Carbon dots as carriers for the development of controlled drug and gene delivery systems

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Carbon dots as carriers for the development of controlled drug and gene delivery systems

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Suresh Kumar Kailasa*, Jigna R. Bhamore*, Janardhan Reddy Koduru†, Tae Jung Park‡ Department of Applied Chemistry, S. V. National Institute of Technology, Surat, India* Department of Environmental Engineering, Kwangwoon University, Seoul, South Korea† Department of Chemistry, Institute of Interdisciplinary Convergence Research, Research Institute of Halal Industrialization Technology, Chung-Ang University, Seoul, Republic of Korea‡

Chapter Outline 1 Introduction ....................................................................................................... 295 2 Structure and optical properties of carbon dots .................................................... 296 3 Carbon dots as carriers for drug delivery ............................................................. 298 4 Carbon dots as probes for gene delivery .............................................................. 305 5 Conclusions and prospectives ............................................................................. 310 References ............................................................................................................ 314

1 Introduction Carbon is a unique element, and its chemical diversity is unmatched with any other element in the periodic table. Pure carbon is an inorganic material and exhibited multiple allotropes with a large variety of material properties (Hirsch, 2010). Graphite, diamond, and amorphous carbon are the main forms of macroscopic carbon that is based on the different degrees of hybridization (sp2 vs sp3). Apart from these forms, a wide variety of carbon’s nanoscale allotropes, including 0-D carbon nanoallotropes (fullerenes and onion-like carbon, carbon dots (CDs), graphene quantum dots, and nanodiamonds), 1-D carbon nanoallotropes (carbon nanotubes, carbon nanofibers, and carbon nanohorns), 2-D carbon nanoallotropes (graphene, multilayer graphitic nanosheets, and graphene nanoribbons), and 3-D carbon nanoallotropes (graphite, 3-D graphenic hybrid superstructures, aerogels, nanofoams, spongelike nanoarchitectures, and hollow 3-D microspheres fullerenes), have been discovered in the past Biomedical Applications of Nanoparticles. https://doi.org/10.1016/B978-0-12-816506-5.00006-1 # 2019 Elsevier Inc. All rights reserved.

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few decades (Georgakilas et al., 2015). Among the carbon-based nanomaterials, the CDs have become the focus of attention due to their unique physicochemical, electronic, and luminescent properties (Yuan et al., 2016). Furthermore, the CDs have considered as the most important 0-D carbon nanostructures and are of particular interest due to their potential attractive merits, including size- and wavelengthdependent luminescence emission, resistance to photobleaching, nontoxicity, and ease of bioconjugation. First, Xu et al. (2004) discovered accidentally a new class of fluorescent carbon nanoparticles, and these particles are the side products while during the separation of single-wall carbon nanotubes (SWCNTs) by gel electrophoresis from carbon soot produced by arc discharge. Later on, Sun et al. (2006) prepared 0-D fluorescent carbon nanoparticles (<10 nm) and termed them as carbon dots. Now, the CDs have become as a new family of quantum dots (QDs) and attracted widespread attention and emerged as an excellent fluorescent nanomaterial in multidisciplinary research areas (Lim et al., 2015). Literally, CDs are typically zero-dimensional (0-D) carbon nanoparticles, which are <10 nm across with an obvious crystal lattice parameter of
0.34 nm corresponding to the (002) interlayer spacing of graphite (Yuan et al., 2016). The surfaces of CDs usually possess numerous functional groups such as carbonyl, hydroxyl, amino, epoxy, and carboxyl, which give rise to their high hydrophilicity and readiness for their surface modifications with various organic, polymeric, or biological species. As a result, CDs have proven to be as potential fluorescent probes in optical sensing (Bhamore et al., 2017), bioimaging (Mehta et al., 2014a,b, 2015; Kasibabu et al., 2015a,b), drug delivery (D’souza et al., 2016a,b; Mehta et al., 2017), photocatalysis (Yuan et al., 2016), and electrocatalysis (Yuan et al., 2016), since they exhibit unique optical properties such as highly tunable photoluminescence (PL) from deep ultraviolet to near-infrared (NIR) and efficient multiphoton upconversion, remarkable quantum confinement effect (QCE), surface effect, and edge effect. In recent years, the number of scientific publications increases exponentially due to their several merits, such as simple synthesis, low cost, and excellent biocompatibility. Until now, the carbon dots have been widely used as potential materials in sensing, bioimaging, optoelectronics, nanomedicine, catalysis, and energy conversion/storage. Even though a number of reviews have described the synthesis, properties, and applications of the CDs (Zhang and Yu 2016; Shen and Liu 2016; Zheng et al., 2015), a more focus overview on carbon dots as probes for bioimaging and controlled drug delivery is still missing. In this book chapter, we start to emphasize properties of CDs. Then, we discuss the origins of their PL, and we will also provide a representative account of their applications in bioimaging and controlled drug delivery systems using carbon dots as drug carriers. As concluding remarks, some perspectives on the future developments of the CD-based bioimaging and drug delivery are to be highlighted.

