Haifeng Dong and Yu Cao
Beijing Key Laboratory for Bioengineering and Sensing Technology, Research Center for Bioengineering and Sensing Technology, School of Chemistry & Biological Engineering, University of Science & Technology, Beijing, PR China
Cancer is one of the most deadly diseases facing humanity [1]. The current standard cancer management includes stage determination, chemo/radiation therapy, and surgical resection. Despite great process has been made in the past few decades, early diagnosis and efficient treatment of cancer are still challenging to overcome [2]. Molecular imaging is a useful tool to monitor in vivo biochemical events and the development of nanomaterials as biomedical imaging agents is a very promising method to obtain detailed images in living systems [3, 4]. It is beneficial for the researchers to follow the distribution of the drug inside the organism and gives further hints for the optimization of disease treatment to combine the drug delivery features with imaging techniques [5, 6]. The combination of diagnostic tools such as optical imaging with therapeutical approaches such as chemotherapy and phototherapy gives rise to promising theranostic nanomaterials [7–9]. As the forefront of theranostic nanomaterials for cancer therapy, the unique optical properties of carbon nanomaterials such as fullerenes [10], nanodiamonds (NDs) [11, 12], carbon nanotubes (CNTs) [13, 14], graphene and its derivatives [15, 16], and carbon quantum dots (CQDs) [17] (Figure 3.1) have inspired extensive studies due to their great potential applications in the field of optical bioimaging analysis and phototherapies [18, 19].
In this chapter we will cover the recent progress in optical biological imaging analysis and phototherapies using carbon nanomaterials.
Since most pristine carbon nanomaterials are highly hydrophobic due to the sp2 carbon nanostructures, proper functionalization is necessary to improve their water solubility and biocompatibility before their biomedical applications. Generally, there are two primary strategies of functionalization: covalent or noncovalent. Covalent modifications usually involve introduction of hydrophilic functional groups (e.g. hydroxyl groups, carboxyl groups, and amino groups) for the further conjugation of protecting polymers (such as polyethylene glycol, PEG), targeting ligand, or drug/gene cargos [20–24]. The covalently functionalized carbonaceous nanomaterials are usually stable. However, the limitation lies in that this type of functionalization method inevitably destroys partial material structure, causing the loss of certain intrinsic properties (e.g. photothermal capacities). Compared with covalent functionalization, the reaction condition of noncovalent functionalization is comparatively mild, which involves coating the carbonaceous nanomaterials with amphiphilic molecules [25]. The hydrophobic motifs of the amphiphilic molecules could be anchored onto the material's surface with the hydrophilic ends extending to the aqueous solution and maintaining the stability of the whole material. Noncovalent interactions include electrostatic forces, π–π interactions, hydrogen bonding, and van der Waals forces. However, lower stability of noncovalent conjugates is the major concern for this type of functionalization. A lot of thought about stability and design of the nanomaterial must be taken into account before choosing an appropriate functionalization method for any carbonaceous nanomaterial. A balance between stability and structural integrity must be maintained before any further biomedical applications.
Different carbon nanomaterials require different strategies of surface functionalization to make them soluble in aqueous environment and compatible with cells and tissues. Fullerenes are typically covalently functionalized through chemical reactions directly with the carbon atoms in the sp2 carbon shell [26], and a library of standard chemical reactions have been developed for fullerene chemistry. CQDs and graphene quantum dots (GQDs) are by nature rich in ─OH and ─COOH functional groups, which can easily form hydrogen bonds with water molecules and thus endow them with good solubility in aqueous environment; nonetheless, it is still desirable to further functionalize them with PEG or other functional groups to increase biocompatibility. CNTs [21, 27] and graphene [28, 29], both of which feature continuous graphitic honeycomb structures expanding over submicrometer to micrometer scales, can be either covalently or noncovalently functionalized to impart water solubility, depending on the need for specific biological applications. Nanodiamond [22], on the other hand, is similar to CNTs and graphene that both noncovalent and covalent functionalizations have been reported to increase their water solubility and biocompatibility.
