Flavonoid-Based Nanomedicines in Alzheimer’s Disease Therapeutics: Promises Made, a Long Way To Go

Abstract

Alzheimer’s disease (AD) is characterized by the continuous decline of the cognitive abilities manifested due to the accumulation of large aggregates of amyloid-beta 42 (Aβ42), the formation of neurofibrillary tangles of hyper-phosphorylated forms of microtubule-associated tau protein, which may lead to many alterations at the cellular and systemic level. The current therapeutic strategies primarily focus on alleviating pathological symptoms rather than providing a possible cure. AD is one of the highly studied but least understood neurological problems and remains an unresolved condition of human brain degeneration. Over the years, multiple naturally derived small molecules, including plant products, microbial isolates, and some metabolic byproducts, have been projected as supplements reducing the risk or possible treatment of the disease. However, unfortunately, none has met the expected success. One major challenge for most medications is their ability to cross the blood–brain barrier (BBB). In past decades, nanotechnology-based interventions have offered an alternative platform to address the problem of the successful delivery of the drugs to the specific targets. Interestingly, the exciting interface of natural products and nanomedicine is delivering promising results in AD treatment. The potential applications of flavonoids, the plant-derived compounds best known for their antioxidant activities, and their amalgamation with nanomedicinal approaches may lead to highly effective therapeutic strategies for treating well-known neurodegenerative diseases. In the present review, we explore the possibilities and recent developments on an exciting combination of flavonoids and nanoparticles in AD.

Alzheimer’s disease (AD) is the most prevalent form of dementia that develops through a progressive deterioration of cognitive functions of individuals of all ages. It steadily progresses from neuronal losses and brain atrophy to the reduction in quality of life, culminating in death. The precise origin of the disease pathology and subsequent death of postmitotic neuronal cells in the brain’s hippocampus and cerebral cortical areas is not very well understood. Several genetic mutations and abnormal processing of specific proteins underlie as the causative factor toward the disease initiation. The primarily described genes having close association with AD are amyloid precursor protein (APP), beta-secretase (BACE1), presenilins (PSEN1and PSEN2), and apolipoprotein E (APOE). In recent years, several genome-wide mapping studies have identified many new candidate genes that may have possible roles in disease pathology, for example, TREM2, UNC5C, ADAM10, CLU, BIN1, ABCA7, PICALM, SORL1, etc.^1,2 Nonetheless, two distinct lines of hypotheses pose amyloid-beta 42 (Aβ42)-laden plaques and neurofibrillary tangles formed of hyperphosphorylated microtubular protein tau as the possible causes of the disease; however, a direct correlation between their deposition is largely missing in most of the studies done so far.^3,4 Several lines of thought also consider many other factors as the possible causes of disease pathogenesis and cell death, e.g., oxidative stress and neuroinflammation that occur in many other neuronal disorders.^5,6

The mechanisms of cognitive impairments are primarily known, which include widespread synaptic dysfunctions and loss of neuronal signaling in several brain areas associated with memory and behavioral attributes.^7,8 Currently, available therapeutic options for treating AD are merely symptomatic and unsatisfactory due to limited clinical application attributed to a very short half-life and fast degradation of drugs.^9,10 Applying higher doses to obtain optimized and consistent effects in providing relief from the pathological conditions may result in adverse systemic effects, which may include nausea, vomiting, headache, confusion, dizziness, hallucinations, and depression, along with constipation, hepatotoxicity, and diarrhea.^11,12 A major challenge in effective AD chemotherapy is the accessibility of drugs across the blood–brain barrier (BBB).¹³ Unsuitable lipophilicity, molecular weight, or charge of drug moieties restrict the effective drug delivery and passage to the CNS through the BBB that maintains the homeostasis in the brain and restricts the diffusion of different kinds of foreign components through the blood vessels to the nervous tissues.¹⁴

Over the past decade, nanotechnological approaches to improve drugs’ administration without causing adverse drug reactions have been widely explored. Over the time, many relevant strategies have emerged that can help the drug formulation cross the BBB and manipulate or interfere with the production, aggregation, and clearance of Aβ.¹⁵ The nanoparticles (NPs) can be exercised as the carriers of pharmaceutical molecules ranging from drugs, antibodies, genes, and peptides. Several attempts of targeted drug delivery with the help of engineered NPs have been reported successfully. For instance, the development of polysorbate 80 coated poly n-butylcyanoacrylate (PBCA) and poly lactic-co-glycolic acid (PLGA) nanoparticles loaded with drug molecules like rivastigmine have shown significantly increased drug delivery to the brain with decreased side effects.^16,17 When applied on tacrine, similar approaches enhanced its brain uptake by four-fold compared to the free drug.¹⁸ Encapsulated chitosan nanoparticles of rivastigmine showed enhanced drug uptake and bioavailability in the brain.¹⁹ The encapsulation in NPs can protect bioactive molecules from systemic degradation, prolong their sustenance in the circulation, enhance the bioavailability, and allow an effective passage through the BBB with minimal side effects. Drug-loaded multifunctionalized NPs can be targeted to specific diseased areas of the brain. Moreover, newly developed biocompatible nanomaterials with improved optical and magnetic properties may also act as excellent alternatives for an early disease diagnosis.²⁰

As a plethora of changes in the aging brain may lead to multiple pathologies, an ideal chemotherapeutic agent is required to be particularly effective for blocking multiple pathways leading to AD development.²¹ Compounds with multitudinous biological activities, e.g., flavonoids, can possibly affect various age-associated changes in the human brain, which otherwise may contribute to disease development and progression.²² A recent study examining the relationship between intake of flavonoids and risk of AD provides convincing pieces of evidence that long-term intakes of a flavonoid-rich diet are associated with reduced risks of developing AD.²³ Several other studies highlight the potential of flavonoids to reduce or prevent all of the major changes associated with the aging brain underlying the development of AD.^24,25 These changes include intensified oxidative stress, altered protein processing, decrease in neurotrophic factor signaling, accompanied by synaptic dysfunction, aggressive neuroinflammation, and activation of cell death pathways, which may altogether contribute to behavioral impairments and cognitive dysfunction.²⁶

Low bioavailability and the highly unstable nature of flavonoids obstruct their use as possible therapeutics for neurodegenerative disease treatment.^27,28 Therefore, developing new generation approaches for drug administration or delivery, for example, nanomedicinal formulations, can serve better for clinical success.²⁹ The present review discusses nanotechnology-enabled approaches that are being developed to enhance the biomedical potential of flavonoids for therapeutic intervention and AD prevention. In addition to a detailed report on causative factors and summarizing currently available nanotechnology-based drug delivery systems for AD treatment, we highlight the impact of flavonoids on various AD hallmarks to delay or suppress the progressive neuronal loss. Although the therapeutic potential of flavonoids and nanomedicine for the treatment of AD has been discussed individually several times, collective literature of amalgamation of the interface of flavonoids and nanotechnology is not available. Foreseeing the progress in nanotechnological research, we can assume that flavonoid-based nanoformulations may lead to highly effective therapeutic strategies for treating AD in the near future.

