The revolutionary development of functionalized carbon nanotubes (
In the past few decades, carbon nanotubes (CNTs) have received tremendous attention from the science community for fundamental research as well as their applications in various fields of study such as catalyst supports [
Since the discovery of CNTs in the early 1990s [
Basically, CNTs are long, tubular fullerene structures in which the walls of the CNTs comprise of hexagonal carbon and the end tips are pentagonal rings [
Carbon nanotubes: single-walled carbon nanotubes (SWCNTs) on the left; and multiwalled carbon nanotubes (MWCNTs) on the right. Adapted from [
There has been significant progress in the research of
The covalent functionalization is more advantageous than noncovalent functionalization if a strong bond is required between the nanotubes and the biomolecules. This covalent binding relies very much on the grafting of chemically reactive molecules onto their inert sp2 carbon structure of the
Purification techniques such as acid oxidation modify CNTs surfaces by inducing the opening of the tube caps and formation of holes at the sidewalls resulting in CNTs with tips and sidewalls decorated with oxygenated functionalities (e.g., carboxylic, carbonyl, and hydroxyl groups). Carboxylic acid functionality is the most commonly used surface defect-derived moieties to connect CNTs with amines site on the biomolecules [
Noncovalent functionalization is not destructive to the sp2 bonding compared to covalent functionalization and, therefore, preserves the functional properties and native structure of CNTs more effectively than covalent methods. The functionalization mechanisms for the noncovalent dispersion of CNTs are often straightforward with the use of sonication, mixing, mixing followed by sonication, centrifugation, or filtration. Nevertheless, the chemical interactions of noncovalent approaches may still incur significant surface doping effects in the nanotubes due to the electronic transitions [
Pristine CNTs are very difficult to disperse in solution due to the formation of big bundles held strongly together by the van der Waals forces. In order to separate the nanotubes from self-aggregation, various dispersion agents such as ionic-complementary peptides [
An in-depth study on the surface modifications by covalent and noncovalent method for the effective dispersion of CNTs is extensively reviewed by Kim et al. [
Some of the recent development with covalent and noncovalent functionalization of CNTs for
Carrier | Surface modifications | Drugs/biomolecules/imaging agents | Type of bonding between CNTs and cargo | Cell lines | Remarks | Reference |
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SWCNTs, MWCNTs | Acid oxidation | Soybean peroxidase | Covalent | Nontumorigenic human bronchial epithelial cells (BEAS-2B) | Acid oxidation increased the number of functional groups and improved CNTs biocompatibility in aqueous environments. | [ |
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MWCNTs | Acid oxidation followed by acylation and subsequently grafted with polyamidoamine (PAMAM) dendrimers | Cadmium sulfide and silver sulfide quantum dots (QDs) as fluorescence labelling probes | Covalent | Gram-positive bacterium |
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MWCNTs | Refluxed in acid nitric followed by thionyl chloride and then functionalized with PEG diamine | Doxorubicin (DOX) and fluorescein isothocyanate (FITC) as fluorescence labelling probes | Covalent | Human adenocarcinoma cells (HeLa), human hepatocellular carcinoma cells (HepG2) and human leukemia cells (K562) | The PEGylated MWCNTs penetrated into mammalian cells without damage plasma membrane and its accumulation did not affect cell proliferation as well as cell cycle distribution. It was found to accumulate in the multidrug-resistant cancer cells as efficient as in the sensitive cancer cells. | [ |
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SWCNTs | Acid oxidation | None | Covalent | Mouse fibroblast cells (NIH3T3) | The |
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MWCNTs | Oxidized in concentrated sulfuric acid/nitric acid mixture and further functionalized with cationic polymer polyethylenimine (PEI) | Paclitaxel (PTX), folic acid (FA) as targeting ligand and QDs | Noncovalent/covalent | HeLa cells and human umbilical vein endothelial cells (HUVEC) | The |
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SWCNTs | Encapsulated with chitosan (CHI) | DOX and FA | Noncovalent | Hepatocellular carcinoma cells (SMMC-7721) | The obtained |
