2.1 Neuronal dysfunction in the PD

The characteristic features of PD include the loss of neurons in the substantia nigra and the presence of Lewy bodies in the neuronal cytoplasm. There are multiple molecular pathogenesis and pathways related to neuronal function involved in the pathogenesis of PD, such as misfolded α-synuclein aggregation, MAO-B catalyzes oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, dysfunction of the lysosomal and proteasome systems, abnormal metal ion homeostasis, neurotransmitter deficiencies, abnormal axonal transport and neuroinflammation.¹⁸ Besides, genetic factors are usually considered to be the etiology of familial PD, and there are around 5%–10% PD caused by genetic factors.³ For example, point mutation (A53T) at the SNCA is the most common familial inherited gene associated with PD.¹⁹ Meanwhile, the most interested genes of mutations are the SNCA, LRRK2 (which encodes leucine-rich repeat serine/threonine-protein kinase 2), PRKN (parkin RBR E3 ubiquitin protein ligase, PARK2), PINK1 (PTEN-induced putative kinase 1), GBA (glucosidase beta acid gene, which encodes glucocerebrosidase) and DJ-1genes,¹⁸ of which SNCA and LRRK2 are associated with dominant PD cases, while PRKN, PINK1, GBA, and DJ-1 are associated with recessive PD cases.²⁰ In addition, environmental factors, including industrial or agricultural pollution (e.g., pesticides and herbicides), may also cause the PD.

As a key pathogenic protein, the intracellular aggregated α-synuclein has been found in all PD patients. In the pathogenic process, soluble α-synuclein monomers initially form oligomers, then gradually aggregate into small fibrils, and finally form large insoluble α-synuclein fibrils to produce pathological aggregates (Lewy bodies).³ In the healthy human brains, only about 4% of α-synuclein is phosphorylated at residues serine-129 (Ser-129). However, approximately 90% of α-synuclein phosphorylated at Ser-129 was detected in the Lewy bodies of PD patients.²¹ Extracellular α-synuclein oligomers are able to target cell and organelle membranes to disrupt their integrity because the N-terminal region of α-synuclein is able to strongly bind with lipid bilayers.²²

It has been known that α-synuclein could not only enrich in the presynaptic terminal of neurons, but also strongly interact with plasma membranes and mitochondrial membrane. The accumulation of α-synuclein is also associated with endoplasmic reticulum stress and defects in organelles such as mitochondria and lysosomes.²³ For example, González-Rodríguez et al.²⁴ found that the dysfunction of mitochondrial complex I could initiate PD. In addition, the dysfunction of lysosome may also promote the aggregation of soluble α-synuclein oligomers, because the aggregated and misfolded α-synuclein cannot be timely and efficiently degraded through macroautophagy and chaperone-mediated autophagy (CMA). Reduced lysosomal glucocerebrosidase hydrolase (GCase) activity in the brains of PD patients caused by GBA mutations is also a common risk factor for PD.^{25, 26} Recent studies have shown that pathogenic α-synuclein species accumulated in neuronal lysosomes in the mouse brain, and the neurons released pathogenic α-synuclein via SNARE-dependent lysosomal exocytosis.²⁷ The NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) is a central driver for neurodegeneration, and its activation could trigger the neurodegeneration and cause the death of neurons.²⁸

2.2 Microglia in the PD

As the primary innate immune cells in the brain, microglia act as a homeostatic sensor by monitoring the surrounding environment, clearing cell debris, and providing neurotrophy.²⁹ In addition to brain immunity, microglia support brain development by pruning the synapses of developing neurons.³⁰ Accumulating evidence indicates that activated microglia are the sources of multiple neurotoxic factors, including inflammatory factors and reactive oxygen species (ROS). They promote the progression of neuronal damage.³¹ Although low-concentrated ROS is the key signaling molecule under normal conditions, but neurons are particularly vulnerable to oxidative stress caused by excess ROS.^{32, 33} In addition, microglia cells can generate and release toxic oxygen and nitrogen species by modulating several enzyme systems, such as nicotinamide adenine dinucleotide phosphate oxidase, inducible nitric oxide synthase (iNOS), and myeloperoxidase. Furthermore, the damaged microglia exacerbate lipopolysaccharide-induced proinflammatory responses, NLRP3 activation, and astrocyte proliferation, thus increasing degeneration of dopaminergic neurons and motor dysfunction.³⁴ Microglia enhance the phagocytosis of α-synuclein aggregates through the interaction between their toll-like receptor 2 (TLR2) and α-synuclein.³⁵ As a return, they can be activated by α-synuclein through either the TLR4 or TLR2 pathway.³⁶ Many studies have reported that microglial activation and neuroinflammation may be the key regulators for the loss of dopaminergic neurons in PD patients.³⁷

