Dendrimers in Neurodegenerative Diseases

Abstract

Neurodegenerative diseases (NDs), such as Parkinson’s Disease (PD), Alzheimer’s Disease (AD), Multiple Sclerosis (MS) and amyotrophic lateral sclerosis (ALS), are characterized by progressive loss of structure or function of neurons. Current therapies for NDs are only symptomatic and long-term ineffective. This challenge has promoted the development of new therapies against relevant targets in these pathologies. In this review, we will focus on the most promising therapeutic approaches based on dendrimers (DDs) specially designed for the treatment and diagnosis of NDs. DDs are well-defined polymeric structures that provide a multifunctional platform for developing different nanosystems for a myriad of applications. DDs have been proposed as interesting drug delivery systems with the ability to cross the blood–brain barrier (BBB) and increase the bioavailability of classical drugs in the brain, as well as genetic material, by reducing the synthesis of specific targets, as β-amyloid peptide. Moreover, DDs have been shown to be promising anti-amyloidogenic systems against amyloid-β peptide (Aβ) and Tau aggregation, powerful agents for blocking α-synuclein (α-syn) fibrillation, exhibit anti-inflammatory properties, promote cellular uptake to certain cell types, and are potential tools for ND diagnosis. In summary, DDs have emerged as promising alternatives to current ND therapies since they may limit the extent of damage and provide neuroprotection to the affected tissues.

1. Introduction

Neurodegenerative diseases (NDs), such as Parkinson′s disease (PD), Alzheimer’s disease (AD), multiple sclerosis (MD) and amyotrophic lateral sclerosis (ALS), are a group of progressive and chronic pathological conditions derived from neuronal loss. There are several common features of these syndromes. On the one hand, the cause of these disorders seems to be a combination of genetic factors and environmental conditions, such as aging. On the other hand, these pathologies are characterized by neuroinflammation and the accumulation of aberrant proteins in toxic aggregates responsible for clinical symptoms [1]. Moreover, the prevalence of these disorders is expected to increase in the coming years due to the lack of effective therapies and the increasing life expectancy of the population. Currently, the treatment available for these disorders is symptomatic. Pharmacological and non-pharmacological approaches do not modify the pathological causes of these diseases or their progression, resulting in a loss of effectiveness and clinical benefits over time. Therefore, NDs represent one of the world’s greatest health challenges and claim the development of new therapeutic approaches. New emerging strategies for the treatment of NDs are known as disease-modifying therapies (DMT), specially designed to stop or slow down the pathological course of these diseases and improve the quality of life of patients. DMT includes small molecules and non-drug therapies, such as gene therapy approaches, neurotrophic factors or cell-base treatments, which are in different phases of preclinical and clinical trials, extensively reviewed (for PD [2,3,4] for AD [5,6,7,8] and for MS [9,10]).

Dendrimers (DDs) are well-defined polymeric structures that offer a multifunctional platform for developing different nanosystems with multiple applications. They have been proposed as interesting drug delivery systems with the ability to cross the blood–brain barrier (BBB) and increase the bioavailability of classical drugs in the brain. In addition, DDs have demonstrated their efficacy in the delivery of genetic material by reducing the expression of specific target proteins. Moreover, DDs have shown promise as anti-amyloidogenic systems, exhibit anti-inflammatory properties, promote cellular uptake to certain cell types, and are potential tools for the diagnosis of ND. Finally, DDs have demonstrated greater aqueous solubility, biocompatibility, polyvalence and precise molecular weight than traditional polymers, making them suitable for targeted drug delivery to the brain [11,12,13,14,15,16,17].

In this review, we will focus on different therapeutic approaches based on DDs specially designed for the treatment and diagnosis of NDs. We will describe the most promising dendrimer nanoparticles currently employed as gene delivery systems, drug carriers or DDs with inherent anti-inflammatory properties, and finally, we will also summarize their use as diagnostic tools.

2. Nanomedicine

Nanotechnology is an innovative branch of science and engineering that involves the design, synthesis, characterization and application of materials and devices at atomic, molecular and macromolecular scales with nanometric size (10⁻⁹ to 10⁻⁶ m). It is a multidisciplinary field that integrates physical, chemical and biological sciences with engineering. It is a recent cutting-edge technology with high potential in several areas, such as electronic, chemical engineering, environmental and food industry, pharmacology, and medicine, due to the possibility of creating novel products with new features. One of the most attractive fields of application of nanotechnology is nanomedicine, which aims to prevent, diagnose and treat different diseases [11]. Nanomedicine has been persistently and exponentially growing over the last decades. The role of nanotechnology in biomedical sciences is mainly focused on improving the properties of diagnostic or therapeutic approaches by means of nano-size compounds, also called nanoparticles (NPs), which are used for a variety of purposes. For example, NPs are excellent drug carriers with the ability to increase the bioavailability of therapeutic compounds by different mechanisms, such as increasing water solubility [12], targeting the desired tissue [13], increasing blood circulation time [14], avoiding enzymatic degradation, evading immune system [15] or allowing more efficient administration routes. This is the case with intranasal delivery instead of oral intake for the treatment of NDs [16]. Moreover, NPs are also an excellent tool for delivering genetic material [17] with the ability to protect it from degradative enzymes inside the human body [18]. On the other hand, NPs can be used as diagnostic compounds due to their chemical nature, such as magnetic nanoparticles (MNPs), which act as contrast agents for imaging [19] or as theragnostic compounds that function as both therapeutic and diagnostic tools [20,21]. There are several types of NPs with potential biological applications in nanomedicine, such as liposomes (LP), DDs, gold NPs, polymeric micelles, metallic NPs, fullerenes or quantum dots [22]. The particle size and surface area of these NPs are critical factors that determine their interaction with biological systems. Apparently, as the size of the materials decreases, there is an exponential increase in surface area relative to volume, and more atoms are on the surface compared to those in the interior. This makes nanomaterials more chemically reactive. For this reason, particle size and surface area determine not only the biological function of NPs but also their toxicity [23] and biodistribution [24]. Despite their excellent properties, NPs inevitably bind to biomolecules in biological fluids, forming the “protein corona” when they are introduced into living systems [25]. In addition to the bio-nano interaction, NPs are also recognized as foreign substances by the human body, and they are usually sequestered, degraded and eliminated by the mononuclear phagocytic system, leading to rapid clearance, low efficiency, and high liver accumulation. As a consequence, few NPs have been included in phase 1 clinical trials due to the limited characterization of several aspects, such as pharmacokinetic parameters or immune response [26]. To overcome this issue and improve their pharmacokinetic profile, NPs are usually decorated on their surfaces. Different molecules, including polymers and biomolecules, have been linked to NPs to reduce their interactions with serum proteins and increase their availability in the bloodstream. For example, polyethylene glycol (PEG) addition is usually employed to increase blood clearance. However, these structural modifications may also affect the biological function of NPs and hinder their uptake by the target cells [27]. Therefore, an in-depth knowledge of the pharmacokinetic, pharmacodynamic and toxicity profile of functionalized NPs is required prior to their use in humans.

