Authors: Sevim Isik, Sedra Alhelwani, Aya Sahsahi, Hilal Balcilar, Bercem Yeman-Kiyak
Categories: Review Article, Alzheimer's disease, Engineered exosomes, Neurological disorders, Parkinson's disease, Personalized medicine, Plant-derived exosome-like nanovesicles
Source: Drug Delivery and Translational Research
Authors: Sevim Isik, Sedra Alhelwani, Aya Sahsahi, Hilal Balcilar, Bercem Yeman-Kiyak
Exosomes are nano vesicles secreted by the cells that play an essential role in intercellular communication, enabling the transport of bioactive molecules, including proteins, lipids, and nucleic acids. Among them, plant-derived exosome-like nanovesicles have attracted considerable interest due to their prospective therapeutic implications, especially for neurological disorders. This article provides an overview of the biogenesis of plant-derived exosome-like nanovesicles, compares their characteristics with mammalian-derived exosomes, and investigates their bioavailability and chemical composition. The article also discusses the mechanisms through which they are uptaken by cells, highlighting several cellular uptake pathways and their significance for targeted drug delivery. Moreover, it explains the molecular basis of neurological disorders and investigates how plant-derived exosome-like nanovesicles regulate intracellular signaling pathways, providing potential therapeutic benefits. Finally, it provides the latest advancements in engineering research, emphasizing biochemical modifications on the exosomal surface, loading therapeutic molecules into exosomes, and exosomes derived from genetically engineered plants, for more effective therapies in neurological disorders.
Exosomes are small nano-scaled membrane-bound vesicles secreted by eukaryotic cells and have gained significant attention for their potential as therapeutic vehicles, particularly in neurological disorders [1]. Plant-derived exosome-like nanovesicles (ELNs), which are naturally occurring nanoparticles secreted from plant cells, have emerged as a promising alternative to mammalian-derived exosomes and conventional nanocarriers. They have several benefits rendering them appeal for neurological therapy. Plant ELNs can cross the blood–brain barrier (BBB), have minimal immunogenicity, and are inherently rich in bioactive substances, including antioxidants and small RNAs. They exhibit biocompatibility, cost-effectiveness, and suitability for oral or nasal administration, which is atypical for traditional delivery methods. They are scalable and morally advantageous, overcoming manufacturing and safety limits associated with mammalian-derived vesicles. These characteristics establish plant ELNs as versatile tools for therapeutic administration and modulation of neuroinflammatory and neurodegenerative pathways [2]. The biogenesis of these ELNs involves the formation of intraluminal vesicles within multivesicular bodies (MVBs), which are released into the extracellular environment upon fusion with the plasma membrane, encapsulating proteins, lipids, DNAs, RNAs, and metabolites [3]. This makes them ideal for targeted drug delivery and intercellular communication. One of the key advantages of plant-derived ELNs is their superior biostability compared to mammalian exosomes, largely due to their robust lipid membranes, enabling them to withstand environmental stresses and maintain their integrity in gastric and intestinal fluids [4]. This stability is crucial for overcoming challenges such as the ability to cross the BBB, which often limits the delivery of therapeutic agents to the central nervous system (CNS), and to withstand gastric fluid [5–7].
Neurological disorders are a highly diversified category of conditions including Alzheimer's Disease (AD), Parkinson's Disease (PD), spinal cord injury (SCI), ischemic brain injuries, and brain tumors, which are all characterized by complex pathologies involving neuronal degeneration, neuroinflammation, oxidative stress, and impaired cellular communication in CNS. These disorders may result from genetic, environmental, and immunological factors, resulting in complex pathological processes. Many neurological disorders, despite their diversity, exhibit common molecular processes such as neuroinflammation, oxidative stress, mitochondrial dysfunction, and synaptic abnormalities. Furthermore, neurodegeneration, glial cell activation, and compromised neural transmission are fundamental to the pathophysiology of these disorders [8, 9].
Various current strategies are being explored to treat neurological disorders, including the use of nanogels and liposomes. Although these methods can deliver drugs and support neuronal regeneration, several significant challenges persist. For instance, nanogels may struggle to penetrate deeply into the brain tissues and might cause issues such as burst drug release, biocompatibility concerns, and neurotoxicity [10]. Liposomes may accumulate in non-target tissues because of their limited specificity for brain regions, potentially leading to long-term toxicity. Additionally, large liposome particles often have difficulty in crossing the BBB. They also exhibit low bioavailability, poor water solubility and scalability issues, all of which can diminish their effectiveness in treating certain brain areas [11]. Moreover, Long Non-Coding RNAs (lncRNAs) have been explored for their therapeutic potential in neurological disorders, owing to their role as gene regulators. However, their application remains constrained by challenges such as off-target delivery risks and immunogenic responses induced by synthetic RNA molecules [12, 13]. Therefore, there is an urgent need to develop highly specific delivery systems capable of preventing unintended interactions and reaching deep brain structures for long-term treatment—potential solutions include natural delivery vehicles or plant-derived ELNs.
Plant-derived ELNs have emerged as alternative promising therapeutic agents against many disorders, demonstrating their ability to modulate inflammatory pathways, reduce oxidative stress, regulate autophagy, and promote neuroprotection. Notably, plant-derived ELNs exhibit antioxidant, anti-inflammatory, antimicrobial and anti-cancer properties [14, 15]. Also, these ELNs play a critical role in targeting pro-inflammatory proteins which are linked with neuroinflammation, for instance, ginger rhizomes-derived ELNs, have shown anti-inflammatory properties by inhibiting NLRP3 inflammasome activation in macrophages, suggesting plant-derived ELNs could target proinflammatory proteins involved within relative signaling pathways in other neurological diseases [16]. These findings highlight the considerable potential of plant ELNs in therapeutic applications, from cancer and aging to gut health and inflammation. Alongside this, surface modifications of plant-derived ELNs enhance their targeting specificity, stability, and ability to cross the BBB. The functionalization of plant-derived ELN surfaces involves PEGylation [17], which is the insertion of maleimide lipid group, to act as bridges and connect with other targeting ligands e.g., peptides (RVG, RGD and aptamers), and coating of small molecules like folic acid, all of which improve ELNs targeting and interaction with various specific receptors of targeted cells, ensuring the therapeutic cargo reaches the desired site of action [18]. These modifications also reduce exosome immunogenicity, making them suitable for long-term therapies without eliciting unwanted immune responses [19].
This review seeks to provide an in-depth exploration of plant-derived ELNs, focusing on their biogenesis, stability, cellular pathways associated with neurological disorders, the signaling pathways which exosomes have the potential to regulate in neurological disorders, and recent advancements in engineered exosomes such as surface-decorated, cargo-loaded and source-based modified (transgenic) exosomes and their interactions with neuronal and glial cells in the brain, to enhance their therapeutic potential in treating complex neurological diseases.
