The re-rank scores and H-bond scores for the best docked conformations of CPT and CPT/8 × 8 graphene, and CPT/8 × 8 GO sheets, respectively.
Carbon nanotubes (CNTs) have attracted substantial research interest in biomedical sciences and bionanotechnology, rendered from its unique structure, electronic, mechanical, and optical properties. Despite the diverse potential applications, the integration of CNTs in biomedical research is one of the most challenging areas where nanotubes fall under much scrutiny. Pristine nanotubes are highly hydrophobic, and non-dispersible in most of the common aqueous and organic solvents and to render nanotubes biocompatible, functionalization is one of the key prerequisites. In this regard, covalent and noncovalent functionalization are the two widely adopted approaches for co-tethering biologically active molecules on the CNTs. Likewise, the hollow cavity of the nanotube facilitates in the endohedral encapsulation of biomolecules, peptides, DNA oligonucleotides, and proteins, thereby retaining the physiological attributes of the biological molecules. The chapter focuses on the emerging approaches to the functionalization of single-wall CNTs (SWCNTs) and the potential application of functionalized SWCNTs in tuberculosis and cancer chemotherapy using state-of-the-art density functional theory, molecular docking and molecular dynamics simulation methods.
- carbon nanotubes
- drug delivery
- molecular dynamics
- density functional theory
1.1. Carbon: The fundamental building block of life
Carbon is the most versatile element in the periodic table that forms the basis of all kinds of life on earth. Elemental carbon displays a complex allotropy depending on the nature of hybridization; diamond (
CNTs are hexagonally arranged, honey-combed lattice of carbon atoms formed by the rolling of graphene into seamless cylindrical structures (see Figure 2a). Nanotubes like graphene have a high diameter to length ratio (aspect ratio)  and demonstrate high electrical, mechanical, and thermal conductivity along with structural stability [4, 5, 6, 7]. CNTs are broadly classified as single-wall CNTs (SWCNTs) and multi-wall CNTs (MWCNTs). The SWCNT comprise of a single graphene sheet, with diameter ~ 0.5–1.5 nm and length of ~100 μm , while MWCNT is formed from the co-axial stacking of SWCNTs, with diameter ~1.4–100 nm, length between 1 nm-μm, and internuclear distance of 0.3–0.4 nm between the co-axial tubes. The representation of a zigzag (
The unique electronic properties exhibited by CNT are governed by the quantum confinement of electrons where the periodic boundary conditions come into interplay. Because of the quantum confinement, electrons can propagate along the tube axis: forward and backward, along with the conservation of energy and momentum. Unlike metals which have a smooth density of states (DOS), CNTs are characterized by many van Hove singularities , and the DOS depends on diameter and chirality of the nanotube . The conducting properties of CNT is an inverse function of its diameter, that is, with increase in diameter, band gap between the valence and conduction bands decreases and at a certain point both the bands overlap to give rise to metallic nanotubes. Semiconducting nanotubes on the other hand (with similar diameter as metallic nanotubes) possess similar van Hove singularities near the Fermi level .
These unprecedented properties have largely contributed to the extensive biomedical research, especially as nanocapsules for therapeutic drugs, proteins, and gene delivery . CNTs find application in bionanotechnology and pharmaceutical sciences, and current drug delivery modules have been incorporating CNTs for improved target specific detection and treatment of diseases. Although the potential application of carbon nanomaterials, particularly CNTs are farfetched, the concerns raised over the effect of large-scale synthesis of CNTs to the environment, biocompatibility, toxicity, biodegradation and remediation cannot be undermined and needs to be addressed thoroughly. Hence, a detailed
Some of the questions that underlie the importance of the study are (i) understanding the mechanism of CNT uptake followed by the subsequent release of therapeutic molecules, (ii)
The chapter in a very comprehensive yet succinct way addresses the potential applications of SWCNTs in drug delivery, managing to draw a fine line between the scopes of application and practical viability of integrating carbon nanomaterials in biomedical research. Herein, we report the theoretical aspects of modeling novel SWCNT-based drug delivery systems using the covalent and noncovalent functionalization schemes. Nanotubes of varying chirality and length are considered for functionalization, drug loading, and targeting onto the active binding sites of receptor proteins. With the successful incorporation of CNTs in cancer therapy, we propose a novel approach toward integrating CNTs in Tuberculosis (TB) therapy. To the best of our knowledge, theoretical studies on the potential application of CNTs in pharmaceutical sciences pertaining to TB and other bacterial diseases have not been discussed extensively. We address the recent theoretical advancements using the state-of-the-art density functional theory (DFT), molecular docking, and molecular dynamics (MD) simulation methods. Molecular docking serves as an instrumental tool in computer-aided drug design for predicting the preferred binding mode of a ligand to a receptor (protein). Docking studies help characterize the protein binding cavity, understand the orientation of ligand with respect to the receptor protein, and the nature of interaction between the protein with functionalized nanomaterial, which can aid in the structure-based design of novel drug delivery systems for future experimental studies.