Many organic fluorophores absorb and emit light in the visible spectral range, which can have some drawbacks. In complex biological systems like cells, or especially in living organisms, the absorption and autofluorescence of the tissue and body fluids are a major problem for optical imaging. The absorption reduces the transmission of the excitation light, and also the emitted fluorescence signal is significantly weakened, or even completely quenched. To overcome this issue, researchers have found that in the near‐infrared (NIR) region between 650 and 950 nm, the so‐called NIR I, hemoglobin and water, as the two main absorbers, have low molar extinction coefficients [30]. NIR light can penetrate the tissue, and the use of NIR‐emitting dyes with emission maxima in the NIR I as fluorescent labels allows for deep tissue imaging. Another optical window, the NIR II, has been identified later in the spectral range between 1000 and 1350 nm [31].
Several spectroscopic techniques can be utilized for biological in vitro and in vivo imaging. One of the most common techniques is fluorescence imaging. Carbon nanomaterials can be used for their intrinsic fluorescence properties or can be tagged with fluorescent molecules.
A fullerene nanoparticle is made of a closed shell of graphene that contains conjugated double bonds [32]. Therefore, electronic transitions are expected to take place between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of the large π‐conjugated system, corresponding to strong absorption mostly in the ultraviolet (UV) region (<400 nm), which should be followed by fluorescence emission in the visible window. However, the fluorescence quantum yield of unmodified, pristine fullerene is very low, on the order of 10−5 –10−4 , and the photoluminescence decreases at increasing temperature with very weak emission at room temperature due to nonradiative recombination of excited nanoparticles coupled with phonons [33].
Semiconducting single‐walled carbon nanotubes (SWCNTs) show a structure dependent fluorescence in the biological important NIR II window that have been widely studied [34, 35]. The band gap fluorescence of semiconducting SWCNTs originates from light absorption at a photon energy of E22, creating an electron–hole pair. This initial excitation is followed by a nonradiative relaxation of the electron to the conduction band and then the fluorescence event with a photon energy of E11. The fluorescence quantum yields of single‐walled nanotubes (SWNTs) are low, typically lower than 0.01 for macroscopic samples of SWCNT [36, 37], and show a strong dependency on environmental factors. Recent advance in chemical doping of SWCNTs through oxygen or sp3 incorporation has led to largely increased fluorescence quantum yield [38]. Also in the visible region, fluorescence of well‐dispersed CNTs was reported, which showed a large dependence on the type of the CNT functionalization [39].
GQDs and CQDs are another example of fluorescent carbon nanomaterials, which are widely researched for biological imaging applications. The mechanisms of the GQD fluorescence are currently under investigation and still, the physical origin of the fluorescence seems not to be fully understood [40, 41]. However, structurally perfect graphene sheet is a zero‐band gap semiconductor with the conduction band and valence band touching at the K points and thus show no luminescence upon photo‐excitation. One common strategy to create fluorescent graphene derivatives is the introduction of defect sites to open the band gap predictably due to quantum confinement and edge effects. Besides, the modification of heteroatoms (such as O, N, P, and S) in the GQDs and CQDs also plays a crucial role in determining the nonradiative decay rate and even the band gap. An interesting feature of GQDs is their excitation wavelength dependent emission. In line with modulation of the excitation wavelength, the emission wavelength undergoes a shift.
The distinctive optical properties of graphene and its derivatives allow them for biomedical imaging in vitro and in vivo [42]. Although pristine graphene is a zero‐gap semiconductor as aforementioned, due to the highly heterogeneous chemical and electronic structures of graphene oxide (GO), strong fluorescence emission has been observed from GO in a broad spectrum, ranging from the UV to NIR, the exact mechanism of which remains to be fully elucidated and has largely been believed to arise from the electronic transition between the nonoxidized, pristine sp2 carbon domain and the oxidized boundaries of GO sheet.