2 Structure and optical properties of carbon dots In general, CDs may be nanocrystallites or amorphous nanoparticles knitted up via sp2 bonding. The height of CDs ranges from 0.5 to 5 nm depending on the precursor and preparation methods. Typical high-resolution transmission electron microscopy

2 Structure and optical properties of carbon dots

(HRTEM) studies illustrated that the CDs show an obvious fringe spacing around
0.34 nm, which corresponds to the (002) interlayer spacing of graphite. Interestingly, during the carbonization, CDs are indigenously functionalized with multiple functional groups, especially oxygen-related functional groups, such as carboxyl and hydroxyl, which impart excellent water solubility and suitable chemically reactive groups for surface passivation and derivatization of various organic, polymeric, or biological materials. The emission colors of semiconductor quantum dots (QDs) are due to the quantum confinement effects and correlate to the nanoparticles’ sizes (i.e., diameters); however, the underlying properties responsible for the luminescence properties of the CDs are different. The origin of the distinct colors observed in the CDs is due to energy states associated with surface defects upon the graphitic nanoparticles. In addition, the large sp2 π-conjugated structure endows CDs with some excellent characteristics, such as good photostability, high surface area, and robust surface grafting through either π-π stacking or their surface functional groups. The theoretical studies revealed that the quantum confinement effect (QCE) plays a key role in the fluorescent properties of CDs, since the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of CDs shift to higher and lower energy, respectively, with increasing dot size, thereby reducing the HOMO-LUMO (H-L) gap (Mandal et al., 2012). Meanwhile, different functional groups (either electron withdrawing or electron donating) strongly modulate the HOMO and LUMO levels; however, no significant change in the H-L gap has been observed. For example, an electron-donating group can raise both the HOMO and the LUMO levels to higher energy, while an electron-withdrawing group would lower the HOMO and LUMO energies relative to dH-passivated CDs. Despite the diversity in the CD structures, the CDs exhibit optical properties (absorption and fluorescence). It was observed that the CDs have an absorbance band in the UV region between 230 and 320 nm assigned to the π-π* transition of C]C bonds with sp2 hybridization and, sometimes, a weaker shoulder at 270–400 nm attributed to n-π* transitions of the C]O bonds, with a tail extending into the visible wavelengths. At the same time, the surface functional groups also play some roles in determining the absorption wavelength of the CDs (Liu et al., 2013). Furthermore, it has been suggested that the two main PL mechanisms may be observed in the CDs, which are defect-state emission (surface energy traps) and intrinsic-state emission (electron-hole recombination and quantum size effect/zigzag sites) (Zhu et al., 2012). It was noticed that the intrinsic-state emission causes bluecolor emission (shorter wavelength) and the defect-state emission generates greencolor emission (longer wavelength), which were confirmed by the PL colors of CDs grafted with amino functional groups (m-CDs) and CDs reduced by NaBH4 (Roy et al., 2015). The luminescence decays of CDs revealed multiexponential PL decays with an average excited-state lifetime of 5 ns for emission at 450 nm. The multiexponential nature of the lifetime suggested the presence of different emissive sites of PL process. It was suggested that PL from intrinsic state decayed (t < 5 ns) faster than that of the defect states (10 ns > t > 5 ns) (Zhu et al., 2012; Liu et al., 2012). Important from a mechanistic standpoint, the multicolor properties of the CDs are due to the relative abundance of functional groups upon the CDs’ surface, particularly aromatic residues, C]N, C]O, and CdN bonds, facilitating to exhibit different distributions

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of the chemical units upon the surface, which results changes in the emission colors (Yuan et al., 2016). As an important role in almost all areas of fluorescent nanomaterials, the excellent optical properties of CDs mainly include high fluorescence stability, nonblinking, tunable excitation, and emission wavelengths (Roy et al., 2015). However, the emitting mechanisms of CDs are still not clear, only keeping the phenomenon levels. The in-depth quantum interpretation needs to be established. Summarizing the above, the unique optical and physicochemical properties of the CDs can be tuned by their size, shape, heteroatom doping, and surface functional groups. As a result, the CDs possess various interesting and useful properties such as good water solubility, good material stability, high fluorescence efficiency, nontoxicity, tunability and stability, good biocompatibility, and easy functionalization, allowing them to open up a broad application prospect in biomedicine and optoelectronics.

3 Carbon dots as carriers for drug delivery Effective drug delivery systems are necessary to achieve sufficient drug bioavailability and facilitate clinical use because these systems can reduce side effects by targeting drugs to required pathological sites in the body and controlling drug release; these systems can also increase the bioavailability of drugs by increasing drug solubility and protecting drug molecules from degradation (Wang et al., 2014a). Ideal drug delivery materials should satisfy the following requirements: nontoxicity, good biocompatibility, high stability, suitable mechanical strength, controlled release of the active ingredients, and ease in incorporating bioactive factors. Several types of materials, including metal nanoparticles, vesicles (liposomes), semiconductor quantum dots, amphiphilic gels, nanodisks, polyelectrolyte capsules, and colloidosomes, have been used to carry various drugs. However, these carrier types do not meet all of those requirements, and the encapsulation of drugs by nanostructures remains unclear. Over the past few years, the CDs have extensively evolved as promising materials to fabricate biomimetic nanostructures and macroscopic functional biomaterials with high levels of biocompatibility, biodegradability, surface functionality, and great efficiency. The use of CDs as drug carriers has drawn great attention because of their good pharmacological properties in drug delivery applications (Yuan et al., 2016), since they have high specific surface area, π-π stacking, and electrostatic or noncovalent interactions, which can be exploited to achieve high drug loading of poorly soluble drugs without compromising potency or efficiency. Zhang et al. (2009) developed targeted drug (doxorubicin (DOX)) delivery system using SWCNTs as drug carrier. The drug binds at physiological pH (pH 7.4) and is only released at a lower pH, for example, lysosomal pH and the pH characteristic of certain tumor environments. It was noticed that the DOX was effectively released from the modified nanotubes and damaged nuclear DNA and inhibited the cell proliferation. Huang et al. (2011) described the use of a new family of folate-decorated and carbon nanotube (CNT)-mediated drug delivery system for controlled release of anticancer drug

