ratiometric fuorescent probe. fabricated with gold, silver, iron, zinc, and copper, all of Some endogenous substances have been also described which provide diferent properties to the fnal nanocar- as pharmacological tools for AD. In that sense, Jeon et al. rier [187]. MNPs have demonstrated anti-microbial, anti- explored the therapeutic potential of Vitamin D-binding cancer, anti-infammatory, and anti-oxidant properties, protein (DBP) [182]. DBP is a glycoprotein that is highly among others [188]. expressed in a wide variety of cells and tissues and has Diferent methods are employed in the fabrication of been shown to play important roles in several physiologi- MNPs, with top-down methods (e.g., mechanical mill- cal processes. Likewise, several studies have highlighted ing, laser ablation, and ion sputtering) and bottom-up that DBP levels are altered in the serum and CSF of AD methods (e.g., solid-state methods, liquid-state synthe- patients and possess optimal properties for Aβ binding, sis methods, gas-phase methods, and electrochemical and thus peripheral clearance [183, 184]. However, pre- deposition). Note that the selected fabrication method vious studies have shown that plasma DBP has a rela- signifcantly conditions the fnal physicochemical prop- tively short half-life. Jeon et al. aimed to encapsulate DBP erties, morphology, and long-term stability of MNPs into PLGA matrices to prolong the presence of DBP in [189]. Green synthesis methods, an emerging trend of the bloodstream and evaluate their efectiveness in an nanotechnology, can overcome the toxicity problems, Aβ-overexpressing mice model of AD. Te developed high cost, and reaction complications of conventional nanocarrier inhibited the polymerization and accumula- fabrication methods, so they have attracted much recent tion of Aβ in both in vitro and in vivo models, amelio- interest. Green chemistry incorporates novel techniques rated Aβ-associated neuroinfammation and neuronal to reduce the health and environmental burdens of Cano et al. J Nanobiotechnol (2021) 19:122 Page 20 of 30

conventional techniques. Some of the most used green detectable and quantifable. Te exposure of this nanode- synthesis methods involve biological methods with dif- vice to both Aβ-enriched artifcial CSF and the human ferent microorganisms and their enzymes and using plant serum of AD patients demonstrated that the developed extracts. Te main advantages of these novel techniques detection method exhibits high sensitivity and specifc- are reduced costs, ease of scaling up for large-scale pro- ity and requires simple manipulation, less time expense, duction, and the complete elimination of energy, high lower expenses, and lower detection limits than those pressure and temperature, and toxic chemicals [190]. achieved by previously reported methods. Much current research focuses on MNPs because Te use of MNPs as therapeutic tools for AD is also of their multiple benefts and versatility for use in dis- important. For example, Liu et al. explored the therapeu- ease diagnosis and treatment, labeling optoelectronic tic potential of quercetin-modifed gold–palladium NPs recorded media, cosmetics, and sensor technology, to promote the clearance of intracellular Aβ by autophagy among others [189]. In addition, the magnetic properties induction and, consequently, reduce Aβ-induced neuro- of some of these MNPs can be exploited to enhance the toxicity [194]. As explained above, quercetin is a potent accumulation of these NPs in specifc organs since they natural antioxidant with limited ability to cross the BBB can be detected and manipulated by remote magnetic and easy elimination. Recent studies have shown that felds [191]. Table 5 displays relevant studies involving quercetin can accelerate the elimination of abnormal Aβ the latest smart MNPs and AD preclinical models. by enhancing the autophagy efect of brain cells [194]. Currently, using MNPs for diagnosis purposes is being Liu et al. reported that the developed MNPs are not widely studied. For example, da Silva et al. recently devel- toxic in both in vitro and in vivo and efectively cross oped a highly sensitive AChE electrochemical biosen- the