Functionalised Carbon Nanotubes: Promising Drug Delivery Vehicles for Neurovascular Disorder Intervention

Neurovascular diseases are linked to the brain’s blood vessels. These disorders are complicated to treat due to the strict selective characteristics of the blood–brain barrier. Consequently, the potency of the pharmacological treatments for these conditions is immensely diminished, leading to a rise in neurovascular-associated morbidity and mortality. Carbon nanotubes are regarded as essential nanoparticles with a promise of treating neurovascular disorders. Current findings have demonstrated the effectiveness of carbon nanotubes as vehicles for ferrying drugs to the site of interest. This review accentuates the theoretical utilisation of carbon nanotubes as drug nanocarriers equipped with the penetrating capability to the blood–brain barrier for treating neurovascular disorders such as ischemic stroke. The success of the carbon nanotube system may result in the development of a new and highly relevant drug delivery procedure. This review will also cover carbon nanotube functionalisation for applications in the biomedical fields, toxicity, in vitro and in vivo drugs and biomolecule delivery, and the future outlook of carbon nanotubes.


Introduction
The brain is the body's control tower and requires efficient and adequate protection from invading microbial pathogens, xenobiotics, and neurotoxic agents.The blood-brain barrier (BBB) plays a crucial role in the safety of the brain from extrinsic factors.The BBB constitutes endothelial cells that line the cerebral vasculature to separate the brain extracellular fluid from the blood [1].This barrier is highly sensitive and selective, and these properties hinder the delivery of neurotherapeutics.Based on this, the clinical failure of a plethora of potentially effective neurotherapeutics is linked to the shortcoming in their delivery mechanism and not as a result of a deficiency of the efficacy of the drugs [2].Current therapeutics in the market are ineffective in treating neurovascular disorders since they cannot be transported and sustained successfully in the brain.This challenge resulted in invasive approaches for treating brain disorders, as shown in Fig. 1.
There were about 12.2 × 10 6 stroke cases, 143 × 10 6 disability, and 6.6 × 10 6 deaths worldwide in 2019 (Global Burden of Disease).Stroke is the second leading cause of death and the third leading cause of disability worldwide based on these statistical data.The total global costs of stroke due to the treatment, rehabilitation, social care, and income losses in 2017 were estimated to be US$891 billion.The continuous escalation of the global burden of stroke leads to innumerable challenges to governments and healthcare systems globally.There is a dire need to address this to curb the burden of stroke [3].Vascular disease is a medical condition that adversely affects the blood vessels, leading to various diseases [4].Stroke is a vascular condition in which brain cells die within a few minutes due to a lack of glucose, nutrients, and oxygen supply to the brain [5].Atherosclerosis may trigger stroke as blood flow to the brain is interrupted by the build-up of cholesterol, fats, and other substances inside and around the arterial wall.A brief interruption of blood circulation to the brain may trigger transient ischemic stroke, leading to more severe strokes [6].The current treatment of ischemic strokes includes oral aspirin and an i.v administration of tissue plasminogen activator (tPA).These therapeutics have been demonstrated to be effective if they are administered within 3-4.5 h of the blood clot detection [7,8].
Nonetheless, these treatments have a plethora of side effects following their entrance into the systemic circulation.Oral medication has a shortfall due to first pass hepatic metabolism, which slows down the onset of action of the drugs leading to the decrease of drug doses to the diseased site [9].Based on these challenges, there is a dire need for the effective and efficient transport of neurotherapeutics to treat neurological diseases successfully.Nanotechnology is a promising strategy for solving the current drug delivery challenges.Nanomaterials are materials with one side with a nanoscale size of 1 to 100 nm, which could influence the frontiers of nanomedicine.The nanostructures can penetrate the tissues to facilitate cellular drug uptake and enable efficient targeted drug delivery.The development of nanomaterials plays a crucial role in circumventing the shortcomings of free drugs.
Furthermore, nanomaterials can navigate biological obstacles such as BBB and CSFB [10].The nanomaterials that can penetrate BBB will improve drug delivery against brain diseases.Carbon nanotubes (CNTs) can be employed as drug nanocarriers due to their cell-penetrating ability and high aspect ratio [11].CNTs are emerging and promising nanocarrier systems of therapeutic agents in various neurological diseases.The chemical functionalisation of CNTs enables easy access to the brain through the BBB.By so doing, the formation of the interface with neurons and the uptake of the CNTs is enhanced at the diseased site [12].Despite all the beneficial effects of the CNTs, they have some limitations, such as protein corona formation around them and cytotoxic effects.CNTs and other nanoparticles may interact with the proteins and lipids in a biological aqueous milieu, which could lead to the development of protein corona [13].The hydrophobic character of the nanoparticle will adsorb the hydrophobic site of the proteins, and the cationic surface of the nanoparticle will attract the protein anionic site.This process forms the hard corona layer, the innermost corona, as depicted in Fig. 2 (1).
Protein corona patterns depend on nanoparticle physicochemical properties, such as shape, charge, nanomaterial, size, and surface functional groups.The corona patterns also depend on environmental conditions, including temperature, dynamic shear stress, media components, pH, and exposure period [14].The soft coronas are formed by the protein-protein interactions on the surface of the hard corona, as depicted in Fig. 2 (2-4).The outermost layer formed on the soft corona's surface by loose proteins is called a loose corona, as illustrated in Fig. 2 (5).Protein corona may alter the physicochemical properties of nanoparticle (NP), which may impact NP distribution, cellular uptake, and immunological responses and toxicity.Protein corona may contain endogenous ligands that coat reactive NP surfaces to block cell membrane receptors.This mechanism may block NP intake and protect NPs from opsonisation by phagocytic cells [15].Garcia-Hevia and colleagues studied carbon nanotube corona and functionalisation to prevent biofouling.Following protein functionalisation, they demonstrated the increase in carbon nanotube diameter using SEM and AFM.The study revealed a significant variability of proteins in the corona as a function of the type of nanotube, functionalisation temperature, and exposure time to serum [16].CNT exposure to the general population has increased significantly as the demand is enormous, and they are being produced at an industrial scale [17].This skyrocketing production of CNTs has triggered safety anxiety linked to the toxicity of CNTs on humans.Despite the extensive work on the cytotoxicity of the CNTs, the findings are conflicting, and the data must be aligned [18].The studies confirm the cytotoxicity of CNTs, whereas other studies have demonstrated their biocompatibility.The cytotoxicity of CNTs could play a crucial role in cancer treatment in disguise [19][20][21].In vitro studies have demonstrated that CNTs elicit cellular responses by triggering molecular signalling linked to stress due to oxidation [22].In another study, it was found that functionalised CNTs were less cytotoxic as opposed to pristine CNTs [17,23].Di Giorgio and colleagues have illustrated that variations in the size, structure, and chemical surface of the CNTs had a more significant influence on the toxicity of the cells [13,24].Fujita and colleagues demonstrated the toxicity of MWCNTs with different physicochemical properties in rat alveolar macrophages.
NR8383 rat alveolar macrophages were exposed to MWC-NTs differing in diameter and length for 24 h, and in vitro, cell-based assays were performed to determine the cytotoxicity.It was found that rigid and needle-shaped MWCNTs with a diameter greater than 50 µm accessed the cytoplasm and triggered cell death without any sign of intracellular reactive oxygen species (ROS) that were generated.Bent MWCNTs with a diameter of less than 20 µm were phagocytosed, reduced cell viability, and generated ROS.Straight and thin MWCNTs with a diameter of less than 20 µm caused an insignificant ROS generation, but cell viability was intact.The in vitro cell-based assays demonstrated high toxicity of MWCNTs with a diameter greater than 50 µm and moderate and low cytotoxicity with a diameter less than 20 µm.These findings confirm the significant contribution of the CNT diameter to the cytotoxicity [25].CNT biocompatibility can be enhanced and toxicity reduced by the functionalisation of the CNTs [26,27].
Sang and colleagues did biocompatibility studies on carbon nanotubes in rat pheochromocytoma (PC-12) cells.To enhance the performance of MWCNTs, they were incorporated in chitosan (CS)/polyethene glycol (PEG) composite scaffolds.PC12 cells were exposed to CS/PEG/CNT composite scaffolds containing 1%, 3%, and 5% MWCNTs.Immunofluorescence analysis confirmed the resemblance of rat pheochromocytoma (PC12) with nerve cells.PC12 cells expressed growth-associated protein 43 (GAP43), nerve growth factor receptor (NGFR), and class III β-tubulin (TUBB3) proteins as confirmed by quantitative real-time polymerase chain reaction (qRT-PCR).The findings have illustrated the exceptional biocompatibility of CS/PEG/CNT scaffold with the potential of being employed in neural tissue engineering [28].Despite their shortcomings, carbon nanotubes have the potential to solve current neurological diseases to improve the mental health of humans.The employment of carbon nanotubes may improve the global economy as the global burden of stroke, such as treatment, rehabilitation, social care, and income loss, will be significantly reduced [3].This review will cover the historical background, synthesis, purification, and characterisation of carbon nanotubes.This review will also cover the functionalisation, cell toxicity, drug delivery, and future outlook of carbon nanotubes.

