Introduction to Cancer Therapy

Cancer has been one of the leading causes of death in various countries, genders, and age groups in the last two decades, with an estimated total of 10 million deaths in 2020. The major types of cancer leading to fatal outcomes are lung cancer, breast cancer, and prostate cancer.1–5 Cancer is a multifactorial genetic disease, and mutations in the cellular genetic material are necessary for its development. These mutations can gradually accumulate over a lifetime, starting from a precancerous condition and developing into a malignant tumor. Malignant diseases can be divided into acquired mutations and hereditary ones, such as the familial form of retinoblastoma.3,6 External agents that trigger malignant transformation can be divided into physical carcinogens (eg, ultraviolet and ionizing radiation), chemical carcinogens (such as asbestos) and biological carcinogens (bacteria and viruses, such as human papillomavirus, hepatitis B virus), human herpesvirus-8 and H. pylori).3,6–8

The disadvantages of conventional cancer treatments, such as chemotherapy, radiation therapy, and surgery must also be taken into account when discussing cancer (Figure 1). Treatment may not only lead to many harmful side effects, but it can also cause cancer resistance. As a rule, side effects occur when healthy cells and the malignant ones are affected. Side effects usually vary according to a given type of malignancy, person and treatment used.9 These topics will be covered in more detail later in this review.

Figure 1 Advantages and disadvantages of traditional cancer treatment techniques. Green boxes represent advantages and red boxes represent disadvantages.

Chemotherapy

One of the classic types of cancer treatment is chemotherapy, the main task of which is to eliminate tumor cells without significantly damaging healthy tissues, which is obviously impossible with classic chemotherapy agents because they are not tumor cell-specific. The history of chemotherapy began in 1940 with the use of nitrogen mustard.10,11 It is usually administered through the mouth or intravenously in regular intervals called cycles so that the organism can recover after the toxic effect.11,12 Different types of drugs are administered for chemotherapy and include alkylating agents that bind to proteins and nucleic acids, antitumor antibiotics that are produced by bacteria and generate free radicals, antimetabolites (disrupt purine or pyrimidine synthesis), topoisomerase inhibitors that are responsible for disrupting the process of DNA replication and many others.11

Radiotherapy

Radiotherapy started in 1895 after the discovery of X-ray and is used in more than a half of cancer treatment regimens.13 Over the years, huge achievements have been reported in the field with the development of 3D conformal radiation methods such as stereotactic (body) radiotherapy (SBRT) and intensity-modulated radiation therapy (IMRT). In addition, accomplishments in imaging systems have minimized radiation exposure to healthy tissue.13,14 This removes some limitations imposed by the maximum tolerated dose.13 Radiation therapy is also associated with ROS production inside the cells via water radiolysis and cytosolic Rac1/NADPH oxidase system.13,15–17 The other mechanism to influence cancer via radiotherapy is through tumor hypoxia.13

Surgery

Surgery is the oldest cancer treatment and the most effective in the case of localized primary tumors.18,19 It can be used to achieve goals such as removing the entire tumor mass, debulking a tumor in case when removing the entire tumor is impossible, or easing cancer symptoms.19 Compared to both chemotherapy and radiotherapy, surgery makes it possible to eliminate all malignant cells.18

Resistance

As was written above, cancers can also outsmart therapy efforts, and the triggered therapeutic resistance will significantly contribute to cancer mortality.20 Unsuccessful treatment will result from combined factors of pharmacokinetics, TME and the resistance mentioned above.21 The vast majority of cancer therapies are chemotherapy, and, in most cases, tumor recurrence and treatment resistance are observed.20,22–25

Non-genetic/epigenetic changes that occur independently of DNA changes play an important role in cancer development.24–27 Many studies have failed to prove the genetic evolution of the disease in a large number of patients with resistance to therapy.24,28 Non-genetic resistance can occur as drug persistence, unstable non-genetic resistance and stable non-genetic resistance.24 Drug persistence for cell culture is similar to antibiotic-resistant bacteria. It occurs in the population of malignant cells with low frequency and exhibits reduced growth and altered metabolism.24 Remarkably, they are genetically identical to the entire tumor mass which proves that epigenetic mechanisms have a curtail part in them. These cells are not mitotically active but can allow other cancer cells to adapt via genetic mutations or epigenetic changes.24,29–31

Epigenetic heterogeneity refers to the variability of the epigenetic state within a cell population as a result of stimulus.24 Two different theories have been proposed for the emergence of acquired resistance to the treatment: Darwinian theory and Lamarckian theory of cancer cell evolution (Figure 2). Darwinian theory of acquired resistance says that natural selection plays the leading role in resistance and there is always a small population of tumor cells that already have therapy-resistant potential. Darwinian theory acts via heritable variability from accidental changes in the genetic material for which positive selection is necessary. It works either by gradually increasing the number of resistant cells or by gradually increasing the stability of resistance. However, in this case, the frequency of advantageous mutations in the cell population is extremely low, and adaptations are limited by these mutations.23,24,32