3 Carbon dots as carriers for drug delivery

(DOX). The synthesized CNT was interacted with DOX via π-π stacking interaction. It was noticed that a number of factors such as pH, particle size, surface properties, degradation rate, interaction force of drug binding to the surface, and the rate of hydration play a key role in drug release. At pH of 7.4, DOX is released at a slow and controlled manner from SWCNT-DOX and SWCNT-DOX-CHI-FA system, and the lower amount of drug release was released at pH of 5.3. Compared with pH of 7.4, the system has a higher drug release at pH of 5.3, indicating that the acidic medium promotes higher drug release because of the reduced interaction between DOX and drug carrier. The fluorescent carbon dots were synthesized using glycerol solvent as a single precursor via a pyrolysis process (Lai et al., 2012). Later on, the CDs were incorporated into mesoporous silica nanoparticles (mSiO2 NPs) to act as a nanocarrier. The as-prepared CDs@mSiO2-PEG nanocomposites were loaded with the anticancer drug DOX, and the controlled release of DOX could be monitored by both time-dependent and spatially resolved ratiometric fluorescence intensity for CDs versus DOX in HeLa cells. To verify the pharmaceutical activity of the released DOX from DOX@CDs@mSiO2-PEG, authors performed two experiments with the free DOX and DOX within DOX@CDs@mSiO2-PEG separately on HeLa cells. The comparative study revealed that the DOX within DOX@CDs@mSiO2-PEG reveals higher cellular toxicity than free DOX, which indicates that the uptake of DOX@CDs@mSiO2-PEG and DOX by HeLa cells might undergo different pathways. As a result, the DOX@CDs@mSiO2-PEG is plausibly internalized into HeLa cells via an active endocytosis process, inducing the cell apoptosis. The synthesized DOX@CDs@mSiO2-PEG has served as an ideal in situ indicator to monitor the efficacy of anticancer drug release via ratiometric changes of the fluorescence for CDs (blue) versus DOX (red) emission at the cytoplasm and the nucleus, respectively. In recent years, the CD-based drug delivery systems play an important role in therapies of the future as nanomedicines by enabling this situation to happen, thus lowering doses required for efficacy and increasing the therapeutic indexes and safety profiles of new therapeutics. The carbon-dot-based nanocarrier systems possess multiple desirable attributes such as the following: (i) When drugs and imaging agents are associated with nanoscale carriers, their volumes of distribution are reduced and have the ability to improve the pharmacokinetics and increase the biodistribution of therapeutic agents to target organs, which will result in improved efficacy (Namdari et al., 2017); (ii) drug toxicity is reduced as a consequence of preferential accumulation at target sites and lower concentration in healthy tissues, and targeting and reduced clearance increase therapeutic index and lower the dose required for efficacy; (iii) the CD-based drug carriers have the desirable advantage of improving the solubility of hydrophobic compounds in the aqueous medium to render them suitable for parenteral administration; and (iv) the CD-based delivery systems have been shown to increase the stability of a wide variety of therapeutic agents such as small hydrophobic molecules, peptides, and oligonucleotides (Zheng et al., 2015). For example, few reports have described the use of CDs as drug carriers for the controlled release of DOX and their confirmation in the cancer cells (Pandey et al., 2013; Tang et al., 2013; Wang et al., 2013; Karthik et al., 2013).

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The gold nanorods (Au NRs) were functionalized with CDs and loaded with DOX, then exposed to Michigan Cancer Foundation-7 (MCF-7) cells for >5 h, and then studied by epifluorescence microscopy. It was noticed that the degree of death of MCF-7 is more when compared with MCF-7 cells under normal conditions, providing an insight toward the strong candidature of the CDs-Au NRs-DOX for photothermal therapy and chemotherapy. It was observed that the pH of the solution plays a key role in releasing the drug molecules from the surface of CDs, which confirms that the CD-based carriers are the pH-dependent drug release. These studies illustrated that the substantial increase of drug release rate was observed at acidic solution, at pH 5.0; the drug release rate is higher than that of pH 6.0; and the percentages of accumulated release in the pH 5.0 solution were 55, 72, and 78% after 24, 48, and 72 h, respectively (Pandey et al., 2013). This indicates that the developed CD-based anticancer drug delivery system minimizes the extracellular loss of drug molecules at neutral environments before reaching the tumor targets and the drug delivery capsules are internalized by tumor cells at low pHs ( 4.5–6.0), allowing effective cancer therapy. Moreover, the intracellular drug delivery and cytotoxicity were investigated on the human embryonic kidney (HEK) 293T cell line (a normal cell line) and HeLa cells using an MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay (Tang et al., 2013). It was noticed that the cell viability percentages of HEK 293T cells are all above 80% at DOX-loaded CDs-folic acid-based nanocarrier system, suggesting that the CDs have negligible cytotoxicity on normal cells. Importantly, the CDsFA-DOX system exhibited less toxicity and increased cell viability percentage for HEK 293T cells when compared with DOX-free drug molecules, confirming that the DOX delivery efficiency inside tumor cells by CDs and the enhanced targeting of tumor cells by surface-functionalized FA can facilitate the cellular uptake by interaction with the overexpressed FA receptors on HeLa cell membranes. Wang et al. (2013) investigated that the internalization of DOX-CDs was by fluorescence microscopy (Fig. 1). Using the excitation-dependent emission of CDs, green emission was observed from hollow carbon dots (HCDs) under blue illumination. After 24 h incubation, the CDs were found throughout the cell cytoplasm (Fig. 1B), surrounding nuclei in particular, which indicates that the CDs can be internalized by A549 cells and mainly localized in the cytoplasm but could not enter the nucleus. However, the bright red fluorescence was noticed in cell nuclei, indicating that DOX was effectively released from the CDs and entered cell nuclei (Fig. 1C and D). The CDs exhibited excellent photostability, suggesting that the CDs can also be used as fluorescent probes for cellular imaging (Fig. 1B). Overall, it was concluded that in the first step, the DOX-CDs entered the cells by endocytosis and formed vesicles and, in the second step, the DOX-CDs carriers were transported into the lysosomes. Finally, the protonated DOX was released and then entered the nuclei due to the acidic environment of the lysosomes. The phototrigger-conjugated anticancer drug (7-(3-bromopropoxy)-2-quinolylmethyl chlorambucil (Qucbl)) was synthesized and loaded on the nitrogen-doped CDs using potassium tert-butoxide in dry tetrahydrofuran (Karthik et al., 2013). The attachment of Qucbl onto the surface of

3 Carbon dots as carriers for drug delivery

FIG. 1 Fluorescence images of A549 cells incubated with DOX-HCDs for 24 h (HCDs 33.3 μg mL1 and DOX 2 μg mL1) observed under (A) bright field, (B) excitation of GFP channel at 475 nm, (C) observed with the Cy3 channel, and (D) merged images. Reprinted with permission from Wang, Q., Huang, X., Long, Y., Wang, X., Zhang, H., Zhu, R., Liang, L., Teng, P., Zheng, H., 2013. Hollow luminescent carbon dots for drug delivery. Carbon 59, 192–199.