Nanotechnology has been widely applied for both diagnostic and therapeutic purposes in several pathologies, such as NDs [28], inflammatory diseases [29], cardiovascular problems [30] and infectious diseases [31]. Currently, the best known use of nanotechnology in the clinical setting is the mRNA-based COVID-19 vaccines, which encapsulate the mRNA of the SARS-CoV-2 spike protein into lipid-base nanoparticles (LNPs) and deliver it into host cells [32,33]. In addition, nanotechnology has been extended to the clinic for oncology therapies [34] and there are some nano-formulations approved for medical use, such as Vyxeos^®, which consist of cytarabine plus daunorubicin encapsulated in LP for the treatment of acute myeloid leukemia [35]. NBTXR3, an NP coated with negatively charged phosphate groups, is currently being studied in a I-II phase clinical trial (NCT02805894) for the treatment of intermediate/high risk prostate cancer. Another example is Patisiran/ONPATTRO^® formulation, a lipid-based delivery system approved for the treatment of hereditary transthyretin amyloidosis [36].

Dendrimers

The word “dendrimer” was created by Prof. Donald Tomalia from the Greek words δέντρo (dendro) and μέροσ (meros), which translates to “tree and “part,” respectively [37]. DDs are highly branched, three-dimensional synthetic macromolecules with a well-defined structure. They are synthesized with precise control of their size, shape and terminal group functionality [38]. In their basic structure, DDs contain three different parts: a central core (either a single atom or a group of atoms having two or more identical chemical functionalities), the external branches emanating from the core as repetition units and the terminal groups located on the surface of the NP. The assembly of units is organized in a geometric progression from the core, which results in a series of radially concentric layers. The sequential growth of DDs is identified with a specific “generation number” (G) and each generation is classified in terms of size, shape, molecular weight and number of surface functional groups. The core is defined as Generation 0 (G0), while the successive addition of branching units gives rise to higher generations such as G1, G2 or G3, e.g., (Figure 1) [38]. The core and the layers of the branching units (generations) affect DD morphology, specifically in the three-dimensional shape. Instead, the internal part determines the hostguess properties of the DDs. Finally, the external charge, the flexibility of the branches and the chemical nature of terminal groups determine their functionality and their interaction with solvents, other molecules or cell structures [39]. Lower generations of DDS are more flexible and have more open and amorphous structures than higher generation DDs, which can adopt spherical conformations. This is known as the “dendritic effect” and is recognized as a unique property dependent on the architecture and size of the DD [40]. The structure of DDs can be precisely controlled and reproduced during their stepwise synthesis by divergent or convergent strategies, hypercore and branched synthesis, “click chemistry,” lego chemistry and double and mixed exponential growth methods [41]. Divergent and convergent methods are the most commonly used. In divergent synthesis, the DD structure initiates at the central core and monomers are added sequentially to create the desired DD generation. However, in the convergent method, DDs start at the periphery and grow to the central core [42]. Consequently, DDs are compounds with well-defined structures, narrow polydispersity and multivalent properties [38]. Unlike other NPs, DDs are characterized by low cytotoxicity, high efficiency to penetrate into the cells and intrinsic fluoresce [43]. Moreover, DDs present additional advantages, such as high stability [44] and the possibility of including a large number of active therapeutic drugs covalently [45]. On the other hand, DD properties can be easily tuned by replacing their terminal groups and modifying the external electrical charge. Consequently, three different families of DDs can be distinguished: cationic, anionic and neutral. Cationic DDs have tertiary amines as terminal functions, which are subsequently protonated, providing a net positive charge to the molecule. Instead, negatively charged DDs are classically obtained by grafting carboxylic acids as terminal functions from which sodium salts are easily obtained. In marked contrast, the use of neutral dendrimers has been poorly explored [39].

DD cytotoxicity depends on the structure and type of terminal groups [46]. Interestingly, the polyvalency or the addition of several chemically different end groups provides versatile functionalization that might produce multiple interactions with specific biological receptor domains. Moreover, the structure of DDs can be easily modified on their surface to provide them with additional multifunctional properties [47]. Functionalization of the periphery by the addition of diverse functional groups can improve interesting properties, such as pharmacological and pharmacokinetic profiles, selectivity for the target, and physicochemical features of the drugs, such as viscosity, solubility and stability [48]. For instance, DD decoration with transferrin facilitates the penetration of the BBB by interacting with specific receptors located on the surface of endothelial cells [49]. Other examples of surface modifications are PEGylation [50] or carbohydrate coupling, which reduce DD toxicity and increase stability in biological media [51,52].

To date, more than 200 kinds of DDs have been synthesized and grouped into different families according to their structure, including poly(amidoamine) (PAMAM), poly(propyleneimine) (PPI), poly(L-lysine) (PLL), poly(carbosilane) (CBS) and phosphorous-containing DDs [53].

The PAMAM family of DDs is one of the most widely used nanomedicines due to its hydrophilic profile and biocompatible properties [41]. This type of NP was defined by Tomalia and co-workers in 1985 [37] and is clearly recognized by a symmetrical structure. They consist of a core, synthesized by the addition of ethylenediamine, diaminododecane, diaminoexane and diaminobutane, followed by the internal branches of methyl acrylate and ethylenediamine and the final amide or carboxilic end-groups [54].

PPI DDs, the oldest class of DDs, were produced by Voegel in 1978. PPI DDs are synthesized by the divergent method by the addition of acrylonitrile, diamine or diaminobutane [55,56]. The most important advantages of PPI DDs include high biocompatibility, high surface area and fast electron transfer rate [57].

PLL DDs are polyaminoacidic structures of PLL organized in linear or cyclic structures. They have gained attention due to their high biocompatibility and easy functionalization of terminal groups [58].

CBS DDs are silicon-containing DDS, first defined in 1992. They are dendrimeric structures containing Si-C bonds at the branches, which provide high stability, flexibility and low polarity to the compounds. Several types of CBS DDS have been synthesized using the divergent method [59].

Phosphorous-containing DDs were defined in 1990. The first example of phosphorous DDs built with phosphonium at each branching point was reported by Engel and co-workers. However, it has never been used for biological purposes [60]. In 1994, Majoral and his group described the first method of synthesis for large phosphorous DDs, which is the most widely used [61]. There are three different families of phosphoDD based on the surface charges: cationic, anionic and neutral. Positively charged DDs were found to be more toxic than negatively charged or neutral DDs [40].