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SWCNTs | Functionalized with carboxylic acid followed by encapsulation with FA-conjugated CHI | DOX | Noncovalent | None | The conjugate demonstrated good stability in aqueous medium due to the encapsulation of CHI and exhibited the characteristics of both targeted and controlled release functions. | [ |
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SWCNTs | Oxidized in oleum and nitric acid and subsequently functionalized with methoxy (PEG) amine | PTX | Non-covalent | It was found that the conjugate was not acutely toxic, primarily accumulated in the liver and spleen and proved to be stable and effective both |
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MWCNTs | Purified using sulfuric acid/nitric acid mixture and further functionalized with polycitric acid-polyethylene glycol-polycitric acid (PCA-PEG-PCA) linear-dendritic copolymers | Cisplatin ( |
Noncovalent | Murine colon adenocarcinoma tumor cancer cells (C26) | The synthesized conjugate was able to be introduced into the colon cancer cells and kill the cells effectively. | [ |
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MWCNTs | Oxidized in concentrated sulfuric acid and nitric acid, followed by conjugation with iron nanoparticles | DOX and FA | Covalent | HeLa cells | The DOX/FA-MWCNT@Fe had a sufficient load capacity (32 |
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MWCNTs | Oxidized in nitric acid and sulfuric acid mixture and subsequently functionalized by 1,2-distearoyl- |
DOX and angiopep-2 as targeting ligand | Noncovalent | Brain capillary endothelial cells (BCEC) and glioma cells (C6) | The conjugate showed a better anti-glioma effect and a lower cardiac toxicity compared to DOX. | [ |
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MWCNTs | Carboxylation, acylation and followed by amidation process to obtain amine terminated MWCNTs | DOX and targeting moiety D- |
Noncovalent | Human breast cancer cells (MCF-7) | The formulation demonstrated enhanced cytotoxicity and mostly taken up by the cancer cells via endocytosis mechanism. It has longer survival span (44 days) compared to untargeted formulation (23 days) and free DOX (18 days). | [ |
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Partially carboxylated MWCNTs | Acid oxidation in nitric acid 70% | Liposomes without PEG-DSPE or drug | Covalent | Human fibroblast adherent cells (HEK 293) | The system can deliver a large amount of drug into cells and further preventing potential cytotoxicity effects of CNTs when administered at high dosage. | [ |
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SWCNTs | Acid oxidation, acylation and thioamidation process | Azithromycin (AZ) | Covalent |
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MWCNTs | Acid oxidation in nitric acid and sulfuric acid mixture | CHI, bovine serum albumin (BSA) and FITC | Noncovalent | HeLa cells | The conjugate obtained good stability and dispersity in aqueous solution over 30 days. It is biocompatible with HeLa cells in which the cell viability is 81% after incubation with concentration 100 |
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MWCNTs | Acid purification in HCl solution | Poly-L-lysine (PLL) | Noncovalent | None | The results showed that the MWCNTs-PLL exhibited good dispersion in water and was found to be pH-dependent. | [ |
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MWCNTs | Poly(acrylic acid) was grafted on MWCNTs through free radical polymerization | DOX, FA and iron oxide magnetic nanoparticles | Noncovalent | Human glioblastoma cells (U87) | The system demonstrated enhanced cytotoxicity toward U87 cells compared with free DOX. It was taken up by U87 cells with subsequent intracellular release of DOX, followed by transport of DOX into the nucleus leaving the nanocarrier in the cytoplasm. | [ |
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MWCNTs | Functionalization with magnetic poly(acrylic acid) | Gemcitabine (GEM) | Noncovalent | Human pancreatic cancer cells (BxPC-3 and SW1990) | The formulation had high anti-tumour activity |
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SWCNTs | Acid oxidation in nitric acid and sulfuric acid mixture | None | Covalent | Primary human umbilical vein endothelial cells (HUVE) | The |
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SWCNTs, SWCNT-COOH | None | Phospholipids (PL) were covalently conjugated to hyaluronan (HA) via amine-coupling chemistry to obtain PL-HA conjugate | Noncovalent | Murine macrophages (RAW 264.7) and the epithelial colon adenocarcinoma cells (HCT 116) | The findings showed that the CNTs-PL-HA internalized into macrophages and exhibited low cytotoxicity. Furthermore, it did not induce pro-inflammatory cytokines or mitochondrial toxicity with leukocytes in contrast to non-modified CNTs. | [ |
