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288 Nicole Remaliah Samantha Sibuyi et al.
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13.2.1 Nanocarriers in drug delivery
The use of nanocarriers as DDS demonstrates good prospects, and can be used to enhance the efficacy and reduce adverse effects of the drugs [19]. Nanotechnology has
alleviated the challenges associated with conventional DDS as well as the drugs by
developing “smart delivery systems”. The theory behind these systems is that they are
easily manipulated by physical, chemical, and biological stimuli; and when subjected
to these stimuli, they can be programmed to release drugs in a regulated manner [4].
The inspiration for introducing nanomaterials to DDS is due to their surface-area-tovolume ratio which enables nanomaterials to adsorb and carry multiple bioactive
compounds [20]. Organic and inorganic nanocarriers, such as liposomes, micelles,
dendrimers, quantum dots (QDs) and CDs, among others, have emerged as promising
drug delivery agents. After conjugation or encapsulation of the drugs on the nanomaterials, they will be delivered to the target site in a controlled manner [5] via passive
or active targeting. An ideal nanocarrier is expected to have the following properties:
mechanic al stability, high drug loading c apacity, biocompatibility, low cytotoxicity,
and a long in vivo residence time [6]. The prolonged circulation or half-life of NPbased DDS in the body provides an added advantage for diagnostic and therapeutic
agents for targeted drug delivery and accumulation at a diseased si te. The NPs can
move freely in the body due to their small sizes [7].
Various types of organic and inorganic nanomaterials have been used in cancer
[21] and diabetes [22] as drug delivery vehicles or as treatments. These include metal
and soft NPs such as QDs, liposomes, and micelles. QDs are semiconductor nanocrystals typically with a diameter of 2–10 nm; they possess a size tunable absorption and
emission bands due to the quantum confinement effects [23]. The size of the QD conjugates can increase to 5–20 nm when functionalized wi th some biomaterials, and
these sizes can passively penetrate solid tissues and be cleared by renal filtration. Due
to long-term toxicity and degradation presented by the heavy metals in the core of the
QDs, the application of QDs is limited to cell-based and small animal studies. Conjugating QDs with non-toxic compounds such as silicon and carbon can also mask the toxicity of the QDs and enhance their biocompatibility [24].
Liposomes and micelles are among the soft NPs that have bee n widely used as
drug delivery agents, due in part to their biocompatible and biodegradable nature.
Liposomes are made up of double phospholipids layers and steroids and are already
used in pharmaceuticals as DDS [25]. Amphotericin and daunorubicin are examples of
the drugs that were transported by liposomes [26]. The membrane structure of liposomes is analogous to the cell membranes, making them better DDS. They have also
been proven to stabilize therapeutic compounds, and improve drug biodistribution.
Positively charged liposomes are currently being used in gene therapy as D NA DDS
[15]. As with other foreign particles that enter the body, liposomes encounter immunogenicity, opsonization, and reticuloendothelial system defences [27] which can possibly lead to their physical instability [15].
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Micelles are made of surfactant amphiphilic block polymers that are spontaneously self-arranged into spherical aggregates [28]. They have hydrophobic cores
which can be loaded with hydrophobic drugs such as docetaxel and camptothecin.
This core assimilation with the drugs results in an enhanced drug bioavailability and
stability [25]. Micelles are non-toxic, have high drug-loading capacity and a faster rate
of clearance, and this makes them suitable for intravenous drug delivery [29]. Despite
these advantages, the stability of micelles is reduced when they encounter changes in
environments and when the concentration is low, they can dissociate [30].
