Theranostic applications of stimulus-responsive systems based on carbon dots

Abstract

Over recent years, many different nanoparticle-based drug delivery systems (NDDSs) have been developed. Recently the development of stimulus-responsive NDDSs has come into sharper focus. Carbon dots (CDs) possess outstanding features such as useful optical properties, good biocompatibility, and the ability for easy surface modification. Appropriate surface modification can allow these NDDSs to respond to various chemical or physical stimuli that are characteristic of their target cells or tissue (frequently malignant cells or tumors). The present review covers recent developments of CDs in NDDSs with a particular focus on internal stimulus response capability that allows simultaneous imaging and therapeutic delivery (theranostics). Relevant stimuli associated with tumor cells and tumors include pH levels, redox potential, and different enzymatic activities can be used to activate the CDs at the desired sites.

GRAPHICAL ABSTRACT

1. Introduction

Over recent decades, there has been an ongoing effort to improve cancer diagnosis and therapy. Although conventional anti-cancer cytotoxic drugs may be powerful, they often possess inadequate tumor targeting, low cellular uptake, and poor concentration in tumors^[1], leading to insufficient overall clinical efficacy.

In order to increase the efficiency of therapeutic drugs and decrease the unwanted side effects, nanoparticle-based drug delivery systems (NDDSs)^[2], which can effectively control drug release in a spatiotemporal manner have been developed. Several different nanoplatforms and methods have been proposed^[3-5] that can be loaded with naked therapeutic molecules, including metal-based and carbon nanoparticles, nano-sized polymers and liposomes. The ability to target the therapeutic agent to a particular site or cell type, whereas at the same time lessening the toxicity to normal cells^[6,7] makes nanomedicines a promising strategy for cancer treatment.

Recently, there has been a great interest in stimulus-responsive NDDSs, sometimes called “smart delivery vehicles.” These NDDSs can respond to specific pathological characteristics of tumors and malignant cells^[8,9] such as pH levels, redox potential, and different enzyme activities acting as a stimulus to trigger drug release.

These stimulus-responsive nanomaterials can either directly respond to biological cues or lead to activation of drug release in an indirect manner. Generally, bioresponsive systems can classified into three categories: direct activation systems, sequential activation systems, and self-regulation systems. In a direct activation system^[10] the biomaterial directly responds to the stimulus and releases the drug. In a sequential model the biomaterial sequentially responds to multiple stimuli such that the exposure to one stimulus results in activation to another stimulus^[11,12], and finally drug release occurs. In self-regulating systems, the released drug can regulate the surrounding environment to exert a feedback effect which subsequently controls the release of the drug^[13].

A growing area of research is the use of stimulus-responsive NDDSs for both therapeutic and diagnostic intentions simultaneously in a so-called “stimulus-responsive theranostic approach”^[8].

The combination of therapeutic and imaging functions into a single nanovehicle^[14,15] may lead to more accurate diagnosis, monitoring drug delivery, and reporting and analysis of the treatment outcome.

Carbon dots (CDs) are a new kind of fluorescent nanomaterial, with unique tunable optical properties, that have demonstrated versatile utility in theranostic applications. There is a large number of functional groups or “handles” on the CD surface, which not only enhances their stimulus sensitivity after functionalization but also facilitates drug-carrier loading. In addition, due to their outstanding photoluminescence properties^[16], CDs can serve as a powerful imaging reporter which together provide the potential for use in theranostic systems.

In this review, we summarize the utilization and potential of CDs in diagnostic and therapeutic fields with a particular focus on biological stimuli-responsive theranostic systems.

2. What are carbon dots?

Carbon dots are a new carbon-based nanomaterial with a very small diameter of 2–10 nm. On such a size scale, CDs experience the quantum confinement effect which leads to the formation of electron-hole pairs with dimensions smaller than exciton Bohr radius^[17-19]. According to the “particle in a quantum box theory,” these mobile electron-hole pairs are confined in all three axes. As the size of the nanoparticle decreases, the exciton energy levels will become increasingly discrete^[20,21] until they can be considered to be a “pseudo atom.”

Since the first report on CDs in 2004, they have been reported to be useful in many biomedical applications due to their impressive properties such as intense photoluminescence (PL), broad optical absorption, good solubility, high conductivity, and good biocompatibility. In comparison with traditional metal-semiconductor based quantum dots and organic fluorescent dyes, CDs have several advantages^[22-27], such as lower toxicity, facile ability for modification, non-blinking PL, and high resistance to photobleaching.

A variety of synthetic methods for CDs have been developed, which can be generally divided into two main approaches^[18,28,29], including “carving up” different bulk carbon materials through top-down routes or carbonization of small molecules using bottom-up methods.

In the bottom-up methods, such as hydrothermal and microwave^[30,31], it is easier to predict the final product and assume the functionality of the CDS because the carbon sources are verified. However, the chemical structure of the CDs generated by top-down synthesis routes, like applying strong oxidizing acids on natural sources^[32], are difficult to be precisely defined due to the chemical complexity in carbonization of the natural sources. Therefore, the physicochemical properties of CDs^[33] that affect their practical applications cannot be accurately controlled in top-down methods.

Although the exact chemical structure of the entire CD is still unclear^[34], the main carbon core consists of diffuse sp² hybridized islands embedded in an sp³ hybridized matrix.