Among their biological applications, DDs are emerging as promising candidates for many applications in nanomedicine and deserve attention as solubility enhancers, carriers for drugs and biological entities and imaging agents [47]. In particular, DDs are interesting drug delivery systems [62,63] in cancer [64,65], NDs [66], ocular problems [67], infectious diseases [68,69] and fungi contamination [70]. DDs are efficient transfection agents for genetic materials, with special attention as carriers in the nervous system. Different ligands can be coupled to DDs to use as transfection reagents. For example, DDs are able to transport small interfering ribonucleic acid (siRNA), deoxyribonucleic acid (DNA) or plasmids into different cell types [64,71,72,73]. The transfection efficiency of DDs depends on the chemical nature of the terminal groups and their size, with the G3, G4 and G5 generations being the most efficient. Similar to other NPs, DDs can be easily modified on their surfaces to improve their therapeutic efficacy [74]. Apart from its use as a gene delivery system, DDs are powerful bioimaging and diagnostic tools for cancer [75,76]. Moreover, DDs offer additional applications in nanomedicine due to their versatility. Several studies have demonstrated its potential as drugs per se as promising anti-cancer agents or powerful anti-inflammatory molecules [29,77], even as key components included in the formulation of biomaterials, such as biochips [78] and vaccines [74].

3. Use of Dendrimers in Neurodegenerative Diseases

3.1. Alzheimer’s Disease

Since Alois Alzheimer described AD in 1907, it has been considered the most prevalent ND worldwide and the major cause of dementia [79]. AD is a progressive and chronic disabling pathology characterized by cognitive impairment, behavioral disturbances, and loss of memory and functional ability to perform daily tasks [80]. Its incidence increases proportionally with the aging of society, especially those over 75 years of age, and it is estimated that the current 45 million people affected will increase to triplicate the number of patients by 2050 [81]. The pathogenesis of AD is not fully understood, but neuronal loss and hippocampal and cortical atrophy are related to two main pathological features: the deposition of extracellular amyloid plaques and intracellular neurofibrillary tangles (NFT) [82]. The main component of amyloid plaques is the aberrant folded amyloid beta (Aβ) peptide with 42 amino acids [83], while hyperphosphorylated Tau protein (p-Tau) is the predominant element of NFT [84]. According to the amyloid cascade hypothesis, Aβ formation and subsequent aggregation in amyloid plaques is the initial step in the onset of AD and determines the following deposition of p-Tau in NFT. Cognitive impairment and progression of clinical symptoms correlate better with NFT accumulation than amyloid pathology [85]. Instead, progressive synaptic dysfunction and neuronal degeneration are attributed to neuroinflammation due to microglia activation [86].

Classical therapy for AD aims to improve progressive cognitive decline and impairment of functional abilities, such as memory. Pharmacological treatment includes acetylcholinesterase (AChE) inhibitors (donepezil, galantamine and rivastigmine) [87] and antagonists of N-metyl-D-Aspartate (NMDA) receptor (memantine) [88], in combination with non-pharmacological therapies to improve the performance of daily tasks [89,90]. However, current AD therapy is only symptomatic and manages the clinical manifestations of the disease for a short period of time. Available treatments do not correct the primary causes of the pathology and are unable to prevent neuronal loss and brain atrophy that lead to cognitive impairment. This is the main reason supporting the development of new strategies aimed at modifying the pathological course of the disease.

3.1.1. Dendrimers for AD Therapy

Dendrimers for Decreasing β-Amyloid Aggregates

In the amyloidogenic pathway, Aβ peptide is initially found as a monomer after the processing of the amyloid precursor protein (APP) by β-secretases, such as β-amyloid converting enzyme 1 (BACE1) and γ-secretase enzyme. The length of Aβ peptide depends on the cutting site of γ-secretase. The peptide composed of 42 amino acids is the major compound and the most toxic species due to its tendency to aggregate, although Aβ 40 is also found in amyloid deposits [91,92]. The progressive self-aggregation of Aβ peptide forms larger harmful species, such as oligomers, protofibrils, fibrils and the final insoluble amyloid plaques, which are the major hallmarks of the brains of AD patients [93]. Therefore, the reduction of toxic amyloid plaques is proposed as one of the most promising therapeutic strategies for slowing the progression of AD. To this end, several mechanisms involving DDs have been described, including direct interaction of DDs with Aβ oligomers, inhibition of Aβ aggregation and degradation of existing fibrils (Figure 2).

Several studies support the use of DDs as candidates for avoiding Aβ-induced toxicity. For example, Chafekar and colleagues described a G1 cysteine DD couple to the KLVFF peptide to form monomeric (K1) or tetrameric (K4) structures. Both complexes prevented Aβ 42 aggregation into amyloid fibrils, but the dendritic K4 form showed a better inhibitory profile than the K1 structure, and it was also able to disaggregate existing fibrils and reduce amyloid deposits, supporting its use for AD therapy [94].

Additionally, PAMAM DDs have also been employed as interesting anti-amyloidogenic tools. Three different generations of PAMAM DDs (G3, G4 and G5) avoided amyloid deposition of the 28 aa Aβ peptide (Aβ 1-28) and additionally promoted the disaggregation of existing fibrils in amorphous structures. The magnitude of the effect increased in a concentration-dependent manner and was also proportional to DD generation. The larger PAMAM DD generations were the most effective in disrupting the amyloid aggregation process [95]. In addition, the same group described the effect of G3 PAMAM DDs on the aggregation of Aβ 1-28 peptide. G3-PAMAM DD inhibited the formation of amyloid plaques due to a direct interaction between DD and the peptide. While low DD concentrations affected the nucleation process, high DD concentrations modified the elongation rate and the interaction of monomers with existing small aggregates [96]. Additionally, bio decorated PAMAM DDs have been explored as powerful tools to decrease the accumulation of senile plaques. G0 PAMAM DDs attached to helical β-peptide KLVFFAE or foldamers recognized Aβ oligomers avoiding their aggregation. Foldamer-DD complexes showed protective properties against Aβ toxicity in murine hippocampal slices by reducing long-term potentiation [97].

Although DDs have been demonstrated to be promising anti-amyloidogenic systems, cationic dendrimers are often cytotoxic because of the interaction with negative charges on cellular membranes [98]. One strategy for reducing the cellular toxicity of DDs is to decorate their surface with glycidic structures, such as maltose, to form glycodendrimers. This approach has been explored by several groups. For example, in a first study, Klementieva and colleagues showed that G4 and G5 maltose-PPI DDs reduced the aggregation of the amyloid peptide fragment of 40 aa (Aβ 40) in a generation-dependent manner. Moreover, they were biocompatible with PC12 and SH-SY5Y cell lines [99]. The G5 DDs avoided amyloid toxicity by forming amorphous structures, whereas the G4 conjugates decreased Aβ deposition without affecting preformed fibrils. A similar neuroprotective effect of PPI glycodendrimers on aberrant amyloid fibrillation has been observed in a second study. Different neutral and cationic G4 and G5 PPI DDs, coupled to maltose or maltotriose, protected SH-SY5Y cells against Aβ 42 toxicity, but only neutral PPI glycodendrimers reduced the toxicity of Aβ peptide obtained from human AD brain extracts. Moreover, both cationic and neutral G4 and G5 conjugates decreased Aβ levels in transgenic mouse models of AD, although they did not reverse memory impairment [51]. The protective effect of PPI DDs against Aβ 1-28 aggregation was also confirmed by modulation of amyloid fibrillation in a pH-dependent manner. At higher DD concentrations, the amyloidogenic process was totally inhibited, but at low DD concentrations, the amyloid aggregation process depended on pH. At acidic pH values, PPI dendrimers interacted more strongly with the peptide and interrupted the fibrillation process, reducing amyloid aggregates [100].