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MWCNTs | Acid oxidation in nitric acid and sulfuric acid mixture | Carvedilol (CAR) and PAMAM dendrimers | Covalent | None | PAMAM-MWCNTs enhanced the drug-loading capacity as well as drug dissolution significantly and, hence, it could be developed as potential nanocarrier for poorly water-soluble drug. | [ |
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SWCNTs | Functionalization with Sgc8c aptamer (targeting agent) | Daunorubicin (Dau) | Noncovalent | Human T lymphoblast cells (Molt-4) and human myeloma cells (U266) | This tertiary complex was found to be pH-dependent with controlled release function and able to selectively deliver Dau to target Molt-4 cells. | [ |
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SWCNTs | Functionalization with FA | None | Noncovalent | Human monocytic cells (THP-1) | The conjugate had low toxicity, good water solubility and was internalized by THP-1 cells. | [ |
Prior to designing a novel delivery system, there are several critical parameters which need to be taken into consideration for the preparation of a functional targeted drug delivery and this will be discussed in the section below.
For an effective delivery, the administered delivery system must be adsorbed and distributed, reach the targeted site, and then react with the desired tissue and exert a response. The uptake and biodistribution of a delivery system is heavily dependent upon its physical properties such as length, diameter, shape, size, and aggregation [
In the case of CNTs, if the length of the tubes is more than 15
Surface modification of pristine CNTs by either covalent or noncovalent surface functionalization will increase the solubility of CNTs and, hence, renders them more biocompatible in physiological aqueous environment. This is because the delivery system is administered into the human body and highly hydrophobic pristine CNTs may cause aggregation which further leads to characteristic cell changes and death (apoptosis) [
In line with this, several groups of researchers carried out
In addition to that, there were also reports which indicated that the size of the functional group (molecular weight > 60 kDa) may cause toxicity in cells [
During the fabrication of CNTs, organic materials such as amorphous carbon, extrinsic defects like catalyst residue (Fe, Ni, and Co), or supporting materials (typically silica, alumina, or magnesium oxide) embedded in the nanotubes could be harmful to biomedical applications. These transition metals can interact and catalyse oxidative species in cells through free radical generation, causing oxidative stress and morphological changes to the cellular structures [
In order to identify the issue related with impurities within the CNTs, a group of researchers have conducted a systemic study of immunological responses in mice by using iron-contaminated and extremely pure MWCNTs [
It is generally believed that the CNTs nanocarriers are dose- and time-dependent in many
A group of researchers performed an
On the other hand, Patlolla et al. investigated the cytotoxicity of oxidized MWCNTs in normal human dermal fibroblast cells, which would be among the first exposed cell types that these engineered nanomaterials can enter the human vascular system through open wounds [
There are a number of useful techniques which could be employed to characterize the physicochemical structure and morphological properties of the drug-loaded
Infrared (IR) spectroscopy is commonly used to gather information about the structure of unknown compounds or impurities remaining from chemical synthesis or molecules functionalized on the surface of the nanotubes. In the case of CNT-mediated drug delivery system, Fourier transform infrared spectroscopy (FTIR) is an appropriate technique to identify and confirm the chemical interaction between the CNTs and drugs through detailed investigation [
Fourier transform infrared (FTIR) spectra of (a) as-received SWCNT; (b) SWCNT functionalized with carboxylic acid; (c) DOX; (d) DOX-loaded SWCNT, and (e) DOX-loaded SWCNT encapsulated with chitosan-folic acid conjugate, respectively. Reproduced with permission from [
On the other hand, spectrofluorometer coupled with fluorescence microscope modified for near-IR (NIR) imaging can be used to investigate the cellular internalization of biocompatible
Raman scattering measurement is another important nondestructive technique used to evaluate the quality (e.g., purity and defect density) of SWCNTs and to assess their functionalization. It has been widely used for the characterization of CNTs to obtain useful information about the changes in band width, intensities, and frequency of the radial breathing mode (RBM), disordered mode (D-band), and tangential mode (G-band) [