The fabrication method and the type of precursor dramatically affect the surface properties, and together determine the functional groups that can be used for attachment of other moieties to the carbon backbone of the dot. There is a large number of active functional groups on the surface including amino groups, hydroxyl groups, and carboxyl/carbonyl moieties which have important effects on the CD properties^[35], particularly optical properties, and moreover can be used as handles for attaching other molecules.

Due to their versatile nature, CDs have great potential to be employed in different biomedical applications such as biosensing, bioimaging, and drug delivery systems.

3. Optical properties of carbon dots

3.1. Optical absorbance properties

Generally, CDs have a maximum absorption peak in the UV region^[36] assigned to π→π* transition with an extended tail into the visible region assigned to n→π* transition (Figure 1).

Figure 1.

A schematic representation of the possible mechanism of PL (photoluminescence) derived from π-conjugated domains. This mechanism is due to the recombination of electron–hole pairs in confined π–π* domains of the sp² sites lying between the σ–σ* states of the sp³ matrix which leads to a visible emission.

However, they only emit light at a particular wavelength that is tunable according to their size. This allows a single wavelength to be used for simultaneous excitation of mixed CDs^[37] with emissions of multiple colors. In comparison to conventional organic dyes^[24], CDs show less scattering and low autofluorescence.

Also, CDs possess high photostability^[38,39] even under extended exposure to UV light showing neither blinking nor photobleaching.

3.2. PL mechanism in CDs

One of the most notable features of CDs is their outstanding photoluminescence. Although the exact mechanism of PL is still an open debate^[40], the two most reasonable mechanisms to explain the PL are called intrinsic and extrinsic effects. PL emission arises from the excited state of CDs and can originate either from π domains confined to the interior (intrinsic effect)^[35,41] or from the exterior surface state (extrinsic effect) (Figure 2).

Figure 2.

Illustrating the possible mechanism of surface derived PL. The imperfect sp² domains create energy traps on surface. The sp² and sp³ carbons along with other surface defects lead to emission.

The excitation dependent PL behavior leads to a tunable multicolor emission^[42]; therefore, the emission color can be precisely chosen based on a certain excitation wavelength.

3.2.1. Conjugated π domain derived PL (intrinsic)

As the size of the CDs decreases, the energy bandgap of the sp³ matrix increases. The σ→σ* transition of the sp³ matrix that arises from the bandgap affects the π→π* transition of the confined sp² clusters which leads to the formation of the electron-hole pairs in such strongly confined clusters^[18,19,23]. Eventually, the radiative recombination of these electron–hole pairs^[41] results in the production of intrinsic photoluminescence in CDs.

The PL derived from confined electrons is size dependent and the emission color can be precisely controlled by adjusting the size of the dot. The bandgap energy is increased inversely proportional to the size. Therefore, smaller CDs need higher energy photons to reach the excited^[43,44] state, and subsequently the emission color will be shifted toward the blue.

3.2.2. Surface derived PL (extrinsic)

The abundant chemical groups on the surface of CDs can act as another PL center. Different surface functional groups such as C─O and C═O create different energy levels on the surface which also constitute an emission center. Since each type of surface group can be excited with a specific wavelength, CDs with different functional groups^[41] will show excitation-dependent emission with different colors. The radiative recombination of confined electrons in the surface groups results^[34] in a bright PL. Surface modification is carried by passivation and doping^[45,46] in order to enhance the PL intensity in CDs. Passivation is accomplished^[47] using different kinds of polymers, proteins and other organic molecules. The surface features responsible for the emission (excitons) can be stabilized^[48] and this eases the recombination of the electron-hole pairs. In addition to increasing the quantum yield (QY)^[49], passivation may also improve biocompatibility (Figure 3).

Figure 3.

Carbon dots with various surface states show an excitation dependent PL. Surface passivation with amino rich groups will decrease the surface traps and an excitation independent PL is occurred.

Doping with different heteroatoms such as nitrogen, phosphorous, sulfur, and oxygen which can increase the energy levels of the traps (electrons or holes)^[50,51] and may increase the QY. Co-doping is also possible^[23] via using an appropriate precursor that contains both heteroatoms.

3.3. Fluorescence upconversion

Fluorescence upconversion is an optical phenomenon in which the emission wavelength is shorter than the excitation wavelength^[52] with an anti-Stokes shift. Upconversion usually involves either multiphoton absorption or the use of lanthanide elements^[53] as nanophosphors with a long-lived intermediate excited state (Figure 4). Upconversion is particularly valuable in bioimaging applications in vivo because long-wavelength excitation light can penetrate deeply into the body^[54,55] due to the near-infrared (NIR) biological window, and also in molecular bioimaging due to higher resolution, lack of background autofluorescence, and less phototoxicity. To obtain fluorescence upconversion using CDs, they can be excited with a femtosecond pulsed laser^[56] which leads to simultaneous absorption of two or more photons.

Figure 4.

Up-conversion fluorescence emission mechanism where the emission wavelength is shorter than the excitation wavelength. The multi-photon excitation leads to up-conversion florescence emissions through the relaxation of electrons from high energy π orbitals to the σ orbitals.

4. Bioresponsive-theranostic systems

Bioresponsive systems can provide nanosystems with two broad goals. First, providing a biocompatible vehicle for targeted delivery of drugs/genes that release their cargo as a response to the biological trigger^[8,57,58]; the second important goal is to design a stimulus-responsive system^[59,60] that allows real-time imaging.