Among the potential molecules that can interfere with Aβ fibrillation, the effect of PLL DDs of the third (G3) and fifth generations (G5) was also characterized in the SH-SY5Y cell line. Only G3 DDs avoided amyloid plaque formation in solution and toxicity of Aβ 42 in neuroblastoma cells [101].

Oligomeric species of Aβ are considered the most neurotoxic species in the pathological course of AD. Phosphorous DDs have been proposed as powerful tools for controlling the fibrilization of intermediate oligomers and avoiding subsequent toxicity in AD. Specifically, G3 and G4 cationic phosphorus-containing DDs inhibited the aggregation cascade of Aβ 1-28 and reduced cytotoxicity in the N2a cell line [102,103].

A different useful option to prevent Aβ 1-28 toxicity is gallic acid-triethylene glycol (GATG) G3 morpholine-decorated DDs (mor-G3). Interestingly, although this kind of DDs accelerated the aggregation of Aβ 1-28 and increased the amount of fibrils, the complexation of mor-G3 DDs with fibrillary species showed a protective effect by reducing Aβ prefibrillary forms and cytotoxicity in B14 fibroblast [104].

Dendrimers for Decreasing Tau Aggregation

Tau is a neuronal protein that promotes the assembly of tubulin and contributes to the stability of microtubules. The deposition of Aβ peptide promotes the hyperphospho-rylation of Tau, which modifies its physiological function and determines its accumulation in NFTs [105].

DDs have been proposed as good candidates for reducing NFT deposition (Figure 2). For example, G3 and G4 phosphorus-containing DDs were able to interact with Tau and promote its assembly into amorphous structures, thereby reducing its aggregation in NFTs. Both DDs modified the length of fibrillary structures, and additionally, G3 derivatives also influenced the amount of fibrillar aggregates [102].

Dendrimers for AD Treatment with Different Mechanisms: Miscellaneous

In addition to their role as nanocarriers or anti-amyloidogenic systems, DDs are considered per se potential drugs to slow or delay the progression of AD. For example, G4 PPI DDs decorated with histidine and maltose (G4-PPI-HisMal) showed neuroprotective properties to address the cognitive decline associated with AD. The treatment of AD APP/PS1 transgenic mice with G4-PPI-HisMal increased the expression of synaptic markers and prevented memory impairment without affecting the amount of amyloid plaques [106]. The use of DDs to improve cognitive decline in AD has also been performed with smart DDs, such as ALZc3. ALZc3 is a Mg-coupling DD at its core with chelating properties and antioxidant activity, useful for improving spatial memory in a rat model of AD based on Aβ-peptide toxicity [107]. The neurodegenerative process of AD is also a consequence of neuroinflammation and the reduction of cholinergic activity. Apart from their role in the modulation of Tau aggregation, cationic phosphoDDs were proposed as interesting drugs for AD therapy due to the combination of several mechanisms, such as the reduction of oxidative stress and the inhibition of AChE activity, to enhance cholinergic transmission [103].

3.1.2. Dendrimers as Vehicles of Therapeutic Genes

Another potential strategy for AD is based on targeting Aβ production by inhibiting the enzymes responsible for its synthesis, such as BACE1 and γ-secretase. One approach to downregulating protein expression levels focused on the delivery of siRNA using DDs as vehicles (Figure 2). Zhang and colleagues developed an siRNA delivery system formed by dendrigraft poly-L-lysines (DGL) coupled with NL4 peptide and apolipoprotein A-1 to enhance cellular uptake. This biocompatible gene carrier allowed the silencing of the BACE1 protein in PC12 neuronal cells [108]. On the other hand, the inhibition of γ-secretase activity has also been performed using a different approach. G0 and G1 lysine dendrons were employed as vehicles for the γ-secretase modulator Flurbiprofen. Both types of complexes successfully increased the penetrance of the drug through an in vitro model of the BBB based on bEnd.3 endothelial cells and reduced the activity of γ-secretase in C6 glial cells, [109]. A multifunctional carrier consisting of PEGylated DGL has been studied for the treatment of AD. This vehicle was designed as a dual delivery system of a specific peptide to target NFTs (TLKIV peptide) and short hairpin RNA (shRNA) molecules to knock down the BACE1 enzyme. The carrier showed effectiveness in crossing the BBB in double APP/PS1 transgenic mice of AD and decreased the accumulation of NFT in the hippocampus and blocked the activity of BACE1, ameliorating memory decline [110].

3.1.3. Dendrimers as Vehicles of Drugs in AD

The successful treatment of AD requires the transport of drugs to the target tissue, the brain (Figure 2). DDs have been proposed as interesting drug delivery systems with the ability to cross biological barriers, including the BBB, and to increase the bioavailability of drugs in the brain. Current therapy for AD includes AChE inhibitors and modulators of glutamatergic excitotoxicity [111]. To support drug penetration across the BBB, different G3 PAMAM DDs conjugated with lactoferrin have been designed to vehicle the NMDA receptor antagonist memantine. The complex improved the pharmacokinetic properties of the drug, increased its bioavailability in the brain and ameliorated the memory decline in AD murine models [112]. The PAMAM-lactoferrin complex was also employed as an effective strategy to increase the brain bioavailability of rivastigmine, an AChE inhibitor. This system was more effective than rivastigmine alone in improving motor and spatial learning and slowing memory loss in in vivo models of the disease [113]. G4 and G4.5 PAMAM DDs have been reported to modify the bioavailability of carbamazepine. While the drug poorly dissolves in water, the complexes increased solubility properties and reduced cytotoxicity in human red blood cells and the N2a line, as well as the side effects in zebrafish [114].

Apart from improving drug pharmacokinetic properties, DDs have been demonstrated to be interesting tools for reducing the toxicity of classical treatments for AD. For example, tacrine treatment restores impaired cholinergic activity in AD patients but induces severe side effects, such as liver damage. G4 and G4.5 PAMAM DDs reduced tacrine-induced hepatotoxicity. The electrostatic interaction between DDs and tacrine reduced the cytotoxic effect of the drug on human red blood cells and the N2a cell line and the hepatotoxicity in zebrafish without altering its pharmacological activity [115].