Synthesis of Carbon Nanotubes
Carbon nanotubes were first reported by Iijima in 1991 [29].CNTs are rolled-up graphene sheets with internal diameters of 0.2-2 nm for single-walled nanotubes and 2-100 nm for multiwalled nanotubes (Fig. 3).They have a high aspect ratio and hollow cavities terminated by two end caps [30].
The main methods employed in CNTs synthesis are laser ablation, arc discharge, and chemical vapour deposition (CVD), as depicted in Table I [32].
Compared with other methods, chemical vapour deposition (CVD) in Fig. 4 is the most effective method, with broad prospects for large-scale CNT control of CNTs at low ambient temperature and pressure due to its simple equipment, simple operation, high purity production, and lower cost [34].

Purification of Carbon Nanotubes
Carbon nanotubes are often contaminated by metals, amorphous carbon, and multishell during synthesis [21,35].Based on the number of impurities introduced into CNTs during the synthesis stage, CNTs must be purified before use.The purification strategies that could be used to produce pure CNTs include the following techniques.

Air Oxidation
The average purity of carbon nanotubes is about 5-10%.CNTs require purification before the attachment of drugs on their surfaces.Air oxidation at 400°C for 40 min reduces the impurities, including catalyst particles and amorphous carbon produced during the CNT synthesis.The main shortcoming of this method is the oxidation of the CNTs and the contaminants being oxidised [36,37].

Acid Refluxing
If the as-synthesised CNTs are refluxed in strong acids, the amorphous carbon, metal particles, and other impurities are effectively eradicated from the CNTs.The most commonly used acids in the purification of the CNTs are HCl, HNO 3 , and H 2 SO 4 .HCl is the most suitable refluxing acid used in the purification process of CNTs [21,35].

Surfactant-Aided Sonication, Filtration, and Annealing
Refluxing introduces the CNTs' entangling, leading to impurities entrapment between the nanotubes.Surfactant-aided sonication is performed to untangle the CNTs to set the impurities free from the CNTs.Sonication of CNTs in sodium dodecyl benzene sulphonate (SDBS) in ethanol increases the dispersion of the CNTs.Following sonication, the mixture is subjected to an ultrafiltration system and annealed at 1000°C in N 2 for 4 h [35].