Figure 2 Models of cancer resistance include Darwinian cancer resistance theory and Lamarckian cancer resistance theory. The Darwinian theory of cancer resistance is based on principles of natural selection, survival advantage and genetic diversity, resulting in highly resistant population. The Lamarckian theory, proposed by Jean-Baptiste Lamarck, suggests that cancer cells can acquire traits during their lifetime and pass them on to their offspring.24–27

The second widely known theory is Lamarckian theory which postulates that the environment plays a crucial role in developing therapy resistance. During the therapy, epigenetic changes force the drug-refractory phenotype, and selection is not involved in the spread of adaptive changes.23,32–36

Nevertheless, recent research showed us that these two theories do not exclude each other and can co-exist in the same cancer cell population, what leads to the existing cancer cell plasticity theory which is very different from healthy tissue plasticity and stem cell plasticity.23

Also, the new theory “use-it or lose-it” by Catania et al combined different driving factors like environment, phenotypic plasticity, mutations, genetic drift, and others. In this theory, positive selection is not necessary and evolved adaptations stem from existing genetic features that are activated in the specific environment, while genes that are not used are being silenced or even possibly physically lost.32

Also, Charles C. Bell suggested that cancer cells adapt via “the path of most resistance”, which includes a mix of both non-genetic changes and genetic changes.24 In addition to these theories, it has been established that information between cancer cells and TME is transferred by tumor-derived exosomes which are vesicles ranging from 30 nm to 150 nm. They contain the noncoding RNA (ncRNA) which is responsible for treatment resistance and metastasis phenotypes.37,38 The cytochrome P450 enzymes could be associated with drug resistance as well, and they are usually overexpressed in some solid tumors.39 It should also be noted that during radiotherapy, immunosuppressive pathways are activated, which can lead to accumulation of tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs), which are all radioresistant.40,41

Photodynamic Therapy in Cancer

On the contrary, photodynamic therapy (PDT) is one of the contemporary non-conventional methods for cancer treatment. In this field, PSs are used along with light of a specific wavelength which will activate them (Figure 3). It focuses on retention of these specific drugs at the tumor site after local or systemic administration.42,43 The antineoplastic effect of PDT originates from three different effects on the human body which area direct cytotoxic effect, damage to tumor blood vessels, and activation of both innate and adaptive immunity.44

Figure 3 PDT is a treatment method that uses a photosensitizing agent and light to destroy abnormal cells. PDT can be divided into two main types of reactions: Type I and Type II reactions which describe the mechanisms through which the ROS are generated to induce cell damage. In Type I reaction, the PS, after absorbing light, transfers an electron to a substrate molecule without participation of molecular oxygen. This creates a highly reactive radical species, often mentioned as a superoxide anion radical which leads to cell damage. It can react with cellular components, triggering oxidative stress and damage to proteins and DNA. This type of reaction is typically non-specific and can damage numerous cellular components. In Type II reaction, after light activation, the PS transfers its energy to molecular oxygen directly producing singlet oxygen, a highly reactive and cytotoxic species. Unlike Type I reactions, Type II reactions are highly specific and primarily target cells containing the PS. The balance between these two reaction types can be influenced by the type of PS used, the presence of oxygen in the target tissue, and the local environment. It is important to consider the generation of singlet as it is mainly responsible for the therapeutic effects of PDT.42

PDT is a promising cancer treatment. However, it is not without limitations as cancer tissue may develop resistance to PDT due to factors such as multiple treatment sessions, changes in protein expression, and alterations in gene expression after irradiation.45–47 PDT can also enhance many intrinsic survival pathways, such as NF-κB, autophagy, anti-apoptotic signals, p53 and many others.46 Also, tumor hypoxia contributes to resistance to oxygen-dependent treatments such as PDT.48,49

Nuclear factor-kappa B (NF-κB) is a transcription factor that plays an important role in both inflammatory and immune responses and cannot be easily considered a target to fight PDT inhibition as it can educate the immune system to fight neoplastic cells but can also help these neoplastic cells to survive the stress arisen by ROS.44,50

It is worth noting that PDT can also trigger autophagy that can add to either resistance or susceptibility to the cancer treatment. It is not a homogeneous process and consists of macroautophagy, microautophagy and chaperone-mediated autophagy.51,52 Autophagy can increase resistance to apoptosis by reusing dysfunctional organelles and cellular components damaged by PDT-induced ROS (Figure 4). This would maintain cellular homeostasis by providing enough energy for cellular vital functions and suppressing anticancer immune effector mechanisms.53