Both metal and soft nanomaterials are, however, plagued by some cha lleng es
which limit their application in in vivo drug delivery. Inorganic or metal NP-based
systems are non-biodegradable and their accumulation in vital organs such as the
heart, kidn eys, liver, and splee n could induce cytotoxicity [31]. The orga nic NPs are
not reproducible and their loading efficiency cannot be ascertained, which then results in reduced drug efficacy [32]. Moreover, the polymeric NPs and liposomes can
induce toxic effects on healthy tissues and trigger immune responses. Thus, many of
the NP-based drug delivery technologies are restricted by the impact they have on biological processes resulting in poor biocompatibility, bystander toxicity, non-specific
adsorption, and formation of protein coronas [33]. To be effective, a nanocarrier must
not only facilitate systemic and intracellular drug delivery but must also retain the
drug’s original features with high precision and sensitivity. This chapter focuses only
on the CDs as the drug delivery agents.
13.3 CDs as potential drug nanocarriers
CDs are the newest and among the sought-after nanomaterials which meet the needs for
targeted drug delivery. They are the most promising drug delivery vehicles, mainly due
to their bio-friendly properties. They are considered to be less toxic compared to other
nanomaterials with similar properties such as fluorescent metal-based QDs [9, 34]. And
unlike others, CDs possess unique and distinct mechanisms of actions to their cargoes,
able to retain their original properties and mode of actions; and therefore regarded as
capable nanocarriers for delivering cargoes to the target sites. CDs can be used to regulate and monitor their own distribution, dispersion, and clearance from various parts of
the body, concurrently doing the same for the molecules they carry [35]. Through their
tunable PL properties, CDs together with their cargoes can be monitored in real time [9].
CDs were shown to enhance the drug solubility and uptake [36] through either passive
or active targeting. Drug release from CDs usually occurs via desorption, diffusion, or
tethered to be released through external or internal stimuli [10, 36–38]. The size and
charge of the CDs influence drug permeation, where smaller sizes are able to pass
through cellular barriers including the blood–brain barrier (BBB) [39, 40].
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13.4 CD drug loading
CDs have a large surface-area-to-volume ratio providing more room to functionalize
them with various biomolecules; these can be targeting moieties, diagnostic and therapeutic agents; individually or all combined depending on the downstream application.
This is made possible by the carbon sources used in the synthesis of CDs; they contain a
lot of functional groups such as NH
surface creating multiple binding sites on the CDs. These functional groups make it easy
to use dif ferent chemistries to load biomolecules onto the CDs, which may include
drugs or contrast agents [3, 10]. Several chemical interactions were explored to load bioactive molecules on the CDs as shown in Figure 13.2A. The most commonly used chemistries include electrostatic interaction, covalent bonding, pi–pi conjugation, and the
1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide
(NHS) covalent crosslinking. Surface functionalization using EDC and NHS chemistry is
a common practice for biomolecules containing either carboxyl (COOH) or amine (NH
groups to covalently conjugate drugs containing these functional groups (Figure 13.2B).
The COOH group on one of the biomolecules (CDs or the drug) allows for carboxyl-toamine covalent bonding with the biomolecules that have NH
, OH, or COOH which are then conferred on their
2
group [37].
2
)
2
Figure 13.2: Different chemistries (A) for modification of CDs through surface functionalization and/or
encapsulation (B) to develop stimuli-responsive CD–drug conjugates.
CDs are flexible and drugs can be loaded either through surface functionalization or via
encapsulation or both (Figure 13.2B) to obtain CDs for a desired function. Additionally,
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the CDs can be designed to release the drugs following an encounter with either external or internal stimuli as highlighted in Figure 13.2C [37] such as cellular enzymes
[38], environmental pH [10], light [36], or temperature. CD–drug conjugate formed by
non-covalent bond between the COOH and NH
release at the diseased tissues as it was shown to be sensitive to changes in pH [3]. Of
importance, a promising dru g nanocarrier must be able to reach the tar get area to
facilitate drug release and function. Although CDs have exhibited many excellent drug
loading and targeting properties, not all CD formulations are able to target the bones
and the BBB. Their targeting is often improved by attaching targeting moieties that are
specific for certain disease biomarkers [41, 42] to reduce drug side effects, and in the
process enhance drug efficacy [42].