Similar to a double-edged sword, the microglia can also alleviate the symptoms of PD by releasing some anti-inflammatory factors³⁸ and phagocytosing α-synuclein aggregates.³⁹ For example, microglia can form functional networks, reduce the load of a-synuclein aggregates by donating healthy mitochondria to each other, relieve the inflammatory response and the cytotoxicity of α-synuclein aggregates, and eventually degrade α-synuclein aggregates in a collaborative way.⁴⁰ In addition, microglia may also play a crucial role in PD immunotherapy by targeting α-synuclein.³ For example, the activated microglia strongly express the major histocompatibility complex (MHC), which is involved in processing and presenting autoantigens to T cells.⁴¹ In a word, microglia play an important role in the progression of PD and can be an important target.

2.3 Astrocytes in the PD

Astrocytes are the most abundant subtypes of glial cells in the brain and are critical for maintaining the normal neuronal function of a healthy brain. As a component of BBB, astrocytes release various NTFs (e.g., glial-derived neurotrophic factor [GDNF]), which are particularly crucial for developing and survival of dopaminergic neurons. Astrocytes can also affect neurons by controlling the concentration of extracellular potassium ions (K⁺) and intracellular calcium ions (Ca²⁺) to prevent their overexcitation. In gray matter, astrocytes control the number of synapses, regulate the blood flow to ensure adequate energy expenditure, and provide lactic acid to neurons for energy metabolism.⁴²

However, increasing evidence suggests that the damage of astrocytes is related to the pathology of PD. For example, reactive astrocytes may contribute to PD pathology through their roles in glutamate-mediated excitatory toxicity, K⁺ buffering, and Ca²⁺ homeostasis.⁴³ The trauma and infection of the CNS may activate astrocytes and exacerbate PD pathogenesis.⁴⁴ There exist two states of reactive astrocytes, which are defined as “A1” and “A2.” They are similar to the “M1” and “M2” phenotype of macrophages.⁴⁵ The activated microglia can secrete tumor necrosis factor alpha (TNF-α), interleukin-1 alpha (IL-1α), and complement component 1q to regulate the transformation of astrocytes into the toxic A1-reactive astrocytes,^{46, 47} as shown in Figure 2. Meanwhile, A1 astrocytes can produce cytokines such as TNF-α, IL-1β, IL-6, and interferon-γ, thus losing the promotive ability for neuronal survival, growth, synaptogenesis, and phagocytosis, and lead to neuronal and oligodendrocyte death.^{48, 49} In contrast, A2 astrocytes play a protective role by secreting NTFs that promote survival and growth, synaptic repair and promote angiogenesis by regulating angiogenesis-related vascular endothelial growth factor (VEGF) and transforming growth factor β (TGFb).⁵⁰

In addition, genetic mutations of DJ-1, α-synuclein, calcium-independent phospholipase A2, ATP13A2 (also known as PARK9), PINK1, and Parkin are involved in the damage of astrocyte functions, including inflammatory response, glutamate transport, and secretion of NTFs.⁵¹ For example, the defects or mutations of DJ-1 in astrocytes impair their neuroprotective effects, including anti-inflammatory function⁵² and mitochondrial quality control.⁵³ The deficiency of the ATP13A2 gene, which encodes transmembrane lysosome type P5 ATPase, exacerbates astrocyte-mediated neuroinflammation through activation of NLRP3 inflammasomes.⁵⁴ PINK1 and Parkin encode mitochondrial targeted protein kinase and E3 ubiquitin ligase respectively, and are involved in mitochondrial quality control pathways for elimination of damaged mitochondria.