Figure 4 How PDT affects autophagy and cancer evolution. (A) Autophagy is an essential biological process that includes degrading and recycling cellular components. Through this process, cells undergo self-digestion, breaking down their organelles, proteins and other components. Throughout autophagy, a double-membrane structure (autophagosome) forms in stages such as initiation, expansion and maturation around the cellular material subjected to degradation. The autophagosome eventually fuses with lysosomes which leads to formation of autolysosomes and degradation of its contents for future recycling. There are different types of autophagy, including macroautophagy (the most common type), microautophagy and chaperone-mediated autophagy. This allows cells to remove damaged or redundant cellular materials, crucial for sustaining homeostasis. (B) As mentioned before, autophagy is responsible for degradation of dysfunctional or damaged cellular components and organelles, thereby providing cells with both energy and building blocks. Cells without proper autophagy mechanisms are vulnerable to PDT which causes apoptosis or cell death. However, in cancer, this can result in forming resistant cell population. (C) Signaling between tumor cells and their microenvironment can induce a temporary, drug-resistant state in malignant cells. Moreover, in cancer-associated fibroblasts, autophagy facilitates proliferation of adjacent cancer cells. (D) Autophagy also triggers the process of epithelial–mesenchymal transition, leading to more stem-like features in cells. In this case, anoikis, which is a form of cell death occurring after cell detachment, is less likely to happen. Additionally, by providing energy to disseminating cells, autophagy also assists with cancer cell dormancy and metastasis.51

PDT can also increase cell resistance to treatment in terms of autophagy by enhancing signaling interactions between cells and microenvironment or by protecting cells from anoikis and promoting metastasis.51 As several studies clearly show, autophagy has cytoprotective and prosurvival features, and depending on a variety of factors such as the cancer type, PS type and tumor stage, it can range from an antitumor effect to a protumor one.54

In accordance with everything written above, there are also different targets to overcome PDT resistance and enhance its therapeutic effect, mainly GRP78-targeting, survivin targeting, PS modification, two-photon absorption and use of NPs.55–57

Glucose-regulated protein 78 (GRP78) is a heat shock protein that is upregulated in tumor cells after PDT, and it was shown that GRP78 could be overexpressed in cancer cells (especially cells in malignant gliomas that are resistant to conventional chemotherapy and radiotherapy) and contribute to metastasis. Reduction of GRP78 concentration sequentially reduces metastasis development in xenograft models.55,58–60 Targeting via subtilase cytotoxin (SubAB) is the most selective targeting as it cleaves and subsequently inactivates GRP78.55 However, GRP78 suppression is under consideration as SubAB could be the reason for the hemolytic uremic syndrome as it is originally derived from Shiga toxigenic Escherichia coli (STEC) strains.55,61,62

The next target could be survivin which is an inhibitor of an apoptosis protein family, and usually the application of PDT results in an upregulation of survivin in a tumor. It plays an important role in stabilizing mitosis and cell adaptation; thus, the suppression of survival may enhance PDT treatment.57 The first antagonist of survivin is a phosphorothioate antisense oligonucleotide which provides a strong anticancer activity.57,63

It should also be noted that one of the limitations and drawbacks of PDT is a limited light penetration to the tissue, as only NIR light can penetrate deeper into the tissue and most of the PS absorbs light at a shorter wavelength than 700 nm. Therefore, one solution can be using two-photon absorption (TPA)-induced excitation as it uses fewer energy photons but a higher wavelength.56

Another drawback of the most commercially used PS is their poor solubility as most of them are hydrophobic and would aggregate in the aqueous environment (cellular cytoplasm and extracellular environment) which would limit their properties for PDT and could harm normal tissue. Nanoparticles could serve as an essential platform for enhancing PTD and drugs in general.56,64,65

Nanoparticles can be different metal NPs (GNPs), super-paramagnetic iron oxide NPs (SPIONs) or even quantum dots (QDs), and they are capable of carrying a large load of PS on their surface, changing their water solubility and its kinetics and also securing them from early degradation.56,66–69 Changes in kinetics usually occur through a specific mechanism, enhanced permeability and retention (EPR) effect.56,70 NP can be functionalized in many different ways, for instance, Master et al worked on PEG-PCL (Poly (Ethylene Glycol)-block-Poly (ε-CaproLactone) methyl ether, with Phtalo- cyanine-4 (Pc4) which further were functionalized with peptide GE11 specific from the epidermal growth factor (EGR) receptor which results in enhanced uptake in an SCC-15 head and neck cell line.56,71 Meanwhile, Gary-Bobo et al noticed the enhanced uptake of mesoporous silica NPs functionalized with galactose-carrying fluorescein by colorectal cancer cells.56,72