in vitro BBB. Furthermore, they increased intracel- sor based on an electrode modifed with iron oxide NPs lular autophagy levels, promoted the degradation of and a deep eutectic solvent [192]. Enzyme-modifed autophagosomes, enhanced Aβ clearance in brain cell electrode sensors ofer a powerful tool for real-time cultures, and reduced Aβ-induced cytotoxicity. Similarly, diagnostics that overcomes the drawbacks of traditional Gao et al. recently evaluated multi-targeted chondroitin analytical approaches, such as time-consuming pro- sulfate /selenium MNPs and evaluated their protective cesses, high cost for measurements, and the need for efect against Aβ-induced neurotoxicity [195]. Chondroi- highly specialized staf [192]. Likewise, their capabil- tin sulfate is a glycosaminoglycan with several biologi- ity to promote the fast electron-transfer kinetics of iron cal functions, such as antioxidation, anti-infammation, oxide NPs means they had optimal properties for use and neuroprotection. Moreover, chondroitin sulfate is in the developed biosensor. Da Silva et al. immobilized involved in cell migration, neurogenesis, axon growth, AChE on a polymeric-based flm pre-formed in modifed synaptic plasticity, neuron regeneration, the inhibition of electrodes, which was composed of iron oxide NPs and Aβ fbril formation, and the blocking of Aβ-induced cell an acid eutectic dissolvent. Tey tested diferent acids to apoptosis [196]. Also, selenium is a potent antioxidant evaluate their infuence on the rate of growth and elec- that plays important roles in detoxifcation, the protec- trochemical properties of the polymer flms and fnally tion of the immune system, and the regulation of cellu- selected ­HNO3. Te authors used chronoamperometry lar redox homeostasis. Furthermore, it also inhibits the to investigate the catalytic activity of the developed bio- formation of Aβ plaques and degrades preformed Aβ sensor for ACh detection. After exposing the biosensor fbers [197]. In this new study [195], Gao et al. obtained to synthetic urine containing known concentrations of promising results. Teir MNPs could reduce damage to ACh, the authors recorded the typical current–time volt- the cytoskeleton and neuronal cells and oxidative stress, age curves and demonstrated that this MNPs-based bio- inhibit the aggregation of Aβ and protect cells from its sensor has high sensitivity, excellent reproducibility, and neurotoxicity, and attenuate the hyperphosphorylation long-term stability, with signifcant recovery ratios. In a of tau protein. Sonawane et al. also explored the efect related study, Xing et al. investigated the development of MNPs in Tau-related pathogenesis. Te authors bio- of an electrochemical technique for detecting Aβ based logically synthesized two diferent protein-capped on silver NPs aggregates as the redox reporters and a cel- MNPs, (i) iron oxide NPs and (ii) cadmium sulfde NPs, lular prion protein ­(PrP95-110) as the bio-receptor [193]. and evaluated their efectiveness in an in vitro model of Te fundamentals of their technique are based on the AD [198]. Te surface modifcation of these MNPs with oscillation of the electrochemical signal promoted by the proteins was performed to inhibit Tau protein aggrega- interaction of Aβ with the PrP­ 95-110-MNPs. Te specifc tion. To complete the biological synthesis of these carri- binding of the Aβ oligomers to the PrP peptide blocks ers, two fungal species were used, Fusarium oxysporum the aggregation of the MNPs’ main electro-transmitter, and Verticillium sp. Te composition and synthesis of which decreases the electrochemical signal that can be both NPs were diferent: iron oxide NPs were synthesized Cano et al. J Nanobiotechnol (2021) 19:122 Page 21 of 30 195 ] [ Gao et al. al. [ 192 ] da Silva et al. 201 ] [ et al. Cai Refs al. 2020 et al. Wahle ‑ - 3b SY5Y cells from SH - SY5Y cells from protect Aβ42 - induced cytotoxicity, okadaicdecrease acid - induced actin cytoskeleton In addition, instability, of ROS the levels decrease the lev ‑ increase and MDA, ‑ els of GSH - Px, and attenu the hyperphosphorylaate ‑ regulating tion of tau by of GSK the expression a highly sensitive AChE AChE a highly