Previous studies have demonstrated that mitochondrial dysfunction is the main cause of astrocytes transforming from the resting state to the neurotoxic A1 state.⁵⁵ Transfer of mitochondria between dopaminergic neurons and astrocytes is a critical way to remove the damaged mitochondria without activating neuroinflammation because the damaged mitochondria in neurons can be released and transferred to astrocytes for processing and recycling.^{56, 57} The astrocytes can provide neurons with functional mitochondria, enhance neuronal adenosine-triphosphate (ATP) replenishment and modulate neuronal antioxidant defenses.⁵⁸ In addition, glutathione (GSH) is one of the main defenses against ROS, depletion of GSH in astrocytes can selectively inhibit mitochondrial complex I, thereby affect mitochondrial function by triggering ROS-induced cascade reaction, leading to the damage of dopaminergic neurons.⁵⁹ It should be noted that reprogramming astrocytes into functional dopaminergic neurons by transcription factors and microRNAs (miRNAs) has recently attracted extensive attention.⁶⁰ However, the long-term viability and functional integrity of neurons derived from them, and the effect of depleting astrocytes on neuronal functions need to be further explored.

4.1 Alleviation of oxidative stress

Substantial evidence supports that oxidative stress is a prominent feature in the pathogenesis of PD. Reduced activity of antioxidative enzymes and nonenzymatic antioxidants in the brain of PD patients could be responsible for the disease progression. For example, it has been demonstrated that the activity of glutathione peroxidase (GSH-Px), which can reduce the oxidized glutathione (GSSG) into GSH in the brain of PD patients, was significantly reduced to result in the accumulation of hydroxyl radical (•OH).⁸⁵ Abraham et al.⁸⁶ demonstrated that the activities of superoxide dismutase (SOD), catalase (CAT), GSH-Px, and glucose-6-phosphate dehydrogenase were significantly decreased in the PD patients.⁸⁶ Dopaminergic neurons are particularly vulnerable to oxidative stress due to the production of numerous oxidants, including H₂O₂, •OH, superoxide radical (•O₂⁻), and DA semiquinone radicals, during enzymatic and nonenzymatic reactions when dopamine is released from synaptic vesicles into synaptic cleavages or cytosol.⁸⁷ Oxidation can cause damages to lipids, proteins, DNA, and RNA in the brains of PD patients⁸⁸ and the accumulation of byproducts from lipid peroxidation has been found in the serum and cerebrospinal fluid of PD patients.⁸⁹

In recent years, nanoparticles with ROS scavenging capability and enzyme-like activity have been developed for PD treatment (Table 1). Carbon, metal, and metal oxide nanoparticles are often used to protect neurons by scavenging ROS. For example, polyhydroxylated fullerene derivative (C₆₀(OH)₂₄) can protect neurons from 1-methyl-4-phenylpyridine (MPP⁺) induced mitochondrial dysfunction and oxidative stress by effectively eliminating superoxide, hydroxyl, and lipid radicals.⁹⁰ Ren et al.⁹¹ demonstrated that graphene oxide quantum dots exhibited neuroprotective effects by effectively scavenging MPP⁺-induced ROS, mitochondrial damage, cell toxicity, and apoptosis. Lei et al.⁹² used ultra-thin graphite-carbon nitride (g-C₃N₄) nanosheets to enhance the peroxidase activity of hemin and restore dopamine-dependent behavior of PD nematode model by reducing ROS-mediated neurotoxicity.

Some inorganic metallic nanoparticles also exhibit excellent antioxidative capability, such as monometallic (e.g., gold nanorods and quercetin encapsulated by mesoporous silica, AuNRs@QCT),¹⁰⁶ and polymetallic nanoparticles (e.g., PtCu nanoalloys).⁹⁵ Ma et al.⁹³ showed that Prussian blue nanoenzyme, as a pyroptosis inhibitor, can alleviate PD by removing ROS to reduce the activation of microglial NLRP3 inflammasomes and caspase-1 (Figure 4A). In addition, metal oxide nanoparticles, including iron oxide,⁹⁶ cerium oxide,⁹⁷ Yb³⁺ and Er³⁺ codoped cerium oxide,⁹⁸ and manganese oxide¹⁰⁷ are also commonly used for neuroprotection due to their excellent enzyme-like activity. Singh et al.¹⁰⁰ reported a Mn₃O₄ nanozyme with SOD-, CAT- and GPx-like activities for protecting SH-SY5Y cells from MPP⁺-induced cytotoxicity. Hao et al.⁹⁴ prepared uniform Cu_xO nanoclusters with ROS scavenging capability to rescue the memory loss of PD mice.