The characteristic features of single-walled carbon nanotubes characterized by Raman spectroscopy. Adapted from [
Characteristic features | Wavenumbers (cm−1) | Comments |
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Radial breathing mode (RBM) | 100–300 | Radial movement of the nanotubes (A1g). When resonating conditions are met, only the Raman spectra of single-walled carbon nanotubes exhibit the RBM peak. |
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Disorder-induced mode |
1300–1400 | Characteristic to nongraphitic materials due to structural defects in the sp3 carbon atoms containing carbonaceous impurities, which is correlated with the extent of side wall chemical functionalization. |
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Tangential displacement mode (G-band) | 1500–1600 | Characteristic of carbon nanotubes, corresponding to a splitting of the E2g stretching mode of graphite, could be superimposed with the G-band of residual graphite. |
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Second order | 2450–2650 | First overtone of the D band, often called G′-band. This second order peak is sensitive to the charge exchanged between CNTs and the guest moiety. |
Thermogravimetric analysis (TGA) is used to quantitatively determine the maximum temperature limit of use for a sample as well as a measurement for the determination of various functional groups attached to the surface of CNTs [
The binding of drug or bioactive molecules to the
Nuclear magnetic resonance (NMR) spectroscopy has been employed to investigate the synthesis and attachment of functional groups to CNTs and the most commonly used are solid state 13C NMR. Solution state 1H NMR is limited for the characterization of SWCNTs due to slow tumbling and low solubility of the SWCNTs results in broad spectra [
Scanning electron microscopy (SEM) is a type of electron microscope and it is primarily used for the evaluation of morphology and suggesting the nature of material quality. SEM can be considered as the most generally used technique when it comes to the evaluation of
Transmission electron microscopy (TEM) is a valuable tool used to characterize the microstructure of samples with very high spatial resolution. TEM provides qualitative information on the morphology (size and shape), structures and defects [
Z-contrast scanning transmission electron microscopy (STEM) is one of the powerful tools for imaging individual heavy atoms in materials science applications and, most recently, this technique is used for imaging the distribution of platinum-based drug molecules attached to SWCNTs [
Atomic force microscopy (AFM) is another option used to study the surface morphology and particle size parameters of CNTs [
The advantage of using scanning tunnelling microscopy (STM) to characterize carbon nanotubes is the generation of a 3D map of the nanotubes surface morphology which is consistent with the images derived from SEM [
In the case of
This nondestructive X-ray diffraction (XRD) method is generally used to obtain information on the interlayer spacing (dhkl), phase purity, and the structural strain of a compound. In the case of CNTs, multiple layers for MWCNTs, chiralities distribution, and diameters are also observed in the XRD pattern. There are two main characteristics that can be studied in the XRD pattern of most CNTs: (i) presence of a graphite-like peak (0 0 2
In another study conducted by Mi et al. on the synthesis of polypyrrole/CNT (PPy/CNT) composites using methyl orange-iron (III) chloride-functionalized CNTs (CNT/MO-FeCl3), they were able to prove that the organometallic-functionalized CNTs were successfully developed via a novel microwave hydrothermal route by using XRD and other characterization methods [
The adsorption property (physisorption and chemisorption), specific surface area (SBET) and porosity (type of pore, pore size, pore volume, and pore distribution) of
Over the past few years,
Applications of
This antibiotic drug has been derived from erythromycin A with improved biological and pharmacodynamics properties over the parent compound. Darabi et al. used two different synthetic approaches (i.e., the acylation and thioamidation process on the nitrile groups) to functionalize SWCNTs with AZ via ester or thioamide bonds [
DOX is a commonly used DNA-interacting drug for treatment of various cancers including ovarian, prostate, breast, brain, cervix, and lung cancers. The clinical application of this anthracycline antibiotic drug is limited due to its short half-life and severe toxicity to normal tissues, particularly the cardiovascular and gastrointestinal system. Therefore, many studies have been carried out in recent years to further enhance the therapeutic efficacy while reducing the toxic effects of the drug.