sensitive biosensor. electrochemical biosensor exhib novel The ‑ limit of detec its the lower that has been tion of ACh Itreported in the literature. good recoveries also shows in the determination of ACh in synthetic urine perform NIR in vivo and magnetic fuorescence of Aβ imaging resonance plaques in the brain of prevent transgenic mice, disag ‑ Aβ aggregation, Aβ preformed gregate fbrils a protective and play of on the toxicity efect cells human neuroblastoma Aβ42 induced by to the MNPs promotes promotes the MNPs to motor changes in forced performance and explora ‑ tory activity motor in mice, ApoE - / and 5xFAD respectively MNPs inhibit Aβ aggregation, inhibit Aβ aggregation, MNPs MNPs were used to fabricate used to were MNPs MNPs can simultaneously MNPs Results The inhalation exposure inhalation exposure The SH - SY5Y cells Synthetic urine APP/PS1 mice Aβ42 induced SH - SY5Y cells In vitro/in vivo Model vivo In vitro/in 5xFAD mice 5xFAD ApoE - / mice Incubation Exposition i.v Incubation Admin. route Admin. i.n for 3–5 h/day for 3 4 mg/m 1.45 µg/mL NA 0.2 mmol Fe/kg 10 ng/mL Dose Zirconium Chondroitin sulfate Chondroitin chitosan NIR Fluorescent probe NIR Fluorescent Surface modifcations clinical studies based on recent MNPs for the diagnosis and treatment of AD and treatment the diagnosis for MNPs clinical studies based on recent - Cerium dioxide Selenium Iron oxide Iron oxide Metal core Selected pre relevant – – – – 5 Table molecule Loaded Cano et al. J Nanobiotechnol (2021) 19:122 Page 22 of 30 al. [ 200 ] Zhang et al. al. [ 198 ] et al. Sonawane al. [ 194 ] Liu et al. Refs al. [ 193 ] Xing et al. - ‑ ‑ oxidative oxidative to recover mitochondrial recover to dysfunction, inhibit Aβ ‑ generation and aggrega tion, block synaptic and and apoptosis neuronal energy neuronal promote eliminat metabolism by and stress ing oxidative activating the anti - consequently pathway, ameliorating the cognitive impairment in AD mice ity of neuroblastoma cells ity of neuroblastoma dual propertiesand show of inhibition and disag ‑ fbrillar Tau of gregation aggregates SY5Y cells, promote the promote SH - SY5Y cells, fusion of autophagosomes accelerate and lysosomes, and the clearance of Aβ, SH - SY5Y cells from protect Aβ - induced cytotoxicity not toxic are MNPs damage. and in vivo both in vitro chemical method for the chemical method for detection of Aβ oligomer with a peptide as the bio MNPs and silver receptor as the redox aggregates reporters Developed MNPs are able are Developed MNPs MNPs do not afect the viabil ‑ do not afect MNPs MNPs activate autophagy of activate autophagy MNPs Results Authors reportAuthors an electro APP/PS1 mice N2a - SW cells Neuro2a cells Neuro2a Nude mice SH - SY5Y cells In vitro/in vivo Model vivo In vitro/in Aβ enriched CSF and serum Stereotaxic injection Stereotaxic incubation incubation i.v Incubation Admin. route Admin. Incubation μg/mL − 100 2µL of MNPs (0.5–2 mg/ml) 2µL of MNPs 6.25–25 µg/ml 5 1 mg/kg 5–40 µg/mL Dose 1.2 nM 95 - 110 proteins cillium sp. PrP Bioactive Hydrogen F. oxysporum and Verti - F. Polysorbate 80 Polysorbate Surface modifcations Silver Palladium Iron oxide sulfde Cadmium Gold / Palladium Metal core (continued) – – – Quercetin 5 Table molecule Loaded Cano et al. J Nanobiotechnol (2021) 19:122 Page 23 of 30 , cellular prion protein; prion protein; , cellular Refs 199 ] [ et al. Yang 95-110 PrP levels levels 2 O 2 H only upon exposure to con ‑ to only upon exposure ditions in which ­ are high, such as those in are inhibit NPs Aβ plaques. and Aβ40 aggregation, the cell membrane reduce disruption, microtubular and ROS defects - mediated apoptosis In this system, CQ is released is released In CQ this system, Results PC12 cells In vitro/in vivo Model vivo In vitro/in ICR mice Incubation Admin. route Admin. i.v 0.5 mg/mL Dose 5, 10, 20 mg/kg – Surface modifcations Gold Metal core (continued) CQ ROS, reactive oxygen species; STZ, streptozotocin oxygen ROS, reactive 5 Table molecule Loaded ­ dye; NIR, phenothiazine-based fuorescent malondialdehyde; near-infrared MDA, peroxidase; fuid; GSH-Px, glutathione cerebrospinal Clioquinol; CSF, metal chelator acetylcholine; CQ, Ach, Cano et al. J Nanobiotechnol (2021) 19:122 Page 24 of 30