Metal chalcogenides NPs also show excellent capability of scavenging ROS. Lactoferrin-modified Au–Bi₂Se₃ nanodots can also improve the memory and activity of PD mice by their multienzyme-like properties, such as SOD, CAT, GPx, and POD, which can maintain mitochondrial function and control ROS levels in cells.¹⁰¹ Quercetin-modified ultra-small Cu_2−xSe nanoparticles (CSPQ nanoparticles), which showed better capability of scavenging free radicals than nanoparticles and quercetin alone, also can alleviate oxidative stress through their multiple enzyme activities. Cell membrane-modified CSPQ nanoparticles can target microglia by the interactions between α4β1–VCAM-1 to polarize microglia into M2 phenotype, and significantly improve the movement disorder of PD mice (Figure 4B).³⁸

Other nanoparticles have been also developed to modulate ROS level for PD therapy. For example, natural compounds curcumin-loaded polysorbate 80-modified cerasome nanoparticles,¹⁰⁸ curcumin analog-based nanoparticle,¹⁰⁹ polydopamine and selenocystine nanocomposite,¹⁰² glycyrrhizic acid-zinc alginate nanogel encapsulated albiflorin.¹⁰³ Zhang et al.¹⁰⁴ developed the BBB-penetrating peptide (rabies virus glycoprotein 29 [RVG29]) conjugating with natural autophagy inducer 4,4′-dimethoxychalcone to form a novel delivery system (RVG-nDMC), which can alleviate oxidative stress in dopaminergic neurons and reverse tyrosine hydroxylase ubiquitination (Figure 4C).¹⁰⁴ Amphiphilic nanoparticles modified natural exosomes, which were derived from human umbilical-cord mesenchymal stem cells (MSCs), also showed excellent capability of capturing chemical attractants (ROS and iNOS) through l-arginine derivatives on nanoparticles (Figure 4D).¹⁰⁵

4.2 Elimination of α-synuclein aggregates

The hypothesis of prion-like α-synuclein suggests that once α-synuclein aggregates were formed in neurons, they would be released into the extracellular space and taken up by neighboring neurons, resulting in the accumulation of α-synuclein in new host neurons.^{110, 111} As an inherent and soluble protein, α-synuclein consists of 140 amino acid residues, mainly locates in the presynaptic terminal of neurons and participates in the regulation of synaptic activity by regulating the release of synaptic vesicles.¹¹² In the healthy physiological state, α-synuclein mainly exists in the form of monomer. Nevertheless, under the pathological conditions, mismodification of α-synuclein (such as nitrification, oxidation, and phosphorylation) leads to degeneration and formation of insoluble oligomers or fibrous structures, even aggregates.¹¹³ α-Synuclein oligomers can bond to lipids and aggravate oxidative stress, increase the permeability of mitochondria, lysosomes and vesicle membranes, and interfere with ion homeostasis and protein clearance pathways.^114-116 Moreover, about 90% of pSer129-α-synuclein was detected in the brain of PD patients. It is the main component of Lewy bodies (the key pathological feature of PD).¹¹⁷

CMA, ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are the key pathways of repairing or eliminating abnormal proteins.¹¹⁸ When α-synuclein is subjected to mutations, posttranslational modifications, or external stimuli (oxidative stress, ultraviolet radiation, toxic compounds, etc.), it usually shows conformational changes. The altered α-synuclein is first recognized and repaired by the molecular chaperones, and the unrepairable α-synuclein is degraded by the UPS system or by the molecular chaperone autophagy. The ubiquitin-proteasome degradation of proteins mainly involves the following steps: (i) ubiquitin activator E1 activates ubiquitin molecules in an ATP-dependent manner; (ii) ubiquitin activator E1 transmits the activated ubiquitin molecule to ubiquitin-conjugating enzyme E2; (iii) ubiquitin ligating enzyme E3 binds the E2 bound ubiquitin to the targeted protein to form a specific ubiquitin protein; (iv) The 26S proteasome specifically recognizes the ubiquitin-tagged substrate protein and promotes its degradation.¹¹⁹ In addition, restoration of 20S proteasome activity in the UPS also promotes the clearance of pathological α-synuclein, thereby protecting neurons.¹²⁰