2. Functionalization of CNTs
Traditional approaches to drug delivery function over a broad spectrum, resulting which, specificity toward drug administration and delivery are rarely accomplished. Development of polymer-based nanocomposite materials has enabled the successful engineering of drug delivery modules via the incorporation of nanomaterials and nanoparticles as nanocapsules for sustained release of therapeutics in a dosage-dependent manner. Nanotechnology, on the other hand, has revolutionized the pharmaceutical sector with the assimilation of functionalized nanomaterials like CNTs in drug, gene delivery, and tissue engineering [14, 15]. CNTs play dual role by rendering directionality in targeting the tumor (malignant) cells and facilitating the controlled mediated release of therapeutic molecules. The application of CNTs as carrier payloads for anticancer drugs cisplatin , carboplatin , doxorubicin (DOX) [18, 19, 20], mechlorethamine , paclitaxel  and antitubercular drugs like isoniazid (INH) , rifampicin, pyrazinamide (PZA)  have been reported.
With the inherent limitations in application of pristine, unmodified nanotubes, functionalization is the collective approach toward tailoring nanotubes electronic properties. Pristine CNTs are generally hydrophobic with low solubility in most of the common aqueous and organic solvents and the hydrophobicity is accounted to the size, structure, and bundling effect which restricts the uptake and assimilation within the biological environment . Functionalization assists in reducing the bundling effect which arises due to the van der Waals (vdW) attractive forces between adjacent nanotube surfaces and is efficient in increasing the biocompatibility thereby facilitating cellular internalization and trafficking. It has been reported that the functionalized nanotubes (
2.1. Solubilization of CNT through covalent functionalization
Some of the alternative schemes to functionalization of CNT is through covalent method using 1,3-dipolar cycloaddition , [2 + 1] cycloaddition of dichlorocarbene, silylene, germylene , hydroboration , arylation, hydrogenation by Birch reduction , carboxylic acid groups , Diels-Alder reaction, esterification of carboxylated nanotubes  and fluorination reactions . Experimental and theoretical studies have shown that the extent of covalent functionalization depends on the curvature of nanotube as an increase in curvature decreases the reactivity toward sidewall functionalization .
2.1.1. 1,3-dipolar Cycloaddition (DC)
The solubility of CNT can be enhanced by the covalent functionalization using 1,3-DC reactions. Azomethine ylide (CH2NHCH2), ozone (O3), nitrone (CH2N(H)O), nitrile ylide (CHNCH2), nitrile imine (CHNNH) are the commonly used functional groups for 1,3-DC reaction. The intrinsic physical properties of CNTs such as photoluminescence and Raman scattering decreases upon covalent functionalization, due to chemical bond formation between the functional group and carbon atoms. Theoretical studies by Lu et al.  reported the reaction energies (
Prato and co-workers  investigated the 1,3-DC reaction of azomethine ylide on both SWCNT and MWCNT. The water-soluble amine functionalized CNT was highly suitable for immobilization of biomolecules, and purification of pristine nanotubes during syntheses. Attachment of peptide molecules onto covalently functionalized SWCNT was reported by Prato and co-workers . The C-terminal group of peptide chain was attached to N-terminal side group to form a supramolecular complex of peptide wrapped nanotubes. Gallo et al. , incorporated
The structure, electronic properties, and reactivity of a series of 1,3-DC functionalized armchair (
2.1.2. Functionalization using organic acids
Covalent functionalization of CNTs using carboxylic (−COOH) group was realized through oxidation with strong organic acids like H2SO4/HNO3 , phosphates, and sulfur-containing units. Acid functionalized CNTs are highly soluble in water under a wide range of pH and exhibit a significant reduction in the aggregation of nanotube bundles. The dispersibility facilitates in the sidewall, endohedral and end tip functionalization of CNT with organic acids and different functional groups. The sidewall functionalization of CNTs via cycloaddition reaction with azide, ozone, transition metal oxides, and carbenes  is illustrated in Figure 3.