Essentials of Alzheimer’s Disease: Genetics, Etiology, Pathways, Symptoms, and Pathophysiology

Alzheimer’s disease (AD) is clinically characterized by a progressive decline in cognition and memory formation due to an irreversible loss of terminally differentiated brain cells, mainly in the cortex and hippocampus regions of the brain.³⁰ It is an acquired disorder of behavioral impairment. Early symptoms of AD include subtle and poorly recognized failure of memory, like failing to recall current conversations, names, or events, which slowly becomes severe due to symptoms like confusion, poor judgment capabilities, noticeable language disturbance, agitation/withdrawal, hallucinations followed by difficulty in speaking, swallowing, and walking.³¹ Occasionally, relapsing seizures, heightened muscle tone, myoclonus, incontinence, and mutism may also occur. Death usually results from general inanition, malnutrition, and pneumonia.³² According to “World Alzheimer Report 2019”, 50 million people have been diagnosed with dementia worldwide, which could grow further up to 150 million by 2050.³³ Alzheimer’s disease (AD) accounts for approximately 70% of all the dementia cases estimated worldwide.³⁴ Given the current projections, AD will have enormous social and economic impacts in the upcoming years. Moreover, unlike other health problems, the number of deaths caused by AD has increased by 89% between 2000 and 2014.³³ Therefore, in light of growing evidence of global prevalence, associated mortality, and economic cost of the disease, increased awareness, improved early diagnosis, and providing a promising cure are a top priority.^35,36

Two major molecular changes characterize the onset of AD: the aggregation of β-amyloid (Aβ) peptide and the formation of neurofibrillary tangles due to tau protein hyperphosphorylation. Simultaneously, other changes such as augmented oxidative stress, mitochondrial apoptosis, inflammation, and synaptic dysfunction may occur, which gradually culminates in the death of neurons.^37,38 Despite decades of research, underlying causes and mechanisms responsible for these molecular changes and AD progression in the brain tissues remain ambiguous.^39,40 Several hypotheses have been proposed to explain the genetics, onset, etiology, pathogenesis, and cure of this neurodegenerative disorder. According to highly worked out amyloidopathy, the aggregation of Aβ or more specifically Aβ42 takes place by the sequential cleavage of the amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase yielding insoluble Aβ fibrils.⁴¹ These fibrils gradually get polymerized into extracellular spaces in the form of senile plaques, which are responsible for the synaptic damage and memory deficit in AD.⁴²

According to the second line of hypothesis, called tauopathy, the intracellular deposition of neurofibrillary tangles (NFT) is due to the rapid phosphorylation of microtubule-associated tau (τ) protein.⁴³ These proteins, Aβ42, total-, and phospho-tau, are primary biomarkers detected in the patients’ cerebrospinal fluid (CSF) and are used in the characterization of the disease.⁴⁴ In addition, several proteins could be used as possible indicators of many other pathological changes associated with AD, indicating the disease progression.⁴⁵ For example, neurofilament light (Nfl) protein shows neuroaxonal degeneration, whereas neurogranin (Ng) indicates possible synaptic degeneration.^46,47 The other CSF biomarkers used to predict the disease pathology are linked with the detrimental changes associated with neuroinflammation. The primary proteins representing this category are YKL-40 and GFAP for astrocytic, and TREM2 and TSPO for microglial activation.^48,49 On the basis of recent meta-analyses and investigations, a number of risk factors have been outlined. They are hypertension, homocysteine levels, heightened inflammation, diabetes mellitus, and obesity.⁵⁰⁻⁵⁴ However, age remains the most crucial risk factor for AD and other disorders of the same class. Even though the disease is more prevalent among the older section of the society (late-onset), the younger population may also develop early onset symptoms of AD pathology that may occur due to various genetic mutations in the primary genes involved in APP processing, e.g., APP, BACE1, PSEN1, and PSEN2.^32,55,56 Alongside, mutations in other genes, such as TREM2, clusterin, BIN1, ADAM10, and apolipoprotein E type 4 (APOEε4), are also considered the possible risk factors for AD.^57,58

APP encodes transmembrane amyloid precursor-like proteins, which generates amyloidogenic fragments under disease conditions, whereas PSEN1 and PSEN2 are responsible for coding γ-secretase enzyme complex.⁵⁹ Overexpression of the APP gene (on chromosome 21) in persons with trisomy 21/Down syndrome has also been found to develop the AD hallmarks after 40 years of age. The presumed reason could be the overproduction of β-amyloid in the brains of individuals with an additional copy of the gene.⁶⁰ On the contrary, late-onset AD is polygenic or guided by multiple factors. Another common hypothesis that runs parallel to the theories mentioned above is the dysfunction of the cholinergic system, causing a reduction in cholinergic markers, memory deficits, and AD progression.^61,62 Genetic and molecular alterations explaining this hypothesis include the presence of APOEε4 and decreased level of phospholipase A2 (PA2) protein, an enzyme responsible for conversion of phosphatidylcholine to choline in the frontal and parietal cortices of AD patients.^63,64 The critical roles of exosomal transport in the progression of many neuronal disease pathologies are now widely established. The exosomes and extracellular vesicles (EVs) may play a wide variety of roles, including the spreading of molecules inducing oxidative/proteotoxic stresses and neuroinflammation.⁶⁵ However, their roles in spreading amyloids, as they do in prion diseases, are reported in several studies, but it is yet to be established.^66,67 In addition, the involvement of EVs in the clearance of disease-associated plaques is also an area of research. Several studies postulate that these membranous structures may play essential roles in the clearance of proteotoxic burden and transport neuroprotective substances. Therefore, the possibilities of their application in AD therapeutics are also under consideration.^65,68

Inflammatory mediators may lead to the activation of microglia. The activated glial cells contribute to AD pathology by increasing the expression of inducible nitric oxide synthase (iNOS), which produces large amounts of nitric oxide that mediates glial-induced neuronal death through inhibition of mitochondrial respiration.⁶⁹ Over the past few years, a novel hypothesis related to inflammation has gained public attention, which provides a connecting link between Aβ aggregation and the NFT formation.⁷⁰ According to this hypothesis, increased inflammation in the brain may be responsible for an increased risk of AD. Surprisingly, consistent inflammatory response facilitates and enhances Aβ and NFT pathologies simultaneously.⁷¹ Although inflammation protects the brain from infection, injury, and foreign threats, a slight imbalance of anti-inflammatory and pro-inflammatory signaling in the brain may give rise to chronic neuroinflammation characterized by activated microglial cells and cytokines release leading to incessant immune response.⁷²⁻⁷⁵ Aging is typically accompanied by a shift of innate immunity toward a pro-inflammatory status, which negatively influences brain pathology.⁷⁶ Previous reports suggest that upregulation of pro-inflammatory cytokine expression (such as tumor necrosis factor-alpha or interleukin-1) by Aβ and activation of astrocytes and oligodendrocytes (the most common types of cells in the brain) can altogether play leading roles in causing neuronal cytotoxicity and generation of reactive oxygen species (ROS).⁷⁷

Neurons are extremely susceptible to oxidative damages due to their high metabolic activity and loss of antioxidant capacity; therefore, excessive free radical generation and mitochondrial dysfunction may collectively lead to neuronal dysfunction and degeneration.⁷⁸ It is well-known that brain-derived neurotrophic factor (BDNF) is essential for cognition and memory formation. It affects synaptic plasticity, and a reduced supply of BDNF to the basal forebrain cholinergic neurons may lead to the emergence of neurodegenerative diseases.⁷⁹ Substantial studies on deficient neurotrophic hypothesis correlate incidences of AD with BDNF mRNA levels, axonal transport deficits, uneven distribution, and dysregulation of neurotrophic factors.⁸⁰ Furthermore, defects in the formation and trafficking of neurotrophin signaling endosomes due to increased APP levels also result in neuronal dysfunction and AD pathogenesis.⁸¹