CDs was confirmed by ultraviolet-visible (UV-vis), Fourier-transform infrared (FTIR), and 13C nuclear magnetic resonance (NMR) spectroscopies and highperformance liquid chromatography (HPLC), respectively. It was observed that Qucbl-CDs exhibited much lower cytotoxicity than chlorambucil. However, the cytotoxicity of Qucbl-CDs toward cancer cells was greatly enhanced after irradiating the Qucbl-CDs, which is due to the efficient photorelease of chlorambucil inside the cell. Thakur et al. (2014) reported a novel microwave-assisted synthetic approach for the synthesis of bright CDs using gum arabic (GA) as precursor. Authors loaded ciprofloxacin on the surfaces of CDs (Cipro@CD conjugate) and studied the release profile of ciprofloxacin. Authors also investigated the antimicrobial activity of bare CDs, ciprofloxacin, and Cipro@CDs on both model gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), suggesting that the Cipro@CD conjugate showed

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enhanced antimicrobial activity against selective Gram’s stain bacteria. These conjugates exhibited high antimicrobial activity against gram-negative P. aeruginosa and relatively less against gram-positive B. subtilis but more than free CDs or free ciprofloxacin. A simple and straightforward approach was developed for the synthesis of green fluorescent carbon nanodots@zeolitic imidazolate framework-8 nanoparticles (CDs@ZIF-8 NPs) at room temperature (He et al., 2014). Authors chosen 5-fluorouracil (5-FU), as a representative anticancer drug to investigate the drug delivery ability of the CDs@ZIF-8 NPs. The release experiments were studied to calculate the cumulative drug release of 5-FU from 5-FU-loaded CDs@ZIF-8 NPs at phosphate-buffered saline (PBS) pH 7.4 and 5.5. It was noticed that the 5-FU was slowly released from 5-FU-loaded CDs@ZIF-8 NPs at neutral PBS solution; however, 92% of 5-FU was effectively released from 5-FU-loaded CDs@ZIF-8 NPs in acidic PBS solution, indicating that the CDs@ZIF-8 NPs can act as a drug delivery vehicle for pH-responsive drug release in cancer cells. Similarly, Wang et al. (2014a) synthesized multifunctional hybrid nanoparticles by combining magnetic Fe3O4 nanocrystals with fluorescent CDs using one-pot solvothermal method in the presence of H2O2. In this method, authors used multifunctional hybrid NPs as a drug carrier for controlled release of DOX. To this, in vitro cytotoxicity of free DOX as a control, free hybrid NPs, and DOX-loaded Fe3O4@CD hybrid NPs was studied against B16F10 cells. These results illustrated that the drug-free Fe3O4@CD NPs are nontoxic to B16F10 cells after 24 h incubation at concentrations of up to 200 μg mL 1. However, the cell viability dramatically decreased when the cells were incubated with the DOX-loaded hybrid NPs, suggesting that the DOX-loaded hybrid NPs exhibited high anticancer activity. Furthermore, the cytotoxicity of DOX-loaded hybrid NPs is very low when compared with the control group of free DOX at the same concentrations, proving that the Fe3O4@CD hybrid NPs demonstrated great promise toward advanced drug carrier nanoplatforms for simultaneous imaging diagnostics and high-efficacy therapy. Mewada et al. (2014) synthesized highly fluorescent CDs using sorbitol and explored their drug-carrying capacity for targeted delivery of DOX. In this study, folic acid (FA) was used as a navigational molecule due to its high expression in most cancer cells, and then, the surfaces of the CDs were protected with bovine serum albumin (BSA), thereby making them more biocompatible and allowing to hold high amount of drug. The drug release studies revealed that the DOX release is first-order kinetics and the DOX@CD conjugate showed an ideal drug release profile at physiological and slightly acidic pH. The DOX@CDs exhibited a higher killing rate of cancer cells than free DOX. The CDs, containing the multifunctional groups, can be used to design drugs that act selectively on a particular tissue. The CD-based drug delivery system is a promising approach for targeted delivery of therapeutics. Several research groups have been focused on the synthesis of fluorescent carbon dots using natural resources and simple organic molecules as precursors through hydrothermal, microwave, and thermal pyrolysis approaches and used as drug carriers for the delivery of various drugs such as cisplatin(IV) (Feng et al., 2016a,b), boldine (D’souza et al., 2016a), DOX (Yang et al., 2016; Zeng et al., 2016; Zhang et al., 2017), flutamide