groups is usually exploited for drug
2
13.5 CDs as a potential DDS
The potential role of CDs as drug delivery vehicles is increasingly being acknowledged
in medicine, and they emerge as the best candidates for disease diagnosis and therapy. CDs have shown many att ributes that qualifies them as putative drug delivery
agents even in difficult to reach areas. Cancer and neurological diseases are amon g
many diseases that stand to benefit from CDs as delivery vehicles, as they are characterized by a highly complex and heavily barricaded microenvironment [43–45], and
BBB [39, 40]. A prerequ isite for any successful disease therapy is for the drugs to
reach the target organs or tissues, intact and in sufficient dosage. However, for cancer, factors such as variable hypoxia, intratumoural pressure gradient, and abnormal
vasculature within the tumours make it difficult to accomplish cancer targeting, thus
reducing the drug efficacy and, at times, causing drug resistance [43–46].
The BBB is another physiological barrier for drug delivery [46] in the brain tissues
for the treatment of brain cancer and other neurological diseases, as it hinders the delivery of drugs from the blood into the central nervous system (CNS) [40, 47]. In fact, the
BBB, blood–brain tumour barrier, and the weak enhanced permeability and retention
effect (EPR) for brain tumour treatment exclude almost 100% of the drugs from targeting the brain tissues [48, 49]. CDs have shown ability to open and cross through tight
junctions of the BBB due to their unique physicochemical properties. CDs as drug carriers can then be used to transport and deliver drugs into the CNS and also to increase
the BBB permeability as shown in Figure 13.3 [50]. There is a growing body of literature
that recognizes CDs to have the ability to cross the BBB in vitro [51] and in vivo [51, 52].
The CDs were able to target the glioma cells and tissue in rats [51] and the BBB in zebrafish and rats [52] without the aid of targeting molecules. Their targeting abilities are
associated with the type of molecules covering their surface, consistent with this hypothesis, CDs synthesized using glucose (GluCDs) as a precursor were shown to target
and be internaliz ed by cells exp ressing the glucose transporters. Evidently so, the
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GluCDs were only localized in glucose transporter-positive yeast strain and not in the
glucose-deficient strains. The GluCDs were able to reach and target the BBB after injection through the heart of the zebrafish and intravenous tail vein in the rats. In the rats,
the GluCDs conjugated with the fluorescein (GluCD-F) were located in the neurons
found in the brainstem, the ventral horn, medial grey, and dorsal grey of the cervical
spinal cord [52]. The CDs also proved to be target-specific, CD-Asp synthesized using
equimolar concentrations of
D-glucose and L-aspartic acid, were able to target the brain
C6 glioma cells and the glioma tissues in the rat brain within 15 min after intravenous
injection through the tail vein. None of the CDs synthesized using the individual materials or those that substituted
methyl group (
L-glutamic acid) were able to replicate these effects or show glioma tar-
L-aspartic acid with an amino acid that differ with a single
geting or specificity [51]. Using passive diffusion transport, Yellow-CDs (Y- CDs) were
able to cross the BBB of the zebrafish. Their uptake inhibited the overexpression of the
human amyloid precursor and the β-amyloid (Aβ) proteins that are responsible for development of Alzheimer’s disease [49]. The ability of the GluCD-F to cross the BBB with
their cargoes and accumulate in the brainstem and cervical spinal cord, and glioma targeting by CD-Asp without any targeting ligands, indicated the feasibility of CD-based
DDS for the CNS-related diseases such as neurodegenerative disease, traumatic injury
[52], and brain tumours [51]. CDs were successfully used as delivery vehicles for chemo therapeutic drugs [9], antipsychotic drug (haloperidol, HaLO) [53], antibiotics [54,
55], as well as mineral supplements. Ferric ammonium citrate (FAC), an iron supplement, was successfully loaded in the CDs (CD-FAC) and improved the biocompatibility
–1
of the FAC on liver (HepG2) cells up to 1,000 μgmL
–1
at ≥100 μgmL
. These suggested that the CDs could do a better job in delivering the
. The free FAC reduced cell viability
iron supplement safely to the cells and enhanced its cellular uptake [56].