Exosomes are small membrane-bound vesicles with sizes ranging from 30–200 nm. They are composed of a unique content of bioactive compounds enclosed in a lipid bilayer. Exosomes biogenesis starts with the plasma membrane invagination to form early endosomes which then mature into late endosomes [20]. The late endosomes undergo a transformation where their membrane folds inward to form smaller vesicles known as intraluminal vesicles (ILVs) within the endosome. As a whole, the endosome is now called a multivesicular body (MVB). Selectively, on the other hand, a unique combination of DNA, RNA, proteins, and lipids is gathered in the ILVs within MVBs. After formation, the MVBs face two fates, either fusing with a lysosome and getting degraded, or fusing with the cell's plasma membrane and releasing the ILVs extracellularly as free exosomes [3, 21]. The biogenesis of exosomes in plants can also take an alternative pathway. Double membrane organelles known as EXocyst Positive Organelles (EXPOs) have been identified in some plants like tobacco. They are similar to autophagosomes except that they are not associated with any degradation pathway, instead, they are involved in a direct secretion pathway [22] (Fig. 1).Fig. 1Biogenesis of plant-derived ELNs
While mammalian exosomes have been extensively studied and despite their biocompatibility, biodistribution, and low immune response, they have faced many challenges such as low stability, homogeneity issues, high cost and low mass production. On the other hand, plant-derived ELNs exhibit intriguing characteristics of higher stability, non-toxicity, uniformity, lower cost and higher mass production, all of which have shed light on their usage in nanomedicine research [2, 20]. These plant ELNs contain phospholipids, glycolipids, sterols, and polysaccharides with high phospholipids levels due to the lack of cholesterol, unlike mammalian exosomes. The presence of cellulose, hemicellulose, and pectin in plant cell walls provides additional protection, enhancing the stability and resilience of plant ELNs against harsh conditions. Their unique phytochemical composition includes polyphenols, phenolic acids, flavonoids, carotenoids, terpenoids, and alkaloids, pool in fostering dietary antioxidants intake, increasing cellular health and metabolic balance [23].
Studies reveal that certain plant extracts, such as onion powder, can inhibit inflammatory pathways, though solubility and bioavailability issues persist [24]. Recently, researchers have noted that even processed plant materials retain extracellular vesicles, which may offer nutritional and therapeutic benefits beyond fresh intake [25].
Free antioxidants, while crucial for alleviating oxidative stress and providing therapeutic and preventive effects against aging, cancer, neurodegenerative disorders, and cardiovascular disease, still confront several difficulties that restrict their stabilities, ranging from inadequate bioavailability, poor stability, and ineffective targeting. Vitamin C, for example, declines during juice processing due to its susceptibility to oxygen and temperature, and because of the intestinal wall's low permeability, necessitating its conversion to dehydroascorbic acid [26]. Similarly, crocins as in extracts face challenges in crossing the in vitro blood–brain barrier (BBB) (PAMPA assay) in their free form due to their relatively large size and susceptibility to degradation. However, crocins may be able to penetrate the BBB if they are metabolized into a simpler form, specifically deglycosylated trans-crocetin, although this process occurs at a slow rate [27]. Therefore, encapsulation into exosomes provides novel delivery strategies to these limitations. Exosomes not only protect their cargo from destruction, but they also allow for effective delivery to specific organs, including the brain, via routes such as the gut-brain axis. Citrus lemon exosomes, for example, increase vitamin C stability and provide safe delivery while avoiding interference with blood coagulation pathways [28]. Similarly, tomafran-derived exosomes naturally enriched with crocins and picrocrocin pass the BBB and reduce oxidative stress and inflammation more effectively than free antioxidants [27]. This encapsulating approach thus opens a promising path for dietary supplements and therapeutic treatments, particularly in neurodegenerative illnesses such as AD and PD.
Besides the unique composition, plant-derived ELNs have rich proteomic and lipidomic profiles, making them significant for intercellular communication and potential therapeutic applications.
General structural characteristics of Plant-derived ELNs are demonstrated in Fig. 2. Most of the protein content in plant ELNs transmembrane and cytosolic proteins among which are proteolysis, aquaporin, and proteases in addition to channels and transporters [29, 30]. They also contain proteins such as glutathione S-transferase, S-adenosyl-homocysteinase, glyceraldehyde 3 phosphate dehydrogenase, annexins, and heat shock proteins or tetraspanins, which are strong candidates to be accepted as plant ELNs markers (e.g., CD9, CD63, CD81, and CD54) [31].Fig. 2Structural characteristic of plant-derived ELNs
Not only their protein content but also the lipid content plays crucial roles in cellular responses and intercellular communication. They maintain exosome stability and structure, facilitate cargo uptake and retention. Studies have shown that plant ELNs acquire higher concentrations of essential components like phospholipids (such as phosphatidic acids (PA), phosphatidylethanolamines (PE), mono- and di-galactosyldiacylglycerol (e.g., MGDG, DGDG)).
miRNAs, non-coding, single-stranded molecules with length around 21 to 23 nucleotides found in eukaryotic cells [32]. Plant miRNAs are specific to plants and plant activities, but lately, it has been found that when ingested and absorbed by animal cells, it may alter the expression of some of the host organism’s genes [33]. Another group of researchers found evidence of plant miRNA in circulating blood of human subject [34].
Plant-derived ELNs are internalized by cells via a variety of cellular uptake mechanisms. Understanding these mechanisms is crucial, as they elucidate how these vesicles deliver their diverse cargo, including RNA, proteins, and lipids to target cells. Cellular uptake pathways and surface interactions are critical determinants of their therapeutic potential, particularly in neurological disorders. Cellular uptake mechanisms can be broadly divided into endocytic pathways, surface interactions, and nanovesicle composition [20, 35, 36].
Endocytosis refers to the process by which cells internalize extracellular materials through the invagination of the plasma membrane. Various pathways, including clathrin-mediated endocytosis, caveolin-mediated endocytosis, macropinocytosis, and phagocytosis, are involved. These pathways effectively deliver plant-derived ELN cargo to target cells [36, 37]. Clathrin-mediated endocytosis involves the recruitment of clathrin proteins to form coated vesicles, internalizing specific cargoes. ELNs may be recognized and internalized by membrane-bound receptors [38, 39]. Receptor-mediated endocytosis ensures selective uptake, triggering vesicle formation and intracellular transport [40]. Caveolin-mediated endocytosis involves lipid-rich invaginations and interactions with membrane components. This pathway avoids lysosomal degradation, preserving structural integrity for cytosolic delivery [41]. Macropinocytosis engulfs extracellular fluid and its contents nonspecifically. Phagocytosis internalizes larger particles, often by immune cells [41]. The endocytic pathway utilized depends on ELN size, composition, and surface properties [38]. Pathway selection influences ELN-based therapeutic effectiveness [23].
Surface proteins on plant-derived ELNs, such as tetraspanins, integrins, and glycoproteins, selectively bind receptors on target cells, enabling receptor-mediated endocytosis. These interactions ensure efficient uptake and intracellular trafficking [42, 43]. Specific proteins may prevent lysosomal degradation, facilitating cargo delivery to therapeutic targets [41]. Selective interactions between ELN surface proteins and receptors, like hippocalcin, enable precise targeting for neurological therapies [44–46]. Lipids and glycoproteins further enhance selective binding and uptake [23]. Modifications to ELN surfaces improve targeting capabilities and therapeutic efficacy [47]. Lipids, such as sphingolipids, influence membrane fusion and transport. These modifications impact uptake mechanisms and distribution, facilitating targeted delivery [6, 7, 36]. Researchers are actively exploring surface engineering to improve biodistribution and therapeutic potential.
Size, lipid profile, and protein composition impact internalization pathways and therapeutic applications of plant-derived ELNs [48]. Those that are larger in size, favor macropinocytosis or phagocytosis, while smaller ones utilize clathrin- or caveolin-mediated pathways [49]. Lipids, like phosphatidylserine, enhance recognition and internalization by phagocytic cells [50]. Surface proteins and glycoproteins trigger receptor-mediated endocytosis, facilitating efficient cargo delivery [35]. Researchers are investigating these features to develop targeted therapies [51]. Plant-derived ELNs contain bioactive compounds, like gingerol or proanthocyanidins, that influence cellular uptake and therapeutic potential [44–46]. Understanding these factors is essential for optimizing these ELNs as drug-delivery systems and therapeutic agents.