Curing Alzheimer’s: A Glimpse of Available Therapeutics

Currently, researchers are mainly focused on drugs that can minimize the synthesis and aggregation of Aβ and/or decrease tau phosphorylation. Molecules targeting cholinesterase and preventing the formation of amyloidic aggregates in the brain tissues could be the other set of possible drug candidates.^82,83 Furthermore, statins, cholesterol-lowering drugs, anti-inflammatory substances, antioxidants molecules, and antibody-based treatment plans have also been investigated thoroughly in the past few decades to lower the symptoms and improve the quality of life of the patients.⁸⁴⁻⁸⁸ Further elucidation of AD etiology to discover novel therapeutic molecules capable of blocking AD progression efficiently and developing strategies to deliver to the regions against BBB may be helpful. Numerous clinical data suggested that impairment of cholinergic–neurotransmitter systems, possibly due to the suppression of acetylcholine (crucial for neural synapse) by acetylcholinesterase (AChE) activity and activation of the glutamatergic system, also plays a very aggressive role in AD pathology.^89,90 In recent years, acetylcholinesterase inhibitors (AChEI) and N-methyl-d-aspartate (NMDA) receptor antagonists are recognized as a potential therapeutic intervention to counter the disease-linked perturbances.⁹²

The FDA-approved drugs for AD treatment include donepezil, rivastigmine, galantamine (reversible AChEIs), and memantine (NMDA inhibitor).^91,92 Interestingly, the mechanism of action of these drugs are dissimilar; for example, the most prescribed and highly selective drug donepezil is a noncompetitive inhibitor of cholinesterases, whereas rivastigmine inhibits the action of both enzymes, acetyl- and butyrylcholinesterases in a pseudoirreversible manner to delay the neurotransmission in the synapses.^93,94 Galantamine interferes with the enzymatic activity of acetylcholinesterase along with the allosteric potentiation of the nicotinic acetylcholine receptors to produce greater amounts of acetylcholine in the brain.^95,96 On the other hand, NMDA receptor antagonists like memantine modulate the flow of glutamatergic neuronal transmission and block the adverse effects of hyperactive glutamatergic activity like impaired synaptic plasticity and damage to the neurons. All these can efficiently manage the glutamate storm in the diseased brains.⁹⁷ To relieve patients from cognitive losses, several combination therapies have also been considered clinically. Unfortunately, except the combination of cholinesterase inhibitors and memantine, the majority of combinations, though successful in preclinical studies, failed to exhibit anticipated efficacy in clinical trials.^98,99

Even though ChEIs are regarded as safe drug candidates, their therapeutic effects are severely compromised due to an array of severe side effects, as discussed previously (Anand and Singh, 2013). Besides, the pharmacokinetic profile of conventional drugs in the biological system has been found to be low with substantial side-effects. This could be attributed to low bioavailability, chemical nature, or volatility of the evaluated drug candidates in the biological system.^100,101 High doses of drugs (e.g., rivastigmine) to obtain an optimum level in the brain may cause adverse effects.⁹³ A short half-life of drug molecules like tacrine, galantamine, and rivastigmine is another challenging aspect that requires further consideration.¹⁰² The oral administration of rivastigmine may lead to adverse gastrointestinal reactions, which is believed to be caused partially due to sudden increase in acetylcholine in the central nervous system. To regulate plasma concentrations of acetylcholine after rivastigmine administration and uneven distribution of the drug in the bloodstream, a transdermal rivastigmine patch formulation was developed to deliver the drug through the skin into the bloodstream.^103,104

Although multiple clinical trials have been conducted in the past few years with several drug candidates designed to directly or indirectly cut the amyloid plaque load, none of these trials have shown promising outcomes.¹⁰¹ The complexity of AD pathology, incomplete understanding of the underlying pathways involved in the development of AD, subsequent neurodegeneration, and the lack of efficacy of available agents are few probable reasons for the failure.⁹⁸ The treatment of AD is particularly complicated due to the presence of the BBB, a physical and biochemical barrier protecting the brain from potentially hazardous substances in the blood flow by restricting the passage of foreign components to the central nervous system (CNS). Most of the conventional drugs fail to cross the BBB and get eliminated by high drug efflux P-glycoprotein. Moreover, drugs carbamazepine are extremely toxic, rendering the therapeutic strategies extremely ineffective.^105,106

Nanomedicinal Approaches for Alzheimer’s Disease: Opportunities with Multiple Challenges

The emergence of nanoscience and engineering is a landmark endeavor in the field of medicinal chemistry. In recent years, the ability to engineer the substances and materials of specific characteristics has gained a level of maturity.¹⁰⁷ The unique physicochemical properties, superior catalytic activities, and improved functionality of nanoparticles (NPs) over their bulk equivalent materials could be attributed to its tiny size with very large surface area, tendency to self-assemble, and quantum effects associated with these materials.^108,109 The versatility of these materials in biomedical applications could be extended to their high stability in biological systems, specific adeptness of targeting tissue, ability to encapsulate drugs, improved sensitivity for imaging and visualization due to enhanced contrast, and better resistance to corrosion over time due to elevated surface to volume ratio.^110,111

Nanoformulations hold great potential to improve a plethora of physical and biological properties of the drugs, which may include but are not limited to enhanced solubility, superior pharmacokinetics, better selectivity, higher efficacy, and least toxicity in comparison to traditional medicines.^112,113 Coupling of nanoformulations, such as polymeric NPs, solid lipid NPs, liposomes, nanoemulsions, nanostructured lipid carriers (NLC), etc., helps in the safe delivery of drugs or bioactive moieties to specific sites of action with an improved pharmacokinetic profile causing minimal adverse drug reactions. These may further assist drugs in overcoming challenges, like passage through the BBB, overpowering gastric pH barriers, increased absorption and bioavailability, prolonged half-life, reaching at the site of action in biologically appropriate therapeutic doses.^105,114 In fact, an equilibrium between the physicochemical and pharmacokinetic properties is required to be maintained to optimize the effectiveness and specificity of therapeutics toward molecular targets.

Widespread applications of nanomaterials in the treatment of both chronic and acute diseases could be attributed to the promises shown in the growing number of studies based on these NPs in diverse kinds of disease models.^20,115 The enormous potential of nanomedicine in the treatment of CNS diseases has been highlighted in the past few years owing to its lipophilicity, lower molecular weight (up to 400 Da), smaller size (in the range of nanometers), appropriate charge with a higher surface-to-volume ratio, which may allow easy access to the brain through the process of simple diffusion.^116,117 Many studies have been done, where nanoformulations have been prepared through polymerization, ionic gelation, coacervation, emulsion solvent evaporation, etc., to target AD pathophysiology in animal models.^15,118−121 There are specific benefits associated with varying preparation methods, e.g., coating NPs with peptides, surfactants, or antibodies can enormously improve the absorption and drug delivery to the brain.¹²² Previously, the concentration of tacrine in the brain and other tissues was improved by nanocoating polysorbate 80, a nonionic surfactant, on the tacrine-conjugated poly(n-butylcyanoacrylate) nanoparticles.¹⁸ In another study, it was found that the chitosan NPs incorporated into rivastigmine may enhance its bioavailability and drug uptake, thus improving the efficacy.¹²³

Surface modification of the nanoparticles tends to facilitate its permeability across BBB without any phagocytic opsonization and provide longer circulation time without unnecessary interactions during their prolonged stay in the biological system.¹²⁴ Nanostructured lipid carriers (NLCs) can significantly improve the bioavailability and facilitate the administration of drugs via the intranasal route.¹²⁵ Such particles can greatly enhance the biodistribution of the drugs with poor water solubility and improve the drug efficacy due to the modified administration strategies, including oral, nasal, etc.¹²⁶ Interestingly, the intranasal route of administration could deliver an increased amount of drugs in the brain as compared to intravenous by accessing the olfactory route and evading the BBB.¹²⁷ These drug-laden NPs have enhanced absorption and delivery to the brain with the least side effects as lower doses could also be effectively administered.¹²⁸ Furthermore, rivastigmine liposomes carrying cell-penetrating peptide (CPP) have shown improved distribution and retention of the drug in the brain, with enhanced pharmacodynamic properties, including lower side effects.¹²⁹ Delivering drugs like donepezil and tacrine using liposomes leads to quick absorption and considerable bioavailability of the drug.^130,131

