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Table 9.1 (continued)
Synthetic methods Merits Demerits
Pyrolysis/carbonization Solvent-free, low-cost,
large-scale production
Chemical vapour
deposition
Controllable morphology
and size, high yield
Non-uniform size distribution
Complicated operation, high cost
9.3 Photodynamic therapy
Photodynamic therapy also known as PDT is an attractive, generally safe, alternative
treatment for both oncological and non-oncological diseases, which is based on selective sensitization of various tissues to light [26–28]. PDT operates on a two-stepped procedure of locally or syste matically applied, tumour-localizing photosensitizing agent
(PS), which then is activated by a specific wavelength and intensity of light, illuminated
through different fibers and endoscopes [26, 28]. Accumulation of light-absorbed photosensitizers in pathological tissue causes a chain of photochemical and photobiological
reactions also known as photodynamic reaction (PDR), which results in photodamage
an eventual death of target cells [26, 29]. Various substances and techniques can optimize PDT’s efficiency such as electroporation and nanocarriers application which can
increase photosensitizer’s tissue selectivity [29]. Photodynamic therapy’s selective process makes it an optimal method in dermatology, oncology, gynaecology, and urology,
furthermore PDT’s good safety profile offers elderly and immune-suppressed patient
who aren’t able to go through with surgery as another option [29, 30].
9.3.1 History
Light has been used as a mean to treat diseases like psoriasis and vitiligo in Egyptian,
Indian, and Chinese culture for more than 3,000 years but it wasn’t until 1900 when a
medical student, Oscaar Raab accidentally discovered the cytotoxic effects of light on
microorganisms. He was studying the interactions of fluorescent dyes on Paramecium
when he realized that intense light applied to acridine can kill the surrounding cells
[31, 32]. In 1901, Neil Finsen used red and UV lights in order to prevent the formation
of small pox pustules and battle for cutaneous tuberculosis, respectfully, and he received the Nobel Prize in 1903 for these endeavors on phototherapy [31]. On the same
year, VonTappier and Jesionek used topically applied 5% eosin solution in combination with white light illumination to treat basal cell carcinoma. Later in 1907, VonTappier and Jodibauer defined the term “Photodynamic therapy ” as interactions between
dynamic light, a PS agent, and oxygen resulting in tissue destruction [28, 31]. Hausman
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reported the phototoxicity of haematoporphyrin on mice skin in 1911 and two years
later first experiment of PDT was done on human by Meyer-Betz who tried 200 mg of
haematoporphyrin on his own skin [31]. For a few decades, PDT and its successes were
forgotten about until in 1955, Schwartz developed haematoporphyrin derivative (HPD)
from acetylation and reduction of crude haematoporphyrin with sulphuric and acetic
acid [31]. HPD was found to be more tissue specific, efficient in smaller doses, and twice
as phototoxic as crude haematoporphyrin [33]. In 1960, Lipson and Baldes realized that
Schwartz’s HPD can be used for photodetection purposes as HPD accumulation in neo-
plastic lesions gave them a visual advantage during surgery [26, 31]. Diamond work on
phototoxicity properties of HDP against gliomas both in vivo and in vitro in 1972 [31].
A big break came through in 1975 when Doughetry and co-workers rediscovered
the value of PDT in oncology. He described a commercially suitable PS agent (HPD) in
junction with a reliable red light source, causing mammary tumour growth eradication in mice [28, 32]. J.F. Kelly and co-workers reported the same result in mice bladder carcinoma in the same year; therefore, the clinical trials begun for both skin and
bladder carcinoma patients in 1976 [28, 31]:
– Doughetry–Skin Carcinoma; 25 patients participated in this trial and 98 out of 113
skin tumours completely responded to PDT.
– Kelly–Bladder Carcinoma; 5 patients were diagnosed with PDT photodetection
andtherewastumournecrosisobserved in 1 patient with recurrent bladder
carcinoma.
– Hayate–Lung and Gastric Carcinoma.
– Balchum–Lung Carcinoma.
– McCaughen–Esophageal Carcinoma.
Numerous successful studies were done over the years on various types of cancers in
their early stages until in 1993, photodynamic therapy (photofrin application against
bladder cancer) was FDA approved in Canada [28]. Today, there are other approved
PS agents used in PDT in order to treat various types of cancers and a number of photosensitizers are in clinical trials, optimizing the efficiency of PDT [31].