Due to the lack of sp2 graphitic carbon, the intrinsic fluorescence emission of nanodiamond can be led back to the presence of nitrogen‐vacancy (N‐V) centers in the structure of the nanodiamond. Its fluorescence is very robust against photo bleaching, making nanodiamond an ideal fluorescence label for long‐term tracking of biomarkers and cells [43, 44].
Fullerene and its derivatives have relatively less bright fluorescence compared to other carbon nanomaterials, and the photodynamic property of fullerene also raises concern on generating active oxygen species after photoexcitation and affecting the cell viability. Nonetheless, there are a handful of reports on using C60 and C70 as fluorescent biomarkers. Prasad et al. have studied the internalization of 750 nm fluorescence emitting C60 aggregates into the cytoplasm of MCF10A cells, which are normal human mammary epithelial cells, with fluorescence costaining of F‐actin using fluoresceinisothiocyanate (FITC) phalloidin [45]. Chung et al. performed the fluorescence imaging of HeLa cells in different excitation/emission channels with distinct colors for C60‐tetraethylene glycol (C60‐TEG) proceeded the tunable fluorescence color depend on the concentration of C60‐TEG in the solution [46]. Lee et al. have reported on using hyaluronated C60 particles for tumor targeting and in vivo fluorescence imaging on tumor‐bearing mice [47]. The hyaluronate‐C60 bioconjugate allows targeted in vivo tumor fluorescence imaging by detecting the deep red fluorescence of hyaluronated C60 at 710 nm under an excitation of 635 nm (Figure 3.2).
GQDs and CQDs are widely applied for optical bioimaging [48, 49]. As early as 2011, Zhu et al. synthesized GQDs through the hydrothermal method from GO, which had a fluorescence quantum yield as high as 0.11, showing excitation‐dependent and solvent‐dependent fluorescence in the visible region (Figure 3.3a) [50]. The results demonstrated that GQDs could be used for fluorescence imaging of cancer cells. Since 2014, high‐quality studies on the preparation of CQDs and GQDs and their application for bioimaging have been published [52–54]. Similar to GQDs, the emission wavelengths of the carbon dots (CDs) shifted, depending on the excitation wavelengths. GQDs and CQDs are also applicable for two‐photon bioimaging [55]. In a two‐photon excitation process, the fluorophore absorbs two photons simultaneously and is excited to a higher energy state, after which the absorbed energy is released by emitting only one photon with a shorter wavelength than the absorbed photons as the molecule returns to the ground state. The group of Sun first observed one‐ and two‐photon fluorescence in CQDs upon excitation with a 458 nm laser or a femtosecond pulsed laser at 800 nm, respectively [51]. When the CQDs were internalized by MCF‐7 cells irradiating with the pulsed 800 nm laser, green two‐photon luminescence was observed (Figure 3.3b). Also, GQDs were successfully used for two‐photon‐induced fluorescence for cellular and deep tissue imaging [56].