Functionalisation of Carbon Nanotubes
CNTs have chemically inert outer layers which could be tagged with functional groups to change their properties for application in various fields such as energy, pharmaceutical, and biomedical industries [13].Functionalisation procedures can do tagging of functional groups.CNTs tend to aggregate, resulting in difficulties in the dispersion of various  solvents.As a result, CNTs are functionalised to avoid conglomeration and bundling to enhance their dispersion in polymeric solvents.The solubility of CNTs in an aqueous milieu is the primary limiting factor of applying CNTs in biomedical fields [38].Functionalisation has been demonstrated to defeat this challenge as the functional groups are introduced on the side chains and caps of the CNTs to enhance the hydrophilicity.Functionalisation reduces CNTs toxicity and enhances biocompatibility, enabling the drug loading, proteins, and nucleic acids for effective and efficient delivery.Functional groups can be coupled to the CNTs by covalent bonding, non-covalent bonding, and electrostatic forces to improve their aqueous solubility, as depicted in Fig. 5 [13].

Covalent Functionalisation
Covalent functionalisation offers a more secure conjunction of functional molecules.Chemical groups such as fluorine carboxylic groups can be coupled to the walls of the CNTs.The shortcoming of this type of functionalisation is the formation of flaws on the CNTs' surfaces [40,41].The main disadvantage of covalent functionalisation is the disruption of the surface conjugated π network [42].There are two types of covalent functionalisation: direct and indirect.Direct covalent functionalisation occurs on the side walls of the CNTs.Indirect covalent functionalisation occurs on the surface of the CNTs with the carboxylic acid group [42].SWCNT functionalisation is much easier as it has a smaller external diameter.MWCNT functionalisation is not easy since they have a considerable outer diameter.

Oxidation of CNTs
Oxidation is the most likely used method for the covalent functionalisation of CNTs.Oxidation is a primary method for covalent functionalisation using strong acids.CNT lengths are reduced, but carboxylic groups are generated, enhancing dispersibility in an aqueous environment [21].The covalent bond between the unsaturated carbon π-bond and other functional groups is formed in covalent functionalisation.Oxidising agents such as nitric acid generate carboxylic or hydrophilic groups at the walls, caps, and sides of the CNTs [43].Oxidation enhances CNT solubility, but an aggregation of the CNTs is activated too.The CNTs are unstable in vivo due to salts in the biological fluid.A hydrophilic polymer such as PEG is attached to the surface of the oxidised CNTs.This attachment makes the CNTs soluble and stable in in vitro and in vivo environments.Oxidation of CNTs is a rosy functionalisation technique for both small-scale and industrial-scale production.Oxidation under an acidic milieu could lead to a reduction of CNTs' length.This effect could benefit the medical field of CNTs in drug delivery [44].

Cyclo-addition Reaction
The reaction occurs on the sides of the wall instead of next to the caps in cyclo-addition.The reaction can be classified as (1) photo-induced cyclo-addition (this reaction involves a photo-chemical reaction with azides); (2) bingal reaction (this reaction occurs in the presence of a strong base); and (3) 1,3-dipolar cyclo-addition reaction (this reaction is commonly used for CNTs functionalisation [45]).

Reaction with Acyl Peroxides or Sulfoxides
This type of reaction functionalises the CNTs' surface and can conjugate functional groups via covalent bonding to the surface of the CNTs.Acyl peroxide has terminal groups for additional functionalisation [46].The reaction between acyl chloride and carboxylic acid forms an amide group.The alkyl or aryl peroxides have been decomposed by thermal methods yielding free radicals, and the reaction of acyl peroxides with CNTs generated carboxyalkyl radicals on the walls of CNTs [41].

Polymer Functionalised CNTs
Polymers play a vital role in facilitating the CNT dispersion and production of CNT-based complexes.The physical and chemical attachments are the principal strategies for CNT modification using polymers [42].Polymer composites are produced from the polymerisation of functionalised carbon nanotubes.Polymerisation augments the chemical, physical, and electrical properties of CNTs [47].

Non-covalent Functionalisation
In non-covalent functionalisation techniques, the chemical properties of CNTs are not adversely affected.Non-covalent functionalisation does not depend on hydrogen bonding, van der Waals forces, or π-π interactions.The introduction of non-covalent surface modifications with natural and synthetic polymers solves the solubility issue of CNTs [24].As a result, the aromatic structure and electronic structures of the CNTs are preserved.In the non-covalent method, long-chain polymers are wrapped up around the CNTs [24].The dispersion processes usually used in dispersing CNTs involve ultrasonic, centrifugation, and filtration techniques.The non-covalent functionalisation involves using surfactants and π-π stacking interactions for the molecules to attach to CNTs [24].Some non-covalent functionalisations include the fluorescence-attached PEG, protein functionalisation, and single-stranded DNA, as depicted in Fig. 5b [24].Polyacrylic acid, pyrene, polyaniline, nucleic acids, and proteins have been successfully coupled to CNTs [36].Surfactants with non-ionic, anionic, and cationic moieties are likely used for the CNT dispersion.Polymers are used as an alternative to surfactants.They are very effective in solubilising CNTs, although they lack dispersion efficiency.Polymers are used as nanocarriers for drug delivery.Moon and colleagues demonstrated serum nucleases sabotaging DNA molecules after their non-covalent functionalisation to the CNTs [36].Surfactant polymers exploit the π-π interactions during the process of CNTs functionalisation.Certain surfactants may yield stable dispersion of CNTs to evaluate spectroscopic properties and improve composite compatibility [36].
Surfactants are very effective in the solubilisation of CNTs.They are also toxic to the biological milieu as they  [39] with permission from Copyright © 2018 Elsevier Inc can penetrate the plasma membranes.The disadvantages of using surfactants as solubilisation agents include cellular protein interactions, a rise in critical micellar concentrations (CMC), and lower stability.Based on these disadvantages, there is a need for the surfactant for the production of stable complexes with the CNTs, non-interaction with the in vivo and in vitro molecules, and non-toxic biocompatible with the biological environment [48].
Phospholipids form part of the plasma membrane, and it could be wise to include them in biomedical applications as they are biologically compatible.Phospholipids are amphiphilic, and they can be used as a functionalising group because a hydrophobic part attaches to the CNTs, and the hydrophilic part attaches to the polymer, such as PEG.The highly stable and biocompatible complexes are generated in this manner.The products can be used in many biological systems, such as biosensing tools, imaging systems, and drug delivery strategies [49].