As the accumulation of unrepaired and undegraded α-synuclein, the proteins become toxic and they are ultimately degraded by ALP (i.e., macroautophagy).¹¹³ In a word, soluble proteins are cleared through UPS and CMA pathways (Figure 5A), while these oligomers and protein aggregates, which cannot pass through the narrow pores of the proteasome, are degraded by the autophagy pathway.¹²¹

The activation of above clearance pathways has significant effects on the treatment of PD. Promoting chaperonin-mediated autophagy can reduce the level of abnormal high-molecular-weight and phosphorylated α-synuclein species, then alleviate α-synuclein-induced neurodegeneration.¹²² During CMA, cellular soluble proteins are recognized by the chaperone heat shock proteins and delivered to the lysosomes for degradation through the lysosomal-associated membrane protein 2A and the lysosomal HSPA8.¹²³ The in vitro studies demonstrated that increasing the activity of heat shock proteins, such as Hsc70,¹²⁴ Hsp70,¹²⁵ Hsp90,^{126, 127} and Hsp104,¹²⁸ can prevent the formation of α-synuclein fibrils, disaggregate fibrils, and even ameliorate α-synuclein induced synaptic deficits.¹²⁹ Some small heat shock proteins, such as αB-crystalline and Hsp27, can prevent α-synuclein aggregation by their transient interaction.¹³⁰ Wu et al.¹³¹ customized a nanochaperone (αS-nChap) to specifically recognize α-synuclein and prevent its oligomerization by dynamically capturing and stabilizing α-synuclein. Meanwhile, oligomeric α-synuclein can be captured to prevent fibrosis, and transported to the lysosomal degradation system (Figure 5B).

Many studies have demonstrated that some nanoparticles can regulate the autophagy (macroautophagy) of neurons, microglia, and astrocytes to promote the degradation of α-synuclein aggregates and the treatment of PD (Table 2). For example, Liu et al.¹⁰⁹ developed a curcumin analog-based nanoscavenger (nano-CA), which can stimulate the major autophagy regulator transcription factor EB (TFEB) to facilitate the clearance of α-synuclein monomers, oligomers, and aggregates in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse.¹⁰⁹ How to controllably enhance the cellular autophagy is of great importance for treatment of PD. In a recent work, Cu_2−xSe-anti-TRPV1 nanoparticles were used as a photothermal switcher to controllably activate autophagy of microglia through the second near-infrared-II laser irradiation. The enhancement of autophagy level by Cu_2−xSe-anti-TRPV1 nanoparticles was regulated by ATG5 and Ca²⁺/CaMKK2/AMPK/mTOR signaling pathways. The enhanced autophagy promoted the phagocytosis and degradation of α-synuclein, and ultimately improved the therapeutic effect of PD (Figure 5C).³⁹

In addition to the above pathways, several other approaches have been shown to clear α-synuclein. Recently, Butler et al.¹³² designed a nanoantibody PFFNB2, which can specifically recognize α-synuclein PFF rather than α-synuclein monomer, to significantly dissociate α-synuclein fibrils. In addition, treatment with PFFNB2 inhibited PFF-induced phosphorylation of α-synuclein serine 129 (pS129) in the primary mouse cortical neurons. Kim et al.¹³³ demonstrated that graphene quantum dots can cross the BBB and protect neurons against dopamine loss induced by α-synuclein preformed fibrils, Lewy body/Lewy neurite pathology and behavioral impairment.

4.3 Chelation of metal ions

Brain imaging and pathological studies have shown that iron accumulation was specifically observed in the substantia nigra of PD patients.¹³⁷ The abnormal accumulation of iron in PD is likely due to the imbalance of iron homeostasis caused by changes in iron regulatory proteins, such as divalent metal transporter 1 (DMT1), ferroportin (FPN), and transferrin.¹³⁸ For example, increased DMT1 levels may lead to the increase of iron input to cells, and FPN is reduced in MPTP and 6-hydroxydopamine (6-OHDA) induced PD models.¹³⁹