3.1.4. Dendrimers for AD Diagnosis

The ability of dendrimers to interact with molecules has been exploited to detect key proteins involved in the pathological course of AD. For example, PAMAM DDs coupled to gold nanoparticles were employed to characterize Tau levels in the plasma and brain samples of AD patients by sandwich immunoassay and amperometry. To this end, Razzino and co-workers designed a 3D-Au-PAMAM covalently attached onto electrografted p-aminobenzoic acid, and the specific capture antibody was immobilized on the amino groups of the DD. This system resulted in high selectivity and a detection limit in the picomolar range [116].

The application of DD for the treatment and diagnosis of AD is summarized in

Table 1. DDs for AD therapy and diagnosis. Abbreviations: AD (Alzheimer’s disease), GATG (gallic acid-triethylene glycol), DD (dendrimer), PLL (poly-L—lysine), DGL (dendrigraft poly-L-lysines), NFT (neurofibrillary tangles), BACE1 (β-amyloid converting enzyme 1), PEG (polyethylenglicol), AChE (acetylcholinesterase).

Strategy	Type of Dendrimer	Mechanism		Loading Cargo/Modification	References
AD therapy	Cysteine-DDs	Reduction of β-amyloid aggregates	Inhibition of Aβ 1-42 aggregation Reduction of pre-formed amyloid deposits	KLVFFGG peptide	Chafekar et al. [94]
	PAMAM DDs		Inhibition of Aβ 1-28 aggregation Disassembling of pre-existing aggregates		Klajnert et al. [95]
	PAMAM DDs		Inhibition of Aβ 1-28 aggregation		Klajnert et al. [96]
	PAMAM DDs		Capture of Aβ 1-42 aggregates	KLVFFAE peptide	Fülöp et al. [97]
	PPI DDs		Reduction of Aβ 1-40 aggregation and toxicity in PC12 and SH-SY5Y cell lines Reduction of Aβ 1-42 levels in SH-SY5Y cell line and APP/PS1 transgenic mice	Maltose Maltotriosa	Klementieva et al. [51,99]
	PPI DDs		Inhibition of Aβ 1-28 aggregation		Klajnert et al. [100]
	PLL DDs		Inhibition of Aβ 1-28 aggregation in solution Reduction of Aβ 1-42 aggregation in SH-SY5Y cell line		Neelov et al. [101]
	Phosphorous DDs		Blocking of Aβ 1-28 aggregation and reduction of toxicity in N2a cell line		Wasiak et al. [102,103]
	GATG DDs		Promotion of Aβ 1-28 aggregation and fibril formation in solution and reduction of prefibrillar toxic forms and toxicity in B14 fibroblast	Morpholine groups	Klajnert et al. [104]
	Phosphorous DDs	Reduction of Tau aggregation	Inhibition of Tau aggregation in NFT		Wasiak et al. [102]
	DGL	Transport of therapeutic genes and reduction of β-amyloid synthesis	Silencing of BACE 1 in PC12 cell line	BACE1 siRNA, NL4 peptide and apolipoprotein A-1	Zhang et al. [106]
	Lysine dendrons		Silencing of γ-secretase in C6 glial cells	Flurbiprofen	Al-azzawi et al. [107]
	DGL	Reduction of β-amyloid synthesis Reduction of Tau aggregation	Silencing of BACE 1 in APP/PS1 transgenic mice Inhibition of Tau aggregation in NFT	BACE 1 shRNA, TLKIV peptide PEG groups	Liu et al. [108]
	PPI DDs	Miscellaneous	Improvement of synapses in APP/PS1 transgenic mice	Histidine, maltose	Aso et al. [109]
	Mg-DDs		Antioxidant activity, reduction of TNF-α expression and caspase-3 activity after Aβ 1-42 toxicity by injection in rat brain		Karimi-Sales et al. [110]
	phosphorus-DDs		Reduction of oxidative stress Inhibition of AChE activity		Wasiak et al. [103]
	PAMAM DDs	Transport of drugs	Improvement of pharmacokinetic properties of memantine	Lactoferrin, drug: memantine	Gothwal et al. [112]
	PAMAM DDs		Increase brain bioavailability of rivastigmine	Lactoferrin, drug: rivastigmine	Gothwal et al. [113]
	PAMAM DDs		Increase solubility of carbamazepine Reduction of toxicity of carbamazepine in N2a cell line and zebrafish	Drug: carbamazepine	Igartúa et al. [114]
	PAMAM DDs		Reduction of toxicity of tacrine in N2a, human red blood cells and zebrafish	Drug: tacrine	Igartúa et al. [115]
AD diagnosis	PAMAM DDs	Evaluation of Tau levels	Quantification of Tau levels in plasma of AD patients	Gold nanoparticles	Razzino et al. [116]

.

3.2. Parkinson′s Disease

More than two hundred years ago, James Parkinson described PD as an involuntary shaking condition [117]. However, nowadays it is considered a complex and chronic neurodegenerative disorder that worsens with age and leads to intellectual and physical disability [118,119]. PD is the second most common pathological condition derived from neuronal degeneration and its prevalence is expected to increase in the following years with the aging of population, until 15 million people worldwide [120,121]. PD is clearly recognized by classical manifestations such as hands shaking, bradykinesia, resting tremor and postural instability. These symptoms arise in the late stages, when neuronal damage is not reversible, and worsen over time due to disease progression. In addition, patients also present non-motor manifestations, such as cognitive impairment, sleep disorders, depression and gastrointestinal or olfactory disturbances in the initial or prodromal phase [122]. Clinical symptoms are related to hypodopaminergic function due to the reduction of dopamine levels in the striatum derived from the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Moreover, abnormal cholinergic activity and widespread neuronal degeneration are also involved in both motor and non-motor symptoms. The second pathological hallmark of the disease is the identification of Lewy Bodies (LB) and Lewy Neurites (LN) as intraneuronal cytoplasmatic protein inclusions enriched in α-synuclein (α-syn) [123].

The current pharmacological treatment of PD is mainly based on dopaminergic therapy. The disease is basically managed with Levodopa, which, since the 1960s, has been the most effective treatment to restore dopaminergic activity, in combination with anticholinergic drugs [124]. However, these therapies are only symptomatic strategies and long-term ineffective because they do not modify the course of the disease. This challenge has been addressed by the development of DMT, which includes several approaches against relevant targets in PD pathology.

3.2.1. Dendrimers for PD Therapy

Dendrimers for Decreasing α-Synuclein Aggregates

Aggregation of proteins is a natural consequence of neuronal aging [125] but in some pathological situations such as PD, accumulation of toxic proteins accelerates and becomes a hallmark of the disease. Initially, protein aggregates play a protective neuronal strategy to isolate dangerous proteins, but the progressive accumulation of proteins, in particular α-syn, in cytosolic insoluble deposits is detrimental to neurons and triggers neurodegeneration [126,127]. Among the physiological functions of α-syn, its role in the control of intracellular vesicle trafficking and synaptic release has been highlighted [128]. The proper functionality of α-syn depends on its structure and correct folding. The protein acquires several conformations in equilibrium, from native monomers or tetramers to more complex structures, such as oligomers. α-syn is composed of three different domains: a hydrophobic central region, flanked by an N-terminal and a C-terminal domain. The central part of the protein is responsible for the acquisition of β-sheet conformation and oligomer formation [129]. Progressive accumulation of oligomers into α-syn fibrils within neurons constitutes the main element of the characteristic LB [130,131] (Figure 3). Aggregation of α-syn into fibrils and its subsequent cell-to-cell transmission represent a pathological gain of function mechanism for the protein, essential for the propagation of PD to surrounding cells [132]. Therefore, α-syn emerges as a key therapeutic target in PD, and decreasing α-syn deposition becomes a major approach to protect neurons from toxic aggregates, prevent neurodegeneration and delay the onset or progression of PD.