Ligand-functionalized nanoliposomes can also enhance the therapeutic effects of galantamine and rivastigmine by lowering the amyloid-beta formation and ameliorating other AD-associated pathological symptoms.^132,133 Drug encapsulation by such nanocarriers could extend their systemic benefits by improving the pharmacological properties of drugs and promoting their retention, bioavailability, and increasing absorption, thus imparting the anticipated therapeutic effects.¹³⁴ In addition, these drug delivery systems have very targeted evasion inside the biological system with lesser chances of random interaction and activation of molecules at unspecific targets, which helps in avoiding the accumulation of possible adverse effects, including abdominal complications, vomiting, nausea, etc., which are common in many treatment plans currently in use.^112,135

Although there is a lack of comprehensive understanding of AD pathogenesis, it is clear that multiple cellular pathways are affected. Targeting a single pathway may relieve the symptoms mildly but will never lead us to a reliable cure. Also, in recent years targeted antiamyloid agents and therapies, including BACE inhibitor drug molecules and anti-Aβ42 antibodies, have failed to meet the expectations in the clinical trials.^136−139 Therefore, in the past few decades, researchers are looking for the beneficial aspects of naturally occurring drug candidates, which may have antioxidative and anti-inflammatory characteristics along with the definite anti-amyloidogenic properties that can prevent aggregation of Aβ42 peptides.¹⁴⁰ Concurrently, it should have high bioavailability, water-solubility, BBB access, with a considerable half-life, absorption, and appropriate distribution in the neuronal cells. Also, lesser toxicity is another primary attribution that the natural molecules provide. Therefore, a thoughtful reconsideration of the amalgamation of natural compounds containing multiple neuroprotective properties as one resort and nanobased approaches to facilitate their delivery to specific brain regions could be a possible futuristic approach in the AD drug development pipeline.

Advantageous Therapeutic Benefits of Flavonoids Against Alzheimer’s disease

Flavonoids are polyphenolic compounds naturally present as colored pigments in higher plants.¹⁴¹ They are synthesized from the amino acid phenylalanine by the phenylpropanoid pathway and are ubiquitously found in fruits, vegetables, nuts, seeds, stems, flowers, and tea.¹⁴² The canonical structure of flavonoids, with more than 10 000 discovered so far, consists of three aromatic rings lying adjacent to each other.^143,144 Two rings named A and B collectively form the 15 carbon flavan nucleus that is further linked with a three-carbon heterocyclic ring C.^145,146 On the basis of the structure, flavonoids have been classified into six broad categories. They are flavanols, flavonols, flavones, flavanones, isoflavones, and anthocyanidins.¹⁴⁷ Flavonoids have been primarily explored for their cytoprotective, antiviral, antiallergic, anti-inflammatory, antidiabetic, antiobesity, antitumor, and neuroprotective properties, and thus are considered as molecules of significant medicinal importance as they exhibit tremendous opportunities to explore and exploit their pharmacological properties.^{142,148−150}

As discussed before, AD pathology is a clinical manifestation of multiple pathways altered due to loss or gain-of-functions of one or more genes, causing a rise in oxidative stress, weaker neurotrophic support, altered energy metabolism, deposition of neurofibrillary tangles, and plaque aggregation followed by inflammation and cellular damage in the specific brain regions.^151,152 The plethora of AD brain changes put a challenge before researchers and clinicians to address them simultaneously; however, our limitations in understanding the primary cause of disease onset further toughens the chances of a finding possible cure soon. However, therapies directed to delay the symptoms and provide symptomatic relief are the current paradigm of AD treatment. Countering multiple age-associated changes in the brain require a combination of drugs simultaneously directed against wide range targets. Flavonoids provide a safer alternative group of compounds, which have been explored widely. Further research is continuously encouraging the idea that some of the dietary flavonoids may have a wide range of therapeutic benefits.

Figure 1.

Flavonoids: A Brief Introduction. (A) Backbone structure of flavonoids consisting of an aromatic ring A fused with an oxygenated heterocyclic ring C further connected to another aromatic ring B. (B) Pharmacological properties and roles of flavonoids in prevention of multiple pathological conditions. (C) Classification and example of flavonoids with their chemical structures. (D) Major plant parts from which flavonoids are extracted and some well-known examples of flavonoids purified from these sources.

Administration of rats with a citrus flavanoglycone hesperidin also resulted in cognitive protection against l-methionine induced oxidative stress and neurotoxicity.¹⁵³ In a mammalian animal model, treatment with (−)-epigallocatechin-3-gallate (EGCG) has shown protective effects against ischemia-induced toxicity in hippocampal neurons.¹⁵⁴ It has also shown beneficial effects in Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) mice models.^155,156 Quercetin interferes with the enzyme proline hydroxylase (PHD), which facilitates the ubiquitination of hypoxia-inducible factor-1 alpha (HIF-1α) by the protein of von Hippel–Lindau, an E3 ubiquitin ligase, and thus stabilizes it under normal oxygen condition.¹⁵⁷ Similarly, another transcription factor associated with redox homeostasis, nuclear factor erythroid 2-related factor 2 (Nrf2-), is reported to be activated by multiple flavonoids, e.g., pinocembrin, eriodictyol, and naringenin, under oxidative stress like conditions.^158−160 Not only the suppression of the oxidative damage but getting better of the microglia- and astrocyte-mediated neuroinflammation is another possible mechanism that flavonoids have primarily been tested for their potential as anti-inflammatory neuroprotective agents.

Previous studies suggest that the downstream effects of activation of microglia-mediated inflammatory changes in the central nervous system (CNS) can initiate a detrimental chain of events leading to the death of neurons. Therefore, the positive effects of many classes of small natural molecules over neuroinflammation have been investigated in the past.¹⁶¹ The major flavonoids with neuroinflammation modulatory potential are baicalein, catechin, EGCG, fisetin, genistein, quercetin, and wogonin; however, they may have different mechanisms of action.^162−166 Interestingly, flavonoids can regulate inflammatory events by decreasing the level of prostanoids and leukotriene with the help of eicosanoid generating enzymes like phospholipase A2, cyclooxygenases, and lipoxygenases.¹⁶⁷ Other possible mechanisms could be the regulation of the cellular metabolic alterations by regulating multiple kinase pathways altered under various disease-associated stress conditions.¹⁶⁸ Some flavonoids, like baicalein, epicatechin, kaempferol, and quercetin, also suppress c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases (ERK)-dependent apoptotic pathways to protect neurons against multiple kinds of stresses and injuries.^169−171

Growing evidence reinforces the notion that flavonoids play active roles in slowing down cognitive decline and dementia.^172−174 Their inherent potential to subdue the progression of neurological disorders has gained special attention in recent years. The elemental role of flavonoids in stimulating cognitive functions, learning, and memory is apparently associated with their ability to induce neuronal regeneration, encourage neurogenesis, antioxidant capabilities, and protect vulnerable neurons through interaction with the molecular counterparts of the brain and intracellular neuronal pathways.^175,176 Multiple lines of investigations proposed different mechanisms to explain the efficacy of flavonoids in learning and memory, clearly highlighting their potential as a possible remedy to cut down the disease severity in the patients.¹⁷⁷ Fisetin, a natural polyphenol, can restore the long-term potentiation (LTP) by inducing ERK signaling and inducing phosphorylation of cAMP response element-binding protein (CREB) in rat hippocampi.¹⁷⁸ Another potent memory enhancer flavonoid is luteolin, which improves the synaptic transmission and, via activation of CREB, stabilizes memory.¹⁷⁹