Fluorescent CNTs have been widely studied as in vitro and in vivo imaging agents. The first report of NIR‐emitted SWCNTs for cellular imaging dates back to 2004, when Weisman et al. incubated macrophage cells with SWCNTs [34]. The SWCNT NIR emission remained intact upon cellular uptake but underwent a slight bathochromic shift. This study rendered the microscopic detection of fluorescent SWCNTs inside of cells as a powerful method to investigate the interactions of CNTs with cells. Neves et al. investigated the internalization and fate of CNTs inside cells in detail [57]. The results revealed that an endocytic pathway was involved in the internalization of RNA‐wrapped, oxidized double‐walled CNTs (oxDWNT‐RNA) (Figure 3.4a,b). The nanotubes were found in clathrin‐coated vesicles, after which they appear to be sorted in early endosomes, followed by vesicular maturation, become located in lysosomes. The first in vivo research was proceeded on the larvae of Drosophila melanogaster which were fed with food‐containing SWCNTs [58]. The NIR fluorescence signal could be detected in the living larvae and was used for imaging (Figure 3.4c–e). Dai et al. for the first time studied the NIR fluorescence of SWCNTs on mice in 2009 [59]. They utilized different kinds of NIR fluorescent phospholipid‐polyethylene glycol (PL‐PEG) coated SWCNTs for targeted cell imaging by attaching an arginine‐glycine‐aspartic acid peptide, also known as an Arg‐Gly‐Asp, or RGD peptide. In vivo studies were carried out with nude mice and tumor bearing LS174T mice. After injection, the PL‐PEG coated SWCNTs showed bright NIR fluorescence imaging with good contrast (Figure 3.4f,g). The SWCNTs could be identified in the vasculature under the skin and also in deeper organs such as liver and spleen. With time, the SWCNTs were cleared from the body. As discussed above, SWCNTs have great promise for use as effective biomarkers for in vitro and in vivo imaging due to their excellent properties such as low cytotoxicity, high photostability, absence of quenching, and photo bleaching if functionalized properly.
Graphene and its derivatives have found applications in bioimaging owing to their intrinsic optical properties. The Dai’s group for the first time utilized the NIR photoluminescence of nGO‐PEG for cell imaging [60]. It was found that GO and nGO‐PEG exhibited fluorescence broadly from visible to NIR‐I window could be used for cell imaging with little background. Although the quantum yield of nGO‐PEG fluorescence was rather low, cell imaging using its intrinsic fluorescence has been successfully realized owing to the extremely low auto‐fluorescence background of biological tissues in the NIR‐I window.
Nanodiamonds have been used for bioimaging for some time and the emission wavelength of nanodiamonds is located in the visible region, with characteristic green to red emission between 550 and 800 nm [61]. Chang et al. reported the application of fluorescent nanodiamonds as nonbleaching imaging agents for human 293T kidney cells with no observed cytotoxicity in early 2005 [62]. After that, so many researches have been studied signaling the huge potential of nanodiamonds as high‐quality imaging agents. In consecutive work, the uptake mechanism of nanodiamonds was further studied showing that nanodiamonds entered the cells mainly by endocytosis, but also on a clathrin‐mediated pathway [63]. While larger nanodiamond nanoparticles localized in vesicles, small nanodiamonds appeared to be in the cytoplasm. Nanodiamonds were also used as fluorescent probes for in vivo studies. The interaction of two different kinds of nanodiamonds (with 5 and 100 nm diameter) with the ciliated eukaryotic unicellular organisms Paramecium caudatum and Tetrahymena thermophila was studied. The nanodiamonds revealed low toxicity and excellent fluorescence properties. The effects of fluorescent nanodiamonds were also investigated on Caenorhabditis elegans and zebrafish [64, 65]. It was found that nanodiamonds could be delivered to the embryos of the next generation providing great capacity for fluorescent in vivo imaging (Figure 3.5). Cheng et al. investigated the long‐term stability and biocompatibility of 100 nm diameter nanodiamonds on mice [66]. The in vivo whole‐body imaging and ex vivo examination of various body tissues suggested that no toxic effects of the nanodiamonds on the mice, rendering promising application of nanodiamonds for in vivo imaging.
Due to their excellent chemical and physical properties like a high surface area, CNTs are widely employed in biomedical applications such as drug and gene delivery. Fluorescein derivatives are widely used as fluorescent labels in combination with CNTs. In 2004, Prato et al. used fluorescein‐tagged SWCNTs to study the internalization of CNTs by human 3T6 and murine 3T3 fibroblasts [67]. Then the cellular uptake mechanisms of functionalized CNTs were studied in detail. It was shown that covalently functionalized CNTs, bearing different fluorescein‐based labels for imaging in addition to other functional groups, were taken up by a variety of different cell lines. The group of Dai exhibited one example for noncovalently functionalized fluorescein labeled SWCNTs [68]. Fluorescein functionalized PEG was physiosorbed onto the surface of SWCNTs, affording stable aqueous suspensions. The fluorescein labeled DNA was also used to functionalize SWCNTs noncovalently to monitor the cellular uptake of the carbon nanomaterial [69].