3 Carbon dots as carriers for drug delivery

(D’souza et al., 2016b), and lisinopril (Mehta et al., 2017), respectively. A tumor extracellular microenvironment-responsive drug nanocarrier based on cisplatin (IV) prodrug-loaded charge-convertible CDs was developed for imaging-guided delivery. It was noticed that cisplatin-loaded CDs-Pt(IV)@PEG-(PAH/DMMA) exhibited high ability for controlled delivery of highly cytotoxic cisplatin under reductive cytosol of cancer cells upon the incubation at tumor extracellular pH 6.8 for efficient cancer therapy. The in vivo results demonstrated that CDs-Pt(IV)@ PEG-(PAH/DMMA) provides a great therapeutic efficacy without any side effects (Feng et al., 2016a). The MCF-7 cells were incubated with boldine-loaded CDs for 48 h, and their uptake ability was measured by fluorescence microscopy, indicating the effective uptake of boldine-loaded CDs by MCF-7 cells through receptormediated endocytosis. Therefore, boldine from boldine-loaded CDs can be reached to the nuclei of MCF-7 cells with increasing time, while the CDs were still outside of the cell nuclei, suggesting that the boldine was released from the boldine-CDs. The cellular uptake of DOX-CDs was also investigated against A549 cells by flow cytometry and visualized by confocal laser scanning microscopy (CLSM), separately (Yang et al., 2016). It was noticed that the CDs were localized in both nucleus and cytoplasm after 0.5 h of incubation, but with increasing incubation time, CDs are localized predominantly to the nucleus. Importantly, both DOX-CDs and free DOX suppressed tumor growth, while the DOX-CDs exhibited higher inhibition activity than the free DOX group, and the tumor growth inhibition rates for free DOX and DOX-CDs were found to be 41.6 and 60.9%, respectively. In order to confirm the DOX-releasing ability of DOX-loaded CDs to cancer cells, two other cancer cell lines (MCF-7 from human breast adenocarcinoma and HeLa from human cervical carcinoma) and two other normal cell lines (cardiomyocytes (H9C2) and human umbilical vein endothelial cells (HUVECs) were also employed for therapeutic studies (Zeng et al., 2016). It was noticed that free DOX showed obvious declining trend in cell viability, evidencing the therapeutic effect of the DOX drugs. However, the DOX-loaded CDs showed the selective therapeutic effect of the CD-DOX drugs in cancer cells and exhibited good cell viability in normal cells, confirming that the CDs proved to be a smart drug carrier for cancer therapy, affording both trackability and targeted release. Recently, Chiu et al. (2016) fabricated S, N, and Gd tri-element doped magnetofluorescent carbon quantum dots (GdNS@CQDs) by one-pot microwave method. The synthesized GdNS@CQDs were functionalized with folic acid (FA-GdNS@CQDs) for targeting dual-modal fluorescence/magnetic resonance (MR) imaging. The in vitro and in vivo studies confirmed that the fabricated CDs exhibited high biocompatibility and low toxicity nature and their targeting capabilities were confirmed in HeLa and HepG2 cells using in vitro fluorescence and MR dual-modality imaging. Furthermore, DOX was loaded on the surfaces of FA-GdNS@CQDs and used as targeted drug delivery system. Authors noticed that various physicochemical interactions, such as π-π stacking and electrostatic and hydrophobic interactions, play a key role for the conjugation of DOX on the surfaces of FA-GdNS@CQDs (Fig. 2A). In vitro fluorescence imaging results revealed that the DOX was effectively entered into the

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nucleus of the HeLa cells, suggesting that the FA-GdNS@CQDs-DOX system was efficiently internalized and localized in the cytoplasm of HeLa cells via receptormediated endocytosis. Subsequently, the DOX was detached from the surfaces of FA-GdNS@CQDs-DOX due to the slightly acidic environment in the lysosomes (pH 5–6), which facilitates to enter easily into the nucleus (Fig. 2B), which suggests that FA-GdNS@CQDs-DOX acts as an efficient theranostic carrier. These CD-based drug delivery systems seem to be viable and promising approaches for the controlled release of various drugs without any toxic effect to the normal cells, which indicates that they have advantages over conventional drug delivery systems. In addition, the CD-based nanocarriers provide ingenious treatment by enabling targeted delivery and controlled release.

FIG. 2 Schematic illustration of (A) the synthesis procedure of FA-GdNS@CQDs-DOX and (B) the possible mechanism of drug delivery in HeLa cells. Reprinted with permission from Chiu, S.H., Gedda, G., Girma, W.M., Chen, J.K., Ling, Y.C., Ghule, A.V., Ou, K.L., Chang, J.Y., 2016. Rapid fabrication of carbon quantum dots as multifunctional nanovehicles for dual-modal targeted imaging and chemotherapy. Acta Biomater. 46, 151–164.

4 Carbon dots as probes for gene delivery

4 Carbon dots as probes for gene delivery Carbon-dot-based nanocarriers have received much attention in biomedical applications due to their biocompatible and physicochemical properties. Due to their biocompatibility and a broad variety of surface functional groups, the CDs have opened avenues for gene delivery applications. Many currently employed gene carriers utilize positively charged polymeric materials as conduits for the delivery of (negatively charged) DNA fragments. For example, Kim et al. (2013) described the use of CD-Au NPs for the delivery of DNA to cells. In this system, the fluorescence emissions resulting from the assembly of CD-Au NPs were quenched by plasmid DNA (pDNA); thus, pDNA release was probed by the recovery of the fluorescence signals. These fluorescence changes were effectively useful for monitoring the association and dissociation of carrier/pDNA easily in real time without any labeling of pDNA and further facilitate to achieve efficient gene delivery (Fig. 3). It was noticed that the assembly entered into the cells with the CDs located in the cell cytoplasm and the pDNA released entered the cell nuclei, achieving critical transfection efficiency. Liu et al. (2012) synthesized the fluorescent CDs using a positively charged polymer—polyethylenimine (PEI)—as the carbon source for gene delivery platform. The DNA fragments were effectively attached on the surfaces of positively charged CDs, yielding to form the CD-DNA complexes, which

FIG. 3 Fabricated CDs as probes for the gene delivery and real-time monitoring of cellular trafficking utilizing CD-PEI/Au-PEI/pDNA molecular assembly of nanohybrids. Reprinted with permission from Kim, J., Park, J., Kim, H., Singha, K., Kim, W.J., 2013. Transfection and intracellular trafficking properties of carbon dot-gold nanoparticle molecular assembly conjugated with PEIpDNA. Biomaterials 34, 7168–7180.