A wide range of flavonoids has been reported to avert cognitive impairments and inhibit AD development through anti-amyloidogenic properties.^180,181 Interestingly, multiple flavonoids have shown direct or indirect anti-amyloidogenic potential in various in vitro model systems. The most studied and widely explored anti-amyloidic flavonoids are didymin, myricetin, morin, prunin, poncirin, quercetin, and rosmarinic acid.^182−184 More interestingly, in a cell system based study, EGCG has shown enhancing the release of soluble nonamyloidogenic form of APP in protein kinase C (PKC) dependent mechanism.¹⁸⁵ Multiple lines of studies have proposed flavonoid-mediated reduction in secreted Aβ levels and inhibition of BACE-1 activity in primary cortical neurons or involvement in tau phosphorylation through suppression of the activities of several kinases including GSK-3β (tau phosphorylation) and interaction with various neuronal signaling pathways through various protein kinase- and lipid kinase-signaling cascades.^186−189 Luteolin and diosmin administered mice models of AD pathology showed inhibition of GSK-3 and altered processing of presenilin-1, resulting in reduced amyloid formation and thus improve the disease condition.¹⁹⁰

Figure 2.

An overview of Alzheimer’s disease pathophysiology: Visible morphological atrophy in the brain resulting from a cascade of events triggered by various genetic and environmental factors leading to Alzheimer’s disease. Major AD hallmarks include amyloid plaques, neurofibrillary tangles, neuroinflammatory changes, and microglial activation. Several therapeutic strategies have been proposed in the past to prevent disease progression and relieve the symptoms.

Inhibition of acetylcholine is one of the preliminary changes in AD pathology; therefore, inhibition of AChE has been one primary drug development strategy for a long time. Several independent research groups have identified a large number of flavonoids with AChE inhibition potential; tiliroside, quercetin, and rutin are prominent natural molecules of this class.^191,192 Apart from these, genistein and silibinin are other flavonoids with strong in vitro inhibitory potential against both AChE and BChE.¹⁹³ Multiple in silico studies have identified similar binding and inhibition of both the cholinesterases by galangin, kaempferol, quercetin, myricetin, and morin.^194,195 Several flavonoids have been found upregulating the secretion of different types of neurotrophic factors in the brain cells; for example, 7,8-dihydroxyflavone (7,8-DHF) is a potent agonist of tropomyosin-receptor-kinase B (TrkB), a receptor for the most abundant neurotrophin, brain-derived neurotrophic factor (BDNF). Therefore, it may compensate for the loss of the BDNF in AD pathology by improving the branching and synapse formation by alleviating Aβ deposition and thus rescues 5xFAD mice from memory loss.^196,197 We have summarized available research outcomes in the form of Table 1 that reflects the therapeutic potential of various flavonoids.

Table 1. Flavonoids and Their Therapeutic Applications in CNS.

flavonoid	model	biological target	therapeutic effect	ref
EGCG	TgCRND8 mice	soluble Aβ	improved learning and memory	(264)
	SAMP8 mice	Aβ1–42, BACE-1, tau,	rescued spatial learning	(265)
	APPsw mice	Aβ1–42, tau	cognitive benefits	(266)
	CUMS rats	Aβ1–42, mTOR, p70S6K	enhanced spatial learning and memory	(267)
anthocyanins	APdE9 mice	APP processing	alleviated behavioral and memory deficits	(268)
curcumin	THP-1 cells	Egr1, TNFα, IL1β, MAPK	ameliorate inflammation	(269)
	PC12 cells	Aβ42	protection from Aβ insult	(270)
nobiletin	APP-SL7–5 mice	Aβ42, ERK	improves memory impairment	(271)
	3xTg-AD	Aβ1–40, ROS	reversal of impaired learning and memory	(272)
	SAMP8 mice	p-tau, SOD1, GPx	ameliorates learning and memory deficits	(273)
	AD rats	Aβ42, PKA, CREB	rescues memory deterioration	(274)
resveratrol	AD patients	Aβ40, MMP9, IL-4, FGF-2	inhibits inflammation and cognitive decline	(275)
baicalein	Tg2576 mice	APP	improved cognitive performance	(276)
myricetin	AD rats	AMPK, SIRT1	suppresses apoptosis, improves learning	(277)
quercetin	3xTg-AD	Aβ42, tau	improves learning performance	(278)
	3xTg-AD	Aβ42, BACE1	strengthens spatial memory	(279)
kaempferol	AD rats	SOD, GSH, TNFα, MDA	improves spatial learning and memory	(280)
silibinin	Aβ treated rats	BDNF/TrkB, autophagy	ameliorates anxiety/depression behaviors	(281)
apigenin	APP/PS1 mice	APP, BACE1, SOD1, GPx	relieves learning and memory impairment	(282)
rutin	rat model	ERK1, CREB, and BDNF	rescues amyloid induced neurotoxicity	(283)
wogonin	3xTg-AD mice	APP, tau, cleaved PARP	enhances neurite outgrowth and memory	(284)
	SH-SY5Y cells	APP, tau, mTOR	may improve cognitive function	(285)
fisetin	2x FAD mice	Cdk5, ERK	improves learning and memory deficits	(286)
	SAMP8 mice	Arc, GFAP, p35, SAPK/JNK	delays aging and cognitive decline	(287)
catechin	PC12 cells	Aβ	reduces apoptosis and neurotoxicity	(288)
7,8-DHF	5xFAD	BACE1, Aβ, TrkB	attenuates memory deficit	(289)
	5xFAD	TrkB	restores synapses and memory	(290)
morin	3xTg-AD mice	GSK3β, tau, Aβ	increased neuroprotection	(291)
luteolin	Tg2576 mice	GSK3β, PS1, Aβ	reduces Aβ toxicity	(292)
diosmin	Tg2576 mice	Aβ42	reduces Aβ toxicity	(292)
	3xTg-AD mice	Aβ42, tau	suppresses inflammation and memory loss	(293)
diosmetin	3xTg-AD mice	GSK3β, γ-secretase	reduces inflammation and cognitive loss	(293)
hesperetin	Neuro-2A cells	IR, Akt, GLUT3, GLUT4	regulates autophagy and metabolism	(294)
hesperidin	Neuro-2A cells	IR, Akt, GLUT3, GLUT4	regulates autophagy and metabolism	(294)
	APP/PS1 mice	iNOS, TNFα, IL1β	restores behavioral/social interaction	(295)
puerarin	SAD mice	GPx, SOD, MDA	regulates oxidative stress/memory decline	(296)
	SAD mice	caspase-3, p38, JNK, Bax	reduces oxidative damage and apoptosis	(297)
	APP/PS1 mice	Nrf2, HO1, Akt, GSK-3β	alleviates cognitive impairment	(298)
naringenin	rat model	GSK-3, PPARγ, IR, Tau, Aβ42	facilitates spatial learning and memory	(299)
naringin	AD rats	Ach, IL1β, mitochondria	mitigates cognitive decline	(300)
genistein	SAD rats	APP, Aβ42, tau	restores autophagy, behavioral normalcy	(301)

Advances in Nanodrug Delivery Systems and Their Benefits in the Context of Flavonoids

Flavonoids have the capability to target multiple pathways associated with several complex neuropathological conditions. Despite multifaceted benefits and very few known side-effects at the prescribed doses, they are not yet considered a suitable class for drug development, owing to their inherent instability, inefficient delivery models, and low retention in target tissues. These limitations of flavonoids have been addressed thoroughly in the past few years to attain clinical success. Nanobased formulations of many standard drugs have been successfully applied to counter the challenges and abolish the major pharmacokinetic barriers. The advanced nanotechnological approaches have shown a vast potential to narrow down the gaps between the physiological effects of the drug molecules and their possible applications in therapeutics. Many flavonoid-based nanoformulations have been prepared and examined for the promising results on cell-based systems and animal models in recent years. A detailed overview of some major studies with such nanomedicinal strategies explored widely in the context of AD is presented in Table 2.