Excess iron can induce oxidative stress through the formation of •OH, leading to the peroxidation of DNA, proteins, and lipids. This is closely associated with α-synuclein accumulation, mitochondrial dysfunction, neuroinflammation, and disruption of the ubiquitin-protease system.¹⁴⁰ The abnormal iron accumulation-induced cell death is most likely due to the imbalance of iron homeostasis caused by the dysfunction of iron regulatory proteins.¹⁴¹ This iron overload-dependent cell death is usually known as ferroptosis. Iron chelation therapy of early PD patients has been clinically tested to evaluate the pathophysiological roles of iron in the PD occurence.¹⁴² Another study showed that decrease of reactive iron by transgene expression of ferritin or oral administration of the metal chelator chloroquine protected mice against the neurotoxicity of MPTP.¹⁴³ Hence, chelating iron ions, or inhibiting ferroptosis by iron chelation has shown the potential in treatment of PD. For example, therapeutic iron-chelating nanoparticles were constructed by using a zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) and HIV-1 transactivating transcriptor, the experimental results showed that these nanoparticles can reverse the pathological and behavioral defects of PD mice.¹⁴⁴ You et al.¹⁴⁵ reported a type of novel polymeric nanoparticles containing deferoxamine modified by brain-targeting peptide RVG29. As shown in Figure 6A, these nanoparticles effectively reduced dopaminergic neuronal damage and reversed neurobehavioral deficits by significantly reducing iron levels and oxidative stress in the substantia nigra and striatum of PD mice (Figure 6B–G).¹⁴⁵

In addition to iron, the imbalance of zinc, manganese, and copper ions are also associated with PD. As the second most abundant transition metal in the brain, zinc ions (Zn²⁺) can directly inhibit the activity of tyrosine hydroxylase and antagonize the binding of Fe²⁺ ions, thus negatively regulate dopamine synthesis.¹⁴⁶ Chelation of zinc ions can alleviate PD-associated lysosomal alterations and dopaminergic neurodegeneration by inhibiting zinc-mediated cytotoxicity.¹⁴⁷ The disorder of manganese metabolism is also associated with PD, because manganese can affect neurotransmitter synthesis, and release, particularly in the basal ganglia.^{148, 149} For example, manganese may compete with Ca²⁺ influx, increase neurotransmitter release and the rhythmic firing activity in dopaminergic neurons, which may cause their dysfunction.¹⁵⁰ Xin et al.¹⁵¹ demonstrated that Slc39a14 (manganese transporter) knockout mice showed a high accumulation of manganese in the brain and motor neurobehavioral dysfunction, which can be alleviated by Na₂CaEDTA (a manganese chelator).

4.4 Delivery of NTFs

NTFs, including GDNF, brain-derived neurotrophin (BDNF), cerebral dopamine neurotrophic factor, and mesencephalic astrocyte-derived neurotrophic factor, are protein molecules that support neuronal development, maturation, and survival.^{152, 153} NTFs regulate the functional activity of neurons through axonal regeneration, synthesis and release of neurotransmitters, and expression of their transporters.¹⁵⁴ NTFs-based therapies hold great promise for treating neurodegenerative diseases such as Alzheimer's disease, PD, amyotrophic lateral sclerosis, and Huntington's disease. However, most NTFs face the problems of short half-life, poor pharmacokinetics, and low efficiency of crossing the BBB in vivo.¹⁵⁵ Systemic administration of NTFs usually results in severe side effects because of their difficulties in crossing the BBB, and free NTFs are readily removed from circulation by extracellular peptidases, tissue binding, receptor-mediated clearance, and thus rapid degradation by glomerular filtration.¹⁵⁶ The clinical treatment with NTFs is therefore high cost and high risk, because NTFs are injected directly into the brain of PD patients.¹⁵⁷ Given the outstanding performance of NTFs in the treatment of PD, the development of effective strategies for delivery of NTFs is crucial for their clinical application.

A promising approach is the use of nanocarriers, which can not only stabilize NTFs, prolong their blood circulation time and half-life in the brain but also control the release of NTFs. Functionalization of NTFs with targeting ligands can improve their capability of crossing the BBB and their efficiency in the brain.¹⁵⁸ The following factors should be considered for efficient delivery of NTFs into brain by nanoparticles, such as the stability of NTFs in body fluids, the capability of NTFs crossing the BBB and diffusing in the brain, and their bioavailability for targeted cells.¹⁵⁵ In the past few decades, many studies on the delivery of NTFs by nanosystems have shown promising results. For example, Xu et al.¹⁵⁹ used a nanocapsule to deliver nerve growth factor (NGF) into the CNS of healthy mice and nonhuman primates to promote NGF-mediated nerve regeneration, tissue remodeling, and functional recovery.  Wu et al.¹⁶⁰ found that GDNF liposomes can be effectively delivered to the brain for repairing dopaminergic neurons under the assist of small molecular agonist SC79, which can cross the BBB. In another translational study, a single administration of microencapsulated NTF was used to sustainably control the release of GDNF in the brain. This method protected GDNF from degradation, and promoted the survival of nigral dopaminergic neurons and recovery of motor in Parkinsonian monkeys.¹⁶¹ In addition to delivering exogenous NTFs, the use of nanotechnology to increase the endogenous NTFs has also shown great potential.¹⁶² In conclusion, NTFs delivered by nanotechnology is a promising approach for the treatment of PD.