Characterisation of Carbon Nanotubes
Much work on carbon nanomaterials is focused on preparation, characterisation, functionalisation, and application.Characterisation plays a vital role in classifying nanomaterials' dimensions, morphology, and composition [50].The physicochemical characterisation of the CNTs is performed to attest to the product quality and to elucidate their physicochemical structure.There are two characterisation classes, namely, physical and biological characterisation.

Physical Characterisation
Physical characterisation could also be further divided into qualitative and quantitative characterisations.Qualitative methods are employed if the compounds of interest are present in the sample, and quantitative methods are then used to determine the amount of the compound in the sample.The methods in Table II are both qualitative and quantitative [51].

Safety of Carbon Nanotubes
The skyrocketing usage of CNTs in many fields, such as pharmaceutical, biomedical, biological, electrical, and engineering, has triggered the mass production of these nanomaterials.CNTs are now offered in commercial-scale quantities.The global commercial market of the CNTs was around 2000 tons and was expected to skyrocket over 10 × fold (i.e.> 20 000 tons) with a total value of over US $ 3.42 billion this year [52].China, Japan, India, and the USA have increased the research funding on CNTs.The soaring production rate of CNTs will lead to environmental and human exposure.The inhalation of the nanomaterials through the respiratory tract is the most plausible route of exposure during the production and application of the CNTs [38].Nanosized particles have a sky-scraping deposition efficiency rate in the deep lung areas as opposed to micro-and macro-particles at the same concentration [52], as depicted in Fig. 6.
The exceptionally high aspect ratio and low solubility of the CNTs, with their additional physicochemical properties, especially the fibre type, raise a concern that CNTs may trigger various diseases [54].The health and safety issues of different kinds of carbon nanotubes have been discussed for the last two decades without an agreement being concluded.Besides the respiratory route, humans will likely be exposed to carbon nanotubes through dermal and ingestion routes [55].When carbon nanotubes are synthesised, handled, and used, preventive safety measures must be employed.Airborne levels of carbon nanotubes at the manufacturing facility and personal breathing zone (PBZ) should be monitored and brought to a minimal level or eliminated if possible by employing engineering controls such as robust ventilation systems and wearing effective and efficient PPE [56].
Aerosolised MWCNTs could be collected by cascade impactor to measure aerosol particle size, concentration, and chemical identification [56].A cascade impactor is a multi-jet, multi-stage device operating at a constant flow rate to allow aerosol characterisation in particle size distribution [57].As demonstrated in Fig. 7, the first stage contains a plate filled with a matrix (brown).The second stage has smaller nozzles and drops to the smallest until the last stage.The reduction in nozzle size creates higher particle impaction velocities onto the matrix.The aerosolised particles (red) flow from the top into the first stage.The particles with aerodynamic diameters greater than 7 µm impact the matrix.The particles smaller than 7 µm flow to the second stage and are collected on the matrix until the last stage [57].Some cascade impactors are equipped with eight stages.This system can also be used for MWCNT particle size distribution measurement to monitor the exposure levels of the CNTs in production facilities.

Drug Loading and Release from Carbon Nanotubes
Functionalised CNTs form part of the targeted drug delivery system to ferry the drug to the diseased site.CNTs have a high aspect ratio to cater for many therapeutics.Multifunctionalisation can enhance the hydrophilicity and biocompatibility of the CNTs [21].The ultrasonication technique performs drug loading by dispersing the functionalised CNTs in deionised water.The drug is also dissolved or dispersed in a solvent, added dropwise to the ultrasonicated functionalised CNTs, and incubated for 24 h under room temperature.Following incubation, the mixture is centrifuged at 1 × 10 4 rpm for 15 min to separate the standalone drug from the conjugate.The concentration of the drug is determined, and the drug loading efficiency is calculated as follows: DLE is the drug loading efficiency, WAD is the weight of the added drug, and WFD is the free drug in the supernatant.
It is imperative to have an understanding of the mechanism of drug release following loading and administration.Stimulants such as pH changes, drug solubility, drug desorption, drug diffusion, magnetic field, electric field, and temperature changes trigger the drug release from the CNTs [21].Ji and colleagues functionalised MWCNTs with polyethene oxide (PEO) and pentaerythritol triacrylate (PETA) polymers.Ketoprofen was loaded on the functionalised MWCNT.Ketoprofen was released from the CNTs by high electric voltage (HEV) exposure.The PEO was dissolved, and the drug was released from the CNTs [58].Prajapati and colleagues loaded gemcitabine on hyaluronic acid PEGylated CNTs.Various pHs were used as stimulants to release drugs from CNTs [59].Drug release also depends on the nanoparticle type [59,60].
A 93% entrapment efficiency of gemcitabine (GEM) was observed when MWCNTs were used without functionalisation with hyaluronic acid (HA) and PEG.A slight drop to 90.9% and 89.9% in the entrapment efficiency was observed when HA and PEG were introduced, respectively.The efficiency increased when both HA and PEG were used (Fig. 8a).This confirms that functionalisation plays a crucial role in the loading of drugs to MWCNTs.The release of gemcitabine from HA-MWCNTs was most effective at pH 5.3 and least effective in PEG-MWCNTs at pH 7.4, as demonstrated in Fig. 8b.
Khoshoei and colleagues conducted a molecular dynamics (MD) study to develop an innovative and responsive drug delivery system.To enhance DOX and PAX targeted delivery by SWCNTs and graphene to cancer cells, carboxyl groups and trimethyl chitosan (TMC) were employed as functionalising and grafting agents.It was found that PAX with functionalised graphene-TMC displayed a more firm carrier than DOX with functionalised SWCNTs-TMC.PAX graphene had the most proper drug adsorption and release behaviour.PAX was adsorbed much faster and released at a prolonged rate, increasing the drug's half-life [61].DOX and PAX are some of the anticancer drugs and have side effects.The side effects can be decreased by improving the solubility and biocompatibility of the drug by adding TMC to DOX and PAX to SWCNTs and graphene and functionalising with the carboxyl group.The pH is essential in drug loading and release from the carrier.The drug is adsorbed strongly to the carrier at pH 7 and released at pH less than 7 [61].Harlepp and colleagues designed a novel NIR lightresponsive nanocomposite system, as demonstrated in Fig. 9.This system, CNTS@MS@IBAM-DOX@HSA, is composed of mesoporous silica shell (MS)-coated CNTs loaded with an anticancer drug, doxorubicin (DOX), and capped with human serum albumin (HSA) and isobutyrate (IBAM) as a binder of HSA to the CNTs@MS.This nanocomposite was incorporated into the hydrogel (Matrigel), and the cancer cell line was cultured on this scaffold.The culture was exposed to NIR, and DOX was released, killing the cancer cells.This mechanism is synergistic, as the cells were killed by both the NIR light and DOX [62].