Li et al. employed a difunctionalization approach to covalently link both folic acid (FA) and iron (Fe) nanoparticles to the oxidized MWCNTs [
A targeted dual delivery system to brain glioma based on PEGylated oxidized MWCNTs (O-MWCNTs) modified with angiopep-2 (ANG) as targeting ligand was successfully prepared by Ren et al. [
To explore the cancer targeting potential of the MWCNTs, Mehra et al. had developed a nanoformulation containing DOX and MWCNTs functionalized with D-
Another group of researchers, Karchemski et al., presented a new platform for drug delivery that is based on DOX-loaded liposomes covalently attached to MWCNTs [
In view of the advantage of MWCNTs and iron oxide magnetic nanoparticles, Lu et al. synthesized a dual targeted delivery system using folate-attached magnetic MWCNTs for the delivery of DOX [
Heister et al. studied the oxidized SWCNTs with DOX via PEG functionalization [
PTX is an antimitotic agent used for the treatment of various solid tumours such as metastatic breast cancer, nonsmall cell lung cancer, and drug-resistant ovarian cancer. The clinical administration of PTX is greatly limited by inefficient distribution, lack of selectivity and poor aqueous solubility.
To improve its application in clinical therapy, Tian et al. designed an efficient targeting delivery system comprising MWCNTs, FA (targeting ligand), and QDs (fluorescence labeling probes) [
Berlin and coworkers formulated PEG-hydrophilic carbon clusters (HCCs) system based on the noncovalent sequestration of the unmodified drug, paclitaxel (PTX, anticancer drug) [
PTX is a drug with bulky structure and, hence, it has poor absorption on CNTs when conjugated to CNT-dispersing polymer for drug delivery. As such, Shao et al. had constructed a novel approach for targeted delivery of the drug using SWCNTs-lipid-drug methodology in which a long chain lipid molecule (nontoxic docosanol) is covalently linked to the drug molecule [
GEM is a nucleoside analogue and a S-phase specific cytotoxic agent for chemotherapy treatments like nonsmall cell lung cancer, haematologic malignancies, pancreatic cancer, head and neck squamous cell cancer, and tumours of the breast, ovary, germ cell, cervix, bladder, and biliary tract.
To improve the efficiency for the treatment of cancer lymph node metastasis, Yang et al. developed magnetic functionalized MWCNTs as drug vehicle for delivering GEM into the regional lymph nodes under the guidance of magnetic field [
DAU is a commonly used anthracycline antibiotics for the treatment of leukemia (acute myeloid leukemia and acute lymphocytic leukemia). However, its therapeutic efficacy is limited by cumulative myelosuppression and cardiotoxicity which leads to unwanted side effects.
In order to maximize the efficacy of DAU and at the same time minimize their negative effects, sgc8c aptamer (the use of this aptamer is to target leukemia biomarker protein tyrosine kinase-7) had been used to functionalize onto SWCNTs and the resulting water soluble aptamer-SWCNTs complex was further loaded with DAU [
CIS (
In view of this, a group of researchers used hydrothermally oxidized SWCNTs as delivery vehicle for the loading of platinum (Pt)-based drug prepared differently using water or dimethylformamide (DMF) as solvents [
Another group employed a different methodology by using SWCNTs functionalized with drug CIS and epidermal growth factor (EGF) to design an effective drug delivery system that specifically targets squamous cancer cells [
The developing potential of
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors are grateful to the Ministry of Science, Technology and Innovation of Malaysia (MOSTI), for funding this project under National Nanotechnology Directorate, Grant no. NND/NA/(1)/TD11-010 (UPM vot no. 5489100) and MyPhD scholarship under the MyBrain15 program for Julia M. Tan.