extracellularly by the fungal species with transient ferro- coupled to a phenothiazine-based near-infrared (NIR) magnetic properties and capped with hydrolytic proteins. fuorescent dye for both the diagnosis and treatment In contrast, cadmium sulfde NPs were synthesized from of AD [201]. Tis compound was shown to efectively the extracellular sulfate-reducing enzymes secreted by inhibit the self-aggregation of Aβ and disaggregate pre- the fungus when provided with mixture of salts, so the formed Aβ fbrils, as well as exhibit high fuorescence surface proteins were composed of a mixture of four dif- enhancement upon binding to aggregate Aβ proteins ferent proteins that probably belonged to the group of [201]. Tanks to their small size, amyloidogenic proteins, these sulfate-reducing enzymes. Both nanocarriers did such as Aβ peptides, tend to be absorbed on the surface not afect the viability of cultured cells. Furthermore, of USPIONs, thereby reducing circulating Aβ. Likewise, both MNPs efciently inhibited Tau aggregation, and brain cells have been shown to be highly sensitive to cadmium sulfde NPs exhibited a signifcant dissembling USPIONs and their responsiveness to an external mag- of Tau NFTs. netic feld is an important advantage to promote brain As described above, oxidative stress is one of the most cell uptake. Moreover, the moderate heat emitted from important molecular hallmarks in AD development. In USPIONs under the action of a low radiofrequency feld AD pathogenesis, the exacerbated formation of H­ 2O2 is represents a signifcant advantage since it increases the favored by generalized brain oxidative stress. Metal che- BBB’s permeability without disturbing its integrity [201]. lators can block the detrimental efects of ROS, but their Cai et al. revealed that developed USPIONs can prevent non-specifc interactions with metal ions of normal cel- Aβ aggregation, disaggregate preformed Aβ fbrils, simul- lular processes and their BBB-reduced permeability sig- taneously perform in vivo NIR fuorescence and MRI of nifcantly limit their therapeutic success [199]. Yang et al. Aβ plaques in the brain, and perform a neuroprotective designed an H­ 2O2-sensitive detection system composed efect against Aβ42-induced toxicity. of Clioquinol-doped gold NPs-capped mesoporous silica Importantly, not only is the theragnostic potential of [199], and revealed that the conjugation of Clioquinol- MNPs being studied, but also their potential neurotoxic MNPs on the surface of mesoporous silica leads to selec- efects. Increasing evidence shows the relevance of toxins tive and sustained Clioquinol release under an increased in AD development, such as ultrafne air pollution parti- ­H2O2 environment (e.g., surrounding of Aβ plaques). cles. Terefore, and since nanomedicine has emerged as Furthermore, their nanodevice efciently crossed the a promising alternative for AD management, concerns BBB, decreased Aβ self-assembly, and reduced microtu- have been raised about the potential neurotoxic efects bular defects, cell membrane disruption, and ROS-medi- of diferent nanocarriers, especially MNPs. Teir small ated apoptosis induced by Aβ40. size and easy penetration into human tissues by difer- Hydrogen ­(H2) has been shown to selectively scav- ent access routes allow their high accumulation in the enge highly cytotoxic ROS. Furthermore, Hydrogen’s brain. Cerium dioxide NPs have gained much interest in bio-safety and bio-difusibility position it ahead of many recent years because of their radical-scavenging proper- compounds in AD drug discovery. However, the high ties. For these reasons, Wahle et al. recently evaluated solubility of H­ 2 and traditional hydrogen administration the neurotoxic efects of zirconium-doped cerium diox- routes, such as the oral intake of hydrogen-rich water, ide NPs in two diferent mice models of AD [202]. Te inhalation of hydrogen gas, or injection of hydrogen-rich continuous inhalation of these nanocarriers promotes saline, do not ensure its accumulation in the brain for changes in exploratory motor activity and forced motor the time necessary to exert its activity [200]. Zhang et