Table 2. Advantages of Flavonoid Laden Nanoformulation against Alzheimer’s Disease Pathology.

flavonoids	experimental model	pharmacological advantages	biological activity	ref
Liposomes
baicalein	SHSY5Y, PC12 cells	slow release, high internalization	protects against oxidative stress	(205)
resveratrol	NIH-3T3	improved solubility	presents no cytotoxicity	(302)
quercetin	rat model	enhanced solubility	anxiolytic and cognitive benefits	(303)
	SK-N-MC cells	enhanced access through BBB	suppression of neuronal apoptosis	(201)
EGCG	N2a cells, rats	increased oral bioavailability	enhances α-secretase activity	(304)
Silica Nanoparticles
catechin	hippocampal cells	high solubility	enhances cell survival	(305)
chrysin	hippocampal cells	improved drug entrapment	suppresses Aβ toxicity	(306)
quercetin	hippocampal cells	retains physicochemical integrity	improves antioxidant activity	(307)
naringin	hippocampal cells	entraps higher drug doses	reduces Aβ toxicity	(231)
Selenium NPs
EGCG	PC-12 cells	high affinity for Aβ	disaggregates Aβ fibrils	(206)
curcumin	5x FAD mice	increased binding with plaques	inhibits Aβ aggregation	(308)
Polymeric NPs
EGCG	APP/PS1 mice	increases drug stability	increased synapses	(309)
EGCG	rats	superior bioavailability of drug	improves neurobehavioral deficits	(310)
anthocyanin	SH-SY5Y cells	slow and sustained drug release	abrogates oxidative stress	(243)
quercetin	SH-SY5Y cells	sustainable release	disassembles Aβ fibrils	(311)
curcumin	SK-N-SH cells	increased solubility and stability	suppresses ROS and Nrf2	(312)
Lipid Core Nanocapsules
resveratrol	hippocampal cultures	restricts drug photosenstivity	reduces Aβ induced inflammation	(313)
Protein Nanoparticles
quercetin	SAMP8 mice	greater bioavailability	improved cognition and memory	(255)
Solid Lipid Nanoparticles
curcumin	5× FAD mice	enhanced affinity to Aβ-plaques	may interfere with Aβ fibrillation	(212)
resveratrol	in vitro BBB model	enhanced cellular uptake	inhibits amyloid beta aggregation	(314)
chrysin	rats	improved bioavailability	mitigates Aβ induced stress	(315)
quercetin	rats	higher drug entrapment	improved memory retention	(249)
	Danio rerio	improved drug release	attenuates cognitive impairments	(316)
Nanostructured Lipid Carriers
baicalein	rat model	higher drug accumulation	superior brain targeting	(317)
quercetin	Caco-2 cells	increased drug residence	improved stress resistance	(318)
Metal Nanoparticles
curcumin	Tg2576 mice	enhanced affinity to Aβ-plaques	may interfere with Aβ fibrillation	(319)
	neuro2a cells	high solubility and Aβ affinity	dissolves amyloid fibrils	(320)
anthocyanins	AD mice model	better release and bioavailability	inhibits neuronal apoptosis	(204)
	AD mice model	instability, bioavailability	lowers Aβ induced inflammation	(321)
quercetin	rat model	high plasma concentration	prevent neural cell apoptosis	(227)
	SH-SY5Y cells	high BBB permeability	enhances autophagy clearance	(322)

We can understand this amalgamation of natural drugs and nanomedicine by a few studies performed in recent years, which have successfully addressed the clinical challenges against many such flavonoid based drug molecules. Naringenin protects neurons against the free radicals and suppresses the inflammation but has limited permeability across membranes; however, naringenin nanoemulsions delivered via a intranasal route have shown promising effects in Alzheimer’s and Parkinson’s disease models.^198,199 Similarly, the neuroprotective potential of highly active quercetin could be masked by constraints, such as low solubility, fast metabolism in the system, and low accumulation in the brain.²⁰⁰ However, a quercetin-coated liposomal formulation was found to permeate BBB effectively and protect cellular AD models from Aβ-induced toxicity.²⁰¹ Selective targeting by conjugating liposomal surface to specific receptors, e.g., lactoferrin, could be another novel strategy, which can specifically target bioactive molecules, e.g., quercetin, to the brain and enhance its accumulation through the receptor-mediated transcytosis.²⁰¹ Another example of a receptor-mediated permeation approach was developed through bradykinin B2 receptor agonist RMP-7, which increases the access of hydrophilic drugs across the tight junction of the BBB.²⁰²

Anthocyanins are very unstable at gastrointestinal pH; therefore, their encapsulation in nanoemulsions has displayed increased retention with an estimated half-life of 385 days, enhancing their antioxidant potential manifold.²⁰³ Additionally, polyethylene glycol (PEG)-ylation of nanoparticles assists in preventing nonspecific interactions of flavonoids with blood proteins and promote their stability. PEGylated gold NP formulation of anthocyanin may suppress amyloid beta-induced neurotoxicity and reduce neuroinflammation through dysregulation of PI3K/Akt/GSK3β signaling and inhibition of tau hyperphosphorylation.²⁰⁴ Nanomodification strategies facilitate several favorable modifications to these neurologically important molecules without losing their original properties providing many advantages over traditional drug delivery schemes. A major challenge before flavonoid-based drug formulations is their sparingly soluble nature in aqueous solutions, affecting their availability in the bloodstream, followed by rapid elimination. Entrapment of baicalein, an aglycone derivative having very low aqueous solubility and stability under biological conditions, in a nanoliposomal preparation decorated with cholesterol and PEG significantly reduced its cytotoxicity and augmented its oxidative stress management, cell uptake, and neuroprotection capabilities in SH-SY5Y and PC12 cell lines.²⁰⁵

Increased uptake and greater accumulation of biodrugs have been achieved in recent years through several novel formulation types, e.g., solid lipid nanoparticles, nanostructured lipid carriers, and selenium nanoparticles of resveratrol, EGCG, and curcumin-like bioactive drug compounds.^206−208 Theracurmin, a colloidal nanoparticle consisting of curcumin, glycerine, and a water-soluble polysaccharide, has shown encouraging outcomes in a randomized clinical study, where the solubility and oral bioavailability of curcumin was increased up to ∼16 times in comparison with unformulated curcumin when administered in healthy volunteers.²⁰⁹ Similarly, LongVida, a solid lipid curcumin particle (SLCP)-based formulation, improved bioavailability of curcumin in addition to protecting it from fast metabolism and elimination in osteosarcoma patients reflecting the importance of solid lipid particles in overcoming clinical constraints of traditional medicine curcumin.²¹⁰ Solid lipid curcumin particles have also been designed and successfully tested against neuroblastoma cells, AD mouse and rat models, and have shown encouraging results, like improved stability and delivery across BBB.^211−213

Incorporation of flavonoids to metal nanoparticles (e.g., gold) benefits maintaining the biocompatibility of NPs that ranges from a reduction in cytotoxicity caused by metal ions to preventing aggregation in an aqueous environment and providing additional stability to the nanoparticles.²¹⁴ Over many years, there has been a steep increase in synthesis and development of several novel nanoformulations, which have paved the way for many drugs to be revisited and applied in neurotherapeutics. Here, we are providing a summarized overview of the specific evolution of different nanoformulations in the field of AD research.