The low quantum yield of GO fluorescence and the auto‐fluorescence interference of biological tissues have limited the applications of GO in bioimaging especially in vivo animal imaging. Therefore, a number of groups used external fluorescent dyes to label GO‐PEG for in vitro and in vivo imaging. Liu et al. labeled nGO‐PEG with a NIR dye, Cy7, by conjugating the dye molecule at the PEG terminal via covalent modification to avoid significant fluorescent quenching [70]. Increasing fluorescence signals were found in the tumor over time, indicating efficient tumor passive targeting of nGO‐PEG in several different xenograft tumor models (Figure 3.6). In order to avoid fluorescence quenching or photo bleaching, radiolabeling is a much more sensitive method for in vivo bioimaging. Liu’s group developed a method to label nGO‐with 125 I by anchoring iodine atoms on the defects and edges of GO [71]. Then they fabricated 64 Cu labeled nGO‐PEG for in vivo positron emission tomography (PET) imaging [72]. Furthermore, by noncovalent modifying with DNA or miRNA detection probe, graphene and its derivatives can be used for in vitro or intracellular miRNA imaging.
Besides the intrinsic fluorescence of nanodiamonds, some reports focus on nanodiamonds functionalized with fluorescein and other dyes for bioimaging. In 2008, the group of Hwang modified fluorescein on the surface of the nanodiamonds for cell imaging [73]. The bright green luminescence of the fluorescein dye was observed inside the cell. Zhang et al. developed a nanodiamond‐based drug delivery system by functionalizing with an anti−epidermal growth factor receptor (EGRF) antibody for targeting and a fluorescein labeled drug‐oligonucleotide conjugate for therapy [74]. The fluorescence of the fluorescein could trace the nanodiamonds inside the cells. As a kind of commonly used anticancer drugs, doxorubicin (DOX) with fluorescence was loaded on nanodiamonds to investigated in vitro and in vivo cancer therapy efficiency [75].
Chemotherapy and radiotherapy are major therapeutic approaches for the treatment of a wide variety of cancers in recent year. However, one of major disadvantages of chemotherapy and radiotherapy is their limited specificity to cancer cells and could lead to undesired side effects to normal tissues and organs. Phototherapies, mainly including photothermal therapy (PTT) and photodynamic therapy (PDT), are able to destruct cancer cells upon specific light irradiation [6, 76–78]. With the help of nanotechnology, phototherapeutic nanoagents could specifically target cancer via either passive or active tumor targeting. Therefore, phototherapies exhibit remarkable advantages in terms of enhancing cancer killing specificity and reducing side effects, in comparison to conventional cancer therapies. In the past few years, phototherapies based on the unique optical and chemical properties of carbon nanomaterials have aroused increasing interest [2, 16, 79, 80].
PTT uses external light‐induced hyperthermia on malignant tissues to kill abnormal cells while avoiding damage to healthy tissue [81, 82]. Carbon nanomaterials such as CNTs and graphene selectively accumulate in tumors through the enhanced permeability and retention (EPR) effect as well as demonstrate extremely high levels of intrinsic absorbance in the biological transparency windows located within the NIR window (750–1700 nm) [16, 83]. Additionally, the strong fluorescence of carbon nanomaterials with emission wavelengths ranging from visible to NIR‐II window allows for simultaneous imaging and treatment. Compared to other nanomaterials including various gold nanostructures [84–86], copper sulphide (CuS) nanoparticles [87–89], and organic nanoparticles [90], the proper surface modified carbon nanomaterials can circulate in vivo with longer half‐lives.