AD is a progressive neurodegenerative disorder characterized by key pathological hallmarks, including synaptic dysfunction due to tau protein hyperphosphorylation, reduced neuronal metabolism, and the loss of multiple neurotransmitters such as acetylcholine, noradrenaline, serotonin, and dopamine. A major structural issue is the disruption of microtubules, caused by hyperphosphorylated tau proteins that destabilize them, leading to neuronal breakdown [52]. AD is marked by extracellular amyloid beta (Aβ) accumulation, a byproduct of abnormal amyloid precursor protein (APP) processing via β-secretase and γ-secretase, and intracellular neurofibrillary tangles formed from tau protein. Aβ binds to degenerated axons, apolipoprotein E, microglia, and astrocytes, impairing synaptic signaling and inducing inflammation [53, 54]. Monoamine oxidase (MAO) plays a role by breaking down neurotransmitters like dopamine, serotonin, and noradrenaline, producing hydrogen peroxide which diffuses through the cell membrane, elevating oxidative stress and promoting lipid peroxidation, which damages neuron structures, triggering inflammatory cytokine release [55]. Oxidative stress also exacerbates Aβ and tau tangle aggregation, creating a feedback loop of increasing free radicals. High reactive oxygen species (ROS) levels further damage cellular components, like mitochondria, leading to disrupted Ca^2+^ homeostasis through N-methyl-D-aspartate (NMDA) receptor overactivation and endoplasmic reticulum (ER) Ca^2+^ overload [56]. Elevated intracellular Ca^2+^ influx triggers pathways such as calcineurin and Bcl-2-associated death promoter activation, releasing cytochrome c, which initiates apoptosis. Aβ also interacts with caspases, breaking down essential cell components and leading to neuronal death [57]. Additionally, ROS activates pathways like stress-activated protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), both of which hyperphosphorylate tau, contributing to neurofibrillary tangle formation which in turn leads to alterations in neuronal signaling and neuronal loss especially in the hippocampus [58], resulting in significant acetylcholine depletion due to reduced choline acetyltransferase activity [59]. Overall, the accumulation of Aβ plaques and neurofibrillary tangles, along with oxidative stress and neuroinflammation, forms a vicious cycle, disrupting neuronal function and communication, eventually resulting in memory and cognitive decline.
Current treatments in AD are commonly targeted towards easing the symptoms and retarding disease advancement rather than curing it, as there is none. Cholinesterase inhibitors and glutamate regulators are mostly used as the pharmacological approach [60]. However, although debated, monoclonal antibodies targeting the amyloid plaques have also been a recent option of treatment with ongoing clinical trials [61, 62]. On the other hand, non-pharmacological approaches such as cognitive stimulation, exercise, and diet are also included in the treatment protocol in order to improve patients’ quality of life [63–65]. Even though these therapies have improved, challenges still remain. These include side effects, high expenses, accessibility difficulties, limited slowing of disease progression due to late diagnosis, and individual variability. Therapies with personalized methods are needed in order to improve patient outcomes. In this sense, stem cell and exosomal therapies have become prominent to promote brain regeneration and repair.
PD is a neurodegenerative disorder marked by the loss of dopaminergic neurons in the substantia nigra, a brain region responsible for producing dopamine. The disease manifests through motor symptoms affecting movement and balance and non-motor symptoms including pain, sleep problems and mental health issues [66, 67]. The death of dopaminergic neurons reduces dopamine levels in the synapses and leads to the formation of Lewy bodies (LB), primarily composed of aggregated alpha-synuclein (α-Syn) [68]. These abnormal protein deposits also contain neurofilament proteins and proteins involved in cellular breakdown, like ubiquitin. The main cause of cell death in PD is thought to be the breakdown of the nuclear membrane, releasing aggregated α-Syn, which interacts with nuclear elements such as histones [69]. α-Syn is primarily found in presynaptic terminals in the CNS, where it binds to synaptic vesicles [70]. α-Syn contains an amphipathic N-terminus, which helps it bind lipids and potentially aids in aggregation. Its non-amyloid component at the C-terminus binds calcium and inhibits aggregation [71]. The protein aggregates, known as amyloids, consist of misfolded proteins rich in β-sheets, which are associated with cellular damage and neuronal dysfunction. Amyloid deposition is linked to issues such as mitochondrial dysfunction, oxidative stress, and disruptions in the ER and proteasomes, contributing to neuronal degeneration [69]. Currently, there is no cure for PD. The standard treatment involves dopaminergic medications like levodopa, COMT inhibitors, anticholinergics, dopamine agonists, and MAO-B inhibitors, which aim to manage symptoms and improve patients'mobility and life expectancy [68]. However, these treatments provide only symptomatic relief without addressing the disease's root causes, highlighting the need for a more comprehensive approach to PD treatment.
Brain cancer, also known as a CNS tumor, is one of the most aggressive and challenging forms of cancer to treat. It is basically a group of abnormal cell growth, can be benign or malignant, that originates within the brain or the spinal cord. The main types of brain cancer include gliomas, meningiomas, and pituitary tumors, each with distinct characteristics and prognoses. Gliomas, which originate from glial cells, are the most common type of brain cancer, accounting for over 80% of all malignant brain tumors [72]. The biological and molecular basis of brain cancer is complex, involving genetic mutations and alterations in signaling pathways that drive uncontrolled cell growth and proliferation [73]. Furthermore, recent research has highlighted the significance of the HGF/MET (Hepatocyte growth factor/Mesenchymal-epithelial transition factor) signaling pathway in the pathogenesis and progression of malignant brain tumors [74]. Treatments for brain cancer have historically been limited, with surgical resection, radiation therapy, and chemotherapy being the primary options. However, there are limitations in the effectiveness of these standard treatments, as brain tumors can often be resistant to therapy and can recur even after aggressive treatment [75]. Newly emerging treatment options, such as targeted molecular therapies, immunotherapy, and personalized medicine approaches, hold promise in improving outcomes for patients with brain cancer [76, 77]. Targeted therapies aim to disrupt specific molecular pathways involved in cancer progression, while immunotherapy seeks to harness the body's own immune system to recognize and attack tumor cells [78]. Additionally, personalized medicine approaches, which involve genetic profiling of individual tumors, can guide the selection of the most effective treatment regimen for each patient. Despite these advances, the challenge of treating brain cancer remains significant, as the BBB can limit the delivery and efficacy of these novel therapies [79, 80]. However, plant exosome-based delivery methods are innovative approaches to overcoming the BBB that are currently being explored to improve the delivery of therapeutic agents to brain tumors. These exosome-based delivery systems have shown promising results in preclinical studies, demonstrating enhanced permeability across the BBB and targeted delivery of therapeutic payloads to brain tumor cells [81, 82].
Ischemic stroke, a progressive and life-threatening condition, accounts for 85% of all stroke cases. It occurs when an artery in the brain becomes blocked, cutting off the oxygen-rich blood supply to the brain, known as cerebral arterial occlusion. This blockage is typically caused by either a clot forming directly in the brain (thrombosis) or one that forms elsewhere and travels to the brain (embolism) [83, 84]. The resulting damage unfolds through a cascade of events that progressively deteriorate brain tissue. The initial trigger is the loss of oxygen and glucose due to blood flow obstruction, which leads to hypoxia in neurons. This deprives them of ATP, as oxidative phosphorylation in the mitochondria halts, resulting in an energy failure. Consequently, harmful substances such as glutamate, ROS, and reactive nitrogen species (RNS) are released, exacerbating oxidative stress. As Na⁺ levels rise, causing an influx of calcium ions (Ca^2^⁺), triggering excitotoxicity, which further damages cells. The excessive oxidative stress also stimulates the release of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, which trigger an inflammatory response and activate microglia and macrophages. As ischemia continues, the tissue becomes more acidic causing nearby neurons to depolarize, extending the injury into the peri-infarct region. This cascade inhibits neuronal repair and leads to apoptosis. In areas where ATP depletion is severe, necrosis occurs, while autophagy may either protect or contribute to further cell death depending on the severity of the ischemia. Prolonged ischemia also damages the BBB, allowing immune cells, proteins, and other substances to leak into the brain, which accelerates inflammation and causes brain swelling (edema) [83]. The current gold standard treatment for ischemic stroke, recombinant tissue plasminogen activator (rt-PA), was approved by the FDA in 1996. Administered intravenously, rt-PA is effective by dissolving the clot obstructing blood flow. However, its limitations were witnessed when it was not administered within the time window, delays in administration can lead to complications such as hemorrhagic transformation, neurotoxicity, and increased mortality [85, 86].