Introduction to Graphene

Graphene is a relatively new nanomaterial, discovered in 2004, which has attracted considerable attention in the scientific community due to its unique physical and chemical characteristics and which plays a vital role in various fields of science.73 Graphene has properties such as high specific surface area, good electrical conductivity, zero bandgap, biocompatibility, and high drug-loading efficiency.74–77 Graphene material is also used for lithium-ion batteries (LIB) due to its electrochemical properties.74,78,79

Graphene is a flat sheet one carbon atom thick (a monolayer). The atoms in its composition are sp2 hybridized and arranged in a honeycomb lattice.80–86 These atoms have four valence bonds, among which one is s and three other p orbitals.87 It can be obtained in various ways, in particular, mechanical exfoliation, chemical vapor deposition (CVD), chemical reduction of graphite oxide, epitaxial growth on SiC, liquid-phase separation, and unzipping of carbon nanotubes (Figure 5).80,86,88,89 CVD method is considered one of the most efficient methods for obtaining monolayer graphene or graphene films with only a few defects. However, this method requires a large number of high-purity gases and high energy costs.74

Figure 5 The example of graphene preparation methods.22,80,88,89,291

Graphene-based materials are often developed as smart platforms for nanocarriers and targeted drug delivery (Figure 6). Such carriers may be sensitive to the tumor microenvironment (TME), in particular, acidic pH and elevated levels of glutathione. In addition, the carriers can be activated by light, magnetic, or ultrasonic stimuli (“exogenous stimuli”). It was also shown that graphene can serve as a heat-conducting basis for increasing local temperature.87 Electricity can also be an exogenous stimulus. Servant et al developed electrosensitive scaffolds based on graphene for polymer implants for drug delivery.91

Figure 6 The versatility of graphene use. As a highly sought-after material, it is used for a wide range of applications in electronics, energy storage, water filtration, biomedicine, and composites.73,74,78,79

Graphene is also selectively absorbed by a tumor. In addition, a high yield of ROS products is characteristic.81 This material has delocalized π bonds responsible for the unique electronic properties that give graphene the ability to heat under NIR (near-infrared) irradiation photothermally. This property is used to ablate tumors.81 Furthermore, graphene is able to adsorb aromatic compounds on its surface due to π-π-electron interaction.81,87,92 Graphene nanomaterials can penetrate the skin, get into the lungs when inhaled and overcome the hemato-tissue barriers when injected, and accumulate in the tissues. Graphene itself accumulates in the kidneys, lungs, and liver when injected intravenously. Nanoparticles are also easily absorbed by mitochondria and cell nuclei. One of the most severe toxic effects of graphene is DNA fragmentation by cellular endonucleases. Moreover, elevated concentrations of heme oxygenase 1 (HO-1), heat shock protein 90 (HSP90), active caspase-3, and endonucleases such as deoxyribonuclease I and endonuclease G are observed.93,94 In addition to cytotoxicity, it is noteworthy that graphene particles can induce stem cell osteogenesis.93

Imperfections such as hydrophobicity and high cost had to be overcome, thus many graphene derivatives have been created. Such derivatives are graphene oxide (GO), reduced graphene oxide (rGO), graphene quantum dots (GQD), graphene nanoribbons (GNR), graphene nanoplates, and many more (Figure 7).80,81,95

Figure 7 The classification of carbon allotropes encompasses several distinct forms, including carbon dots, fullerenes, carbon nanotubes, graphene (a single layer of graphite), graphite (two-dimensional layers), diamonds, and other related variants.96,128

Graphene-based materials can also be used for visualization. Single-Wall Carbon Nanotubes (SWNTs), for example, have NIR photoluminescence and low autofluorescence, making SWNTs promising new NIR fluorophores.98,99 This review describes graphene derivatives such as GO, rGO, GA, and quantum dots in more detail.

Graphene Derivatives Graphene Oxide and Reduced Graphene Oxide Graphene Oxide

GO is a budget material with a lattice of carbon atoms bound by sp2 bonds with sp3 defects, well dispersed in water and other solvents due to functional groups on its surface. GO contains functional groups such as hydroxyl, epoxy, carboxyl, carbonyl, phenol, lactone, and quinone groups. These functional groups increase the hydrophilicity of the surface, which means that biochemical reactions and bioconjugation reactions can occur on its basal plane as well as on its edges.26,81,96,100 These functional groups create active sites for covalent or non-covalent modifications, which makes it easy to functionalize GO further using polymers, drugs, and other molecules. GO also showed a high adsorption capacity for proteins. The adsorption mechanism depends on GO morphology, oxidation state, and hydrophobicity. Polypeptides can be adsorbed on the surface of GO by the following interactions: hydrophobic–hydrophobic interaction, van der Waals interactions, electrostatic interactions, and also π–π stacking due to the large number of π-electrons on the basal plane of the GO surface.100 It also exhibits photoluminescence, the wavelength of which varies from near-UV to NIR.101