SWCNTs were the first carbon nanomaterial used for PTT. In 2009, Choi et al. reported photothermal ablation of human epidermoid mouth carcinoma KB tumor in mouse intratumorally injected with PEGylated SWCNTs using 808 nm laser irradiation [91]. A significantly reduced tumor volume was found for the treated mice compared to the control. In 2010, Dai’s group reported the use of SWCNTs noncovalently functionalized with PL‐PEG and C18‐PMH‐PEG to achieve a high passive tumor uptake based on the EPR effect for efficient PTT [92]. This was the first work to image tumors in the NIR‐II window with SWCNTs and the first systemic injection of these nanomaterials for PTT. Since then, a lot of studies have been done for PTT to decrease the injection dose and irradiating power density [93, 94].
Besides SWCNTs, graphene, and its derivatives have also been successfully applied for PTT owing to their strong absorbance in the NIR window [95]. In 2010, Liu et al. for the first time studied the behaviors of PEGylated nano‐GO in mice after intravenous (i.v.) injection by a fluorescent labeling and imaging method and revealed the EPR effect of cancerous tumors [70]. Owing to the high uptake inside the tumor, the photothermal agent nGO‐PEG achieved efficient tumor ablating with 100% of tumors eliminated under 808 nm laser irradiation at a power density of 2 W cm−2 for five minutes (Figure 3.7a). While the injected doses (20 mg kg−1 ) and NIR laser densities (2 W cm−2 ) are comparable to that of other photothermal agents such as gold nanomaterials, showing that nGO‐PEG appeared to be an excellent in vivo tumor NIR PTT agent without exhibiting noticeable toxicity. It has been well known that the reduction of GO could lead to dramatically enhanced optical absorbance in the NIR region. In order to reduce the requisite dosage and laser power density for PTT, Dai et al. reported that ultra‐small reduced nanographene oxide (nRGO) could be prepared by the reduction of PEGylated GO, indicating a significant increase of absorbance in the visible to NIR window (Figure 3.7b) [96]. By further coating with PEGylated phospholipid and conjugating with an RGD‐targeted peptide, the nRGO‐PEG could act as a highly efficient targeted photothermal agent for in vitro selective cancer cell ablation.
Different from PTT, PDT relies on reactive oxygen species (ROS) such as single oxygen (1 O2) produced by photosensitizer (PS) molecules to kill cancer cells under the irradiation of light with appropriate wavelengths [97–99]. Three pivotal factors account for PDT therapeutic efficiency: (i) PS photo‐transferring efficiency, (ii) light of the wavelength that activates the PS, and (iii) molecular oxygen. While less research has been conducted on using carbon nanomaterials as PS for PDT, the synergistic effects of combining carbon nanomaterials with PS significantly boost the efficacy of cancer treatment. PS can be noncovalently loaded onto the aromatic surface of carbon nanomaterials coated in biocompatible polymers through π–π stacking for activation after laser excitation.
Fullerenes such as C60 and C70 have been found as PSs with a high efficiency of ROS generation, allowing for efficient PDT using fullerenes without loading additional photosensitizing molecules [100]. Hamblin et al. reported PDT using N‐methylpyrrolidinium‐fullerene for the treatment of colon adenocarcinoma by intraperitoneal injection of the functionalized fullerene into the abdomens of the tumor‐bearing mice and found significantly slowed tumor growth compared to the control group [101]. The unique property of fullerenes in the efficient conversion from photons to ROS has made fullerene derivatives competitive PSs for PDT applications in in vivo animal and even preclinical settings [102].