Spinal cord injury (SCI) is a severe neurological condition arising from primary injury of both traumatic causes—such as falls and road traffic accidents—and nontraumatic factors, including infections and tumors, which could all lead to glial membrane and axonal network damage as well as immediate damage to neuronal tissues. As the population ages, the incidence of SCIs, particularly from falls, is expected to rise [87]. In the secondary injury phase, SCI triggers a cascade of complex, interconnected pathological changes at the injury site due to excessive glutamate, which causes neuronal excitotoxicity with calcium (Ca^2^⁺) and sodium ions (Na⁺) influx, ionic imbalances, inflammation and necrosis. First, ischemia and hypoxia initiate edema and mitochondrial dysfunction, reducing cellular energy and ATP and inducing alterations in the morphology of neuronal cells [88]. This leads to an overproduction of ROS and RNS, causing widespread damage to cell membranes by mediating lipid peroxidation, proteins, and DNA. As oxidative stress intensifies, pro-inflammatory cytokines are released, creating a vicious cycle of inflammation that stimulates microglia activation and macrophages infiltration. These processes contribute to glial scar formation, further hindering recovery, increasing tissue necrosis, and decreasing neuronal regeneration, ultimately leading to extensive neuronal apoptosis. The result is paralysis, disruptions in sensory function, and even potential organ failure due to the extent of damage to the nervous system [87, 89]. After SCI, several factors limit tissue regeneration at the lesion core. Non-neuronal cells (fibroblasts, microglia, endothelial cells) contribute to the formation of a fibrotic scar. Similarly, astrocytes form an astroglial scar, which acts as a barrier to protect the surrounding tissues from further damage, although it also hinders regeneration [87]. Recent research indicates that commonly used anti-inflammatory such as corticosteroids and neurotrophic medications in SCI treatment do not adequately target early oxidative stress. Therefore, they are ineffective in significantly improving SCI symptoms [90, 91]. While surgery can help improve motor function in traumatic SCIs, it often results in complications like respiratory and cardiovascular problems [92]. Despite medical advancements, there is still no effective drug to reduce the intense oxidative stress following SCI, highlighting a significant gap in current treatment options [93].
The gut-brain axis refers to the bidirectional communication between the gastrointestinal tract and the CNS, which plays a crucial role in regulating various physiological and neurological functions. This intricate network involves neural, endocrine, and immune signaling pathways that allow the gut microbiota to influence brain function and vice versa. The four major pathways of the gut-brain axis include the vagus nerve, the neuroendocrine system, the immune system, and the gut microbiota-derived metabolites and signaling molecules [94, 95]. Dysregulation of this axis has been linked to a range of neurological and psychiatric disorders, including AD, PD, multiple sclerosis (MS), autism spectrum disorder, depression, and anxiety [96]. PD, for instance, can be triggered by gut microbiota dysbiosis and inflammation [97]. Conversely, AD has been associated with gut microbiome alterations that may contribute to neuroinflammation and the development of pathological hallmarks in the brain [98]. In MS, gut dysbiosis and intestinal barrier dysfunction can lead to increased permeability and the entry of inflammatory molecules into CNS, exacerbating autoimmune responses [99]. Moreover, the gut-brain axis has been implicated in the pathogenesis of autism spectrum disorder, with disruptions in the microbiome-gut-brain signaling potentially contributing to the core symptoms of the condition [100]. Similarly, depression and anxiety have been linked to imbalances in the gut microbiome, which can influence neurotransmitter production, neural plasticity, and the stress response [101]. The key symptoms and signs of gut-brain axis-related disorders often include gastrointestinal issues, cognitive impairments, mood disturbances, and neurological deficits. These diseases typically progress through stages of gut dysbiosis, increased intestinal permeability, neuroinflammation, and the accumulation of pathogenic proteins, leading to the manifestation of clinical symptoms [102, 103]. The biological and molecular basis of these disorders involves complex interactions between the gut microbiome, the immune system, and the CNS, with ongoing research aimed at elucidating the precise mechanisms underlying these connections [104]. Key mechanisms include the production of neuroactive metabolites, the modulation of the immune system, and the regulation of the hypothalamic–pituitary–adrenal axis, which governs the body's response to stress. These pathways can influence neurotransmitter synthesis, neuronal excitability, synaptic plasticity, and brain-derived neurotrophic factor expression, all of which play critical roles in neurological and psychiatric conditions [105, 106]. Current treatment options for gut-brain axis-related disorders include dietary interventions, probiotics, antibiotics, anti-inflammatory medications, and neurotransmitter-targeting therapies, but their effectiveness is often limited by the complex and multifactorial nature of these disorders [96]. Challenges in effectively treating these diseases include the need for personalized approaches, the difficulty in modulating the gut microbiome, and the lack of a comprehensive understanding of the precise mechanisms linking the gut and the brain [107]. Nonetheless, promising discoveries about plant-derived ELNs offer hope for the development of more effective treatments in the future.
Neurological disorders such as neurodegenerative diseases including AD and PD, as well as acute injuries, such as strokes, have complex, interrelated molecular mechanisms specific to them. The most crucial pathways involved in the progression of these diseases include neuroinflammation, neuroprotection and synaptic plasticity, autophagy and protein homeostasis, antioxidant and calcium signaling pathways [9]. The operational flow of these pathways is necessary to maintain healthy, functional and responsive neurons against oxidative stress, damage or inflammation. In recent studies, it has been shown that plant-derived ELNs have a therapeutic potential in neurological disorders through modulating the above-mentioned pathways. Accumulating more knowledge about the regulation and overlapping of these pathways can shed light on possible therapeutic approaches for various neurological disorders.
Neuroinflammation is a series of interwind and complicated cascade of events that happens in the central nervous system and it can be triggered by injuries, exposure to toxins or infection. The detection of the previously mentioned risks leads to the upregulation of inflammation signaling pathways including NF-κB, MAPK, and JAK/STAT pathways. Microglial cells are then activated leading to elevated secretion of pro-inflammatory cytokines, chemokines, ROS and nitric oxide [9]. This reaction is initially protective but in a chronic state it contributes to the progression of neurological diseases. Studies revealed that Allium Tubersum and Atractylodes lancea ELNs (A-ELNs and ALR-ELNs respectively) both exhibit anti-inflammatory effects by inhibiting inducible nitric oxide synthase (iNOS) expression, and reducing pro-inflammatory cytokines [108, 109]. Another notable study reported that exosomes isolated from Lycium Barbarum L. when loaded with isoliquiritigenin (ISL), significantly impact the neuroinflammatory pathway by reducing pro-inflammatory cytokines like IL-1β, IL-6, TNF-α, while increasing the expression of anti-inflammatory cytokine IL-10. Additionally, it inhibits the expression of iNOS, and activates the pAKT/AKT pathway which is critical for survival. Lastly, it promotes neuroprotection and synaptic plasticity by upregulating Tuj1, MAP2, GAP43, NF200, and MBP markers [110–112].