Metallic Nanoparticles (MNPs)

Metal may induce excessive ROS production leading to an exaggeration of AD symptoms; therefore, the application of MNPs in AD pathology is debatable. Also, the accumulation of MNPs such as Ag, TiO₂, ZnO, etc., in delicate organs like the brain may have profound implications leading to permanent damage.²¹⁵ However, a decreased neurotoxicity has been observed in the presence of antioxidants, such as N-acetyl-l-cysteine, selenium, etc., which has neuroprotective effects.^216,217 Selenium, a trace element, and its NPs have gained special attention in recent years due to its lower toxicity and antioxidant properties along with exceptional antiaggregatory properties.^217,218 A formulation of selenium NPs and sialic acid-functionalized with BBB permeable peptides B6 has demonstrated exceptional results.²¹⁹ Toxicity issues can also be undermined through the biological synthesis of these nanoparticles where phytochemicals and other biological sources replace harmful chemical reagents to reduce and stabilize metal ions, an approach generally referred to as green synthesis.^220,221 Capping of metal nanoparticles (iron oxide, cadmium sulfide) with proteins of fungal origin also has shown disaggregation of tau without affecting the cell viability of neuroblastoma cells.²²²

Figure 3.

A concise overview of multifunctional nanoparticles: Schematic representation of two major approaches of NP synthesis (in the central part): Top-down and bottom-up, accomplished with the help of physical, chemical, and biological methods of synthesis (left). A representative nanoparticle is decorated with multiple ligands, antibodies, peptides, nucleic acid, etc., for targeted drug delivery (in the oval). Different types of nanoformulations commonly used for drug delivery purposes, and a few critical parameters of classifying these nanomaterials (right).

Flavonoids with immense antioxidant potential, e.g., EGCG, quercetin, anthocyanin, and curcumin, have been combined with selenium, iron oxide, and gold nanoparticles. Silver and gold NPs and various modifications of these metallic nanoparticles have been tested in multiple disease therapeutics in the past.^115,223,224 Modification to these nanoscale moieties with functional groups, ligands, and macromolecules like peptides, proteins, and DNA may facilitate the greater accumulation of therapeutic substances at the target tissue.²²⁵ In a recent study, EGCG-functionalized selenium NPs were coated with Tet-1 peptides for higher cellular uptake and were able to inhibit amyloid aggregation and also to disaggregate the preformed toxic aggregates.²⁰⁶ Similarly, PEG stabilized gold nanorods, when dual-functionalized with Angiopep 2 and D1 peptides, had promising efficiency of reaching the CNS respectively and targeting Aβ aggregates, without compromising neuronal cell viability.²²⁶ In another study, conjugation of quercetin with super-paramagnetic iron oxide nanoparticles enhanced bioavailability and improved learning and memory in healthy rats.²²⁷

Silica-Based Nanoparticles

The therapeutic potential of silica-based nanoparticles, a promising delivery platform for targeted brain therapy, is severely compromised by its neurotoxic behavior attributed to excessive generation of oxidative stress and neuroinflammation that might increase the risk of developing AD.^228,229 These adverse effects could be ameliorated by organic modification and functionalization of silica nanoparticles, consisting of stabilized monodispersed aqueous nanosuspension with surface-functionalization using amino groups.²³⁰ Silica nanoparticles synthesized as nanocarriers of flavonoids like quercetin and naringin provided beneficial results in terms of enhanced neuroprotective capacity against Aβ-induced oxidative stress. The nanoparticles were more competent in loading capacity, antioxidant activity, and release profile.^231,232 A separate study involving catechin-loaded silica nanoparticles showed enhanced cell survival and protective activity in primary rat hippocampal neurons against Cu²⁺-induced oxidative stress.²³³

Liposomes

Liposomes are vesicle-like structures made up of phospholipid bilayers. They are essential in the context of AD because of their capabilities of crossing BBB and safely delivering both hydrophilic and hydrophobic molecules to their target tissues without causing much side effects.²³⁴ In some cases, liposomes can be administered intranasally, resulting in improved penetration into the brain while protecting their contents from degradation.²³⁵ Their surfaces can be modified for selective targeting using antibodies against the transferrin receptor, which increases its affinity toward the brain and amyloid-β peptides.²³⁶ They are preferred over other nanomaterials because of their biodegradability and increased cellular uptake without eliciting any immunological response.²³⁷ Liposomal formulations of curcumin have been prepared and examined both in vitro and in vivo, have shown a very high affinity for Aβ_1–42 fibrils, and were significantly effective in ameliorating Aβ-induced neurotoxicity in AD mice.^238,239 These formulations exhibited enhanced solubility and cellular uptake without causing toxicity. Additional PEG coating of liposomes may prolong the circulation time of the bioactive curcumin.²⁴⁰ In another study, liposomal formulations of quercetin showed cognitive enhancement at a much lower dose than the conventional form of the drug.²⁴¹

Polymeric Nanoparticles

A number of polymeric nanoparticles in the form of either nanocapsule, where the drug molecules are enclosed inside a polymeric membrane, or nanospheres in which therapeutic agents are evenly dispersed throughout the polymer matrix, have been designed and successfully tested on AD mouse models.¹⁵ The polymer often used in such preparations PLGA can readily encapsulate hydrophobic compounds to form PLGA derived nanoparticles. These nanoparticles can be further modified to add favorable characteristics like solubility or receptor-mediated targeting of therapeutic molecules.²⁴² For example, the efficiency of anthocyanins, curcumin, and naringin was found to be highly improved when encapsulated in PLGA or PEG alone or in combination.^231,243,244 These formulations enhanced bioavailability and neuroprotective capacity of flavonoids manifolds by protecting them from degradation, oxidation, and unfavorable interactions with plasma proteins, along with their enhanced capacity of diminishing oxidative stress and destructing Aβ aggregates.

Solid Lipid Nanoparticles (SLNs)

SLNs are aqueous colloidal nanoparticles suitable for delivering both hydrophilic and lipophilic drugs to their target tissues or sites.^245,246 They provide better tolerance than polymeric nanoparticles and, at the same time, appear to resolve stability and drug release issues of lipid nanoemulsions and liposomes.²⁴⁷ Several SLNs encapsulating chrysin and curcumin have been useful in AD animal models with increased uptake, bioavailability, permeability, and greater neuroprotection without loss in its antioxidant capabilities in rat AD models.^211,248 In a different study, resveratrol has been encapsulated in an anti-transferrin receptor monoclonal antibody functionalized solid lipid nanoparticles, which has substantially enhanced uptake by human brain-like endothelial cells as compared to normal SLNs or the ones functionalized with an unspecific antibody.²⁰⁷ SLN-encapsulated quercetin also showed increased memory-retention in mice and attenuating effect in neurocognitive impairments along with amelioration of biochemical changes in zebrafish.^249,250

Protein Nanoparticles

Protein nanoparticles could potentially transform properties of materials in terms of size and surface area, prompting them to be more reactive and available for surface modifications.²⁵¹ The significance of protein-based nanostructures has recently been realized due to their wide range of applicability attributed to very high biodegradability and biocompatibility.²⁵² A plethora of protein nanoparticles made up of proteins, like whey protein, gelatin, zein, soy protein, and milk protein, have been explored and reported by researchers.²⁵³ Flavonoids like quercetin and resveratrol encapsulated with the help of zein, a maize protein, to produce colloidal nanoparticles have been reported recently.²⁵⁴ Quercetin encapsulated zein nanoparticles have shown improved oral bioavailability (∼60%) with sustained plasma levels of quercetin, besides improved cognition and reduced astrogliosis in the Samp8 mice model.²⁵⁵ Similarly, resveratrol encapsulated zein NPs offered high and prolonged plasma levels of resveratrol with significant oral bioavailability improvement (∼50%) in mice models. Further, a pH-independent release of resveratrol from the nanoparticles and moderate augmentation in anti-inflammatory effects was observed.²⁵⁶ Improvements in the oral and topical bioavailability of resveratrol were also achieved with the help of encapsulated casein nanoparticles.²⁵⁷

Figure 4.