Such materials have found their way into many areas of science, including drug delivery, corrosion protection, sensors, and water treatment.82,102–104 GO also finds its application in electrochemistry and energy storage: supercapacitors, solid-state electrolytes, and GO in fuel cells.105,106 Moreover, GO can be used to fabricate composites in various forms, such as nanoparticles, hydrogels, films, and fibers.107

This material is easily obtained by oxidizing graphite with concentrated acids and strong oxidizing agents such as H2SO4, HNO3, or KMnO4.82,108,109 This method of obtaining GO was introduced in 1859 by Brodie who oxidized graphite in the presence of potassium chlorate KClO3 and fumed HNO3 + NO2 (Figure 8).109 Then, in 1937 Hofmann and Koenig made several improvements, as well as Hummers and Offeman.110 Marcano et al improved the Hummers’ method by eliminating sodium nitrate (Figure 8).110–113 This change eliminated the formation of toxic nitrous gases. Since these methods use potent oxidizing agents, the resulting GO sheets have significant defects in their crystal network.114 In addition, industrial waste can be utilized as a source of GO. It is possible to extract graphene from industrial waste, namely, the synthesis of GO from waste containing graphene precursors. Such waste may include Li batteries, biowaste (food, grass, insects), charcoal, soot, and others.115–118

Figure 8 Methods of preparation of GO.82,108,109

Mechanical characteristics of GO include internal strength, ductility, brittleness, and others. However, destroying sp2 bonds also decreases internal strength and Young’s modulus compared with graphene. To analyze the mechanical properties of GO, various methods can be used, such as tensile atomic force microscopy.81 The thermal conductivity is also relatively low, and it can be increased by imposing a polymer on the GO surface or by combining GO with metal oxide nanoparticles (for example, TiO2 or ZnO).81

Reduced Graphene Oxide

rGO is a sheet of sp2 carbon atoms with a restored π-electron graphene network and a minimum number of oxygen-containing groups.114,119 Thanks to π conjugation, improved optical absorption, and conductivity, rGO is even more suitable for PTT than GO. GO is reduced using chemical agents or physical methods, during which the carboxyl (-COOH), hydroxyl (-OH), and epoxy (-O-) groups are removed by the reducing agent, which in turn reduces the solubility in water.96 The properties of rGO are similar to those of graphene, and it is possible to change them depending on the reduction method and reduction degree. The most effective and simplest method is chemical reduction.119,120 Reductants such as hydrazine, hydrazine hydrate, dimethylhydrazine, or strong alkalis can be used, as well as green reductants such as honey, tulsi (Ocimum sanctum) leaf extract, cinnamon extract, and green tea extract.114,121,122 The following methods are available: photoreduction, solvothermal reduction, and microwave reduction (Figure 9).114,123

Figure 9 Connections between Graphite, Graphene, GO, and rGO.

Biological Properties and Bio Applications

The data on the biocompatibility of GO are rather contradictory. More and more studies show that GO has a small cytotoxicity, but its manifestation depends on the method of obtaining GO, as well as on the form of GO.81,100,124 For example, GO flakes have rough and sharp edges, which allow them to disrupt the integrity of a bilipid layer of a membrane, disrupt the membrane potential, and are widely distributed in the whole volume of tumors (in particular, tumors of the nervous system such as glioblastoma). These data demonstrate the potential of graphene as a delivery vector for both drugs and various proteins.100,119,125 This is especially important for potent aromatics that are insoluble in water. Modification of GO with polyethylene glycol (polyethylene glycol) makes it possible to create a biocompatible GO-PEG conjugate, stable in biological solutions, which can add hydrophobic aromatic molecules like SN38 (analogous to camptothecin) via π-π-stacking.126 GO-PEG can also be loaded via π-π stacking with doxorubicin (DOX), which is hydrophobic. Thus, altered GO exhibits more potent cellular toxicity.97,127 The release of imposed drugs is possible with the help of various stimuli, as discussed above. One of these incentives can be electricity. In their work, Weaver et al showed that it is possible to release the anti-inflammatory molecule, dexamethasone, in response to voltage stimulation with a linear release profile.128