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can easily internalize into cells. Similarly, a facile and green system was fabricated for the preparation of DNA-CDs using genomic DNA isolated from E. coli (Ding et al., 2015). The DNA-CDs have proved to be high biocompatible materials that can serve as a new type of fluorescent vehicle for cell imaging and drug delivery studies. In another report, branched polyethylenimine-based CDs were synthesized using branched polyethylenimine by oxidation and a modified hydrothermal reaction (Hu et al., 2014). The size of synthesized CDs was found to be <4 nm and exhibited a graphitic structure with lattice spacing of 0.30 nm. The biocompatibility of the CD was investigated using a cell-counting kit-8 (CCK-8) assay in MCF-7 cells and 293T cells (Fig. 4A). It was noticed that the branched polyethylenimine-based carbon dots exhibited lesser cytotoxicity than branched polyethylenimine in both of the two kinds

FIG. 4 (A) Cell cytotoxicity of the PCD and bPEI (MCF-7 cells). (B) Agarose electrophoresis assay of DNA/PCD complexes at different weight ratios. (C) Fluorescent images of EGFP expression obtained by a confocal laser scanning microscope in 293T cells transfected with DNA/PCD complexes at weight ratio of 5. Incubation time, 48 h; scale bar, 50 lm. Green (light gray in print versions), green fluorescent protein; blue (dark gray in print versions), PCD; gray, bright field. (For colour interpretation see online.) Reprinted with permission from Hu, L., Sun, Y., Li, S., Wang, X., Hu, K., Wang, L., Liang, X.-J., Wu, Y., 2014. Multifunctional carbon dots with high quantum yield for imaging and gene delivery. Carbon 67, 508–513.

4 Carbon dots as probes for gene delivery

of cells. In order to confirm CDs as the gene carrier, the CDs were mixed with DNA with different weight ratios (DNA/PCD) to form DNA/PCD complexes. As shown in Fig. 4B, with the decrease of weight ratio, the fluorescence of the CDs was increased, and the intensity of DNA migration band was decreased significantly and finally vanished at or below the weight ratios of 100, 50, 20, 10, and 5. To visualize the gene expression in vitro, transfection experiment in 293T cells was performed by using enhanced green fluorescent protein as the reporter gene (Fig. 4C). The strong green fluorescence was noticed from the 293T cell, suggesting the successful delivery and expression of EGFP. Furthermore, the green fluorescence protein and blue CDs indicated that the fluorescence of CDs was used as the efficiency label for the gene delivery. The high transfection efficiency and bright blue fluorescence illustrated that the branched CDs act as excellent carriers for both cell imaging and gene delivery. In recent years, the CD-based delivery systems are widely used to carry and image on small interfering RNA (siRNA) in vivo and in vitro due to their unique physicochemical and excellent optical properties. The CDs are the ideal tool for discovering and validating cells and small animals, and their large surface area with multifunctional groups provides plenty of opportunities for further biofunctionalization while maintaining a high siRNA loading efficiency. As a result, the CDs have proved to be as scaffolds for intracellular delivery of other molecular cargoes, such as small interfering RNA (siRNA, also known as “silencing RNA”). The siRNA technology is one of the promising strategies for turning off the expression of target genes. However, transporting the (negatively charged) siRNA across the cell membrane is among the most formidable challenges for practical applications of the technology. To overcome from this challenge, Wang et al. (2014a,b) synthesized fluorescent carbon dots using citric acid and tryptophan as carbon and nitrogen sources. The CDs were functionalized with polyethylenimine (PEI) and used as gene carriers for the delivery of survivin siRNA into human gastric cancer cell line MGC803. In this approach, PEI-CDs act as cell transfection agent, which enables transport across the membrane and uptake of the CD complexes into the cell. Furthermore, PEI behaves as a scaffold for docking of the siRNA molecules through electrostatic affinity to the positively charged particles’ surface, resulting to form positively charged siRNA-PEI-CD complex for cell uptake. Wu et al. (2016) developed multifunctionalized, integrated theranostic nanoagent based on folate-conjugated reducible polyethylenimine-passivated carbon dots (fc-rPEICDs) and emitted visible blue photoluminescence under 360 nm excitation, showing high ability to encapsulate multiple siRNAs (EGFR and cyclin B1) followed by releasing them in intracellular reductive environment. As a result, the above CDs were used as good siRNA gene delivery carriers for targeted lung cancer treatment. Moreover, fc-rPEI-CDs/pooled siRNAs were selectively accumulated in lung cancer cells via receptor-mediated endocytosis, facilitating a better gene silencing and anticancer effect, which indicates that the fabricated CDs with multifunctional groups may benefit clinicians to monitor real-time response on the effect and track the development of carcinomatous tissues in diagnostic and therapeutic aspects. Similarly, one-pot synthetic approach was adapted for the preparation of CDs with zwitterionic functional groups using β-alanine as a passivating and zwitterionic