CNTs can either act as PSs themselves or load exogenous PSs for PDT. Imahori et al. found semiconducting SWCNTs could generate ROS more efficiently than metallic SWCNTs, opening up the possibility of using chemically separated, pure semiconducting SWCNTs as PSs for PDT [103]. Zhang et al. have demonstrated that photodynamic effects of SWCNTs highly depend on the modification method of the nanotubes, and effective in vivo tumor destruction has been realized based on PDT using polyethyleneimine (PEI) and polyvinylpyrrolidone (PVP) modified SWCNTs [104]. SWCNTs can also be used as nanocarriers to deliver PSs to target tissue such as the tumor, owing to the high accumulation of SWCNTs inside tumor tissue based on the EPR effect. Lee group reported PEGylated SWCNTs loaded with Chlorin e6 (Ce6) could be applied for PDT‐based tumor treatment in mice [105].
By taking advantage of the ultrahigh loading of molecules through π–π stacking, nano‐GO has been used to deliver PSs in vitro at high concentrations [106]. The first study of the graphene‐based PDT was reported by Shi et al. In that work, zinc phthalocyanine (ZnPc), a widely used PS molecule, was loaded on the surface of nGO‐PEG via p–p stacking and hydrophobic interactions. The obtained nGO‐PEG‐ZnPC exhibited significant cytotoxicity toward cancer cells under Xe light irradiation. In a later work, Huang et al. reported that folic acid‐conjugated GO could load Ce6 for targeted delivery to fluorescence resonance (FR) positive cancer cells and achieved effective cancer cell photodynamic destruction using a 633 nm He─Ne laser irradiation [107].
Recently, GQDs have been reported as PS to efficiently generate singlet oxygen (1 O2) for PDT [108, 109]. Ge et al. presented GQDs as a new PDT agent to produce 1 O2 via a multistate sensitization process, resulting in a quantum yield of about 1.3 [110]. The as‐prepared GQDs exhibit a combination of properties, including broad absorption from the visible to the NIR, deep‐red emission, good aqueous dispersibility, high photo‐ and pH stability, and favorable biocompatibility, which enable them to act as a multifunctional nanoplatform for the simultaneous imaging and highly efficient in vivo PDT of cancer (Figure 3.8).
Carbon nanomaterials with exciting chemical, mechanical, and optical properties have had a tremendous impact on the development of theranostic methods for in vitro and in vivo biomedical applications, including imaging, drug delivery, and phototherapies. Although all made of the same chemical element, these nanomaterials with different allotropic forms of carbon exhibit distinct properties and behaviors, depending on how the carbon atoms are bonded to form the larger structures on the nanoscale and on the size of the nanostructures. With a library of well‐established surface functionalization and passivation methodologies, it is now possible to make water‐soluble and biocompatible carbon nanomaterials with minimum in vitro and in vivo toxicity, and to use these nanostructures for a myriad of biomedical imaging and therapeutic applications.
To achieve effective in vivo live animal imaging, PTT and PDT, the light penetration depth is the main challenge to overcome. Carbon nanomaterials like SWCNTs and GOs processed NIR fluorescence have shown great potential for deep tissue imaging and phototherapy. But it still remains highly challenging for deeper imaging and therapy to centimeters in a living organism. Several possible directions for deeper fluorescence imaging and phototherapy are given: first, carbon nanomaterials can be engineered to have even longer fluorescence emission wavelength to afford deeper in vivo fluorescence imaging and phototherapy; second, improving the fluorescence quantum yields for deeper in vivo imaging and increasing the photothermal conversion efficiency for more efficient PTT; third, combining other diagnostic tools and therapeutic approaches for synergetic diagnostic and therapy.
The focus of previous research efforts was on CNTs, nanodiamonds and graphene‐based nanomaterials. However, the future clearly lies in the development of sophisticated, multifunctional theranostic nanomaterials with properties for specific purposes. Other carbon nanomaterials like carbon nanohorns (CNHs), carbon nanoonions (CNOs) and graphdiyne, are highly promising for future developments. We believed that with innumerable exciting opportunities existing down the road, more environmentally friendly and human friendly products and techniques of carbon nanomaterials would be developed in the future.