Graphene itself and rGO, on the other hand, exhibit high cytotoxicity, which can also be explained by their geometry and spatial structure. rGO also has a large number of delocalized electrons due to low oxygen content, which leads to disruption of signaling pathways in the cell.81 Wang et al showed in their work that exposure to GO below 20 μg/mL on cells (Human Fibroblast Cells, HDF) exhibits low cytotoxicity with cell survival over 80%.76 At concentrations above 50 μg/mL, GO exhibits obvious cytotoxicity.76 It has been shown that GO is internalized by cells and is mainly localized within endoplasm and organelles, such as lysosomes, and mitochondria. The adhesiveness of HDF cells treated with GO was also analyzed. Western blot results showed that cells cultured with GO have markedly reduced expression levels of laminin, fibronectin, focal adhesion kinase, and the cell cycle protein cyclin D3 compared to untreated cells.76 Regarding the effects of GO on living organisms, namely mice, the injection dose of 0.1 and 0.25 mg GO per mouse did not cause death in the exposed animals and showed no clinical signs of toxicity. However, Wang et al also showed that in the group of mice treated with 0.4 mg per mouse, 4 out of 9 died, and their death was usually preceded by lethargy, inactivity, and weight loss.76

GO can also be used for work in the field of regeneration and tissue engineering, especially to restore bone tissue in severe lesions.129,130 One of the modern materials to be applied in this field is GO aerogels. These aerogels are strong, have a porous structure, and can imitate bone tissue. Another advantage is their ability to absorb growth factors on the surface.129,130

Moreover, GO and rGO show high antibacterial activity. As already mentioned, these materials mainly affect bacteria through direct contact with sharp and superoxide anion-independent oxidation.131–133 Lipid peroxidation plays an important role as well; GO nanosheets can also trap bacteria and are able to extract phospholipids from cell membranes due to dispersion interactions between GO and lipids.134,135 Also, when modifying GO and rGO with silver nanoparticles, it is possible to achieve a synergistic effect.120,134

Graphene Acid

Graphene acid (GA) is a graphene derivative that contains evenly spaced carboxylic acid groups directly bonded to the sp2 carbon backbone and has several advantages over the commonly used GO. Such advantages are aromaticity and a large number of homogeneously distributed COOH groups on the basal plane. GA luminescence has a maximum of 500nm. This product has excellent conductivity and biocompatibility and can be used as a catalyst and an electrocatalyst.136–139 By oxidation with permanganate, GA can be obtained according to Tour’s method. During the first oxidation, GO is obtained, and during the second one graphene acid. The total volume of the sample decreases by about three times, which indicates the oxidation of GO to CO2.137

Further, one of the most important applications of GA is environmental cleaning, in particular removing heavy metals, since this plays a key role in the global issue of drinking water availability. In this case, GA with 33% by weight carboxyl groups is one of the solutions to this problem, as it has proved to be able to remove highly toxic metals such as Cd2+ and Pb2+.139

It is also possible to modify GA via carboxylic acid groups. One option is covalent functionalization.136,140 For instance, Mosconi et al functionalized GA surface with ferrocene (Fc) moieties through carbodiimide chemistry.140 It allowed the introduction of up to 3.6% at. of iron as Fe2+ ions.140 The next options are non-covalent functionalization, nanoparticles, and single metal atom immobilization on GA.141,142 Bioinspired nickel bis-diphosphine HOR catalyst was grafted on GA by Reuillard et al.141 The immobilization of Sm2O3 particles by Sanad et al could be an example of functionalization with nanoparticles.143

Carbon Quantum Dots (CQDs), Graphene Quantum Dots (GQDs), and Graphene Oxide Quantum Dots (GOQDs) Carbon Quantum Dots (CQDs)

CQDs were mentioned and obtained during the isolation and purification of single-walled carbon nanotubes for the first time by Xu et al144,145 Later, Sun et al named these fluorescent carbon nanoparticles “carbon quantum dots”.144,146 This novel material solved several problems the conventional graphene had, as CQDs have good solubility and strong luminescence, for which they are referred to as carbon nanolights.147,148

CQDs are a mixture of sp2 and sp3 carbons in a quasi-spherical crystalline structure with their properties being directly linked to the π-electron state of the sp2 carbons.149 Their size is up to 10 nm.147 The photoluminescence is size dependent, the size of the conjugated π-domains influences photoluminescence, with changes either promoting or inhibiting the direct transition of electrons from the conduction band to the valence band. This transition is responsible for generating band gap fluorescence.149–151 CQDs also show a clear dependence of photoluminescence on the excitation wavelength.147 When photoexcited, CQDs demonstrate outstanding capabilities as both electron donors and acceptors.147,152 As well, CQDs exhibit optical absorption in the UV region, with a tail extending into the visible range.147

There are various methods of CQD synthesis that are classified into top-down and bottom-up routes. Top-down methods involve the reduction and fragmentation of large sp2 carbon domains into smaller components. These techniques include arc discharge, chemical oxidation, sonication, hydrothermal methods, and others (Figure 10).144,146,153–155 Bottom-up approaches for synthesizing CQDs mean constructing the material from precursor molecules, yielding particles with consistent sizes and control over size distribution. These methods cover hydrothermal treatment, ultrasonic treatment, thermal decomposition, pyrolysis, carbonization, microwave synthesis, and the electrochemical method (Figure 10).156–158