Three categories of biological triggers or stimuli can be defined: (a) triggers that are related to particular characteristics found in different organs (organ level), (b) triggers which arise from pathological conditions or abnormal tissues (tissue level), and (c) triggers which are found in particular organelles inside cells (cell level). Some of these triggers can occur at all levels, for example in pH-responsive systems. pH differences are found within endosomes and lysosomes acting as cellular triggers^[61], the pH values in different parts of the gastrointestinal tract act as an organ level trigger, whereas tumors and inflammatory sites have acidic pH values which act as tissue level triggers. Redox triggers are found at the cellular level, where the response depends on differences in the balance between reduced glutathione (GSH) and glutathione disulfide (GSSG)^[62] and/or^[63] concentration of reactive oxygen species (ROS). The difference in the concentration of GSH occurs in the cytosol within cells and in the extracellular fluid, but also between normal tissues and tumor tissues (tissue-level). On the other hand, some triggers mainly occur on the cellular level^[10] such as enzyme activity, hypoxia and the concentration of glucose or inorganic ions.

The fabrication of multifunctional nanoplatforms, which are able to incorporate bioresponsive elements, therapeutic agents and imaging agents in a single construct can be called “bioresponsive-theranostics.”

Bioresponsive-theranostic nanoplatforms will generally have three or four different components: a targeting moiety or a responsive trigger, a nanoplatform that can either be a pre-formed nanoparticle or can be self-assembled, a therapeutic cargo (drug or gene), and an imaging agent. For example, the interaction of paclitaxel (PTX) and human serum albumin (HSA) occurs through hydrophobic binding^[64], and when the PTX is PEGylated it forms the FDA approved anti-tumor agent called Abraxane. The PTX also prompts the self-assembly of HSA and chlorin(e6) (Ce6)—a photosensitizer. The optical imaging is provided by the fluorescence properties of Ce6. Another component is required to add a targeting property to this platform, Qian Chen et al. used the Arg-Gly-Asp (RGD) peptide^[65] to pilot nanoparticles to the tumor endothelium via binding to integrins (αvβ3). Their research indicated high efficiency to eliminate tumors in animal experiments^[66].

Another bioresponsive-theranostic platform was designed by Maiti et al. They used the conjugation of gemacitabine (cytotoxic drug), coumarin (fluorescence imaging agent) together with biotin (targeting ligand). Respectively, the anticancer property comes from the gemacitabine (GMC)^[67] while the stability and bioresponsive features are related to the disulfide bridge that forms the link between GMC and substrate.

In many studies, mesoporous silica nanoparticles (MSN) have been employed, due to their well-defined surface properties, large surface area, tunable pore size, and the ability for the pores to be closed by a “gatekeeper” molecule that can be then be opened by a stimulus-responsive trigger, thus releasing the cargo. Several methods have been deployed for fabricating MSNs such as encapsulation of other agents into a silica shell or assembling them on a silica surface. Dual imaging functionality has been provided by the inclusion of a fluorescent dye and attaching magnetic nanoparticles (for MRI^[68]) onto silica nanoparticles.

In a study by Chen et al.^[69], a multifunctional drug carrier that could also allow imaging was designed based on MSNs. They provided smart release and imaging by capping MSN with gadolinium-bovine serum albumin while targeting was achieved with the addition of hyaluronic acid.

As mentioned above, one of the main constituents of the bioresponsive-theranostic systems is a fluorescent reporter dye. Since many organic dyes suffer from^[70] low quantum yield, toxicity, nonspecific binding, and poor light emission there is a need for an alternative, improved fluorescent reporter such as CDs.

5. CD-based imaging systems

In recent years, CDs have been used as fluorescent imaging agents, without any need to doped or labeled with dyes or other fluorescent molecules for biomedical applications. Their advantageous features include^[71] ease of synthesis from abundant precursors, low cost, biocompatibility, and tunable photo-responsiveness. Favorable biological properties include cell uptake and permeability, weak interaction with serum proteins^[70], and generally not being recognized by the immune system.

Furthermore, as mentioned above, their high surface-to-volume ratio has provided researchers with the possibility to conjugate a wide range of ligands to modify the CDs in order to provide bioresponsive properties. The emission color of CDs can be tuned according to the synthesis conditions, and the emission and excitation spectra extend all the way from the UV to IR. Also, the pH-dependent emission of CDs can be used in a bioresponsive platform.

CDs are becoming widely utilized as image-guided nanocarriers because it is becoming increasingly possible to take advantage of the high sensitivity and specificity of fluorescent imaging. They can also be used for the delivery of anti-cancer agents to the tumor site. In the following sections, we will summarize some recently published papers that explore innovative bioresponsive-theranostic systems based on CDs.

5.1. CDs-based pH-responsive systems

pH values are substantially different in different tissues and organs such as the stomach, and intestines (for oral delivery) and in the liver or lungs (for systemic delivery). Moreover, pH values are different in tissues affected by diseases; for example, in cancer, the high rate of metabolism and relative lack of oxygen leads to metabolic glycolysis that produces acidic conditions (lactic acid) around tumors (ranging from 5.7 to 7.2) compared to 7.4 to 7.6 in normal tissues. Due to these tumor-specific pH changes, pH-sensitive DDS have been designed to produce a stimulus-sensitive drug delivery approach.

An effective pH-sensitive DDS must be stable at physiological pH until it reaches the pH trigger point at its desired target. There are three strategies to design pH-responsive nanocarriers: (a) protonatable groups; (b) acid labile bonds; (c) pH-sensitive PEG detachment.