DDs have emerged as effective tools to block α-syn fibrillation (Figure 3). Two independent studies have shown the anti-aggregating properties of PAMAM DDs in PD. In the first of these, Rekas and colleagues demonstrated that G3 to G5 PAMAM DDs inhibited α-syn fibrillation and disaggregated preformed fibrils. The dual effect is dependent on the concentration and generation number [133]. In a second study, a further characterization of PAMAM DDs’s structure suggested that only G4 DDs, with terminal amino groups, were able to inhibit α-syn fibrils formation, unlike G3.5 DDs, functionalized with carboxyl groups on the surface [134].

On the other hand, a different DD system, based on phosphorous addition, has been proposed as a promising candidate to reduce α-syn aggregation. Milowska and colleagues showed that G3 and G4 phosphorous DDs blocked α-syn fibrillation until a 0.5 µM concentration [135]. These authors also demonstrated that phosphorous DDs with antiviral properties, termed viologen-phosphorous DDs, are suitable compounds for controlling α-syn aggregation and toxic fibril formation [136].

Alternatively, CBS DDs have been described as effective therapeutic agents in a rotenone-induced mouse model of PD. DDs exerted a dual neuroprotective mechanism due to both their ability to encapsulate and complex rotenone and to prevent fibrillation of α-syn [137]. Interestingly, CBS DDS prevented rotenone damage more efficiently than PAMAM, phosphorous and viologen-phosphorous DDs [138]. The effectiveness of CBS DDS in modulating the aberrant accumulation of α-syn has also been explored in induced pluripotent stem cell (iPSC) lines obtained from PD patients carrying the LRRK2 G2019S mutation. These results highlight the potential of CBS DDs for PD therapy [139].

3.2.2. Dendrimers as Vehicles of Therapeutic Genes

It is widely accepted that PD has a multifactorial origin. PD is the result of a complex relationship between individual genetic susceptibility and environmental factors that increase the risk of developing the disease [140]. Although most PD cases are sporadic forms, a small proportion of PD patients receive a pathological genetic variant through a familial hereditary pattern. Among the main genetic causes linked to PD it has been described mutations in several genes such as SNCA, LRRK2, PINK1, DJ-1, VPS35 and Parkin [141]. Loss-of-function studies have been designed to reduce protein expression levels derived from key genes to minimal amounts, mimicking the effect of protein silencing. In this context, DDs have emerged as potential carriers for introducing genetic materials, such as nucleic acids, into the cells (Figure 3). In physiological conditions, positive charges on the surface of DDs (generally amine groups) can react with negative charges of nucleic acids to form dendriplexes, which are internalized through different cellular mechanisms [142]. Among the genetic causes related to PD, the discovery of mutations in the gene encoding α-syn (SNCA) was the first characterized [143]. In particular, missense point mutations and duplications or triplications of the SNCA gene are linked to autosomal dominant forms of PD [144]. One possible explanation for the toxicity is the increase in protein levels. A potential therapeutic option for managing α-syn load is the reduction of protein synthesis, although downregulation of α-syn has typically been accomplished by viral carriers rather than by dendrimeric structures [145,146,147].

Loss-of-function studies can also be designed to modulate cellular pathways that are modified during the pathological course of the disease. Restoring the activity of specific targets through RNA technology is an alternative to ameliorate the symptoms. For example, a delivery system based on a DGL vector, coupled with rabies virus glycoprotein peptide and complexed with caspase-3 shRNA, successfully improved motor performance in rotenone-treated rats as a murine model of PD. This strategy reduced caspase-3 protein expression and dopaminergic neuronal loss [148].

3.2.3. Dendrimers as Vehicles of Drugs or Trophic Factors

Among the main reasons supporting the use of DDs as vehicles for therapeutic compounds are the improvement of pharmacokinetic properties and the safety profile [149]. A DD system based on G4.5 of carboxyl decorated PAMAM DDS was successfully used to complex the drug carbamazepine. This formulation improved specific drug properties, such as solubility and safety profile, due to the reduction of the dose required and consequent toxicity in human red blood cells, the N2a cell line and zebrafish [114].

However, the main challenge in the treatment of NDs, including PD, is the transport of drugs into the brain. Drugs are combined with DDs to improve pharmacodynamic properties and enhance the access of the active compounds to specific biological targets (Figure 3). For example, the gene encoding for the trophic factor human glial-derived neurotrophic factor (hGDNF) was loaded into an angiopep-coupled DGL system to increase BBB penetration. Angiopep binds to low-density lipoprotein receptor-related protein (LRP), which is expressed at a high level in the BBB. The system showed increased gene expression in rat brains and neuroprotective effects in the rotenone-induced PD model, with recovery of the parkinsonian phenotype, motor activity and amelioration of dopaminergic loss [150]. In another study, the neuroprotection of hGDNF encapsulated in lactoferrin-decorated PAMAM DDs was confirmed. After treatment with the complex, GDNF overexpression increased neurotransmitter levels, prevented neuronal loss and improved motor activity performance in a rotenone-induced rat model [151]. On the other hand, specific antagonists of adenosine receptors (AR), such as theophylline or caffeine, have shown therapeutic potential for the treatment of PD [152]. A study by Kecskes and colleagues described that G4 PEGylated PAMAM DDs conjugated to xanthines enhanced receptor affinity and increased the interaction with binding sites [153].

The application of DD for the treatment and diagnosis of PD is summarized in

Table 2. DDs for PD therapy and diagnosis. Abbreviations: CBS (carbosilane) DDs (dendrimers), α-syn (α-synuclein), DGL (dendrigraft poly-L-lysines), hGDNF (human glial-derived neurotrophic factor), AR (adenosine receptor), mHippoE-18 (embryonic mouse hippocampal cell line), GATG (gallic acid-triethylene glycol) PLL (poly-L-lysine), DGL (dendrigraft poly-L-lysines), NFT (neurofibrillary tangles), BACE1 (β-amyloid converting enzyme 1), PEG (polyethylenglicol), AChE (acetylcholinesterase).