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ligand, allowing cytoplasmic uptake and subsequent nuclear translocation of the CDs ( Jung et al., 2015). The fluorescence images revealed that the fluorescence of CDs began to appear in the cytoplasm after 2 h treatment, indicating that the CDs permeated the cell membrane. After 6 h incubation, the CDs were internalized into the nucleus, a partially delocalized fluorescent signal was observed at perinuclear region, and similar levels of fluorescence were monitored on both sides. Noticeably, significantly strong fluorescence of the cells after longer incubation periods (24 and 48 h) originates mainly from the nuclei, suggesting that the CDs were successfully internalized into the cells and the nucleus, which is attributed to the zwitterionic surface state and small size of CDs. The positively charged surface group on the CDs was effectively interacted with the negatively charged cell membranes to enter the cytoplasm via clathrin- and caveola-mediated pathway along with the involvement of passive diffusion. Similarly, multifunctional CDs were synthesized using alginate and 3% hydrogen peroxide through an inexpensive one-step hydrothermal carbonization route (Zhou et al., 2016). Confocal microscopic results illustrated that the cellular uptake process of the CD/pDNA complexes involved a combination of caveola- and clathrin-mediated endocytosis, suggesting that multiple factors including particle size and shape, surface charge, and the type of particles and cells can significantly play a key role to the internalization of CDs into the cells. In order to confirm the localization of exogenous DNS, confocal microscopic studies were performed using CQDs/pDNA-YOYO-1 as a fluorescent probe (green fluorescence) (Fig. 5). The images were measured at excitation wavelengths of 405 and 488 nm at different time intervals (4, 6, and 8 h) after transfection with CQDs/pDNA-YOYO-1 complexes. It was noticed that some of the CQDs/pDNA-YOYO-1 complexes entered the cells and a few were located within the nucleus (green fluorescence); however, the fluorescence intensity was gradually increased with increasing time, which indicates that more CDs were located at the periphery of the nucleus (blue fluorescence). Meanwhile, the green dots (light gray in print versions) were observed in the nucleus, suggesting that an elevated amount of pDNA (labeled with YOYO-1) was released from CQDs/pDNA-YOYO-1 complexes into the nucleus where the pDNA can achieve effective and stable expression. These results indicate that the CD/pDNA complexes act as efficient gene carriers for high transfection efficiency via timely release of pDNA in the periphery of the nucleus, which can be useful for effective expression of the target proteins. Dou et al. (2015) designed and synthesized multifunctional CDs using glucose and polyethylenimine (PEI) as precursors, used as both antibacterial agents and gene delivery systems. In order to confirm the gene delivery ability of CDs, the plasmid DNA (pDNA) was condensed by quaternized CDs via electrostatic attraction and then transfected in HEK 293T cells. These results indicated that the branched PEI-modified CDs exhibited higher gene transfection efficiency than linear PEICDs and the PEI-CDs exhibited good gene transfection capability with 104 times higher efficiency than naked DNA delivery. Kim et al. (2017) described the use of CDs as a functional siRNA delivery system that induces efficient gene knockdown in vitro and in vivo. It was noticed that the CDs greatly enhanced the cellular uptake

4 Carbon dots as probes for gene delivery

FIG. 5 Process of entering cells. Laser scanning confocal microscopy images showing the process of CQDs/YOYO-1-pDNA complexes entering 3T6 cells and the nucleus. The images were captured 4, 6, and 8 h after transfection (excitation wavelengths of 405 and 488 nm for blue and green fluorescence, respectively). YOYO-1, which emits a green (light gray in print versions) fluorescence, was used to label pDNA and identify its location in the nucleus. Scale bars ¼ 20 lm. (For colour interpretation see online.) Reprinted with permission from Zhou, J., Deng, W., Wang, Y., Cao, X., Chen, J., Wang, Q., Xu, W., Du, P., Yu, Q., Chen, J., Spector, M., Yu, J., Xu, X., 2016. Cationic carbon quantum dots derived from alginate for gene delivery: one-step synthesis and cellular uptake, Acta Biomater. 42, 209–219.

of siRNA, via endocytosis in tumor cells, and exhibited low cytotoxicity and unexpected immune responses. Fluorescence images demonstrated that the CDs can be favorably localized in cytoplasm and successfully released siRNA within 12 h. The CD-PEI-mediated functional siRNA delivery system was successfully used as an in vivo mouse model, showing the remarkable gene knockdown efficacy and siRNA protection from degradation in vivo. Very recently, Yang et al. (2017) reported a simple and one-step hydrothermal carbonization approach for the synthesis of the positively charged CDs using PEI and FA as carbon sources. Cytotoxicity results revealed that the cytotoxicity of PEI was greatly decreased in the presence of

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CDs and applied as a probe for selective imaging of folate receptor (FR)-positive cancerous cells from normal cells. The surfaces of synthesized CDs/pDNA have positive charges, which facilitates to interact with the weak negative charges of cell membrane, suggesting that the CD surfaces efficiently capture pDNA via the electrostatic interactions between positively charged amine groups in PEI and negatively charged DNA molecules. To visualize the gene expression in vitro, they performed transfection experiment between the CDs and pDNA in 293T and HeLa cells, respectively, and EGFP complementary DNA in plasmid (pEGFP) as a reporter gene. These results indicate that the positively charged CDs can efficiently transfect the cells, which confirms the CDs as potential candidates for gene delivery and gene therapy. Table 1 represents the applied synthetic approaches, precursors for the preparation of CDs, their optical properties, and the CDs as nanocarriers for drug and gene delivery systems.

5 Conclusions and prospectives In recent years, carbon dots have emerged as a promising scaffold for drug and gene delivery that provide a useful complement to more traditional delivery vehicles. Their combination of biocompatible, nontoxic, multifunctional groups; small size; high surface area; and tunable stability provides them with unique attributes that should enable new nanocarriers for drug and gene delivery strategies. In this chapter, we summarized the latest advances in the use of CDs and their corresponding derivatives for biomedical applications during the past several years. Utilizing their excellent physicochemical and optical properties, the CDs and their derivatives have been found to be efficient nanocargoes for the delivery of various drugs and gene. The recent investigations revealed that the CDs have great potential for developing drug and gene delivery systems. Though the currently observed therapeutic effects of the CD-based nanomaterials are promising, further improvements are needed by enhancing their drug and gene cargo properties via the attachment of targeting moieties or modifying with suitable chemical moieties to tumor microenvironments or external physical stimuli. Even though cytotoxic studies have revealed that the CDs and their derivatives did not induce any obvious toxic effects to the tested cells and animals, more efforts and depth investigations are still needed to clarify their toxicology profiles and biodegradation behaviors in detail and more systemically. Though these possibilities have been implied, unfortunately, real applications regarding these are limited. Therefore, more effort needs to be made to clarify the feasibility of using these CDs as carriers for controlled drug and gene delivery systems. Up to now, several precursors have been used for the preparation of CDs, but to tune their physicochemical and optical properties, more efforts are needed, since the synthesized CDs do not exhibit fluorescence emissions in the NIR (750–900 nm) and NIR-II (1000–1700 nm) spectral regions, which is more important for bioimaging. Furthermore, more efforts are essentially required to design and fabricate CDs for labeling of stem cells, even the nucleus of stem cells,