The Antioxidant signaling pathway is a cellular defense mechanism against elevated ROS levels. The detection and response to oxidative stress involve a wide network of genes, enzymes, and many regulatory molecules [113]. The core regulator of this pathway in the CNS is the nuclear factor erythroid 2–related factor 2 (Nrf2), which controls the production of key enzymes including heme oxygenase-1 (HO-1) and superoxide dismutase (SOD) [114]. It is responsible for maintaining redox homeostasis and neutralizing ROS, which eventually protects the cells from oxidative damage [115]. In this context, the previously mentioned ALR-ELNs also promote neuroprotection through pathways involving immunoresponsive gene 1 (IRG1) and Nrf2 [109]. Drynariae rhizoma ELNs target oxidative phosphorylation and mitochondrial dysfunction in Huntington's disease, while modulating Nrf2/ARE signaling to provide antioxidant and neuroprotective benefits [116]. Lycium Reuthenicum ELNs (LRM-ELNs) reduce oxidative stress by enhancing nuclear Nrf2 translocation and boosting HO-1 and NQO1 expression in an in vitro AD model [117, 118]. Furthermore, a study showed that nanoparticles derived from carrot reduce 6-OHDA-induced oxidative stress and apoptosis in SH-SY5Y cells by suppressing caspase-3 cleavage and modulating Nrf-2, Heme oxygenase (HO-1) as well as NAD(P)H Quinone Dehydrogenase 1 (NQO-1) [119]. Onion-derived exosomes also provided protection against oxidative stress by activating KEAP1/NRF2/ARE signaling pathway, reducing ROS levels by enhancing HO-1 expression and translocation of Nrf-2 [110–112].
The brain's apoptosis and neuroprotection pathways are two contrasting but related biological mechanisms that regulate neuronal survival and death in response to a range of pathological and physiological events [120]. Key neuroprotective pathways include the MAPK/ERK, PI3K/Akt, and Nrf2 signaling cascades, while apoptosis is regulated through intrinsic (mitochondrial) and extrinsic (death receptor) pathways. Both pathways eventually converge on caspase-3 activation [121]. A recent study shows that LRM-ELNs reduce Aβ-induced apoptosis in PC12 cells by downregulating MAPK and enhancing the PI3K/Akt pathway [117, 118]. In another study, researchers showed that Pueraria Lobata Roots derived exosomes enhance PINK1-Parkin-mediated mitophagy and promote SQSTM1/p62-mediated autophagy [122]. Additional research reported that LRM-ELNs maintain mitochondrial membrane potential (MMP) and lower the Bax/Bcl-2 ratio in the setting of Aβ-induced apoptosis in HT22 cells. This lowers Caspase-3 cleavage and, ultimately, inhibits the intrinsic apoptotic pathway [117, 118].
Calcium (Ca2 +) is a very important molecule in the CNS. It works as a secondary molecule that regulates many neuronal processes such as synaptic transmission, gene expression, plasticity, and cell survival through various calcium signaling pathways [123]. Glutamate excitotoxicity on the other hand is a hallmark of various neurological diseases and is mediated by dysregulation of calcium signaling pathway [124]. In addition to iron dysregulation and lipid peroxidation, high glutamate causes an increase in intracellular Ca2 +, which in turn leads to ferroptosis. To mitigate this, a group of researchers proved that green onion-derived ELNs can reduce lipid peroxidation by enhancing the expression of glutathione peroxidase 4 (GPX4) and inhibit glutamate-induced Ca2 + influx in HT22 mouse hippocampal cells [125].
Mechanisms and molecular insights of plant-derived exosomes in neurological disorders is detailed in Table 1. Table 1Mechanisms and molecular insights of plant-derived exosomes in neurological disordersPhyto-exosomes sourceIsolation MethodSize (nm)Zeta Potential (mV)Identified Bioactive ComponentsTherapuetic PotentialStudy TypeDisease TargetsMechanisms of Action/EffectsRefCitrus LemonUC93.77 ± 21.31 nm (DLS)−3.46 ± 1.45 mVVitamin CAntioxidant NeuroproctectiveIn vitro (Neuroblastoma: SH-SY5Y)AD↑ Antioxidant activity↓ Aβ-induced neurotoxicityAbility to cross BBB (PAMPA assay)Maintain neuronal health[28]Tomafran (Bioengineered Tomato)UC169 nm (NTA)-CorcinsAntioxidant Anti-inflammatory NeuroproctectiveIn vitro (Neuroblastoma: SH-SY5Y)AD↑ Neuroprotection↓ Oxidative stress and inflammation↓ Okadaic acid-induced tau hyperphosphorylation[27]Lycium Ruthenicum MurrayPEG6000-based precipitation114.1 nm (NTA)−6.36 mVPolysaccharidesPolyphenolsAnthocyaninsAntioxidant Antiapoptotic NeuroproctectiveIn vitro (Pheochromocytoma PC12)AD↓ Aβ-induced apoptosis↓ MAPK↑ PI3K/AKT pathway↓ Bax ↑ Bcl-2[117, 118]Lycium Ruthenicum MurrayPEG6000-based precipitation114.1 nm (NTA)−6.36 mV-Antioxidant Antiapoptotic NeuroproctectiveIn vitro (Mouse hippocampal neuronal HT22)AD↓ Aβ-induced apoptosis↑ MMP↓ Bax/↑ Bcl-2↓ Caspase-3 cleavage↓ Oxidative stress↑ NRF-2↑ HO-1 ↑ NQO1[117, 118]Salvia Sclarea Hairy RootsUC + SEC116 ± 15 nm (NTA)-Triterpenoids: asiatic acid, ursolic acid, oleanolic acid, and peroxidasesNeuroprotective AntioxidantAnti-apoptotic In vitro (Neuroblastoma: SH-SY5Y)PD↓ 6-OHDA autoxidation and ROS↓ Apoptosis↑ Mitochondrial functionMaintain metabolic homeostasis[126]Pueraria Lobata RootsUC + MF125.0 ± 9.7 nm (DLS)−5.0 ± 0.7 mVPuerarin, daidzein, genistein, and miRNAs: hsa-miR-16-5p, hsa-miR-30a-5p, hsa-let-7d-5p, hsa-miR-15b-5pAntioxidantAnti-inflammatoryNeuroprotectiveIn vitro (Neuroblastoma: SH-SY5Y) andIn Vivo (C57BL/6 mice)PD↑ PINK1-Parkin- + SQSTM1/p62-mediated mitophagy↑ MMP↓ Oxidative stress↓ LRRK2 expressionMaintains MRC complexes I and VRestore ATP synthase[122]CarrotsSEC + UF143.9 nm(NTA)−10.2 mV-Antioxidative Anti-apoptoticIn vitro (Neuroblastoma: SH-SY5Y)PD↓ 6-OHDA-induced oxidative stress↓ Apoptosis↓ Caspase-3 cleavage↑ NRF-2↑ HO-1 ↑ NQO1[119]Drynariae Rhizoma RootdUC + 0.22 μm MF71.67 nm (NanoFCM)-NAD(P)H-quinone oxidoreductase, DNA-directed RNA polymerases, ribosomal proteins, ATP synthase, maturase K, phytochromeAntioxidant NeuroprotectiveProteomics + BioinformaticsAD, PD, HDActivate Nrf2/ARE axis crucial for cellular defenseInvolvement of oxidative phosphorylation could help ↓ Oxidative stress↓ Mitochondrial dysfunctionMaintain neuronal health[116]Green OnionPEG8000-based precipitation167.4 nm(DLS) − 16.06 mV-AntioxidantAntiferropticNeuroproctectiveIn vitro (Mouse hippocampal neuronal HT22)Ferroptosis↓ Glutamate-induced ferroptosis↓ Oxidative stress↓ Lipid peroxidation↓ DMT1 and TfR1↑ FPN1↑ GPX4Restore iron homeostasis and neuronal health[125]OnionsUC156 nm(NTA)−24.8 mV-AntioxidantAnti-inflammatory Antiapoptotic NeuroproctectiveIn vitro (Pheochromocytoma PC12, Mouse microglial BV2)In vivo (C57BL/6 mice)SCIActivate KEAP1/NRF2/ARE