Lu et al.  using two-layered ONIOM (B3LYP/6-31G*:AM1) approach reported the reaction pathway and site selectivity for [2 + 1] cycloaddition of dichlorocarbene, silylene, germylene, and oxycarbonylnitrene onto (5, 5) SWCNT. Dichlorocarbene addition occurs preferentially at the 1,2-pair site. The silylene addition at 1,2-pair site was predicted to be exothermic (−20.7 kcal mol−1) and follows a barrier less reaction pathway. Germylene addition was exothermic by 8.5 kcal mol−1, lower than dichlorocarbene and silylene and proceeds in absence of a transition state pathway. Oxycarbonylnitrene addition onto 1,2-pair site of SWCNT was exothermic by 66.2 kcal mol−1, higher than the other three cycloaddition groups. The transition state had an activation barrier of 7.2 kcal mol−1, which suggested that the cycloaddition reaction was facile in nature.
2.2. Noncovalent functionalization of CNTs
Although covalent functionalization improves the solubility of CNT, it modifies the intrinsic electronic properties by deforming the C-C bond length, perturbing the
2.2.1. Functionalization via π-π stacking
The noncovalent functionalization of pyrene on SWCNT was investigated by Cosnier and co-workers  for application as modified electrodes in biosensing devices. Calomel electrode was taken as the reference and Pt electrode (5 mm diameter) modified by casting 20
Noncovalent functionalization of porphyrin molecules with SWCNTs have been extensively studied as high yield light-harvesting systems with tunable electronic properties for biological and optoelectronic applications [55, 56]. Roquelet et al. , reported an efficient method for the synthesis of porphyrin/SWCNT complex utilizing a micelle-swelling technique in presence of organic solvent. The organic solvent leads to swelling of the micelle facilitating the interaction of porphyrin molecules to the micelle core and SWCNT. Dispersion corrected DFT calculations on the structure, electronic and optical properties of SWCNT functionalized tetraphenylporphyrin (TPP) molecule showed that diameter rather than chirality of the nanotube stabilizes the π-π stacking of TPP molecule . The optical absorption of TPP was not affected by the diameter or chirality of CNT and the optical spectra showed the absorption of π-stacked TPP at almost the same position as the isolated TPP, indicating that the TPP absorption properties were preserved in the complex.
2.2.2. Functionalization using biomolecules and nucleobases
Functionalization of CNTs with biomolecules is useful in the development of nanobio composite devices. Immobilization of DNA in DNA-based biosensors is possible with the incorporation of CNTs in nucleic acid sensing, gene therapy, and biosensor fabrication [59, 60, 61]. DNA because of the base pairing sequence facilitates in the alignment of nanotube assembly . Rodger and co-workers  investigated the interaction of CNT with DNA using linear dichroism (LD) method. DNA/CNT hybrid exhibited higher LD signals, higher than the sum of the LD spectrum of individual DNA and SWCNT. Jung et al.  developed methods for covalent linking of DNA oligonucleotides onto SWCNT films which were later immobilized onto solid surfaces. The carboxylated/aminated DNA oligonucleotides were covalently attached to functionalized SWCNT, the length of which was controlled via oxidation with strong organic acids, leading to the formation of carboxylated SWCNT.