Figure 13.3: Passive (A) and active (B) translocation of CDs in the BBB for drug delivery and imaging
of the brain tumours.
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Passive targeting of CDs is a desirable trait for drug delivery agents; however, not
all CDs possess this property [51] and at times those that have lack target specificity.
To increase their s pecificity and localization, targeting moieties such as aptamers,
antibodies, and nanobodies can be easily attached to the surface of CDs. Active targeting has emerged as a powerful plat form for CD-based DDS for the treatment of diseases, including the CNS-associated diseases [57] and cancer. Transferrin [40] and
folic acid [49] receptors are among the biomarkers that were identified as targets for
the BBB and cancer. And when the targeting ligands for these receptors were covalently conjugated to CDs, the CDs were able to target and pass through the BBB of zebrafish via receptor-mediated endocytosis and accumulated in the brain [40].
The CDs are also compatible with other nanosystems and can be used to create
CD–nanohybrid DDS with synergistic, but superior, activities than the individual
nanomaterials [38, 58]. Mathew and co-workers produced a chitosan (CS)/CD-based
nanocomposite, wherein dopamine was later entrapped in the matrix to form a dopamine@CS/CDs nanocomposite. The originality of their technology was influenced by
the use of a non-toxic carrier to successfully deliver drugs to all diseases; in particular, the CDs permitted consistent dopamine release in neurodegenerative diseases.
The chitosan/CD nanocomposite allowed for efficient transportation of dopamine and
prolonged drug release [58]. Mesoporous silica nanoparticles (MSNs) were developed
into MSNs-CD nanocomposite, with the CDs used as imaging agents [38].
13.6 CDs as a trackable drug delivery agents
CDs are a subset of nanomaterials [59] with multifunctional capabilities and used as
an alternative drug delivery vehicle that enables real-time imaging [3]. CDs have been
studied extensively since their first discovery in 2004; mainly due to their photoluminescent, strong absorption properties, photostability, resistance to photobleaching,
low toxicity, environmental-friendliness, biocompatibility, and facile preparation,
amongst others [60].
CDs stand out as delivery agents due to their PL and fluorescent properties which
could be simultaneously used for both drug delivery and bioimaging. The CDs can help
follow or track the movements and localization of the CDs [3, 9] and indirectly its cargoes
in real time [3]. CDs with a size distribution between 1 and 4 nm could be detected in the
kidneys with a strong fluorescent signal after 30 min, and the intensity of the signal was
reduced over time. Negligible amounts of CDs were also observed in the liver and spleen
after 30 min of treatment, and the signal was completely undetected at the other time
points. Their presence in the kidneys and bladder signifies that the CDs were excreted
through glomerular filtration, and most of them were removed from the system within
3 hr. The fluorescent intensity of these CDs was maintained for 2 weeks on the samples
that were stored at room temperature and 4 °C. Unlike other nanofluorescent agents
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such as QDs and carbon nanotubes (CNTs); CDs are compatible with various biological
media, stable in acidic and basic media at pH range of 3–9, and are resistant to photobleaching [9].Thus, CDs can be used as alternatives to the traditional and semiconductor
fluorophores (QDs), all of which suffer from toxicity, high cost and photobleaching [34].
Wide application of CDs has been recorded in biomedical fields, not limited to imaging,
drug delivery, and theranostics [50]. These applications are inspired by the exceptional
properties of CDs shown in Table 13.2.
Table 13.2: Properties that play a role in bioimaging.