Biomedical Applications of Carbon Nanotubes
One of the shortcomings of the CNTs is poor water solubility due to their hydrophobic nature in their pristine form.This limitation can be dealt with by modification of the CNT  [59] under a Creative Commons Attribution License surface using a functionalisation strategy.The solubility of carbon nanotubes in an aqueous milieu is an essential requirement for the pharmacokinetics and pharmacodynamics of the therapeutic in the mammalian body [13].

CNT Properties to Enable Their Cellular Uptake
Many studies have authenticated the cellular uptake of CNTs, although the mode of action of CNTs' access into the cells is still unclear [17].The CNTs must be modified first to enable further functionalisation with the intended therapeutic.CNTs are purified and functionalised by acids such as nitric and hydrochloric acids and polymers such as PEG to reduce their cytotoxic effects before loading drugs on them.The loaded medicines can then be off-loaded from the CNTs once they have reached their destination.
In addition, recognition units or targeting ligands are coupled to the CNTs to facilitate the uptake of carbon nanotubes by the target cells.Furthermore, a tracking agent, such as a fluorescently labelled ligand, is attached to the CNTs to trace the carbon nanotubes within the body [63].The CNTs' cellular uptake can also be traced by employing state-of-the-art microscopic techniques such as AFM and TEM [17].Functionalised CNTs are effortlessly taken up by cells through passive and endocytosis-independent mechanisms, as depicted in Fig. 10.
Needle-shaped nature of CNTs enables them to penetrate the cells harmlessly.Orientation of the CNTs plays a crucial role in cellular uptake.The CNTs align perpendicularly to the cell membrane to enhance the perforation and diffusion into the membrane to gain access to the cytoplasm [17].Since CNTs have magnetic properties, they can kill a targeted cell by rotational movement from the external magnetic field.
The size and surface charge of the functional groups on the CNT surface enhance the penetration of the CNTs into cell membranes.Carbon nanotubes can be delivered to the body through different administration routes, such as oral, nasal, subcutaneous, abdominal, intratracheal, intramuscular, intracerebral, and intravenous routes.The uptake mechanism is also dependent on the CNTs' length.The carbon nanotubes shorter than 1-µm diameter are easily internalised into the cells [64].Shorter CNTs are easily absorbed into the body through columnar cells of the intestines when administered orally.CNTs administered subcutaneously dawdle in the injection site before diffusion from the point of application to the lymphatic system.

The BBB Structure and CNTs
The idiosyncratic physicochemical attributes of the CNTs proffer them promising targets for neurological diseases.CNTs are cylindrical structures with high specific area and cell membrane penetration power.The additional benefit of the CNTs is their ability to allow multiple conjugations of biomolecules on their walls, caps, and central cavities.These properties enhance the delivery of drugs of interest into the brain [65].The strategies of targeting the drugs in the brain for treating neurological diseases using carbon nanotubes have triggered more interest in recent years [38].
The nanoscale size of carbon nanotubes (≤ 200 nm) allows surface functionalisation and the straightforward penetration of BBB.Furthermore, they easily permit the conjugation of molecules, hence the delivery of chemicals and drugs into the brain.These properties are beneficial in treating brain cancer and neurodegenerative disorders [66].The blood-brain barrier is a selectively permeable layer around the brain to protect it from external forces.The BBB avoids the access of foreign matter, such as giant molecules and toxic substances, into the brain.
This barrier permits the transport of glucose, oxygen, and other beneficial molecules to the brain tissues.The BBB comprises a basement membrane, microvascular endothelium, pericytes, astrocytes, and a neurovascular unit [67,68].CNTs can access the BBB via different pathways, and each route has been designed for a specific role, as depicted in Fig. 10.
CNTs can bypass the BBB by traversing the nasal cavity and nerve endings or diffusing through the glial cytoplasmic membrane [18].CNTs, including other nanomaterials functionalised with ligands like transferrin, can bind to the BBB receptors and traverse through the BBB without causing any damage on the tight junction, as demonstrated in Fig. 10.The BBB is well reinforced with P-glycoproteins (P-gp) on the luminal side, and this efflux transporter removes a broad spectrum of drugs before they reach the parenchyma.The P-gp suppression should be activated by introducing RNAi to suppress its expression, and the MWCNTs will then gain access to the parenchyma [12].
Since native CNTs are highly hydrophobic, they tend to form aggregates with different sizes making them impossible to cross BBB [18].Functionalised CNTs remain in suspension and can cross BBB via energy-dependent transcytosis.The proposed route of targeting BBB is through receptor-mediated transcytosis (RMT).The RMT is a primary neurotherapeutic transport system independent of drug size and lipophilicity properties [69].A carbon nanotube is conjugated to the transferrin, binding directly to the transferrin receptor on the BBB.CNTs may induce oxidative stress disrupting BBB and gaining access into the brain.Short carbon nanotubes, including other nanostructures deposited into the pulmonary tissues, may circulate into the blood circulatory system and gain access to the brain tissue.Intranasal nanomaterials can traverse directly to the brain via the olfactory neurons [18].