Li et al.  investigated the self-assembly of CNT and gold nanoparticles into multicomponent structures using DNA oligonucleotides. The CNT pre-functionalized with -COOH facilitated in the grafting of ssDNA strands and multiple assemblies of nanotubes were thus possible using this technique. In another combined theoretical and experimental study by Sood and co-workers , interaction of DNA nucleobases namely adenine (A), guanine (G), cytosine (C), thymine (T) with (5, 5) SWCNT was reported. The
2.2.3. Noncovalent functionalization using polymers
Polymer wrapping of CNTs mediated via noncovalent functionalization toward the synthesis of highly dispersed, stable and reinforced functional dispersants in aqueous and organic solvents was reviewed by Fujigaya and Nakashima . The polymer wrapping forms a thermodynamically stable coating and any unbound polymer could be removed via dialysis, ultra-centrifugation or chromatographic separation techniques. Similarly, noncovalent functionalization of CNTs using polyethylene glycol (PEG) PEGylated-phospholipid chains forms an effective means of high loading of the drug and biomolecules at the free end of PEG chain and onto nanotube sidewall. PEGylated-phospholipid chain facilitates the high loading (about ~400%) of drug molecules onto CNT and characterizes as a potent carrier vehicle in drug delivery applications. PEG tailored SWCNT exhibit no toxicity for over several months, which was further substantiated from time-dependent assays performed onto mice. Drugs which normally remained insoluble within the biological systems, upon conjugation with PEG modified CNT revealed high solubility as well as retention time within the body. The noncovalent functionalization retained the physical properties of nanotubes without drastically perturbing the overall electronic properties.
First-principles studies on the interaction of conjugated polymers with (8, 0) SWCNT and (10 × 10) graphene sheet was investigated by Jilili et al. , to confirm the experimental observation that polymers are suitable for noncovalent functionalization. The GGA approximation was predicted to be inadequate in describing the physisorbed systems, whereas LDA and vdW corrected GGA yield conclusive results. The electronic structure of SWCNT/graphene was maintained around the Fermi energy with negligible charge transfer between the conjugates. The interaction of polymer-SWCNT/graphene was of weak vdW type with minimal effects on the physical and electronic properties of SWCNT/graphene, important for an effective noncovalent functionalization.
3. Functionalized carbon Nanomaterials in drug delivery
Drug delivery is a process of administering drugs in a controlled, sustained manner to achieve maximal therapeutic efficacy upon transdermal administration. The foremost objectives in developing novel drug delivery systems are to improve the therapeutic competence by  increasing bioavailability,  preventing toxicity, harmful side-effects by increasing the persistence of a drug,  reducing drug exposure toward non-target cells, and  minimizing drug degradation and loss [70, 71, 72, 73]. Drug delivery systems are designed to improve the pharmacological and therapeutic profile of drug molecules with an ability to cross the cell membrane upon administration [74, 75]. The most important characteristic of SWCNT as a drug delivery system is its ability to penetrate the cell membrane , and facilitate the intracellular internalization and trafficking within the cell cytoplasm . A major breakthrough in nanoscience was the advent of CNTs as one of the most sought-after materials for designing novel drug delivery carrier modules to comply with the biotechnological and pharmaceutical objectives. CNT due to its needle-like cylindrical structure can easily penetrate the cell membrane and enter the cell nuclei, while the cell does not recognize it as an intruder.
Functionalized CNTs can act as carriers for antimicrobial agents like amphotericin B [78, 79] and transport it within the mammalian cells. This reduces the antifungal toxicity as compared to the toxicity of free drug (40% of the cells being killed by CNTs-free formulation compared to no cell death by CNTs formulation). The surface-engineered CNTs can capture the pathogenic bacteria in liquid media [80, 81]. In addition, SWCNTs exhibit unique optical properties such as near-infrared region (NIR) fluorescence, and Raman scattering. The fluorescence range spans the entire biological tissue transparent window and is, therefore, promising for drug detection and biological imaging [82, 83, 84].