The introduction of ionizable chemical groups, such as carboxylic acids, amines, imidazolyl groups, and phosphoric acids, into the nanomaterial, is one of the most common approaches to achieve pH-sensitive DDSs. Accepting or donating protons causes a conformational change in the structure that leads to the drug release in three specific ways: (1) precipitation/aggregation; (2) destabilization; and (3) disruption (dissociation).

The second strategy is acid-labile bond cleavage which is able to enhance the extracellular drug release and endosomal escape at the same time^[57]. In this strategy in acidic conditions, hydrolysis of acid labile bonds between the nanoparticle and the drug leads to the drug release.

The third approach is PEG detachment to overcome the “PEG dilemma.” This dilemma involves the fact that PEG inhibits rapid clearance of the carrier by the immune system and inhibits protein adsorption due to the negative charge and electrostatic repulsion, which are beneficial. On the other hand, PEG also prevents cellular uptake of the drug carrier, and electrostatic repulsion inhibits the negatively charged carrier from entering into a cancer cell, which is disadvantageous. This strategy can increase cellular uptake and enhance drug release rate from endosomes to improve therapeutic efficiency.

Many investigators have combined the inherent properties of CDs with pH-sensitive strategies to introduce a novel versatile platform. Niladri Sarkar et al.^[72] fabricated a stimuli-responsive hydrogel using carbonated Aloe vera leaf gel attached to the CDs, calcium alginate (CA) hydrogel film as a carrier, and vancomycin as a model drug to study the release behavior at different pH values. CA film covered the CDs which increased the loading capacity of the drug up to 50% (38–89%). On the other hand, the amount of drug uptake increased by more than 96% by loading vancomycin into a β-cyclodextrin (β-CD) carrier. Vancomycin was released from the vehicle at pH 1.5 (close to stomach acid pH) at a rate of 56% over 120 h, which was related to the hydrogen bonds formed between surface hydroxyl, amine and carboxyl groups of the CDs, and the hydroxymethyl groups of vancomycin. The drug release and loading capacity indicated that this carrier could be used for oral delivery of vancomycin.

In another study, Li Zhout et al.^[73] attached CDs onto the surface of MSNs as caps to construct a smart vehicle for theranostic applications. CDs were functionalized with carboxylic groups to provide direct electrostatic binding to the amine groups of MSNs. These caps allowed the entrapped drugs in MSNs to be stable at neutral pH while improving drug release at lower pH values which led to the increased tumor cell toxicity as shown in Figure 5.

Figure 5.

CDs usage in fabricating pH-sensitive drug carrier based on mesoporous silica nanoparticles (MSNs). MSNs had been loaded with DOX and CDs attached on MSNs as caps. Consequently, not only do CDs add bioimaging feature but also prohibit undesirable drug leakage. Bonding CDs to MSNs can occur through different methods as illustrated above: (a) CDs are attached to MSNs by a linker sensitive to acidic pH; (b) CDs are attached to MSN by electrostatic attraction.

The combination of chitosan and CDs can provide both pH-sensitivity and pH-sensing properties for the nanocomposite. The pH-sensitivity that is attributed to chitosan lies within the range of 5.0–7.4 (Figure 6). This feature can lead to drug release at physiological pH values (5.0–7.4). Furthermore, the non-linear polymers PEG are thermosensitive and can potentiate the triggered drug release. The necessary heat can be supplied by the absorption of NIR laser beams by the CDs. Hui Wang et al.^[74], designed a hybrid nanogel (PEG-chitosan@CDs) which was utilized as a chemo-photothermal treatment. They investigated the efficiency of this dual responsive system using in vitro assays. Due to the synergistic effects of the hybrid nanogels, the therapeutic effects were increased.

Figure 6.

The schematic of CDs different approaches in thranostic delivery systems: (a) CDs/chitosan synthesized through one-pot hydrothermal method, and served as carrier and imaging tool. The synthesized CDs had strong emission which vanished after CDs loaded with DOX. After releasing the drug, CDs emitted weak florescent emission. (b) CDs linked with Fe₃O₄, which had prepared under precipitation method and had loaded with targeting tools(Zn(NO₃)₂/folic acid), to add bioimaging feasibility.

In one study^[55], a fluorescent porous carbon nanocapsule preparation (size ~100 nm) was used for two-photon microscopy imaging in a NIR/pH dual responsive system. Therapeutically this nanostructure could be used both for drug delivery and photothermal therapy due to high CD absorption. The CDs were embedded in FPC-NCs and could be excited by one-photon absorption at a 405 nm laser beam, or by two-photon absorption at 900 nm. The FPC-NCs were loaded with DOX, and due to the presence of amino groups on DOX and carboxyl groups on the FPC-NCs, when the pH was lowered from 7.4 to 5.0, DOX was released.

In 2016, CDs were employed to modify a metal-organic framework. Chowdhury et al.^[75] produced a multifunctional nanostructure by modifying the surface of Fe₃O₄ (acting as a substrate), using folic acid (acting as a targeting molecule), doxorubicin (acting as an anti-cancer drug), CDs (acting as a fluorescent tag), and carboxymethyl chitosan (acting as a pH-responsive molecule). The nanocomposite exhibited better cellular internalization into HeLa cells in comparison with normal cells (L929), as shown in Figure 7.

Figure 7.

A diagram illustrating dual stimuli-responsive drug release, and mechanism of cancer photothermal therapy (PTT) and photodynamic therapy (PDT). The samples were irradiated by laser. And, when the photosensitizers absorb a specific wavelength of light, they produce reactive oxygen species (ROS) (PDT) or heat (PTT) in which cancer cells will die. Additionally, the Doxorubicin will be released in an acidic environment.