CNTs in Central Nervous System
Neurodegeneration is a neurological condition caused by a cumulative dysfunctional nervous system, which could result in the degeneration of specific neurons in the central nervous system.The repair treats some neurological disorders of neurons.The most common neurodegenerative disorders include amyotrophic lateral sclerosis, Huntington's disease, dementia, Alzheimer's disease, and Parkinson's disease [70].CNTs have been investigated for their ability to act as good electrical conductors and scaffolds.This property may trigger neuronal growth and differentiation, as depicted in Fig. 11.
Malarkey and colleagues demonstrated that the specific conductivity level of CNTs plays a crucial role in neuronal growth and differentiation.Cellular adherence, migration, and protein expression were enhanced in cells seeded on the substrates with 0.43-0.9S/cm electrical conductivity [72].Neural stem cells (NSCs) are considered a long-lasting source of neural cells, capable of auto-renewal and differentiation.Kam and colleagues conjugated laminin to SWCNTs to form layer-by-layer thin films.The formulation was conducive to NSC differentiation and appropriate for their successful excitation [73].CNTs can be applied in neuro-electrical interfacing in vivo as they integrate with neural cells to form artificial synapses [74,75].
Moreover, CNTs can be used in tissue engineering as they facilitate the proliferation and differentiation of stem cells [18].These applications may lead to the success of treating currently untreatable diseases.Functionalised CNTs may enhance biocompatibility than native CNTs in vivo environments.However, functionalised CNTs in the brain may be defunctionalised, and this may cause an accumulation of free functional groups and pristine CNTs, which in turn may compromise the safety of CNTs in the brain.Consequently, a clear understanding of the neurotoxicity of CNTs must be sorted out before application of the CNTs in in vivo situations.

Intranasally Delivered CNTs, Distribution in the Brain, and Their Neuroprotective Effects
CNTs should be delivered to the central nervous system (CNS) safely and non-invasively to keep this delicate organ, the brain, intact.Soligo and colleagues investigated the brain distribution and neuroprotection of the intranasally delivered CNTs in their study.MWCNTs delivered intranasally could reach different brain areas, such as the hippocampus, amygdala, thalamus, and hypothalamus.These limbic areas are crucial in the progressive development of central neurodegenerative diseases.Moreover, CNTs were demonstrated to have a neuroprotective effect through neurotrophic factor modulation [76].

CNTs for the Treatment of Ischemic Stroke and Alzheimer's Disease
Ischemic stroke is a swift persistent neurological defect caused by an occlusion or burst of the blood vessels that supply blood to the brain.This defect may lead to neuronal damage inside the brain.Currently, ischemic stroke therapeutic modalities such as radiotherapy and chemotherapy have various shortfalls, including side effects and invasiveness.Lee and colleagues investigated the effect of CNTs on the brain.Amine-functionalised SWCNTs were administered into the right lateral ventricle of the rat stroke model, and this led to the protection of the Fig. 11 CNTs in neurodegenerative disorders.Adapted from reference [71] with permission from Copyright © 2020 Elsevier B.V neurons as attested by a drop in plasma inflammatory markers [77].In a different study, CNTs were infused into subventricular zone neural progenitor cells, followed by intracranial administration into a cerebral ischemic rat model, and subsequently, the affected neurons were repaired [78].
Costa and colleagues also illustrated the healing capabilities of the functionalised CNTs in the Alzheimer's mice model.The functionalised MWCNTs were conjugated with gadolinium L2 and intravenously administered into the Alzheimer's mice model.The formulation traversed the BBB via receptor-mediated transcytosis, and a therapeutic effect was successful [79].Shrestha and colleagues functionalised the MWCNTs with polyurethane and silk fibroin using an electrospinning technique, and an electrospun scaffold was produced for neuronal growth and differentiation.The in vitro studies were performed in Schwann cells, and the development and proliferation were observed in these cells when incubated with the scaffold.Furthermore, the platform enhanced neural cell sensitivity, expression, and axonal growth [80].Based on these findings, CNTs are pivotal in treating neurodegenerative disorders and other neurological diseases.

CNTs in Targeted Drug Delivery System
The main aim of the studies on nanomaterials is to find a solution to the challenges of drug delivery.The challenges include poor drug solubility, cytotoxicity, cellular drug distribution, drug inaccessibility into cell membranes, damage to healthy tissues, and nonspecific cellular selection for the treatment of disease [81][82][83].
A plethora of therapeutic molecules can be incorporated into the functionalised CNTs for delivery to the site of interest, as illustrated in Table III [60,84].

Drug Delivery System
Targeted drug delivery has many benefits as small drug dosage is administered at the site of interest.As a result, fewer or no side effects and good patient compliance (in terms of time, efficacy, and cost) are achieved.The nanoparticle-drug complex delivery system should reach the site of interest, recognise the disease, bind to the diseased area, deliver its drug amount to specific tissues, and reduce drug toxicity to healthy tissues under ideal conditions [60].Conjugating targeting ligands, including small molecules, peptides, antibodies, designed proteins, and nucleic acids on the surface of nanostructures, is the most likely strategy that improves targeted delivery.Small organic molecules are the most likely used targeting ligands due to their easy preparation, stability, and manipulation of conjugation [88].