3.1. SWCNTs in tuberculosis therapy
The science of bacteriology is credited to the contributions of Louis Pasteur and Robert Koch. It was the discovery of
Streptomycin was the first antitubercular drug discovered in 1943  and since then several therapeutic drugs like para-amino salicylic acid (1946), isoniazid (INH) (1952), pyrazinamide (PZA) (1952), cycloserine (1952), ethionamide (1956), rifampicin (1957) and ethambutol (1962) have been discovered. The TB therapy involves a combination of four first-line drugs namely; INH, PZA, rifampicin, and ethambutol administered for a period of 2 months followed by minimum 4 months’ treatment regimen of INH and rifampicin . PZA (pyrazine-2-carboxamide) is one of the first-line drugs used in TB treatment recommended by the WHO. PZA is metabolized into its active form (pyrazoic acid) by the amidase activity of
We performed DFT, molecular docking and MD studies on the SWCNT-mediated PZA delivery onto the active site of
Noncovalent functionalization of SWCNT and boron nitride nanotubes (BNNTs) with PZA was investigated using DFT and MD simulation (see Figure 5) to comprehend the role of nanotube chirality on the electronic properties of the complexes . BNNTs are structural analogs of CNTs with a wide band gap of ~5.5 eV, high chemical, and thermal stability. The potential application of BNNTs is rather limited in terms of its high chemical stability and poor dispersibility. The theoretical results predict the modification in electronic structure of both SWCNTs and BNNTs with the enhancement of electronic states, significant lowering in HOMO-LUMO energy gap and the presence of new dispersionless states within the band gap. Depending on the nanotube chirality, PZA exhibits a preferential selectivity for adsorption, which is further confirmed from the band structure, DOS, total projected DOS, and frontier orbital analysis.
The functionalized nanotube facilitates in the loading and delivery of a PZA onto the active entering pathway of pncA without the nanotube affecting the structural conformation of pncA as shown in Figure 6. The incorporation of nanotube yields better docking scores for PZA then the drug being administered in bare form. Although covalent functionalization aids in achieving target specific delivery of PZA within the active site of pncA, noncovalent functionalization was predicted to be effective for engineering nanotube structure and electronic properties for successful drug delivery applications.
Zanella et al.  performed theoretical studies on the interaction of non-steroid anti-inflammatory drug nimesulide with pristine and Si-doped capped SWCNT. The adsorption of nimesulide on Si-doped capped SWCNT exhibit a higher binding energy of 1.8 eV compared to pristine capped SWCNT (0.32 eV) which was due to the high reactive bonding sites on Si atom. The strong interaction of nimesulide with Si-doped SWCNT served as better drug delivery carriers in comparison to pristine capped SWCNT.
Wang and co-workers  performed MD studies to investigate the mechanism of encapsulation of nifedipine drug within (10, 10) SWCNT. Their studies showed that the internal adsorption of nifedipine was more stable than external adsorption by 5.3 to 7.8 kcal/mol. In solvent phase, the encapsulation of nifedipine was impeded due to competitive vdW and hydrophobic interactions in SWCNT-nifedipine-water complex. Encapsulation of nifedipine orients the distribution of water molecules inside SWCNT accompanied by the H-bond formation between water molecules and oxygen atom of nifedipine. During the encapsulation process, SWNT undergoes weak fluctuations due to the oscillatory behavior of nifedipine encapsulated within the CNT.