Parameter CDs QDs
(semiconductors)
Fluorescence High High High Optically
Solubility High Poor Poor Poor Poor High
Photostability High High High High Poor Varies
Toxicity Low High High Low Low High
Cost Low Low Low Low Low Low
Quantum yield High High Low Low Low (no
References [][, ][–][, ][, ][, ]
Note: Most nanomaterials on the table must be modified to improve their properties, therefore recorded
as “poor”.
CNTs Nanodiamonds Graphene Fluorophores
transparent
No
fluorescence
(originally)
modification)
Low
Moderate
Amongst the different properties of CDs responsible for their recognition in drug delivery, their emission qualities are their most enticing attributes that can aid in tracking the
CDs when used in vivo. CDs absorb light over a wide UV spectrum and emit light with
high intensity in a narrow spectral range [67]. CDs have high tunable PL and excitation
capabilities that are exploited for a variety of applications. The optical properties of CDs
are responsible for their trackable ability when used in biomedical application as either
drug delivery or therapeutic agents. The excellent PL properties of CDs and high fluorescence intensity have been exploited as imaging-guided nanocarriers for the delivery of
drugs [68]. For instance, CDs functionalized with an anticancer drug conjugated to a
photo trigger agent (3-bromopropoxy)-2-quinolylmethyl chlorambucil; Qucbl) demonstrated an adjustable emission range of 325–550 nm and a broad absorption range of
350–450 nm. The emission spectrum of Qucbl-CDs spans from visible to NIR wavelengths
and was not associated with the photo trigger (quinoline moiety) as it does not have this
kind of variable emission spectra. It was then concluded that the tunable emission observed with the Qucbl-CDs must be caused by the CDs [53]. Thus, surface passivation or
modification of CDs can alter the absorption properties of the CDs, which are responsible
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for the fluorescent and the trackable ability of the CDs. Green-emitting CDs (G-CDs) were
used to track delivery and localization of a cancer treatment in a mouse model of liver
cancer. CDs generally have a high fluorescence intensity and durable photostability,
which were exploited in the G-CDs to monitor doxorubicin (DOX) and proved that CDs
provide a smart DDS that allows for both traceability and controlled drug release [3]. The
following properties highlight the mechanisms behind the trackable ability of the CDs:
13.6.1 Absorption
CDs have a unique absorption spectrum with absorption peaks typically in the range
between 230 and 320 nm that extend into the visible region as an absorption tail. The
✶
transition of C–C bonds, the n–π✶transition of C = O bonds, and/or others may
π–π
be responsible for various absorption shoulders. Although this is typic ally the case,
the UV absorption peaks may vary significantly depending on the method that was
employed to synthesize the CDs [69]. The hollow CDs (HCDs) demonstrated bright PL
and were used for DOX loading and release. The HCD-DOX with inherent fluorescence
served as a prototype of anticancer drug carrier with a dual emission. The HCD-DOX
were trackable by a noticeable peak around 490 nm, which was a typical absorption
wavelength of DOX. The HCD-DOX remained stable for numerous weeks without
clumping and generated a red fluorescence when exposed to UV light [70].
13.6.2 Photoluminescence
CDs demonstrated excitation-dependent radiation, which was explored for the PL
mechanism and adapting CDs for biomedical applications. The PL features of the CDs
are the most relevant [69, 71] for monitoring the drug uptake and response in real
time. The PL of CDs can be triggered by photoexcitation once internalized by the cells.
The CD photons or electromagnetic radiation absorption will then result in the emission of an electromagnetic wave in the visible light wavelength range [72]. When compared to the emission of organic fluorophores, the emission peaks of CDs were
frequently wide, with substantial Stokes shifts. This phenomenon could be due to a
wide range of CD sizes and their surface chemistry, distinct emissive traps, or other
factors. Each CD contains a variety of emissive locations, and while the optical properties of small-sized CDs are suspected for their PL properties, their precise mechanism
is not well understood [73].