4.5 Gene therapy

As mentioned previously, gene mutation is one of the pathogeneses of PD. Therefore, introducing therapeutic genes (DNA/RNA), silencing, or correcting the defective genes can be used to treat PD. PD-related genes mainly include SNCA, LRRK2, PINK1, Parkin, and DJ-1. Among of them, SNCA is the first gene encoding α-synuclein, which is the major component of Lewy bodies associated with hereditary PD.¹⁹ LRRK2 mutation is the most commonly inherited gene in PD, accounting for 3%–41% of familial PD cases.¹⁶³ PINK1 and Parkin genes are involved in mitochondrial maintenance, and associated with mitochondrial dysfunction and oxidative stress. DJ-1 mutations cause 1%–2% autosomal recessive Parkinsonism, and oxidative stress damage. The strategy of specific knockdown of target genes by siRNA can be used to treat neurodegenerative diseases such as PD. Meanwhile, nanoparticle-based gene delivery has emerged as a potential treatment for PD (Figure 7A).¹⁶⁴ This method may avoid harmful side effects, such as immunogenicity and carcinogenicity.¹⁶⁵ For example, the siRNA (SNCA) delivered by polyethyleneimine nanoparticles can effectively reduce α-synuclein expression in PD models.¹⁶⁶ In addition, Liu et al.¹⁶⁷ showed the excellent performance of exosome-curcumin-phenylboric acid-poly (2-(dimethyl amino) acrylate) nanoparticles modified with RVG29 in the targeted delivery of SNCA siRNA for clearance of α-synuclein aggregates in the PD neurons.

Unlike siRNAs, miRNAs are a class of short noncoding RNAs that regulate gene expression by binding with cellular transcripts, leading to translational repression or degradation of targeted messenger RNAs.¹⁶⁸ Given the important role of miR-124 in neurogenesis, synaptic morphology, and neurotransmission, as well as regulation of mitochondrial dysfunction, oxidative damage, and neuroinflammation in PD, plasma miR-124 could be a potential biomarker of PD.¹⁶⁹ miR-124 can promote neuroprotection, neurogenesis, and functional motor recovery in preclinical models of PD.¹⁷⁰ For example, miR-124 was delivered by polymeric nanoparticles to improve motor symptoms in 6-OHDA mice.¹⁷¹ Huang et al.¹⁷² prepared RVG29-conjugated poly(lactic acid-co-glycolic acid) (PLGA) NPs to deliver miR-124 to the brain, and the nanoparticles alleviated PD symptoms by relieving neuroinflammation.

NPs could be also used to deliver DNA fragments for monitoring and therapy of PD (Table 3). For example, Miao et al.¹⁷⁹ developed Tb-based luminescent metal-organic framework containing Pt-aptamer for noninvasive monitoring of α-synuclein (Figure 7B). In addition, a novel brain-targeted zinc oxide quantum dot gene delivery system (ZnO@polymer-NpG) was developed to deliver plasmid DNA (pDNA) and NGF into the brain. The released pDNA can silence α-synuclein by suppressing the expression of SNCA (Figure 7C), and protect neurons.¹⁷⁵ Similarly, Gao et al.¹⁷⁶ fabricated polydopamine nanoparticles and coated with magnesium oxide and anti-SNCA plasmids. The nanoparticles exhibited brain targeting capability and antioxidative activity, and showed good neuroprotective effect. In their another study, chitosan-pegylated polylactic acid nanoparticles coated with NGF, acteoside, and pDNA significantly reversed the loss of dopaminergic neurons in the substantia nigra and striatum of PD mice.¹⁸⁰ They also prepared actively targeted gold nanoparticles carrying pDNA, which can inhibit the expression of α-synuclein.^{177, 178}

Another well-studied gene therapy is based on the NTF gene (e.g., BDNF or GDNF gene). For example, brain-penetrating pegylated NPs loaded with GDNF gene were demonstrated to restore dopamine levels and dopaminergic neuronal density in the striatum of 6-OHDA-induced PD rats.¹⁸¹ In addition, a novel microvesicle (MB)–liposome complex was used for delivery of GDNF/BDNF gene to promote normal dopamine secretion and recovery of motor behavior in PD models.¹⁸² All these examples demonstrate that nanotechnology is an attractive approach for delivery of genes to treat PD.