al. performance in AD mice and increases GFAP expression developed reactive hydrogen-doped palladium NPs and in healthy mice. Tus, although MNPs represent a prom- evaluated their efects against oxidative stress-induced ising therapeutic alternative, these results highlight the mitochondrial dysfunction in both in vitro and in vivo importance of exhaustive research into their neurotoxic- models of AD [200]. Tis was the frst time that a nano- ity to ensure therapeutic safety. system was shown to be able to perform a self-catalysis Conclusions of carried ­H2 and realize an in situ sustained release of bio-reductive hydrogen. Tis innovative system efec- Dementias, including AD, are the ffth-leading cause tively stores and releases bio-reductive hydrogen to selec- of death worldwide, accounting for 2.4 million deaths tively ­scavenge.OH in AD cells, inhibit Aβ generation and yearly. Te expected increase in the number of cases of aggregation, reverse synaptic defcits and neuronal death, dementia in the next few decades is even more impor- and ameliorate mitochondrial dysfunction and cognitive tant, given that there are currently no efective disease- impairment in transgenic AD mice. modifying treatments for AD [1]. In addition, even Going a step further, Cai et al. recently developed ultr- when clinical trials are initiated, many of them fail to asmall superparamagnetic iron oxide NPs (USPIONs) discover a new disease-modifying drug to market: Cano et al. J Nanobiotechnol (2021) 19:122 Page 25 of 30

of every 100 molecules in clinical AD trials, only one Diseases (CIBERNED, CB06/05/0024); Portuguese Science and Technology Foundation (FCT) for the strategic fund (UIDB/04469/2020) and European reaches the market, compared to the pharmaceuti- Regional Development Funds. cal industry average ratio of 14.6:1 [1]. Without novel promising treatments in sight, increasing numbers of Authors’ contributions AC: proposal and schematization of the article, bibliography search, content cases will pose an undue burden on patients who have analysis & selection, writing of original full draft, writing of revision full draft, dementia, their caregivers, and healthcare systems in fgure design. PT: content analysis & selection, writing of original draft section, general. Te latest drug development pipeline shows language correction. ME: bibliography search, content analysis & selection, writing of original draft section, table edition. JTD: bibliography search, writ‑ that, in 2020, there were 121 compounds in clinical tri- ing of original draft section, language correction. GT: writing of original draft als for the treatment of AD [10]. Te lack of success in section, writing-review & editing. ESL: bibliography search, writing of original AD drug development reveals the complexity of this draft section. MLG: content analysis & selection, writing-review & editing. AC: content analysis & selection, writing-review & editing. EBS: content proposal, disease and the current challenges of AD neurophar- writing-review & editing, language correction. AR: content proposal, writing- macology research. Suggestions to abandon the amy- review & editing, revision of reviewed manuscript. MM: content proposal, loid hypothesis are increasing and emerging interest is manuscript direction, writing-review & editing, revision of reviewed manu‑ script. MB: content proposal, manuscript direction, writing-review & editing. focused on combination therapies [10]. Progress in AD All authors read and approved the fnal manuscript. management depends on innovation, the assessment of new candidates, and the implementation of new Funding Not applicable. trial approaches. As in other chronic diseases, such as cardiovascular disease or cancer, a learning phase pre- Availability of data and materials ceded periods of sequential incremental success lead- Not applicable. ing to meaningful treatments. Terefore, the increase of studies of combinations of new biomarkers and drug Declarations targets, combined with novel research in state-of-the- Ethics approval and consent to participate art nanocarriers, will pave the future for the more ef- Not applicable. cient delivery of bioactive