Table 1 Overview of Used Carbon Sources, Synthetic Approaches, Optical Properties, and Their Applications in Drug and Gene Delivery Systems

Precursor

Carbonization

Absorbance (nm)

SWCNTs

Refluxed

–

–

SWCNTs

–

–

–

Glycerol

Pyrolysis

–

350/460

Gum arabic

Microwave

215

300/468

Graphite rods

250–350

405/498

Bovine serum albumin Citric acid and urea

Electrochemical method Solvothermal reaction Microwave

230

360/440

–

–

5.5 and 7.4 5.3 and 7.4 7.4 and 6.5 5.8 and 7.2 5.5, 6.5, and 7.4 5.0 and 7.4 –

Gum arabic

Microwave

218 and 264

250/500

7.4

265

420/497

242

440/520

5.5 and 7.4 7.4

CDs@ZIF-8 Ferrocene

Hydrothermal

Fluorescence (Ex/Em) (nm)

pH

Drug Releasing Study

Cells

References

DOX

HeLa cells

DOX

Cancer cells

DOX

HeLa cells

DOX

MCF-7 cells

DOX

HEK-293T cell line and HeLa cells A549 cells

Zhang et al. (2009) Huang et al. (2011) Lai et al. (2012) Pandey et al. (2013) Tang et al. (2013) Wang et al. (2013) Karthik et al. (2013) Thakur et al. (2014)

DOX Quinoline chlorambucil Ferry ciprofloxacin hydrochloride 5-Fluorouracil DOX

HeLa cells Bacillus subtilis, S. aureus, E. coli, and P. aeruginosa HeLa cells Mouse melanoma cells B16F10

He et al. (2014) Wang et al. (2014a) Continued

Table 1 Overview of Used Carbon Sources, Synthetic Approaches, Optical Properties, and Their Applications in Drug and Gene Delivery Systems Continued

Precursor

Carbonization

Absorbance (nm)

Sorbitol

Microwave

212 and 264

350/552

Citric acid

Thermal pyrolysis Hydrothermal

240 and 343

Dried shrimps

Fluorescence (Ex/Em) (nm)

pH

Drug Releasing Study

Cells

References Mewada et al. (2014) Feng et al. (2016a) D’souza et al. (2016a) Yang et al. (2016)

DOX

HeLa cells

–

5.8 and 7.2 6.8

Cisplatin(IV)

321

430/475

5.2

Boldine

A2780 cells and HeLa cells MCF-7 cells and SH-SY5Y cells A549 cells

4Hydrazinobenzoic acid –

–

350

–

5.5 and 7.4

DOX

Microwave

–

–

DOX

Citric acid and diethylenetriamine Vancomycin

Thermal pyrolysis Hydrothermal

240 and 343

360/458

5 and 7.4 6.5–6.8

Cisplatin(IV)

319

338/428

5.2

Flutamide

Milk

Hydrothermal

245 and 302

370/455

5.2

Lisinopril

HepG2 cells and HL-7702 cells MDA-MB-231 or MCF-7 cells MCF-7 and SHSY5Y cells HeLa cells

Citrate, N-acetyl-Lcysteine, Na2S, GdCl3 Glycerol, PEI

Microwave

280 and 375

–

7.4, 6.0, and 5.0

DOX

HeLa cells

Microwave

–

350/465

pDNA

HeLa cells

Pyrolysis

–

350/470

pDNA

HepG2 cells

Glycerol and branched PEI25k mixture

Zeng et al. (2016) Feng et al. (2016b) D’souza et al. (2016b) Mehta et al. (2017) Chiu et al. (2016) Kim et al. (2013) Liu et al. (2012)

DNA

Hydrothermal

280

366/445

4.1

DOX

bPEI

Hydrothermal

335

335/460

–

DNA

Citric acid and tryptophan

Pyrolysis

365

360

7.4

Survivin siRNA

Glycerol, PEI

Pyrolysis

–

360/460

5.5

siRNA

Citric acid and βalanine Alginate, and 3% H 2O 2 PEI and FA

Microwave

–

340/435

–

DOX

Saccharomyces cerevisiae cells and HEK 293 cells MCF-7 cells and 293T cells Human gastric cancer MGC-803 cells and human gastric epithelial GES-1 cells Human non-small cell lung cancer cells HeLa cells

365/450

–

pDNA

3T6 cells

Hydrothermal

272 and 356

370/452

–

pDNA

Glucose and PEI

Hydrothermal

–

–

pDNA

Citric acid and PEI

Microwave

243 and 354

360/466 and 360/473 360/450

293T and HeLa cells HEK 293T cells

–

siRNA

Hydrothermal

HeLa and MDAMB-231 cells

Ding et al. (2015) Hu et al. (2014) Wang et al. (2014b)

Wu et al. (2016) Jung et al. (2015) Zhou et al. (2016) Yang et al. (2017) Dou et al. (2015) Kim et al. (2017)

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which plays an important role of stem cells in cancer progression, metastasis, and drug resistance. In addition, significant synthetic protocols are further needed for the synthesis of CDs for developing dual-modality or multimodality bioimaging agents including magnetic resonance imaging or photoacoustic imaging. Therefore, much significant efforts have to be devoted to address the toxicology profiles of the CD-based materials using different cells and animals and their applications in biomedical fields.

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