In another study, a water-induced shape memory polymer (SMP), composed of polyvinyl alcohol (PVA) and CDs, was used to construct a dual responsive platform by Yang et al. in 2016. When a composite strip containing SMPs was placed at 80 °C and bent into an angle of 180°, the shape was lost after it cooled down. However, the shape resumed within 20–200 s after being immersed in water. The water-induced shape recovery was mainly attributed to the plasticizing effect of water and its competitive hydrogen bonding. Furthermore, the shape recovery was tunable by altering the environmental pH value and temperature. The introduction of CQDs allowed the shape change of SMP to be visualized under UV light. The bending angle of the SMP strip was measured by being immersed in water with different temperature and pH while a digital camera recorded the deformation. These systems were proposed to be applicable to the various medical fields^[76] including smart devices, drug-release, and sensors. Magnetofluorescent CDs (MCDs) are another example of a dual responsive system based on pH/NIR stimuli. In one study, Ming Zhang et al. fabricated FeN@CDs using a green hydrothermal method. This system had the ability to be monitored by MRI (due to the Fe constituent)^[77], folic acid allowed targeting to folate receptors, and riboflavin supplied a photodynamic therapy (PDT) effect. Photothermal therapy (PTT) efficacy was supplied by the CDs (Figure 8).

Figure 8.

Schematic illustration of enzyme responsive DOX-loaded mesoporous silica nanocarrier capped with carbon dots for targeted drug delivery. (a) Drug loading process. The positively charged CDs-PEI nanoparticles prepared by polyethyleneimine grafted on the pore openings of mesoporous silica through disulfide bonds, then DOX and CDs were loaded into the carrier. Hyaluronic acid was coated on the surface because of increasing biocompability and targeted drug releasing by enzyme cleavage. (b) Receptor-mediated cellular uptake by CD44 receptor which is overexpressed in the tumor cells and drug releasing by hyaluronidase (abundant in the tumor environment) resulting in increased tumor cytotoxicity and lower side effects. In addition, after reaching the targeted tumor cells where there is a high concentration of glutathione, the specific enzymatic cleavage of the disulfide bonds can remove the CDs from the surface of nanocarrier and the DOX can be released.

Moreover, in another study Omidi et al.^[78] described a different application of CDs, that combined an antibacterial activity with the optical features of CDs. They designed a wound healing system with a pH-sensitive monitoring ability which was composed of chitosan modified CDs. Their optimized nanocomposite possessed improved mechanical strength and stimulated wound healing due to the antibacterial activity of chitosan-based CDs. Although these researchers indicated the promising role of CDs in pH-sensitive systems, further research is required to reach a clinical application.

5.2. CDs-based redox responsive systems

Redox-sensitive materials can be designed to provide smart delivery taking advantage of redox changes^[79] caused by the difference in reduced glutathione (GSH) concentration between cancer and normal cells (at least three-fold higher in cancer). It is worth noting that the response of such systems is a consequence of electron transfer redox^[8,80,81] reactions. Due to the outstanding properties of CDs, there have been many investigations which have used them in redox responsive systems for theranostic applications which will be reviewed below.

As disulfide bonds are a redox-sensitive linker, CDs are often functionalized with these bonds. S─S bonds are stable at low reducing levels, but at higher reducing levels, such as those which occur at high glutathione concentration, the disulfide bonds will be reduced to two thiol groups. This transition leads to release of the drug^[80,82]; for instance, in one study, Jian Jiao et al.^[83] covered MSNs with fluorescent CDs attached via disulfide bonds to produce MSNs–SS–CDPAA. The DOX cargo (model cytotoxic drug) was located inside the pores. The results showed release of drug under redox condition at both neutral and acidic pH values. The release mechanism was monitored through measurement of the zeta potential and fluorescence spectrophotometry. The zeta potential of MSNs–SS–CDPAA increased from −34.1 mV to −15.8mV in the presence of 10 mM GSH at 37 °C, which allowed disulfide bond cleavage. This study concluded that an increase in the biocompatibility and stability of the MSNs–SS–CDPAA occurred in comparison with unmodified MSNs because the negative charge imparted by CDPAA could prolong the circulation time in blood.

An analogous study used MSNs modified by CD via a disulfide bridge (MCNs). The CDs blocked the mesoporous surface through an electrostatic interaction and prevented the release of the encapsulated DOX, which was loaded inside the drug carrier channels. After reaching the target disease environment, the disulfide linkages were cleaved due to the high concentration of GSH, followed by the opening of the gate, and the leakage of DOX. In addition, CDs acted as a fluorescence probe in this system. Attachment and detachment of CDs to MSNs imparted an imaging capability. The CDs were quenched in the bound (oxidized) state^[84], but fluorescence was restored when the disulfide bonds were cleaved.