Nowadays, photodynamic therapy is employed for breast, gynaecological, intraocular, brain, head and neck, intraperitoneal, pancreatic tumours as well as cutaneous
malignancies [31].
9.3.2 Principle of PDT
9.3.2.1 Photosensitizing agent
A photosensitizer is a natural or synthetic structure which is able to transfer light energy to its surrounding [32]. There are more than 3,000 PS agents (e.g., Chlorophyll)
but only a few numbers are suited for photodynamic therapy. After HPD’s approval in
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1993, several PS agents have been developed. Although PSs differ in photochemical and
pharmacological properties, they must have certain characteristics in common [34]:
– Non-toxic until activated by clinically useful light wavelength.
– Pain-free treatment.
– Chemically pure and stable.
– Easy to synthesis and commercially available.
– Highly selective and with tumour affinity.
– Appropriate absorption (400–800 nm); if the wavelength is lower than 400 nm, it
will cause excessive photosensitivity and if it’s higher than 800 nm, it won’t have
enough energy to excite PS molecules.
– Reliable generation of PDR.
– Hydrophilic or soluble in body tissue for easy systemic application.
– Minimal dark toxicity in the dark.
– Rapidly eliminated from patient’s body and with no toxic degradation product
after treatment [29, 32].
Currently, various appropriate PS agents are being employed for their clinical application. HPD (photofrin, sodium porfimer) is the first approved and the first generation
of PSs, thus it holds the most clinical experience [32]. Photofrin can be employed for
lung, bladder, and esophageal cancer treatment [34]. The treatment time is 20 min per
lesion and is reported to be pain-free, easy, and reliable [32]. Photofrin is a mixture of
60 molecules containing various monomers and oligomers of haematoporphyrin; consequently, it has a low chemical purity [29, 32]. It has a poor tissue penetration due to
maximum light absorption of only 630 nm [29]. HPD’s long half-life and high tissue
accumulation causes skin hypersensitiv ity, and no exposure to direct sunlight is instructed to the patient as sever burn can occur [29, 32]. Photofrin’s disadvantages instigated the development of second-generation photosensitizers.
Second-generation PS agents have a higher light absorption of 650–800 nm, higher
chemical purity, and a higher yield of singlet oxygen, nevertheless their water solubility
is a meagre [29]. M-tetrahydroxophenyl chlorine (Foscan, mTHPC) is synthetically purified plant-based chlorine that can be employed to treat diverse stages of head and neck
cancer [32, 34]. Foscan is very active and produces a rapid PDR, so treatment is extremely effective and is completed in only few seconds. However, due to Foscan’shigh
energy, the patient must be kept in a dark room for 24 h after drug application in order
to avoid severe burn or dark toxicity and the treatment is reported to be painful [32].
Mono-L-aspartyl chlorine e6 (MACE, Fotolon, NPe6, LS11) is also a plant-based
chlorine which is highly effective but with no dark toxicity. Moreover, treatment can
be done only after few hours in the same day and thus is convenient for the patient.
Fotosens, a dye-based photosensitizer, is also activated shortly after application and
the treatment takes 20 min to finish [32].
Aminolevulinic (ALA) is a prodrug altering to IV protoporphyrin via enzyme intermediation and is used generally for cutaneous carcinomas. ALA can be applied topically,
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orally, and intravenously due to its low toxicity but it is reported to be severely painful during illumination [30, 32]. There are other second PS agent that aren’tengaged
as much, fo r instance, texaphyrin, thiopurine derivative, and benzoporphyrin derivative [29].
Today, third PS generation is being deve loped with the attention is on PS-substrate affinity increase. To achieve this goal, PDT’s different modifications are being
investigated. These modifications include but not limited to combination of PS with
target receptor-focussed molecules, nanocarriers application, PS and LDL (Low density lipoprotein) combined to answer tumour cell’s need of cholesterol, PS and cancerous monoclonal antibody conjugation, and more [29].
9.3.2.2 Light
The wavelength of light employed in photodynamic therapy is photosensitizer specific
and unique. ALA, photofrin, MACE, and Foscan can be activated by a wide range of
visible light from red to blue, thus the wavelength can be selected based on clinical
application. Clinical red light (630 nm) has a higher wavelength and therefor a deeper
penetration of 0.5 cm, which can be used for both surface and deep tumours, while
clinical blue light (400 nm) with lower wavelength can be employed only for superficial tumours via its 1 mm penetration [32].