Strategy	Type of Dendrimer	Mechanism		Loading Cargo/Modification	References
PD therapy	PAMAM DDs	Reduction of α-synuclein aggregates	Inhibition of α-syn fibrillation Reduction of pre-formed α-syn fibrils		Rekas et al. [133]
	PAMAM DDs		Inhibition of α-syn fibrillation		Milowska et al. [134]
	Phosphorous DDs		Reduction α-syn fibrils formation		Milowska et al. [135]
	Viologen-phosphorous DDs		Inhibition of α-syn fibrillation		Milowska et al. [136]
	CBS DDs		Inhibition α-syn fibrils formation in mHippoE-18 [137] and iPSC [139]		Milowska et al. [137,139]
	CBS DDs, PAMAM DDs, Phosphorous DDs, Viologen-phosphorous DDs	Reduction of toxicity of rotenone	Complexation of rotenone		Milowska et al. [137,138]
	DGL	Transport of therapeutic genes	Silencing of caspase-3 in rotenone treated rats	Caspase-3 shRNA, rabies virus glycoprotein peptide	Liu et al. [148]
	PAMAM DDs	Transport of drugs or trophic factors	Improvement of pharmacokinetic properties of carbamazepine Reduction of toxicity of carbamazepine in N2a, human red blood cells and zebrafish	Drug: carbamazepine Carboxyl groups	Igartúa et al. [114]
	DGL		Neuroprotection of hGDNF in rotenone treated rats	hGDNF mod: angiopep	Huang et al. [150]
	PAMAM DDs		Neuroprotection of hGDNF in rotenone treated rats	hGDNF mod: lactoferrin	Huang et al. [151]
	PAMAM DDs		Enhance binding properties of AR antagonist	Xanthines	Kecskes et al. [153]

.

3.3. Multiple Sclerosis

MS was defined as a clinicopathological entity more than 150 years ago by French neurologist Jean-Martin Charcot, who coined the concept of “sclerosis in plaques” [154]. Currently, it is described as an autoimmune inflammatory disorder of the central nervous system (CNS). It is characterized by the presence of inflammation and lymphocyte infiltration, leading to damage to the integrity of axons and myelin sheaths and causing neurological disabilities over time. Symptoms and impairments include visual problems, numbness, weakness and tingling pain [155]. MS is one of the most common causes of neurological disabilities worldwide and mainly affects young people in their 20s–40s at disease onset. An estimated 2.3 million people worldwide suffer from MS, with a prevalence of 50–300 per 100,000 population [156].

The underlying etiology of MS is still not known, but CNS inflammation and demyelinated nerve cells are the main hallmarks of the pathology. Initially, disruption of blood vessels in the BBB allows autoreactive T cells against myelinic components to migrate across the BBB and trigger the further recruitment of immune cells from the peripheral blood [157]. T lymphocytes attack myelin in the brain and spinal cord, causing degeneration of oligodendrocytes and gradually leading to neuronal demyelination and the clustering of inflammatory cells around demyelinated plaques [158]. The mutual interaction of microglia, astrocytes and T-cells contributes to chronic demyelination and neuronal loss over time [159].

DMT is the standard medication used to treat MS. There are currently more than 15 FDA-approved DMT that modify, modulate or suppress the immune system, limit neuroinflammation in the CNS, and prevent the relapse and the formation of new lesions. However, DMT only improves disability progression for 2–3 years after the beginning of the treatment, none target cognitive impairment, and produce significant side-effects [160]. Therefore, new therapies that reverse disease progression and reduce adverse events are needed.

3.3.1. Dendrimers for MS Therapy

Dendrimers for Decreasing Neuroinflammation

Neuroinflammation plays a key role in the pathogenesis of MS. The inherent anti-inflammatory activity of certain DDs has been known since the first decade of the 21st century.

In 2008, nonlinear peptide DDs synthesized as octamers of an encephalitogenic proteolipid attached to a central lysine core reduced the course of experimental allergic encephalomyelitis (EAE) in mice by altering antigen-specific cell trafficking and inducing the retention of peptide-specific T cells in the spleen [161]. Shortly later, Chauhan and co-workers unexpectedly found that PAMAM DDs ended by carboxylate, amine or hydroxyl groups exhibited anti-inflammatory properties in vivo. This anti-inflammatory activity was higher for DDs possessing amine or hydroxyl surface groups and lower for carboxylate-ended DDs [162].

Furthermore, anionic phosphorus-based DDs capped by amino-bisphosphonate groups, and especially DDs azabisphosphonate (ABP), activated anti-inflammatory and immunosuppressive human monocytes, reducing the inflammatory response. The effect of phosphorous dendrimers was demonstrated in vivo in different models of peripheral inflammation in mice [163] and were then subject to a patent describing their use as anti-inflammatory agents for the treatment of autoimmune diseases [164]. More recently, anionic ABP DDs have been shown to reduce neuroinflammation by promoting IL-10 producing CD4⁺T lymphocytes and inhibit the progression of EAE, producing results similar to those of approved drugs [165]. In contrast, the anti-inflammatory properties of neutral phosphorous DDs have been poorly explored. In 2013, Blattes and co-workers reported that mannosylated G3 and G4 poly(phosphorhydrazone) (PPH) DDs inhibited neutrophil influx to the site of inflammation and reduced the release of pro-inflammatory cytokines, such as TNFα and IL-12, by binding to the C-type lectin receptor DC-SIGN [166]. More recently, Posadas and co-workers demonstrated the anti-inflammatory activity of neutral PPH dendrimers (G and G4) in vitro and in vivo by inhibiting the NFkB activation pathway [29]. Therefore, phosphorous DDs display powerful anti-inflammatory properties that could be exploited in the treatment of autoimmune diseases, such as MS.

Dendrimers as Vehicles of Drugs

Disruption of the BBB and the presence of neuroinflammation characterized by excessive activation of T-cells and microglia represent the main hallmarks of MS. Interestingly, G4 hydroxyl-terminated PAMAM DDs show a remarkable ability to selectively deliver loaded drugs to activated brain microglia. Although hydroxyl DDs cannot cross the intact BBB, the impaired BBB in MS allows them to penetrate into the brain and be taken up by activated glial cells supporting these structures to selective drug delivery to the CNS [167]. In this regard, hydroxyl-G6 PAMAM DD conjugated with minocycline showed higher efficiency in crossing the BBB and reducing CNS inflammation than the free drug [168]. Similarly, conjugation of 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), a selective inhibitor of the enzyme glutamate carboxypeptidase II, to the surface of G4 hydroxyl-terminated PAMAM DDs resulted in potent brain penetrant and selective uptake in microglia that improved cognitive function [169].

Dendritic polyglycerol derivatives (dPG) are another type of DD with a similar safety to linear PEG or dextran, useful for systemic drug delivery [170]. Recently, dPG microspheres loaded with dimethilfumarate (DMF), a DMT approved by the FDA, and curcumin (CUR), a naturally occurring polyphenol with therapeutic potential in MS, have shown a good long-term release of both drugs with low in vitro toxicity, although future biocompatibility studies in more complex biological models are required [171].