Potential Therapeutic Applications
Guo and colleagues investigated the promotion of apoptosis of nasopharyngeal carcinoma cells by multiwalled carbon nanotubes as an adjuvant nanomaterial for photothermal therapy of cancer.It was found in their study that MWC-NTs and MWCNT-hyaluronic acid composites effectively inhibited the proliferation rate of nasopharyngeal carcinoma cells, CNE-1, by photothermal therapy and enhanced the apoptosis of CNE-1.This study has demonstrated the therapeutic potential of MWCNTs and MWCNT-hyaluronic acid in cancer treatment [89].
Begum investigated the co-loading of doxorubicin (DOX) and gemcitabine (GEM) on pristine and functionalised carbon nanotubes by molecular dynamics (MD) simulation.Functionalisation of carbon nanotubes with carboxyl groups increased the efficiency of DOX loading.Folic acid-functionalised CNTs delivered DOX to cancerous cells effectively.PEGylated CNTs have shown more vital interaction with GEM.Dual delivery systems are more effective and efficient than traditional delivery systems.The findings have demonstrated the therapeutic potential of this dual chemotherapeutic drug delivery system in the treatment of cancer [90].
De Alcantara and colleagues did a preclinical evaluation of PEGylated MWCNTs in tumour-bearing mice.PEG-MWCNTs were radiolabelled with [99mTc] Tc to facilitate biodistribution of the formulation.The findings revealed the developed product's prolonged blood circulation time and increased tumour uptake rates.Toxicological studies have performed the results demonstrated the non-toxic effects of the PEG-MWCNTs.Based on the findings of De Alcantara and colleagues, PEGylated MWCNTs have the potential for future oncologic applications [91].

Role of CNTs in Neurological Disorders
CNTs have specific properties such as good electrical, high aspect ratio, high payload capacity, penetration capability, and good thermal conductivity, enabling CNTs for easy surface modification, making them suitable nanocarriers for drug delivery in tumour tissues and the brain [92].Several studies have illustrated the CNTs' capability of acting as a drug delivery nanocarrier by encapsulating drugs effectively in their central cavities or conjugating drugs on their walls.They can be equipped with targeting moieties to deliver drugs to an area of interest in the brain.CNTs can be used as a drug delivery nanocarrier to navigate the BBB.The formulation can be prepared by decorating the CNTs with anti-inflammatory drugs, tracers, and targeting ligands [35].Functionalised CNTs penetrate the cells through receptor-mediated endocytosis and macropinocytosis processes.Additionally, the needle-like mechanism of the CNTs facilitates their cell membrane penetration of the site of interest.The BBB is the main contributing factor to the inaccessibility of the drug to the CNS [92].
Shityakov and colleagues evaluated the active transport of MWCNTs labelled with fluorescent tag FITC across immortalised murine microvascular cEND cells using the Tran-swell® device (Corning Inc., NY, USA) [93].The distribution and accumulation of FITC-labelled MWCNTs in the target cells were assessed by fluorescence microscopy.Other studies demonstrated the successful intracellular uptake of functionalised MWCNTs in vitro BBB models using TEM and Ɣ-scintigraphy [92].Yang and colleagues investigated the uptake of CNTs in the brain.SWCNTs were administered orally to the mice for ten days.TEM performed detection in various cells, including the brain.In another study, SWCNTs were able to deliver acetylcholine in Alzheimerinduced mice.The learning and memory of the mice were improved [92].
Acetylcholine deficiency triggers Alzheimer's disease leading to cognition and memory loss.Native acetylcholine is hydrophilic and incapable of crossing the hydrophobic BBB.Acetylcholine was non-covalently loaded into SWCNTs to make it penetrable to the BBB [94].Among numerous neurological disorders, neuro-oncology has attracted intense attention.Treating brain tumours is challenging because of their rapid growth, invasive nature, and BBB presence.Based on these challenges, numerous neurotherapeutics cannot contribute their efficacies in clinical treatment.The trans-activating transcriptional activator suppresses occludin in BBB.By so doing, the BBB permeability is improved.
Moreover, the TBCNT@OXA nanocarrier system enhanced the penetration power to the BBB.By so doing, the anti-tumour activity is increased, leading to the successful treatment of glioma [95].Doxorubicin (DOX) was targeted with PEGylated-oxMWCNTs that were conjugated to angiopep-2.DOX-oxMWCNTs-PEG-ANG was more effective in anti-glioma activity as opposed to a standalone DOX.
Based on these findings, the oxMWCNTs-PEG-ANG is regarded as the most promising dual-targeting carrier for DOX delivery [96].Zhao and colleagues functionalised the CNTs and conjugated them to CpG oligonucleotides.The conjugates were tagged with Cy5.5.These CNTs were administered stereotactically into the brain, as shown in Fig. 12.The uptake of the CpG-Cy5.5-CNTs by the glioma cells was immensely enhanced.The pro-inflammatory cytokines were elevated; hence, the tumour growth was lowered [71].
Levodopa is a dopamine precursor.It is regarded as a nitty gritty in treating Parkinson's disease.The shortcoming of levodopa is its rapid decarboxylation following oral administration.A minute amount of the intact levodopa reaches the brain.Tan and colleagues loaded levodopa on carboxylated SWCNT for treating Parkinson's disease.The findings confirmed the synthesised nanohybrid's effectiveness as the cells' viability was not compromised [97].Xiang and colleagues explored the mechanism of SWCNTs in Alzheimer's disease.Their findings confirmed the SWCNTs' potential in neural disorder therapy [98].
Kafa and colleagues demonstrated the transport of MWCNT-NH 3+ across the BBB in vitro co-culture BBB model [99].In a different study, f-MWCNTs were found to cross endothelium and assemble in rodents' brain parenchyma following intravenous administration [99].Carbon nanotubes can be used as a standalone option on the nervous tissue or as a combination of other molecules to ferry drugs to the diseased site (Table III).
Lee and colleagues showed the neuroprotective effects of SWCNTs after the MCAO ischemic rats were given intraventricular administration of SWCNTs-NH 3+ .The neurons were protected from injury, and the recovery of neuronal behaviour was enhanced [100].Guo and colleagues investigated the delivery of dopamine by SWCNTs to the brain for Parkinson's disease (PD) treatment.Dopamine was loaded on the functionalised SWCNTs (SWNCT-PEGs-Lf), and the complex was intravenously administered to the PD mice.PEG was used to protect the complex from opsonisation by the macrophages and to increase the circulation time to increase the dose gradient of the complex.
Parkinsonism was successfully treated as this dual modification of PEG and lactoferrin enabled the delivery of the drug to the affected site in the brain [101].Kafa and colleagues exploited the ability of angiopep-2-targeted functionalised MWCNTs to navigate the BBB in their studies.The uptake of the CNTs by the neural cells was highly improved, which could be applied in treating glioma [94].
Lee and colleagues synthesised carbon nanotube platforms with different conductivity and hydrophilicity.The platforms were used to determine the effect of carbon nanotubes on astrocyte function.Astrocytes are responsible for releasing gliotransmitters to regulate synaptic transmission [102].
Astrocytes on MWCNT platforms illustrated enhanced cell proliferation and adhesion.Integrin and GFAP expression were upregulated.Integrin is a transmembrane receptor involved in cell-cell and cell-extracellular matrix adhesion.Moreover, GABA and glutamate in astrocytes were augmented on MWCNT platforms, as illustrated in Fig. 13.
Soligo investigated the distribution of non-electroconductive MWCNTs-1 and electroconductive MWCNTs-2 in the brain parenchyma of the rats.The rats were divided into two groups: healthy (injected with citrate) and diabetic (injected with streptozotocin).Rats were exposed to intranasal administration of both MWCNTs.The rats were euthanised, and carbon nanotubes were investigated in various brain areas.The neuroprotective potential of both MWCNTs was evaluated.It was found that both MWCNTs are distributed in numerous brain areas, in particular, the limbic region (neurodegenerative disease-prone region), when administered intranasally.In addition, it was also demonstrated that electroconductive MWCNTs were more neuroprotective as opposed to non-electroconductive CNTs [103].
Alvarez and colleagues demonstrated the neuronal activity recording and neurostimulation of the dry-spun carbon nanotube microelectrode in the fly and cockroach.Their electron transport capabilities were compared to noble metals such as gold and silver.CNT fibre was found to have a higher rate of electron transfer leading to lower impedance.Furthermore, these carbon nanomaterials performed well for in vivo electrophysiology, rendering them a promising prospect for neurophysiological applications [104].