4.6 Stem cell transplantation

Due to the progressive loss of dopaminergic neurons in the substantia nigra striatum in PD and the fact that stem cells may differentiate into specific neuronal groups (such as DA neurons), cell replacement therapy has become a viable option for the treatment of PD.¹⁸³ In the 1980s, the approach of transplant fetal midbrain tissue with dopaminergic neurons, which can restore physiologically synaptic release of dopamine in the denervated striatum and reverse motor deficits, aroused great interest in the clinical community.¹⁸⁴

To date, different types of stem cells, mainly including neural stem cells (NSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and MSCs, have been used to improve the symptoms of various neurodegenerative diseases.¹⁸⁵ NSCs are stem cells derived from embryonic brain tissue or from patient cells with the self-renew capability. They can differentiate into neurons, astrocytes, and oligodendrocytes.^{185, 186} ESCs are stem cells with high differentiation and strong self-renew capability of differentiating into any type of cells in principle, including dopaminergic neurons.¹⁸⁷ Similar to ESCs, human iPSCs can differentiate into dopaminergic neurons. However, iPSCs cannot spontaneously form dopaminergic neurons after transplantation, which means that they must be differentiated into sufficient progenitor cells before transplantation.¹⁸⁸ MSCs are a class of adult stem cells with multidirectional differentiation potential, and exist in the interstitium of various tissues and organs. Stem cells used for nerve repair and regeneration can differentiate not only into neurons,¹⁸⁹ but also microglia¹⁹⁰ and astrocytes.¹⁹¹ However, cell replacement therapy is often affected by low graft survival, moderately limited cell integration, and delayed therapeutic effect, which limits its further application.¹⁹²

The rich properties of nanoparticles, including promoting the secretion of various bioactive factors for anti-inflammation, antiapoptosis, and immunoregulation, regulating cell metabolism, protecting dopaminergic neurons, and reversing motor dysfunction, make them very attractive in stem cell regulation. On the one hand, the use of nanoparticles to carefully modulate the microenvironment and regulate stem cell growth is valuable for the treatment of neurodegenerative diseases. On the other hand, based on the unique properties of nanoparticles, the transplanted stem cells could be accurately tracked by in vivo imaging after the stem cells are labeled by nanoparticles, and the therapeutic effects of stem cell transplantation could be evaluated.¹⁸⁵ For example, He et al.¹⁹³ reported that bioactive black phosphorus nanosheets can effectively improve the antioxidative capacity and the survival rate of different types of stem cells by upregulating the nuclear factor erythropoie-like 2-dependent antioxidative pathway, including human placental chorionic MSCs, iPSCs, and NPCs. Shi et al.¹⁹⁴ demonstrated that chiral nanoparticles with high G-factor values significantly upregulated Map2, Yap1, and Taz genes, resulting in mechanical force, cytoskeletal protein action, and accelerated differentiation of mouse NSCs into neurons. Huang et al.¹⁹⁵ synthesized a PBAE-PLGA-Ag₂S-RA-siSOX9 (PPAR-siSOX9) nanoformula with high gene/drug deliverability to promote the neuronal differentiation of NSCs. It has been demonstrated that hydroxyapatite nanorods promoted neural differentiation of NSCs into GABAergic neurons through Ca²⁺/c-Jun/TLX3 signaling, which was further confirmed by the in vivo study.¹⁹⁶ Many innovative studies on the functional nanoparticles for tracking stem cells in PD have also been reported. For example, gold NPs were used to monitor the migration and homing patterns of exosomes derived from bone marrow MSCs (MSC-exo) in various brain pathologies after intranasal administration.¹⁹⁷ However, the safety and controllability of using nanoparticles and stem cells to treat neurodegenerative diseases need to be studied more extensively.