In a study reported by Tao et al. in 2016, CDs were functionalized with RGD and mPEG as an active ligand targeted to α_vβ₃ (a type of integrin) and were then inserted into a pH/redox dual-responsive system to increase cisplatin drug delivery. In this system, PEG was bound to RGD via a benzoic-imine bond that is cleaved at tumor extracellular pH. Also, CDs with their bright fluorescence allowed image-guided monitoring of the drug uptake and release. The efficiency of the system was examined by studying cell viability, flow cytometry, and confocal imaging. Results showed that the nanostructures had low cytotoxicity to normal cells that had generally low expression of α_vβ₃ integrin, and higher cytotoxicity to tumor cells with high expression of α_vβ₃^[107]. In a study reported by Tao et al. in 2016, CDs were functionalized with RGD and mPEG as an active ligand targeted to α_vβ₃ (a type of integrin) and were then inserted into a pH/redox dual-responsive system to increase cisplatin drug delivery. In this system, PEG was bound to RGD via a benzoic-imine bond that is cleaved at tumor extracellular pH. Also, CDs with their bright fluorescence allowed image-guided monitoring of the drug uptake and release. The efficiency of the system was examined by studying cell viability, flow cytometry, and confocal imaging. Results showed that the nanostructures had low cytotoxicity to normal cells^[85] that had generally low expression of α_vβ₃ integrin, and higher cytotoxicity to tumor cells with high expression of α_vβ₃.

5.3. CD-based enzyme responsive systems

In a living organism, all biochemical reactions are basically controlled by enzymes^[86]; and, consequently, biology has supplied a boundless repertoire of targets which may respond to enzymatic triggers. The utilization of enzyme responsiveness in the nanoparticle arena is associated with a growing interest in using naturally derived processes and materials as an inspiration for drug delivery systems. The advantages of enzymes as a trigger^[87] are the selectivity and specificity attributed to the bio-recognition of their substrates, combined with their excellent catalytic efficiency. In the biological environment, enzymes carry out numerous biochemical reactions which control diverse biological processes (e.g., muscle contraction, cell movement)^[88,89] and nanoscale processes^[90-92] (e.g., signal transduction, formation of cellular adhesion complexes, protein expression, etc.). Most enzyme responsive materials rely upon an enzyme-induced change in the macroscale or nanoscale physical or chemical properties of the materials. Enzyme responsive materials can be designed to respond to a specific enzymatic reaction^[93-95], as a result, altering their structure, and releasing their cargo. The release of the cargo may also be dependent on surface modification of the nanomaterials. This modification can be done by attaching specific molecules to the surface of the nanomaterials that are affected by enzymatic reactions^[95] which causes a change in their physical properties. Enzymes may also affect the chemical properties of the nanomaterial^[94], so that loaded drugs may be released by chemically degrading their structure. Not only does enzymatic degradation increase the drug bioavailability but also synergistic effects caused by multi-cargo release could increase the therapeutic index of the DDS. Among all DDS, enzymatic responsive systems (ERS) have provoked a great deal of attention.

Compared to other stimulus-responsive systems, such as temperature, magnetic field, and pH, ERS has been studied in a larger variety of settings. In nature, enzyme activity is finely controlled^[96,97] to regulate biological and biochemical processes. Disturbances in the activity or expression of enzymes can occur in many disease states, and can be taken advantage of using enzyme responsive materials^[98-101] and leading to release of therapeutic cargos. Enzymes can be either over-expressed or under-expressed in diseased sites; therefore, the activity of the enzymes may be higher or lower in different specific disease states.

Enzyme-responsive carbon-based nanoparticles often depend on the cleavage of a short peptide^[102,103] sequence by proteases, or cleavage of ester bonds by esterase enzymes. There are well-known linkers^[102,104] which can be incorporated into nanoparticles like polypeptide linkers which are cleaved by lysosomal enzymes. Overexpression of lysosomal enzymes often occurs in tumor cells^[105,106]. Linkers for attachment of numerous active agents to nanoparticles^[107] such as anticancer drugs like curcumin have been clinically investigated and developed. Besides lysosomal enzymes which mostly lead to drug release in the intracellular mileau, protease activation can release drugs from nanocarriers^[94] at disease locations both in the intracellular and the extracellular environments. Protease enzymes have many roles in a variety of cellular processes, such as proteasomal degradation, cellular differentiation^[108], angiogenesis^[108], tissue remodeling^[108], hemostasis^[109,110], inflammation^[111], immune response^[112], necrosis^[113], apoptosis^[113], and coagulation^[113,114], etc. Due to these vssssarious functions and roles, these enzymes are a suitable target for the design of drug delivery systems for diseases such as inflammation, cardiovascular disease, neurodegenerative disorders, and various cancers.

In addition to protease and esterase enzymes, other enzymes such as hydrolases and oxidoreductases^[94,115] can be involved in enzyme responsive drug delivery systems. Furthermore, moieties involved in the drug-NP linker could also be phosphorylated by kinases or dephosphorylated by phosphatases. Changes in phosphorylation status could be a signal for selective cleavage by matrix metalloproteinases. This approach could be suitable for those DDS which can react to certain enzymes overexpression at disease sites.

Due to the diverse collection of ERS and special characteristic roles of CDs in theranostic applications, there might be a role for CD-assisted ERS drug delivery systems for theranostic purposes. Herein, we will summarize the recent progress in ERS and CDs in DDS approaches.