Figure 10 Various preparation methods can be employed to obtain CQDs, utilizing different carbon sources and synthesis procedures. The most usual division of preparation methods are top/down and bottom-up methods. Commonly used carbon sources include citric acid, glucose, and carbon black, while synthesis procedures range from hydrothermal and microwave-assisted methods to electrochemical and pyrolysis techniques.156–158

Graphene Quantum Dots (GQDs)

GQDs are a new zero-dimensional material with lateral sizes up to 100 nm, most often 3–20 nm, single nanosheets of sp2 carbons with luminescence properties, exceptional optoelectronic properties, and excellent biocompatibility.159–163 Also, GQDs are low-cost, optically and chemically inert, and easy to fabricate. GQDs have applications in areas such as drug delivery, bioimaging, sensors, photovoltaic devices, and catalysis.161,164–166

GQDs have negatively charged carboxyl groups, which can provide good electrostatic properties for further functionalization. Conjugated π–π bonds also contribute to it.167 GQDs are considered non-toxic, particularly to human cell lines. However, cytotoxicity can increase due to the nonspecific adhesion of dots to the cell membrane.166,168

There are various methods for synthesizing GQDs, which are classically divided into top-down or bottom-up ones. The top-down method involves destroying the graphene sheet, CNTs, the graphite using arc discharge, chemical or laser ablation, chemical or electrochemical oxidation, and ultrasound.162,169,170 The bottom-up methods include carbonizing organic precursors such as citric acid, amino acids, carbohydrates, and some aromatic organic compounds using microwave treatment, hydrothermal treatment, solvothermal treatment, or other methods.166,169–171 GQDs were first synthesized by Pan et al in 2010.162 They had a crystalline structure of single or a few layered graphene and had an elliptical or circular shape. However, there may also be quadrate, hexagonal, as well as triangular GQDs.162,169

Graphene Oxide Quantum Dots (GOQDs)

As it was mentioned earlier, GO attracted attention among researchers due to its minimal toxicity, biocompatibility and hydrophilicity. GOQDs are nanoscale carbon-based materials that are derived from GO. These quantum dots possess unique optical and electronic properties due to their small size up to 30 nm and quantum confinement effects.160,172–174

On their basal plane and at the edges, GOQD have oxygen-rich functional groups such as epoxy, carbonyl, hydroxyl, and carboxyl groups, which facilitate further functionalization through electrostatic interaction, π–π stacking and chemical reactions.175

Preparation of GOQDs includes oxidizing, exfoliating, and cutting carbon precursors into nano-sized particles using chemical oxidation, hydrothermal, or solvothermal treatments under harsh conditions, often requiring concentrated acids like HNO3 or H2SO4 for prolonged time.172,176,177

GOQDs have applications in diverse fields, regarding their distinct characteristics. They find utility in removal water pollutants, biological imaging, optoelectronic sensors, LEDs, fluorescent agents, lithium-ion batteries, and many others.160 GOQDs hold promise for biomedical applications due to their non-toxicity, hydrophilicity, and high light-emitting efficiency, which originate from quantum confinement and edge effects associated with their oxygen-functional groups.156,177

Biological Properties and Bio Applications

Due to their properties, CQDs and GQDs can be widely used in various fields, including drug and gene delivery, biological imaging, electrochemiluminescence sensors, electrochemical sensors, and more (Figure 11). Numerous studies have confirmed the DNA fragmentation activity of therapeutic drugs when used in conjunction with GQDs.178 For example, Fang et al developed a multifunctional GQD complex of hollow carbon nanoparticles to encapsulate DOX at the average size of 120 nm. They were also able to generate heat when irradiated with an NIR laser for synergistic photothermal therapy (PTT).169,179 When functionalized with an antibody, selective destruction of cancer cells in vitro is possible.91

Figure 11 The features of CQDs.159,160,162,163,169,180

It has also been reported that GQDs are a promising potential treatment and a way to relieve the symptoms of amyloidosis, the essence of which is the aggregation and deposition of amyloid proteins in plaques around cells, which subsequently causes organ and tissue failure. Misfolded amyloid proteins are also the cause of brain tumors, Alzheimer’s disease, Parkinson’s disease, and stroke. GQDs can act as inhibitors of aggregation and, consequently, toxicity of amyloid proteins.181,182

Moreover, the fluorescence of CQDs and GQDs can be used to visualize living cells in the NIR range and to selectively recognize and bind to cancer cells, such as B-cell lymphoma.91 They are also characterized by their resistance to photobleaching due to their crystal structure. From this point of view, GQDs are superior to CQDs.183,184 GQD could be further functionalized with PEG and can selectively accumulate in the tumor after being injected as an agent for tumor fluorescence imaging.185