Various light sources have been tested and used over the years such as gold vapour laser, Nd/YAG-pumped dye laser, argon-pumped laser, and copper-pumped
laser, but these light systems are expensive and hard to handle. Today, the preferred
light source is different types of light-emitting diodes (LEDs) since they are not only
small, portable, cost-efficient, and easy to use, but also with same results in PDT as
other systems [34].
9.3.2.3 Mechanism
Appropriate wavelength of light transfers l ight energy to PS molecules via electron
transmission producing a PDR [32]. After illumination, photons imitate PS molecules
from a ground energy state (singlet state) to a short-lived excited singlet s tate and
later to a rather long-lived electronically excited state called triple state [31]. The excited triplet state can then source three outcomes (Figure 9.2):
a) Light and heat release: PDR is a fluorescent process and can be detected through
high fluorescent regions (tumour cells) and low fluorescent regions (normal
healthy cells). Loss of fluorescent post-PDT can also indicate that the tumour has
been ablated [32].
b) Type I photochemical reaction: Triplet state PS interacts with the substrate (cell
membrane and molecules) directly and transfers a hydrogen atom or electron,
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generating free radicals. Oxygenated products are formed from free radicals and
oxygen reactions [31]. The resulting oxidative stress leads to cell destruction [29].
c) Type II photochemical reaction: In this assumingly more important pathway, trip-
let state PS transfers its energy directly to oxygen molecules [29, 31], and then
these new-fangled singlet oxygen molecules form reactive oxygen species also
known as ROSs [31].
Figure 9.2: Photodynamic reaction mechanism [29].
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Type I and type II reactions can only occur under non-anoxic areas and can both
transpire simultaneously; the ratio however, depends on PS type and affinity, substrate
concentration, pH, and oxygen concentration [29, 31]. ROS short half-life (< 0:04μs )and
extreme reactivity affects cells in destruction radius (< 0:02μm ) through three different
but linked mechanisms; direct tumour photodamage, tumour-associated vascular damage, and immune response against tumour [31].
ROS can directly cause tumour cell death by both apoptosis and necrosis [31, 32].
Different PSs have diverse ways of arrival mechanisms to tumour site, such as receptor-mediated phagocytosis/endocytosis, low-density lipoprotein receptor binding, uptake by tyrosine kinase/epidermal growth factor receptor, diffusion, biodistribution,
and other experimental ways. Photofrin and MACE take multiple pathways to tumour
location and concentrate in cell and organelle membrane like mitochondria, while Foscan takes Golgi and endoplasmic reticulum to accumulate [32].
Once light is illuminated, cellular and sub-cellular destruction and eventual tumour cell necrosis follows. Calcium and metabolic byproducts release cause cells to
malfunction and overwhelm repair functions. Cytokines and toxic chemicals leakage
from organelles has a lethal bystander effect on nearby cells, furthermore, they can
induce a regional and systemic immune reaction [32]. Tumour apoptosis can also be
initiated by photodynamic therapy when low light dose is employed. As a result, cells
cease to function but there is no bystander effect nor an immune respond [32]. Nonhomogenous drug distribution of PS and oxygen unavailability within the tumour tissue don’t allow this mechanism to eradicate the tumour unaided [31].
Necrosis and apoptosis of vasculature contribute both directly and indirectly to tumour destruction. Tumour cell’s viability relies on nutrition supplies carried through
blood vessels. Then again, blood vessels maintenance depends on growth factors provided by tumour cells [31]. Endothelial vascular cells can concentrate photosensitizers
due to its comparable receptors and pathways. When the accumulated PS is activated
by illumination, PDR sources vascular wall disruption. This disruption can distress the
blood flow resulting in microvascular collapse, hypoxia, and anoxia. Lack of oxygen
and other supplies causes vascular shut-down and necrosis of both nanovasculature
and tumour [31, 32]. Toxic chemicals and calcium release result in collapse of microvasculature as well, blocking feeding of tumour [32]. Vasoconstriction, thrombus formation,
and platelets aggregation have also been reported [31]. Vasculature apoptosis can also
occur, resulting in tumour hypoxia and destruction [32].