4. SWCNTs in cancer therapy
Platinum-based Phase II and Phase III anticancer drugs hold promise in the treatment of cancer with new drugs being discovered, some of which are still under clinical trials. The two main limitations in use of Pt-based anticancer drugs are  the anticancer drugs undergo poor circulation in tissue cells and its activity is reduced with time due to the complex formation with plasma and tissue cells, and  tumor cells demonstrate resistance toward Pt-based drugs under prolonged exposure, rendering them ineffective as potent anti-tumor agents. Lippard and co-workers [16, 17] incorporated capped
We performed density functional studies on the noncovalent functionalization of non-Pt-based anticancer drug camptothecin (CPT) on graphene-based nanomaterials and its prototypes, including graphene oxide (GO) . The noncovalent adsorption of CPT induces a significant strain within the nanosheets and the interaction was thermodynamically favored from energetics perspective. In case of GO, surface incorporation of functional groups resulted in significant crumpling along the basal plane and the interaction was mediated by H-bonding rather than π-π stacking. The molecular docking studies of CPT onto Top1 (Figure 7a) showed CPT to be stacked between the Watson Crick AT and GC base pairs and the interaction was mediated via π-π stacking (Figure 7b). For the binding of CPT functionalized graphene and GO with topoisomerase I (top 1) CPT interacts through π stacking with AT and GC base pairs of DNA. The optimum interacting distance of CPT from AT and GC bases was calculated at 3.87 and 3.38 Å, from the central aromatic rings (Figure 7b). The re-rank score of bare CPT drug was calculated as −89.01 a.u. with an H-bond score of −2.53 a.u. as shown in Table 1.
|System||Re-rank score CPT||Re-rank score nanosheet||H-bond score CPT||H-bond score nanosheet|
|8 × 8 graphene/CPT docked onto Top1||−89.10||95.87||−2.57||0.00|
|8 × 8 GO/CPT docked onto Top1||−90.21||126.21||−2.29||−4.44|
Likewise, for the docking of CPT/8 × 8 graphene with Top1 (Figure 8a) CPT gets docked between the AT and GC base pairs. However, graphene gets docked along the phosphate backbone of the ds-DNA helix as shown from Figure 8b indicating a strong interaction between the polar phosphate groups of the DNA helix. Compared to the docking of bare CPT drug, presence of graphene stabilizes the intercalation of CPT between the AT and GC base pairs, as observed from the increase in re-rank score values.
The docking of CPT/GO with Top1 as illustrated in Figure 9a, depicts CPT to get docked between AT and GC base pairs of DNA, mediated by π-π stacking interaction similar to that observed for bare CPT and CPT/8 × 8 graphene. However, in the presence of GO, GO undergoes strong interactions with DNA bases and gets docked between the DNA helix and the interaction is stabilized by intermolecular H-bond between polar functional units on the basal plane of GO and DNA nucleobases (Figure 9b). The molecular docking studies on bare CPT and CPT functionalized graphene and GO systems showed that the interaction of CPT with Top1 is mediated by π-stacking interaction between the aromatic rings of CPT and the A and C bases of DNA. In presence of graphene and GO, CPT undergoes a similar trend in adsorption while the graphene and GO nanomaterial gets docked along the phosphate backbone indicating a strong preferential interaction with DNA.
Boucetta et al.  investigated the supramolecular MWCNT-DOX-copolymer complex for anticancer activities. Since DOX, a clinically acclaimed anticancer drug belonging to the family of anthracyclines exhibit fluorescence properties, its uptake and interaction with nanotubes upon administration can be monitored using fluorescent spectrophotometry. The copolymer coated MWCNT formed supramolecular complexes with DOX via
Carbon nanotubes have proffered as one of the novel functional materials of the 21st century, broadening the theoretical and experimental perspectives in research to explore its novel and intriguing properties. Due to the conjugated
Although CNTs demonstrate practical applicability in all facets of science, be it biology, physics, medicine, nanotechnology, catalysis, or materials science, its long-term implications need to be assessed from the perspective of human health to environmental risks. The long-term fate of CNTs released into the environment depends on the structural, morphological and synthetic treatments . Methods of reducing toxicity
We investigated the potential application of SWCNTs, graphene-based nanomaterials and its prototypes in TB and cancer chemotherapy using conventional DFT methods supported by molecular docking and MD simulation on the nature of interaction of therapeutic drug functionalized SWCNTs/graphene with the binding site of the protein. The functionalization of SWCNTs with therapeutic drugs using covalent and noncovalent schemes were adopted to investigate the drug binding with the nanotube and the stability of the conjugated complexes. DFT results supported by molecular docking and MD simulation helps in contemplating the feasibility of SWCNT-based novel drug delivery in cancer and TB therapy.