In one early study^[116], a dual-stimulus responsive enzyme and redox DDS with a tracking function, based on CD-capped MSNs, was developed. The positively charged CDs-PEI nanoparticles were prepared by grafting polyethyleneimine (PEI) onto the pore openings of mesoporous silica using disulfide bonds as a linker. CDs-PEI were used as gatekeepers to trap the cargo within the hollow mesoporous silica cavities. Then hyaluronic acid (HA) possessing a negative charged was coated onto the surface of the MSNs to allow controlled drug release, to increase the biocompatibility, and also improve the stability of the nanoparticles. DOX was used as the drug due to its common use in clinical applications. Drug release profiles indicated that DOX-loaded MSN–SS–CDPEI@HA exhibited dual enzyme/redox-responsive drug release. DOX/MSN–SS–CDPEI@HA displayed slower release of DOX at pH = 7.4 buffer compared to pH = 5.0 buffer. The results demonstrated the advantage of DDs to minimize undesirable drug release under normal physiological conditions. In addition, the release of DOX was significantly enhanced in the presence of hyaluronidase both at pH = 7.4 and pH = 5.0.

In the next study^[117], CDs were coated onto MSNs that had been equipped with a pH-switchable zwitterionic surface, combined with enzyme responsive controlled drug release properties, and strong fluorescence. When CDs-MSNs were internalized into cells, the enzymes were unable to reach the cavities of CDs-MSNs to trigger drug release unless the zwitterionic surface has been switched off in the tumor acidic environment. These synergistic effects of switchable surface and enzyme response releasing of the drugs, extend the cycle of treatment, avoid unexpected release, and eventually accomplish accurate drugs transfer to cancer cells. The synthesized MSNs were reacted with 3-propyl methacrylate with MWt of 248.35 to form C─C double bonds on the surface of the CDs-MSNs. Then, two thiol derivatives, cysteamine hydrochloride, and 2-ethanethiol hydrochloride were grafted onto the CDs-MSNs at a suitable ratio. The final product was denoted as CDs-MSNs-DME/DMA PCL.

In the last study^[116], a nanocarrier was described for controlled release of a targeted anticancer drug while simultaneously allowing real-time bioimaging. This took the form of an enzyme-redox dual responsive hybrid with CDs conjugated to MSNs. The CDs with controlled release and targeting properties were produced by a polymerization reaction. MSNs were coated with hylauronic acid and had CDs attached with disulfide bonds. The MSN–SS–CDs@HA were without activity in normal cells that have low levels of reduced glutathione and low hyaluronidase expression. However, after being taken up by targeted cancer cells that have high levels of glutathione and hyaluronidase, the reductive cleavage of the disulfide bonds can release the N-doped CDs@HA from the surface of MSN, and after cleavage of the HA by hyaluronidase, finally the encapsulated drugs can be released. This improved anticancer treatment minimizes the side effects of the drugs.

6. CDs applications in phototherapy

In the last decades, phototherapy has become a common therapeutic intervention and attracted many investigations. Being a noninvasive, simple and safe strategy, phototherapy kills the cancer cells through photoablation by either generating reactive oxygen species (photodynamic therapy) or heat (photothermal therapy). A photosensitizer (PS) as an integral part of both photodynamic therapy (PDT) and photothermal therapy (PTT (is a material that reacts upon laser beam irritation, and produces singlet oxygen or heat. A promising PS for using in cancer therapy is expected to have certain features such as high efficiency of light conversion, inactivity in the absence of light, photostability, and absorption at wavelengths 600–1,000 nm^[118] (i.e., Therapeutic window). On the other hand, nonbiodegradability, poor water dispersibility, and low stability of PS limit their application^[119] in therapeutic systems. Recently, researchers suggest that CDs with unique merits such as lower costs and simple synthesis paths can be considered a suitable photosensitizer in PDT and PTT (Table 1).

Table 1.

A summary of some achievements in the preparation of stimuli-responsive theranostic systems based on carbon dots.

Ref.	Materials and synthesis method	Average size	PL properties	Function	Stimulus agent	Drug	Quantum yield
[120]	Microwave treatment of lignosufonate lignin	4.9 nm	λex = 440 nm λem = 475 nm	Imaging agent/drug carrier	pH	Curcumin	47.3%
[121]	Carbonization of glucose in 1,2-ethylenediamine and concentrated phosphoric acid	10 nm	λex = 403 nm λem = 502 nm	Imaging agent/drug carrier	pH	Doxorubicin	9.6%
[122]	Hydrothermal treatment of magnetite carbon NPs in hydrochloric acid	3–8 nm	λex = 460 nm λem = 520 nm	Imaging agent/PTT agent	pH/NIR	Doxorubicin	8.8 %
[77]	Hydrothermal treatment of chitosan	5.8 nm	λex= 410nm λem= 470 nm	Imaging agent/drug carrier/PTT & PDT agent	pH/NIR	Doxorubicin	13.2%
[116]	Hydrothermal treatment of citric acid	3 nm	λex = 350 nm λem = 450 nm	Imaging agent	Redox/Enzyme	Doxorubicin	39.6%
[123]	Hydrothermal treatment of PEI and citric acid	4–5 nm	λex = 360 nm λem = 450 nm	Imaging agent	Redox/Enzyme	Doxorubicin	—
[85]	Thermal pyrolysis of citric acid	5–8 nm	λex = 360 nm λem = 458 nm	Imaging agent/drug carrier	Redox/pH	Cisplatin (IV)	25.5%
[124]	Hydrothermal treatment of PEI and citric acid	3–4 nm	λex = 350 nm λem = 450 nm	Imaging agent/PTT agent	Redox/NIR	Doxorubicin	—
[83]	Hydrothermal treatment of PAA	2–4 nm	λex = 370 nm λem = 450 nm	Imaging agent	Redox	Doxorubicin	—
[125]	Thermolysis of citric acid	6.3 nm	λex = 360 nm λem = 454 nm	Imaging agent/drug carrier	Redox	Cisplatin (IV)	27.7%

Most of the synthesized CDs indicate excitation/emission peaks in a region lower than 700 nm in the visible light range; moreover, their photothermal conversion efficiency is low. Thus, in order to use CDs as a PS, investigators have to improve these deficiencies using different synthesis methods and precursors or surface modifications. For instance, Jiechao Ge et al. fabricated CDs^[126] through the hydrothermal method; they used polythiophene benzoic acid (PBA) as a precursor. The fabricated CDs indicate 27% singlet oxygen-generating efficiency and photothermal conversion efficiency up to 36.2%. Moreover, the synthesized CDs exhibited a visible to NIR absorption and emission from 640 to 680 nm. They claim that the properties, which make CDs an intrinsic thranostic agent, are regarded to the usage of PBA.