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13.6.3 Up-conversion PL (UCPL)
Up-conversion fluorescence (UCPL) is the process of converting longer wavelength light
into higher energy photons. As a result, the UCPL property of CDs can be linked to
multi-photon excitation, which results in the emission of light at shorter wavelengths
and higher energy than the excitation wavelength due to the simultaneous absorption
of two or more photons [72]. The longer excitation wavelengths and diverse labelling
with distinct emission wavelengths reduce background autofluorescence, and photon
tissue permeability improves because of UCPL, making it an important function in bioimaging [70]. UCPL is a common fluorescence that is excited by flow from the second
dispersion in a single-phase spectrophotometer and can be eliminated by using a passing filter in the excitation route of a conventional fluorescent spectrophotometer. The
UCPL of CDs opens new possibilities for cell imaging with two-photon luminescence microscopy [74]. CDs’ UCPL was evaluated in the dopamine@CS/CD nanocomposite and
confirmed that the PL property observed in the nanohybrid was based solely on the
CDs distinguishing attribute. The dopamine@CS/CD nanocomposite was stimulated at
510 nm with an emission peak generated at 550 nm. As a result of the excitation and
emission in the visible spectra, the CD up-conversion fluorescence property has been
shown to aid in bioimaging, suggesting that CDs can be employed as a bioimaging tracer
for tracking delivery of CDs and their cargoes in neurodegenerative diseases [58] and
other diseases.
13.7 Drug release from CDs
CD–drug conjugates can be tailored based on the disease environment, to help dis -
lodge the drugs from the CDs and activate their functions when they reach the target
site [9]. The release of the drug from CDs can be regulated by one of the following
mechanisms: diffusion of the loaded drug [75] and stimu li-responsiv e drug release
[76]. The latter concept raises concerns, as like any other carbon-based nanomaterials,
the cargoes (drugs) loaded in the CDs may diffuse when placed in an aqueous environment and only a fraction of the drug will reach the targeted tissues [77]. This clashes
with the desired release profile of the therapeutic drugs, hence the design of CDs for
controlled drug release using stimuli-responsive strategies.
In these strategies, the ability of the drug to be released is driven by exerting a stimulus upon the nanocarrier at the target site to unload their cargo. Stimuli-responsive DDS
can prevent premature drug release, which is a common issue with traditional DDS [78].
Thestimuliorthetriggersareclassifiedintoendogenous (internal) and exogenous (external) stimuli (Figure 13.4). The endogenous stimuli are localized and contained either
within the diseased cells or its microenvironment [79]; examples include pH, cellular
chemical (particularly oxidation–reduction, redox), and enzymatic reactions. The pH
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disparity between the pathological tissue’s microenvironment when compared to the
physiological pH in the normal cells is the most widely explored endogenous stimuli for
drug release. The tumour’s acidic extracellular ambiance and low pH conditions in the
endosomal and lysosomal compartments were shown to cause drug release when subjected to the pathological site. Similarly, a change in glutathione (GSH) levels associated
with disease development can also trigger drug release by cleaving the disulphide bonds
used in the conjugation of the drug to the nanocarrier, causing redox s usceptibility
[79]. Furthermore, the enzyme-susceptible nanocarr iers can result in flexible drugrelease kinetics, which can be caused by disease-induced expression of enzymes such
as intracellular proteases or kinases, among others. Aside from these, increased adenosine 5′-triphosphate (ATP) and reactive oxygen species (ROS) levels in cancer cells can
promote an ATP- and ROS-responsive drug release [80].
Figure 13.4: Drug release from stimuli-responsive CDs based on endogenous and exogenous stimuli.
Exogenous stimuli have more clinical applications due to the ability to control the accumulation of the nanocarriers in specified diseased sites or tissues and the precision
with which the load is released when modulated by an external source. This type of
release is independent of the biological environment or circumstances, allowing for
adaptive activation of the drug release [79]. There is a common concept that some tissues such as tumours and infections are associated with elevated temperatures when
compared to normal cells. However, due to the thermo-influence of various cellular
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