signaling pathway crucial for cellular defense↓ Oxidative stress↓ Apoptosis↑ NRF-2↑ HO-1 ↑ SOD↓ Caspase-3 cleavage↓ Bax/↑ Bcl-2[110–112]Lycium Barbarum LUC151.45 ± 3.86 nm (DLS) − 5.34 ± 0.472 mVIsoliquiritigeninAntioxidantAnti-inflammatory Antiapoptotic Neuroproctective NeuroregenerationIn vitro (Neural stem NSCs and microglial N9)In vivo (Rat SCI model, 3D-printed scaffold)SCI↓ iNOS↓ TNF-α, ↓ IL-6, ↓ IL-1β ↑ IL-10↓ Oxidative stressActivate pAKT/AKT signaling pathway to promote axonal repairPromote neurogenesis ↑ Tuj1, ↑ MAP2, ↑ GAP43, ↑ NF200, ↑ MBP[110–112]Momordica CharantiaUC131.6 nm (NTA)-miR5266Anti-inflammatory Antiapoptotic NeuroproctectiveIn vitro (Mouse hippocampal neuronal HT22)In vivo (Male Sprague–Dawley rats)Ischemic Brain Injury↓ MMP-9Maintain the integrity of BBBUpregulate tight junction ↑ claudin-5, ↑ ZO-1Activate PI3K/AKT/GSK3β signaling pathway crucial for cell survival↓ Apoptosis↓ Caspase-3 cleavage↓ Bax/↑ Bcl-2[127]Panax NotoginsengUC151.3 nm (DLS)−8 mVLipids ceramide, phosphatidic acid, diglycerides, polyunsaturated fatty acids, Proteins (206 cytosolic and plasma membrane proteins), miRNAsAntioxidantAnti-inflammatory NeuroproctectiveIn vitro (Primary mouse BV2)In vivo (Male Sprague–Dawley rats)Ischemic Reperfusion InjuryAbility to alter the microglia ↓ M1 phenotype (CD86 + CD206 −)↑ M2 phenotype (CD86 − CD206 +)↓ TNF-α, ↓ IL-6, ↑ IL-10↓ Oxidative stress↓ ApoptosisMaintain BBB integrityActivate PI3K/Akt signaling pathway crucial for cell survival[44–46]Momordica charantiaUC40–150 nm (NTA)-miRNAse.g., miR-5266AntioxidantAnti-inflammatory NeuroproctectiveIn vitro (Mouse brain microvescular endothelial bEnd.3)In vivo (Male Sprague–Dawley rats)Ischemic StrokePrevent ONOO⁻/HMGB1/MMP-9 Signaling Pathway↓ oxidative stress↓ Hemorrhagic Transformation↑ Microvascular IntegrityUpregulate tight junction ↑ claudin-5, ↑ ZO-1[110–112]Atractylodes lanceaUC + exoEasy Maxi Kit50–365 nm (TEM)-Methoxycoumarin miRNAs: ath-miR166f, ath-miR162a-5p, ath-miR162b-5pAntioxidantAnti-inflammatory NeuroproctectiveNeuroregenerationIn vitro (Primary mouse BV2)Neuroinflamation↓ iNOS↓ TNF-α, ↓ IL-6, ↓ IL-1β, ↓ CCL2, ↓CXCL10↑ HO-1, ↑ IRF7↓ Oxidative stressActivate IRG1/NRF-2 essential for maintaining cellular homeostasis and brain's resilience[109]Allium TubersumUC + exoEasy Maxi Kit1145 ± 2 nm (TEM)-Dexamethasone miRNAs: ath-miR166g, ath-miR168a-5pAntioxidantAnti-inflammatory NeuroproctectiveIn vitro (Primary mouse BV2)Neuroinflamation↓ iNOS↓ NO↓ TNF-α, ↓ IL-6,↑ HO-1, ↑ cGMPHO-1 produces CO and cGMP to mediate the anti-inflammatory and antioxidant effects[108]OatOptiPrep gradient purificationExosomes were ranged 30–150 nm-β-glucan, digalactosyldiacylglycerol (DGDG), proteins, lipids, and polysaccharidesAntioxidant Anti-inflammatory NeuroproctectiveIn vitro (Primary mouse BV2) + In vivo (C57BL/6 mice)Neuroinflamation↓ Dectin-1-mediated inflammatory pathway↓ TNF-α, ↓ IL-6, ↓ IL-1β↓ Phospho-NF-κB↓ Phospho-SykReduce neuronal apoptosis ↓ TNFR1, ↓ RIPK1↓ Caspases-3, −8 cleavage[128]Momordica CharantiaUC120 nm (NTA)-miR5813Anti-glioma Anti-metastatic NeuroprotectiveIn vitro (Human glioma U251, T98G, U87; SH-SY5Y) + In vivo (BALB/c nude mice)GliomasInhibit the proliferation of U251 glioma cellsInduce U251 glioma cells cycle arrest ↓ PCNA, ↓ Cyclin D1↑ P21Suppress the migration and invasion of U251 glioma cells ↓ Vimentin, ↓ N-cadherin, ↓ MMP9Prevent epithelial-mesenchymal transition (EMT)Inhibit pAKT/AKT signaling pathwayAbility to cross BBBAccumulate in glioma tissues[129]Panax ginsengUC151.6 nm (NTA)−17.9 mVGinsenosides(Rg1, Re, Rg3, and Rb1)miRNAs (98 types)and proteins (86 types)Anti-glioma Anti-cancer Anti-inflammatory NeuroprotectiveIn vitro (Glioma: C6) + In vivo (male Wistar rat; Balb/C mice)GliomasAbility to cross the BBBAbility to target glioma cellsExhibit non-cytotoxic effectsInduce c-MYC gene silencing through ptc-miR396fModulate Tumor Microenvironment ↑ M1 recruitment—↑ CD86, ↑ IL6↓ M2 polarization—↓ CD206, ↓ IL10↓ Tregs, ↓ CD8 + Regulate Cancer-Associated Fibroblasts ↓α-SMA, ↓ IntegrinsReduce tumor progression mechanism ↓ TGF-β, ↓ IL-10↑ BAX, ↓ BCL-2, ↓ Survivin[130]GrapefruitUC135.3 ± 4.62 nm (DLS)−37.7 ± 0.10 mVDoxorubicin-loaded heparin-based nanoparticles (DNs)Anti-glioma Antitumor AntiproliferationIn vitro (Glioma: LN229, U251, U87) + In vivo (BALB/c nude, C57BL/6 mice, Sprague–Dawley rats)GliomasAbility to cross the BBB↑ Invasion into tumor cellsMediate prolong the circulation time of drugs in the bloodstream↑ IL-6, ↑ IFN-α, ↑ TNF-α, ↑ IP-10Keep other levels of IL-1β, IL-2, or IFN-γReduce glioma cells proliferation ↓ Ki67, ↓ CD34[131]Citrus LemonUC100.21 ± 3.35 nm (DLS)-DOX + Decorated with cRGDAnti-inflammatory Neuroproctective AntitumorIn vitro (Mouse Glioblastoma: GL261; Mouse brain microvescular endothelial bEnd.3) + In vivo (C57BL/6 J mice)GliomasELNs were engineered as structural droplets DOX-loaded “ESDDs”Ability to cross BBB and BBTB↑ Invasion into tumor cells↑ Intracellular Uptake↑ Accumulation of DOX↑ Glioblastoma chemotherapy↓ Glioblastoma growth, ↓ Angiogenesis↓ TNF-α, ↓ IL-6, ↓ IL-1β, ↓ IP-10↑ DOX bioavailability[132]GrapefruitUC87.2 ± 11.3 nm (DLS)−13.9 mVmiR17Anti-inflammatory Neuroproctective AntitumorIn vitro (Mouse GL26-Luc) + In vivo (C57BL/6 J mice)Brain TumorDeliver therapeutic miR17 to folate receptor-positive tumor cells↓ MHCI on glioblastomaActivate NK cellsInhibit tumor growthAvoid immune activation ↓ F4/80 + macrophages↓ Iba-1 + microglia[133]GarlicUC30–200 nm-Phosphatidic Acid (PA)Antioxidant Anti-inflammatory Neuroproctective Anti-obesityIn vitro (Primary mouse BV2; Embryonic primary mouse neuronal E17) + In vivo (C57BL/6 mice)Obesity (gut/bain axis modulation)↓ TNF-α, ↓ IL-6, ↓ IL-1β, ↓ IFN-γSupress cGAS/STING inflammatory pathway↓ MMP↓ ROS↓ Apoptosis↓ IDO1Enhance neurogenesis ↑ β-tubulin II, ↑ MAP2, ↑ Nestin, ↑ OCT4Suppress the IDO1/AHR axisReverse high-fat diet (HFD)-induced obesity and insulin resistance[134]UC: ultracentrifuge, dUC: differential ultracentrifuge, DSL: dynamic light scattering, NTA: nanoparticle tracking analysis, MF: membrane filtration, UF: ultrafiltration, TEM: transmission electron microscopy