Conclusions and Future Perspectives
Neurological diseases are severe problems and significant health concerns globally.Innumerable therapeutic approaches to this class of diseases have been investigated, but various challenges restrict their treatment efficacy.The contributing factor to the low success rate is the active drug molecules' inability to access their target sites of action inside the brain.Nanotechnology proffers various fascinating and promising novel means of treating neurological disorders.Nanotechnology has the potential to entirely alter how the CNS-targeted treatments are executed in delivering neurotherapeutics to the brain.CNTs are some of the novel nanomaterials or nanocarriers in the field of nanotechnology used to deliver the drug of interest to the diseased site in a controlled manner.CNTs have hollow central cavities that can be loaded with the drug to increase efficiency.CNTs are manufactured at an industrial scale, leading to high exposure levels to the communities by direct and indirect means.The high production of CNTs resulted in safety concerns regarding the effects of carbon nanotubes on human health.
Notwithstanding their beneficial effects, controversial issues surrounding the toxicity of the CNTs have restricted the speedy industrial transition and clinical translation.Based on this controversial issue of CNT toxicity, CNT-based delivery systems are still in the pipeline for FDA.Nevertheless, the functionalisation of CNTs enhances the solubility and biocompatibility of the CNTs.Precautionary measures should be considered when CNTs are used based on their toxicities.Briefly, CNTs and CNT-based drug delivery systems have immeasurable potential in medical, biomedical, drug delivery, and therapeutics and would soon be a focal point of research.

Fig. 3
Fig. 3 Carbon nanotube structure.a Single-walled CNTs.b Multiwalled CNTs.Adapted with permission from reference [31] under a Creative Commons Attribution License

Fig. 4
Fig. 4 Synthesis of carbon nanotubes.a Chemical vapour deposition setup demonstrating the gas supply, nebuliser, furnace and temperature control, and exhaust.b CNT synthesis from the catalyst on the substrate using a root growth mechanism

Fig. 6 Fig. 7
Fig.6 Predicted fractional deposition of inhaled particles in the human respiratory tract's nasopharyngeal, tracheobronchial, and alveolar regions[53].Reproduced with permission from Environmental Health Perspectives (EHP)

Fig. 8
Fig. 8 Drug loading to and release from functionalised MWCNTs.a Entrapment efficiency of gemcitabine by MWCNTs.b Gemcitabine release from MWCNTs.Reproduced with permission from reference [59] under a Creative Commons Attribution License

Fig. 9 A
Fig.9A NIR-responsive CNTS@MS@IBAM-DOX@HSA nanocomposite.Reproduced with permission from reference under a Creative Commons Attribution License

Fig. 12 Fig. 13
Fig. 12 CNT mediated delivery of CpG and tumour regression in glioma.Adapted from reference [71] with permission from Copyright © 2020 Elsevier B.V

Table I
Comparison of CNTs Synthesis Methods.Adapted with Permission from Reference [33] Under a Creative Commons Attribution License

Table II Physical
Characterisation of Carbon Nanotubes.Adapted With Permission From Reference [51] Under a Creative Commons Attribution License

Table III
[13]nt Application of Functionalised MWCNTs in Drug Delivery.Adapted With Permission From Reference[13]Under a Creative