Finally, a peptide DD with a precise amino acid sequence similar to glatiramer acetate (GA), one of the most successful first-line treatments for MS, has recently been obtained. This DD has been shown to steer monocytes toward an anti-inflammatory state, providing a suitable point for developing new immunomodulatory DDs [172].

3.3.2. Dendrimers for MS Diagnosis

The ability of DDs to interact with molecules has been exploited to detect two MS biomarkers, myelin basic protein (MBP) and Tau protein, in CSF and serum. A 1G tri-methylolpropane tris[poly(propyleneglycol) was coupled to graphene oxide and the specific antibodies that recognize the target proteins to obtain a nanoimmunosensor quite similar to the commercially available ELISA kit [173].

Furthermore, DDs could represent a new platform for the detection of lesions in the brain since they are systems capable of crossing the BBB and improving the delivery of contrast agents. In addition, metallic nanoparticles such as iron, gold or silver coated on the surface of DDs raise the possibility of their application as contrast agents. These systems, already tested in oncological diseases, could be extended to MS diagnostics [174].

The application of DD for the treatment and diagnosis of MS is summarized in

Table 3. DDs for MS therapy and diagnosis: Abbreviations: MAPs (Multiple Ag peptides), GPBP (guinea pig myelin basic protein), ABP (azabisphosphonate), PPH (poly(phosphorhydrazone), BBB (Blood–brain barrier), 2-PMPA (2-(phosphonomethyl)-pentanedioic acid), dPG (dendritic polyglycerol, DMF (dimethilfumarate), GA (glatiramer acetate), Ab (antibody), MBP (myelin basic protein)), CSF (cerebrospinal fluid).

Strategy	Type of Dendrimer	Mechanism		Loading Cargo/Modification	References
MS therapy	Lysine DDs	Decreasing neuroinflammation	Inhibition of Th17 Lymphocytes activation Reduction of IFN-γ-producing cells	MAPs (GPBP72–84 MAP, PLP178–191, PLP139–151	Wegmann et al. [161]
	PAMAM DDs		Reduction of paw edema in carrageenan-induced rats, inhibition of COX-2, NO production and arthritis phenotype in LPS-induced rats		Chauhan et al. [162]
	Phosphorous DDs		Activation of monocytes, reduction of paw swelling, amelioration of arthritis manifestations, reduction of pro-inflammatory cytokines and increase of anti-inflammatory cytokines		Hayder et al. [163]
	ABP DDs		Increase of IL-10 production		Hayder et al. [165]
	PPH DDs		Inhibition of neutrophils Reduction of pro-inflammatory cytokines (TNFα and IL-12)	Mannose	Blattes et al. [166]
	PPH DDs		Inhibition of NFkB pathway		Posadas et al. [29]
	PAMAM DDs	Transport of drugs	Improvement of minocycline access through the BBB	Drug: minocycline	Sharma et al. [168]
	PAMAM DDs		inhibition of the enzyme glutamate carboxypeptidase II Selective uptake by microglia	Drug: 2-PMPA	Hollinger et al. [169]
	dPG		Improvement of pharmacokinetic profile of the drugs by long-term release Reduction of toxicity of the drugs in CHO-kI cell line	Drug: DMF, curcumin	da Silva et al. [171]
	Peptide DD		Differentiation of monocytes to M2 state Production of anti-inflammatory cytokines (IL-1Ra)	Drug: GA	Erzina et al. [172]
MS diagnosis	Graphene-based DD	Detection of MS biomarkers	Detection of MBP and Tau proteins in CSF and serum	Ab anti- MBP and anti-Tau	Derkus, et al. [173]
	PAMAM DDs	Detection of brain lesions	Administration of contrast agents	Gold, silver, iron	Wang et al. [174]

.

3.4. Amyotrophic Lateral Sclerosis (ALS)

First described by Charles Bell in 1824, it was the neurologist Jean-Martin Charcot, who began using the term ALS in 1874 [175]. ALS is a progressive ND affecting motor neurons in the brain and spinal cord. Unlike MS, symptoms begin with muscle weakness and progress to affect speech, swallowing and finally breathing. Similar to other NDs, misdiagnosis determines disease prognosis and a patient’s quality of life [155].

The annual incidence of ALS is around 1-2.6 new cases per 100,000 persons. This disease is characterized by rapid progression with average survival 3–4 years from onset, whereas the average age of onset nowadays is 59–60 years [176]. Although the etiology remains unknown, a subset of cases (~10%) appears to be inherited. Approximately 1–2% of cases are related to mutations in the gene encoding superoxide dismutase-1 (SOD1), in which more than 130 mutations have been identified. Mutant SOD1 displays an enhanced propensity to misfold and aggregate, contributing to spinal motor neuron toxicity [177].

Functionality loss, haploinsufficiency, RNA toxicity and proteotoxicity seem to be the main pathological mechanisms in ALS. In addition, elevated levels of glutamate from the catabolism of the neuropeptide N-acetyl-aspartyl-glutamate (NAAG) by glutamate carboxypeptidase II (GCPII) hyperactivate NMDA receptors, leading to excitotoxic death [178,179].

Currently, the specific drugs approved by the FDA for the treatment of ALS are Riluzol, a glutamine agonist, and Edaravone, a free radical scavenger. Neither of these prolongs patient survival by more than 2–3 months, highlighting the need for new therapeutic strategies in ALS [180,181].

DDs for ALS Therapy

Although ALS is a devasting ND with no cure to date, the use of nanomedicine remains poorly explored [182]. Regarding DD application in ALS, recently, an hydroxylated G4 PAMAM DD covalently attached to 2-(Phosphonomethyl)pentanedioic acid, a potent GCPII inhibitor, resulted in potent inhibition of this enzymatic activity in activated macrophages infiltrating the muscle and significantly delayed neuromuscular junction denervation and loss of muscle function in a SODG93A mice model of ALS [183].

4. Conclusions

Current therapies available for NDs, such as PD, AD and MS, are able to reduce clinical manifestations and improve the quality of life of patients. The big challenge in the treatment of these pathologies is the development of strategies that prevent neurodegeneration and delay the onset or progression of these diseases. DDs have gained attention in recent decades due to their potential as drug vehicles usually employed for the management of NDs. In addition to being useful delivery systems for improving the pharmacokinetic properties of drugs and reducing their toxicity, DDs have been shown to exert anti-aggregation properties to reduce protein accumulation or promote protein disaggregation, and certain structures display inherent anti-inflammatory properties. DDs are able to interact with key therapeutic targets in AD, PD and MS to decrease aberrant protein deposits and protect neurons from toxic aggregates and to reduce neuroinflammation in NDs. However, DDs are not only useful agents for the therapy of NDs; they have thus far proven to be interesting diagnostic tools for AD. However, although the potential of DDs for the therapy of NDs has been highlighted, further efforts are needed to characterize the behavior of DDs in humans before planning the development of future clinical trials.