Engineered plant-derived ELNs represent a versatile and effective platform for delivering therapeutics to manage neurological disorders. Their inherent lipid composition confers biocompatibility, reducing toxicity and immunogenic concerns, thereby enabling repeated therapeutic applications. They can be engineered through two main post-isolation modifications, including surface functionalization and drug loading, as well as source-based modifications, such as genetically engineering transgenic plants to boost therapeutic potential [27]. Furthermore, these nanovesicles can be purposefully functionalized to enhance targeting specificity, improving precision. For instance, conjugation with DSPE-PEG-RVG augments selectivity towards dopaminergic neurons, facilitating receptor-mediated internalization and enabling treatments for conditions like PD [122]. Plant-derived ELNs can undergo advanced surface engineering through post-isolation modifications, including advanced surface functionalization, such as incorporating folic acid into grapefruit-derived ELNs to target folate receptor-positive tumor cells selectively. Hybridizing these ELNs with polyethylenimine (PEI) enhances RNA encapsulation efficiency while reducing the toxicity of free PEI [133]. Furthermore, employing hydrophobic insertion techniques and click chemistry allows for the addition of targeting ligands, like the R11-3 aptamer, to enhance cellular uptake by binding to specific receptors, such as those on BBB endothelial cells [19]. These surface modifications improve targeting specificity, cellular internalization, and therapeutic efficacy, solidifying plant-derived ELNs as a robust platform for precise drug delivery.
These nanovesicles are also engineered to cross the BBB, surmounting a key obstacle in neurological treatments. Their improved stability and extended circulation enhance bioavailability, while modifications bolster their cargo loading capacity [110–112]. The loading strategy for grapefruit-derived extracellular vesicles employs a biomimetic patching approach, integrating doxorubicin (DOX)-loaded heparin-based nanoparticles (DNs) onto the vesicle surface. This innovative method offers a fourfold increase in drug-loading capacity compared to traditional encapsulation techniques. Additionally, these engineered extracellular vesicle-drug nanoparticle hybrids exhibit pH-sensitive properties, enabling controlled release of DOX under acidic conditions, such as those found in tumor microenvironments. This ensures efficient delivery of the chemotherapeutic agent to glioma tissues, maximizing therapeutic efficacy while minimizing off-target effects [131]. Furthermore, these engineered vesicles enable the regulated release of their therapeutic payloads, facilitating sustained therapeutic impact. PDNVs exhibit immunocompatibility and facilitate the targeted delivery of biomolecules to diseased neurons, thereby modulating mitochondrial function and mitigating oxidative stress [108]. Furthermore, these versatile nanocarriers can encapsulate and transport therapeutic RNAs, proteins, and antioxidants, positioning them as a promising platform for revolutionizing the treatment of neurodegenerative disorders. Their safety, scalability, and non-invasive nature offer viable strategies for promoting brain health.
Pueraria lobata-derived ELNs, represent a novel and effective platform for delivering modified biomolecules for the treatment of neurodegenerative disorders [122]. The natural lipid bilayer structure and biocompatibility reduce toxicity and immunogenicity, allowing drugs to be used more efficiently and effectively. These molecules can cross BBB, allowing the delivery of biomacromolecules to diseased cells. Conjugation of Pu-ELNs membrane surface with ligands such as DSPE-PEG-RVG significantly increases their selectivity, especially for dopaminergic neurons, facilitating recognition and uptake by nicotinic acetylcholine receptors (nAChRs) (Fig. 3). Modification increases the transit time, reduces the immune response, and improves bioavailability, allowing for a long-lasting therapeutic effect. In a model of PD, Pu-ELNs have shown remarkable therapeutic potential by restoring mitochondrial function, reducing oxidative stress, and promoting PINK1-Parkin mitophagy, and reducing neurodegeneration [122].Fig. 3Post-isolation and source-based modifications of plant-derived ELNs
Grapefruit-derived ELNs were surface-modified by incorporating a lipophilic folate ligand into their lipid bilayer during preparation [133]. This enabled the active targeting of folate receptor-positive tumor cells (Fig. 3). Additionally, the nano vectors were hybridized with PEI to enhance the efficiency of RNA encapsulation and reduce the toxicity associated with free PEI. These modifications improved the targeting specificity, cellular uptake, and therapeutic efficacy of the nano vectors for the delivery of miR17 to brain tumor cells (Fig. 3). In another study, grapefruit ELNs have undergone surface functionalization using a hydrophobic insertion technique to incorporate specific lipids, such as DSPE-PEG2000-Maleimide [19]. This modification process enables further changes through click chemistry, allowing the attachment of targeting ligands like the R11-3 aptamer (Fig. 3). The aptamer enhances cellular targeting by binding to specific receptors, including those on BBB endothelial cells, thereby improving uptake. The modified surface of grapefruit ELNs also exhibited alterations in zeta potential, confirming successful ligand attachment, while the PEG chains contribute to their biostability by reducing protein adsorption which further enhanced ELNs specificity, targeting, and therapeutic efficacy. In summary, plant-derived ELNs offer a promising nanoplatform for the treatment of neurological disorders. They exhibit high biocompatibility, effective targeting, and also the ability to deliver therapeutic biomacromolecules.
In addition, in the pursuit of improved glioma therapies, a study group have developed grapefruit-derived ELNs as a drug delivery system that integrates DOX-loaded heparin-based nanoparticles (DNs) with cRGD (cyclic Arginine-Glycine-Aspartic acid) for targeted therapy (Fig. 3) [131]. The DNs incorporate heparin to enhance drug loading capacity, achieving a four-fold increase compared to traditional methods. This allows for more efficient delivery of DOX, leading to improved anti-glioma efficacy. The DNs are designed with a pH-sensitive mechanism to facilitate the release of DOX in the acidic environment of tumor tissues, maximizing its therapeutic impact against glioma cells. cRGD is known to bind specifically to the integrin αvβ3 receptor, which is overexpressed in glioma cells. cRGD is conjugated with the carboxyl group of heparin in the DNs to improve targeting capabilities, allowing the ELNs to preferentially accumulate in glioma tissues via receptor-mediated endocytosis. Heparin-infused biomimetic delivery system enhances chemotherapy efficacy in brain tumors by stabilizing, prolonging circulation, and reducing immune response, improving survival rates and reducing pro-proliferative markers.