Therapeutic potential of flavonoid nanoformulations in Alzheimer’s disease: Combination of therapeutic properties of flavonoids with nanobased delivery systems may provide an effective therapeutic tool for the successful cure of Alzheimer’s disease, which remains one of the most challenging puzzles of the medical sciences after more than a 100 years of its discovery.

Conclusions and Future Directions

Neurodegeneration is a gradual and multifactorial process that starts with genetic or environmental factors and progresses with exacerbation of accumulated oxidative stress, proteotoxic load, compromised cellular energetics, mitochondrial dysfunction, and neuroinflammation.^258,259 The growing number of evidence supports the notion that many natural molecules, especially the dietary flavonoids, could be considered therapeutic alternatives in AD due to their innumerable health benefits, including antioxidative, anti-inflammatory, and anti-amyloidogenic properties. Furthermore, flavonoids can pass through the BBB and positively support the brain from multiple kinds of stresses and damages.²⁶⁰ Also, their multifaceted potential of targeting different subcellular pathways and many systemic changes, like oxidative damage, glutamate storm, inflammation, and compromised proteostasis, has been explored and widely known.

Multiple studies are underway to present flavonoids as possible AD drugs at the preclinical level. Unfortunately, a few clinical studies conducted on curcumin and resveratrol in the past have failed to provide convincing results due to inefficient delivery, and some reported toxicities.^261,262 In several independent studies, challenges like poor pharmacokinetic profile, low permeability across BBB, stability, and aqueous solubility for flavonoids have been observed, which may hinder or challenge these drug candidates’ potential applicability in the context of CNS disorders. However, nanobased delivery systems can greatly enhance the biomedical applications of flavonoids and other natural molecules in the context of brain diseases. The amalgamation of natural products with nanomedicine in the past decade has given enormous hope for the drug discovery paradigm in the context of many critical diseases, including cancer and neurodegeneration. A fusion of flavonoids with the nano interface has been found to resolve existing pharmacokinetic and toxicity issues by modulating NPs surface chemistry to a great extent. The mechanism through which this change occurs is still not adequately understood and needs to be elaborated further. In-depth perusal into the mechanisms by which flavonoids exhibit their neuroprotective effects and alter properties of nanomaterials to reduce side effects, rendering them nontoxic and multifold beneficial in drug delivery is needed.

Further studies are required to decipher the protein targets and flavonoid–protein interactions in AD to successfully translate the experimental results into clinical outcomes. The central focus should also be given to studies focusing on devising the long-term systemic efficacy, dosage concentrations, and a precise route of administering these drugs for their clinical studies usage before we can consider flavonoid-based nanodrugs in Alzheimer’s treatment. Studies on the combination of flavonoids to enhance the synergistic effects in its correct therapeutic dosage in cells and animal models could also be pursued for a superior outcome. In the context of NPs, more focus should be given to the furtherance of enhancing the synthesis processes and physicochemical properties, including size, shape, structure, surface coating, zeta potential, conjugation procedure, and safety concerns. These parameters are crucial in determining the pharmacokinetics, cellular uptake, and cytotoxicity of nanodrugs and optimizing the delivery systems toward their successful translation into clinics. For example, polymeric NPs show pH and temperature specific enhancement in targeting and efficacy to deliver drugs across physiological barriers.²⁶³

These reports altogether suggest that an in-depth understanding of nanomaterials is essential in fabricating appropriate biocompatible nanocarrier corresponding to the targeted site and mechanism of action against AD. The advanced methodologies involved in the enrichment of pharmacological properties of flavonoid nanoformulations with subsequent modifications in their pharmacokinetic and pharmacodynamic profiles need to be deciphered through detailed in vivo studies before we can reach up to some conclusions regarding the future of this amalgamation of natural products and nanomedicine.

Glossary

Abbreviations

7,8-DHF

7,8-dihydroxyflavone

ABCA7

ATP-binding cassette transporter A7

AChE

acetylcholinesterase

AChEI

acetylcholinesterase Inhibitor

AD

Alzheimer’s disease

ADAM10

A disintegrin and metalloprotease 10

Akt

protein kinase B

ALS

amyotrophic lateral sclerosis

AMPK

AMP-activated protein kinase

APOε4

apolipoprotein E type 4

APP

amyloid precursor protein

Aβ

amyloid beta

BACE

beta-secretase

BBB

blood–brain barrier

BChE

butyrylcholinesterase

BDNF

brain derived neurotropic factor

BIN1

bridging integrator 1

Cdk5

cyclin-dependent kinase 5

CNS

central nervous system

CLU

clusterin

CPP

cell-penetrating peptide

CREB

cAMP response element-binding protein

EGCG

(−)-epigallocatechin-3-gallate

Egr1

early growth response protein 1

ERK

extracellular signal-regulated kinases

FDA

Food and Drug Administration

FGF-2

fibroblast growth factor 2

GFAP

glial fibrillary acidic protein

GLUT

glucose transporter

GPx

glutathione peroxidase

GSH

glutathione

GSK-3β

glycogen synthase kinase 3β

HIF-1α

hypoxia-inducible factor-1 alpha

HO-1

heme oxygenase-1

IL-1

interleukin-1

IL-4

interleukin-4

iNOS

inducible nitric oxide synthase

IR

insulin receptor

JNK

c-Jun N-terminal kinase

LTP

long-term potentiation

MAPK

mitogen-activated protein kinase

MDA

malondialdehyde

MMP9

matrix metallopeptidase 9

MNPs

metallic nanoparticles

mTOR

mammalian target of rapamycin

NFT

neurofibrillary tangles

NLC

nanostructured lipid carriers

NMDA

N-methyl-d-aspartate

NP

nanoparticle

Nrf2

nuclear factor erythroid 2-related factor 2

PA2

phospholipase A2

PARP

poly(ADP-ribose) polymerase

PBCA

polysorbate 80 coated poly n-butylcyanoacrylate

PD

Parkinson’s disease

PEG

polyethylene glycol

PHD

proline hydroxylase

PICALM

phosphatidylinositol binding clathrin assembly protein

PKC

protein kinase C

PLGA

poly lactic-co-glycolic acid

PSEN

presenilins

PI3K

phosphoinositide 3-kinase

PKA

PPAR-γ, peroxisome proliferator-activated receptor-γ

ROS

reactive oxygen species

SAPK

stress-activated protein kinases

SIRT1

sirtuin1

SLCP

solid lipid curcumin particle

SLNs

solid lipid nanoparticles

SOD

superoxide dismutase

SORL1

sortilin-related receptor 1

TNF-α

tumor necrosis factor-α

TREM2

triggering receptor expressed on myeloid cells 2

TrkB

tropomyosin-receptor-kinase B

TSPO

translocator protein

Unc5c

uncoordinated 5c

The authors declare no competing financial interest.