Shi et al developed coated GOQD magnetic nanocomplexes with high fluorescence, which can improve the diagnosis of cancer in infected blood using multiphoton luminescence.176 Pramanik et al reported short sequences of artificial RNA conjugated graphene oxide-based for improved two-photon selective imaging of breast tumor cells.186 Also, GOQD can be used as a two-photon fluorescence probe for imaging multiple drug-resistant bacteria (like Methicillin-resistant Staphylococcus aureus).187

Nanotubes and Nanohorns Single-Walled Nanotubes and Multi-Walled Nanotubes

Carbon nanotubes can be divided into two groups: single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs). They are rather different in terms of their physical properties due to their structural differences, namely the number of carbon layers. SWCNTs, respectively, consist of a single layer of graphene with a diameter of 0.4 to 2 nm. MWCNTs, on the other hand, consist of two or more sheets of graphene with a distance between layers of 0.34 nm that form cylinders, so their diameter is from 1 to 3 nm.188 SWCNTs can be further divided into the following three groups: armchair, zigzag, and chiral (Figure 12).189 Carbon nanotubes have properties such as high rigidity (Young’s modulus 1 TPa) and strength with a tensile strength of 60 GPa for SWCNTs and 150 GPa for MWCNTs and stability at high temperatures (in vacuum and air, the limiting temperatures are 2800 °C and 750 °C, respectively). They also exhibit high electrical conductivity and high heat transfer coefficient.189–192 Their application is possible in areas such as electronics, sensors, and biomedicine, including the delivery of drugs to target organs.193–195 They have a large surface area, which enables the conjugation of various molecules on the walls in large quantities. Molecules containing aromatic groups can also be noncovalently bonded due to strong π–π interactions.195

Figure 12 Carbon nanotubes are nanostructures that take the form of cylindrical tubes by rolling up a sheet of graphene. They can be divided into the following three types: zigzag, armchair, and chiral. Zigzag nanotubes have chirality (n,0), hexagons at the tube end with edges resembling a zigzag pattern, and are typically metallic, with no band gap, affecting their electronic properties. Armchair nanotubes have chirality (n,n), and hexagons at the tube ends, with edges running parallel to the tube axis, resulting in a flat, open-ended structure. Depending on the tube’s diameter, they can either be metallic or semiconducting. The tunability of the electronic properties of chiral nanotubes makes them versatile for various applications.189,196

CNTs can typically be synthesized by the following methods: arc discharge, laser ablation, and chemical vapor deposition. The synthesis of individual SWNTs requires catalysts such as cobalt, nickel, iron, and others. In the synthesis by the arc discharge method, a high temperature of more than 3000°C is required, which mediates the evaporation of carbon atoms into the plasma to form various CNTs. Iijima used this method to synthesize MWCNTs. The next synthesis method, chemical vapor deposition, uses such precursors as methane, ethylene, and similar. The laser ablation method uses the evaporation of graphite in an electric furnace heated to 1200°C.188

Carbon nanotubes also have significant antimicrobial activity, which may be due to the synergy of physical and chemical effects. The intracellular content of bacterial cells is released through physical damage to the membrane, as described above. This process, however, depends on the size of the CNTs. Small CNTs can be internalized by bacteria and disrupt the metabolic processes in cells through oxidative stress.89

CNTs have several drawbacks, as well. Firstly, CNTs are inherently hydrophobic and insoluble in most biological media, making them difficult to use as a material for drug delivery or biomolecules. To overcome this problem, CNTs are functionalized for improved solubility and biocompatibility, allowing further drug modification with growth factors, antibodies, and so on. The methods of such functionalization can be noncovalent functionalization outside CNTs, functionalization of defects, covalent functionalization, and encapsulation of bioactive molecules inside CNTs.195,197 Also, functionalization methods can be divided into chemical and physical. The surface of CNTs can be chemically modified by oxidation, cycloaddition, and addition of functional groups such as carbenes, nitrenes, and similar. Physical modification can include methods such as the π–π stacking described above, coating with polymer chains, using surfactants, and adsorption via hydrophobic interactions.190

Secondly, CNTs may exhibit toxicity, which may be caused by their spatial conformation. Thus, carbon nanotubes and other fibrous materials will have an impact on living cells due to their jagged and flat edges.86

Nanohorns

Single-Walled Carbon Nanohorns (SWNHs) were first presented by Iijima in 1999. As a rule, SWNHs do not exist separately; they exist in a spherical aggregate of 80–100 nm in size in the amount of about a hundred.198–200 The cones are formed by cutting a graphene wedge and seamlessly