ELNs derived from grapefruit represent a platform for neurotherapeutic drug development, providing a non-invasive and highly efficient approach to treating neurodegenerative disorders [133]. These toxic and nontoxic transporters can cross the BBB and deliver biomolecules directly to host cells, thereby preventing and alleviating immune suppression. These ELNs can be encapsulated in PEI to increase RNA and DNA encapsulation efficiency while reducing PEI toxicity. Systemic modifications, such as folic acid supplementation, can enhance targeting by activating donor-positive tumor cells. (Fig. 3). This receptor activation promotes the release of therapeutic miRNAs, such as miR17, which can downregulate major histocompatibility complex 1 (MHC1) expression, activate natural killer cells, and inhibit tumor growth. Intranasal administration of FA-coated grapefruit-derived ELNs ensures rapid and efficient delivery, reaching brain regions such as the olfactory bulb and hippocampus within hours, and also exhibits low toxicity and prolonged distribution [133]. The ability of grapefruit-derived ELNs to incorporate and protect biomolecules such as RNA shows its potential in gene therapy, especially for brain tumor treatment.
Grapefruit-derived ELNs can also be utilized for the encapsulation of therapeutic agents through physical techniques like probe sonication. This method enables the loading of drugs, including DOX, into the lipid bilayer structure of the vesicles (Fig. 3). The resulting loaded ELNs of grapefruit exhibit stability and can achieve controlled release of their cargo. Notably, these loaded vesicles demonstrate a pH-responsive release profile, where at physiological pH, approximately 45% of the drug is released within 48 h, while a more sustained release of up to 80% occurs over the same period under acidic conditions [19]. This pH-dependent release behavior ensures the efficient delivery of therapeutic agents to diseased areas, such as tumor microenvironments, while minimizing undesirable off-target effects.
As one of innovative treatment strategies contributing to SCI repair, in a recent study, an anti-inflammatory and neuroprotective flavonoid called isoliquiritigenin (ISL) was loaded into ELNs derived from Lycium barbarum L. (indicated as ISL@PE), then incorporated into a 3D-printed bionic scaffold (Fig. 3) [110–112]. In the in vitro section, ISL contributed to the therapeutic potential of ELNs, where both created a favorable microenvironment for neuronal repair by modulating inflammation, decreasing ROS and pro-inflammatory cytokines IL-1β, IL-6, and TNF-α while upregulating IL-10, promoting the synaptic growth for neurons survival and enhancing the differentiation of neurons in NSCs, as evidenced by increased expression of markers like MAP2 and Tuj1. In addition, ISL@PE supported the phenotype transition M1-to-M2 when delivered to N9 microglial cells. iNOS and oxidative stress were reduced while anti-inflammatory (Arg-1, CD206) was upregulated. ISL@PE-incorporated 3D-printed hydrogels significantly improved motor function recovery, reduced glial scarring, and enhanced neuronal regeneration, with elevated levels of GAP43, NF200, and MBP indicating axonal growth and myelination in the rat model of SCI [110–112].
In the pursuit of innovative treatments for neuroinflammation, a recent study has demonstrated the drug carrying potential of ELNs derived from Allium tuberosum. A-ELNs were utilized as a novel drug delivery system to enhance the anti-inflammatory effects of dexamethasone in BV-2 and MG-6 cells derived from C57/BL6 mice [108]. Dexamethasone (Dex) was encapsulated within A-ELNs, forming Dex-A-ELNs, which demonstrated a significant increase in anti-inflammatory potency compared to either A-ELNs or Dex administered alone (Fig. 3). This synergistic effect was evidenced by a marked reduction in the levels of inflammatory cytokines and nitric oxide induced by LPS stimulation. Specifically, the treatment with Dex-A-ELNs resulted in a more substantial decrease in the expression of pro-inflammatory mediators such as iNOS and TNF-α, and IL-6, compared to the effects observed with A-ELNs or Dex alone. Furthermore, the mRNA expression of HO-1, was significantly elevated following treatment with Dex-A-ELNs, indicating an enhanced cellular response to oxidative stress and inflammation. The encapsulation of Dex within A-ELNs not only improved the translocation of the drug into both BV-2 and MG-6 cells but also altered the particle size, which may have contributed to improved pharmacokinetics and bioavailability.
Studies on transgenic plant-derived ELNs are unfortunately very limited. The only study conducted in this area is that a transgenic tomato was developed by a group to express crocin and picrocrocin genes from saffron; the biosynthetic genes responsible for these antioxidants were extracted from saffron and incorporated into the tomato genome via metabolic engineering (Fig. 3) [27]. This change sought to address the high cost and restricted availability of saffron by providing an efficient, scalable alternative for manufacturing these strong apocarotenoids. Tomafran, the bioengineered tomato, accumulates crocins and picrocrocin in its fruit, both of which have been linked to neuroprotection. Crocin-enriched Tomafran extracts, particularly when naturally encapsulated in purified Tomafran exosomes or loaded into chitosan nanoparticles, greatly increased neuronal survival in SH-SY5Y cells by reducing Okadaic acid-induced oxidative stress and tau hyperphosphorylation. This innovation demonstrates its promise as a low-cost treatment or nutraceutical for slowing AD development and improving brain function.
Although plant-derived ELNs present promising therapeutic potential, several limitations restrict their clinical translation for neurological disorder treatments [18]. A significant hindrance is their restricted ability to cross the blood–brain barrier (BBB) effectively, which is a crucial consideration in neurotherapeutics, particularly when contrasted with exosomes derived from mammalian sources that possess endogenous targeting capabilities [6, 7]. Furthermore, they typically lack inherent targeting specificity, necessitating post-isolation modifications like ligand conjugation or surface engineering to enhance cellular uptake and tissue selectivity [19]. The absence of uniform isolation and characterization methodologies further exacerbates batch-to-batch variations in yield, purity, and bioactive cargo, thereby affecting reproducibility across different research endeavors [47]. While plant-derived ELNs demonstrate enhanced biocompatibility and reduced immunogenicity compared to synthetic drug delivery systems, such as liposomes and polymeric nanoparticles, they generally exhibit diminished stability and less controlled release kinetics [35]. Moreover, their biological activity may differ depending on plant species, growth conditions, and extraction methods. The limited availability of in vivo neuropharmacological data, coupled with inadequate comprehensive safety assessments, raises concerns regarding their scalability and clinical applicability, underscoring the necessity for more thorough investigations in subsequent studies.
Plant-derived ELNs represent a novel and promising drug delivery system for the treatment of neurological disorders, combining biotechnology and pharmaceutical approaches. Due to their biocompatibility, minimal toxicity, and ability to traverse the BBB, ELNs provide a targeted, non-invasive approach for addressing neurological disorders. Specifically, surface modifications and loading with therapeutic molecules that improve therapeutic efficacy present prospects for the development of novel treatment techniques for neurodegenerative diseases and other central nervous system disorders.
Nonetheless, it is crucial to acknowledge that the incorporation of ELNs into clinical applications remains at its very early stages. Further experimental and clinical investigations are required to assess their efficacy, safety, dosage optimization, and long-term biological impacts. Future investigations will further the understanding of the pharmacokinetic features of plant-derived exosomes, their impact on immunological responses, and their therapeutic efficacy in diverse neurological disorders. At present, the majority of research on plant-derived ELNs is in experimental and preclinical stages, with clinical trials yet to begin.
In summary, plant-derived ELNs possess revolutionary potential in the management of neurological disorders and may provide effective therapeutic alternatives in the future. Nonetheless, for this technology to go to clinical use, extensive human research and scientific proof are necessary.