molecules. Furthermore, the Consent for publication clinical development of biomarkers of AD progression The corresponding author of this manuscript, Dr. Amanda Cano, on behalf of to create treatments that are more efcacious will be all co-authors (Patric Turowski, Miren Ettcheto, Jason Thomas Duskey, Giovanni one of the most ambitious challenges for future clinical Tosi, Elena Sánchez-López, Maria Luisa García, Antonio Camins, Eliana B. Souto, Agustín Ruiz, Marta Marquié and Mercè Boada) declares the consent of pub‑ practice. In that regard, recent advances on nanomedi- lication of this manuscript in the Journal of Nanobiotechnology. All authors cine-drug development and novel diagnostic biomark- have read and approved the submitted fnal version. ers could represent a promising alternative in the Competing interests management of AD, as well as other neurodegenerative None of the authors has any conficts of interest including any fnancial, diseases. personal or other relationships with other people or organizations. All authors have reviewed the contents of the manuscript being submitted and approved its contents. Abbreviations AA: Ascorbic acid; Aβ: Amyloid-β; AD: Alzheimer’s disease; APP: Amyloid- Author details 1 precursor protein; BBB: Blood–brain barrier; CDs: Carbon dots; CSF: Cerebro‑ Research Center and Memory Clinic, Fundació ACE. Institut Català de Neuro‑ spinal fuid; CU: Cognitively unimpaired; DALYs: Disability-adjusted life years; ciències Aplicades, International University of Catalunya (UIC), C/Marquès de 2 DBP: Vitamin D-binding protein; EGCG:​ Epigallocatechin-3-gallate; EOAD: Early Sentmenat, 57, 08029 Barcelona, Spain. Biomedical Research Networking 3 onset AD; EPO: Erythropoietin; FABP3: Heart fatty acid-binding protein 3; FDA: Centre in Neurodegenerative Diseases (CIBERNED), Madrid, Spain. Institute 4 Food and Drug Administration; GAP-43: Growth-associated protein 43; LNPs: of Nanoscience and Nanotechnology (IN2UB), Barcelona, Spain. Department Lipid-based nanoparticles; LOAD: Late onset AD; LT: Lutein; MNPs: Metal-based of Pharmacy, Pharmaceutical Technology and Physical Chemistry, Faculty nanoparticles; MCI: Mild cognitive impairment; ND: Neurodegenerative dis‑ of Pharmacy and Food Sciences, University of Barcelona, Barcelona, Spain. 5 eases; NIR: Phenothiazine-based near-infrared; NfL: Neuroflament light; NFTs: UCL Institute of Ophthalmology, University College of London, London, 6 Neurofbrillary tangles; Ng: Neurogranin; NLCs: Nanostructured lipid carriers; UK. Department of Pharmacology, Toxicology and Therapeutic Chemistry, NPs: Nanoparticles; PCL: Poly-ε-caprolactone; PEI: Polyethyleneimine; PLA: Faculty of Pharmacy and Food Sciences, University of Barcelona, Barcelona, 7 Polylactide; PLGA: Poly(lactide-co-glycolide); PNPs: Polymeric nanoparticles; Spain. Nanotech Lab, Te.Far.T.I, Department of Life Sciences, University 8 PS1: Presenilin-1; PS2: Presenilin-2; p-tau: Hyperphosphorylated tau; SLNs: Solid of Modena and Reggio Emilia, Modena, Italy. Umberto Veronesi Founda‑ 9 lipid nanoparticles; sTREM2: Soluble ectodomain of TREM2; TREM2: Triggering tion, 20121 Milano, Italy. Department of Pharmaceutical Technology, Faculty 10 receptor expressed on myeloid cells 2; t-tau: Total tau; USPIONs: Ultrasmall of Pharmacy, University of Coimbra, Coimbra, Portugal. CEB ‑ Centre of Bio‑ superparamagnetic iron oxide nanoparticles; VILIP-1: Visinin-like protein 1. logical Engineering, University of Minho, Campus de Gualtar, 4710‑057 Braga, Portugal. Acknowledgements Authors acknowledge the support of the Instituto de Salud Carlos III (ISCIII) Received: 20 January 2021 Accepted: 17 April 2021 Acción Estratégica en Salud, integrated in the Spanish National R D I Plan and fnanced by ISCIII Subdirección General de Evaluación and the+ Fondo+ Europeo de Desarrollo Regional (FEDER “Una manera de hacer Europa") grant PI17/01474 awarded to Mercè Boada and grant PI19/00335 awarded to Marta Marquié; Spanish Ministry of Economy and Competitiveness (SAF2017- 84283-R); Biomedical Research Networking Centre in Neurodegenerative Cano et al. J Nanobiotechnol (2021) 19:122 Page 26 of 30

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