In another study, solvothermal process was used to obtain theranostic intrinsic CDs^[127] using a cyanine dye and PEG800. These multifunctional CDs do not require multistep preparation, which decreases the reproducibility rate, compared with multicomponent systems. In comparison with the cyanine dye, the obtained CDs indicated high hydrophilicity and photostability. The conversion efficiency of the CDs was about 38%, and in vivo test showed up to 90% tumor inhibitory rate in mice after exposed to laser beam at 808 nm.

Recent investigations paved the way for reaching to CDs with high photothermal efficiency comparable to metal nanoparticles such as gold nanoparticles (60%). For example, Minhuan Lan et al.^[128] prepared S, Se-codoped CDs which enhanced the conversion efficiency. Moreover, NIR emission observed with two peaks at 730 and 820 nm. They claim that the presence of Se (as a heavy element) in the graphitic lattices might have induced the opening of the bandgap, which leads to NIR emission. The QY of CDs severely declined after doping owing to the energy-gap law; however, the NIR emission made in vitro and in vivo bioimaging possible. The doped CDs were unable to produce singlet oxygen, which is regarded to change in the excited energy states. Thus, the absorbed energy converted mostly to heat. The cytotoxicity tests showed no adversity in the presence of CDs without laser irradiation.

Some researchers acclaim that pure red emissive CDs are required to enhance the thranostic photothermal systems. Shan Sun et al.^[129] achieved CDs with pure red emission via microwave-assisted heating. They used citric acid as carbon source and formamide solution which had an essential role for an effective preparation in their avenue. In this study, not only the constructed CDs indicated high photothermal efficiency (44%) but also showed an excellent QY (23% in methanol and 16% in water). The results of optical tests in the different pH showed that the PL emission has relation to surface state; moreover, they attributed the absorption at 550 to n → π* transition of π system in C─N/C═N or C─O structure. The cellular tests indicated the penetration of prepared CDs into the nucleus in MCF-7 and HeLa cells, therefore, enhanced fluorescence observation owning to accumulation in the nucleoli.

Another study designed a chitosan-CDs hybrid nanogel (CCHNs)^[130] loaded with DOX which has NIR fluorescence and NIR photothermal conversion. CHHNS exhibited lower QY (4%) in comparison with that of CDs (9%) which had prepared through the hydrothermal process^[131] by glucose. They state that this decrease might be related to the interactions of chitosan chains with CDs’ surfaces. Although CCHNs passed the cell membrane, they could not enter in the nucleus. Furthermore, investigating on a mouse model indicated that CCHNs can pass through the blood brain barrier, and this is related to the size (80 nm) and surface charges (+20 mV) of CCHNs. The comparative study of CCHNs and chitosan nanogels (without the CDs) demonstrated higher loading efficiency of CCHNs which might have been the results of π→π* interaction between drugs molecules and embedded CDs. The release of drug occurred under acidic pH (endogenous) owing to the presence of chitosan in CCHNs, and laser irradiation (exogenous); CDs converted NIR light to heat leading to ease the release of the drug. However, CCHNs without drug have no cytotoxicity for cells even under laser irradiation. Thus, here, PTT was used as a stimulus for drug release instead of being a therapeutic complement.

Investigators evaluate the capability of CDs for NIR bioimaging and photothermal eliminating of bacteria. For instance, Belkahla et al.^[132] prepared suitable CDs through the alkali-assisted ultrasonic. They used E. coli as the model, and the results indicated the eradication of the bacteria under 680 nm laser ablation with low power.

7. Conclusion and future perspectives

Carbon dots possess unique properties that distinguish them from traditional metal quantum dots and fluorescent organic dyes. CDs have attracted much attention in the past decade, and there has been a great deal of research on their application in different fields ranging from bioimaging, biomedicine, biosensors, optronics, and catalysis. A currently active area of research is the development of CDs as a theranostic agent by the design of image-guided drug delivery systems. To improve the effectiveness of such nanocarriers, researchers have moved toward the design of nanocarriers sensitive to an internal or external stimulus. Due to the specific pathological characteristics of tumors, the stimulus could be internal such as pH, enzyme activity, or redox conditions, or external such as magnetic fields, temperature, laser beams, or ultrasound.

However, the great possibilities that are offered by CDs are often limited by their inadequate surface preparation state and unclear molecular structure. These drawbacks are related to the synthesis method and subsequently lead to a lower than optimum QY. Therefore, the precise chemical reactions involved in CD formation should be studied in more detail.

In this review, we have summarized the use of CDs as theranostic stimuli-responsive nanomedicines. By overcoming the challenges and issues, these multi-functional stimuli-responsive NDDSs currently suffer from, they could play an important role in personalized medicine in the future.