Photodynamic therapy has an immunomodulatory effect and can suppress the
immune sys tem. Cell necrosis cascades an immune response through inflammatory
mediators (e.g., cytokines, growth factors, and proteins) released from pathological tissue. These mediators stimulate several white blood cells’ (including neutrophils and
macrophages) activation that can eventually result in tumour cell death. Macrophages
phagocytize PDR’s damaged cells and debris upon arrival and also activate CD8 cytotoxic T lymphocytes through CD4 helper T lymphocytes. When photodynamic therapy
is completed cytotoxic T-cells can induce cancerous cell apoptosis [32].
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9.3.2.4 Clinical procedure
PS drug application varies in clinical use. While in most PDTs, PS is applied systemically via intravenous injections, few superficial skin cancer, premalignant skin lesions, and oral mucosa can be treated with topically applied PS (usually ALA) [34].
Moreover, maximized PDR is achieved with clinically determined but minimal photosensitizer dosage for the reason that PS molecules have the opportunity to concentrate in tumour tissue only [32].
Drug-light interval or DLI is the incubation period for photosensitizer drug to accumulate in target tissue before light illumination [34]. A short interval (15 min) provides
PS with enough time to concentrate in vascular compartment, causing vascular statis
and thrombus followed by direct tumour kill, but then in a longer DLI (4 h), PS accumulates in extravascular compartment. In conclusion, intervals differ in time based on
their clinical application and tumour destruction but they mostly take 24–72 h [31].
After an appropriate DLI passes, light is illuminated by different techniques:
– Superficial PDT is employed with thin and accessible tumours. Light is delivered
by a fibre with a microlens (applicator) on its tip.
– Interstitial PDT is used to treat tumours thicker than 1 cm. Laser fibre and appli-
cator are implanted into the tumour tissue through needles.
– Intra-active PDT is applied in hollow organ carcinomas like uterus and bronchus.
– Intra-operative PDT is photodynamic therapy and surgery combined and is em-
ployed with anatomically complex areas [34].
Light dosage can also be altered. High light doses result in necrotic pathways and immune response, while low light doses induce apoptosis [32].
Altering drug dose and application, light dose and technique and drug-light interval allows clinicians to optimize a proper PDR providing necrotic pathways, apoptotic
pathways, selective vascular destruction, and selective tumour destruction [32].
9.3.3 Benefits and disadvantages
To better realize PDT’ s advantages, we must compare it with other forms of cancer
therapy. Photodynamic therapy process generally requires one single PS injection (it
can be repetitive if needed), followed by a single clinical illumination. Both steps are
expected to be through in a short amount of time (usually one day), while chemotherapy, radiotherapy, and surgery can take weeks to month to be completed. Furthermore, PDT is cost-efficient, on an out-patient basis and can increas e life expectancy.
Most importantly, it protects normal healthy tissues, creating a safety margin from
photodamaging. Minimal long-standing fibrosis results in a functional recovery without scaring due to a matrix provided by PDR for tissue regeneration since subepithelial collagen and elastin aren’t damaged [28].
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Although PDT is a valuable alternative cancer treatment, it is not without limitation. Photodynamic therapy is a localized treatment; hence it is limited to local, small,
and accessible tumours. However, with fibre-optic technology advancing, appropriate
light can be transmitted to virtually everywhere in the body [28]. Furthermore, PDT
side effects have been reported in different clinical applications. Early onset side effect of photodynamic therapy in cutaneous diseases treatment involves:
– Pain; it is the most frequent (58%) and limiting side effect. Pain starts early during
illumination, picks in the first minute, and decreases from t hen. It can induce
other symptoms like hypertension and can negatively influence patient’slife
quality. Many PDR mechanisms can mediate pain, for example, produced ROSs
stimulate sensory nerves conducting pain also, hypoxia triggers pain signals due
to the low oxygen level around mitochondria-rich nerve endings. Intensity of
pain depends on the depth of singlet oxygen production and therefore differs in
PSs. Pain management measures can be taken via air analgesia, topical anesthe-
sia, nerve blocks, and opioid ingestion (Figure 9.3).
– Photosensitivity; systemic administrated PS leaves residues in skin and as soon as
these accumulated molecules are activ ated by normal sunlight, it can c ause se-
vere tissue morbidity and clinical first or second degree burns. Photosensitivity
differs from one photosensitizer to the other but light pr ecautions must always
be taken.
– LSRs; there is erythema and oedema with 89% prevalence, scaling with 80%, pus-
tules with 6%, and erosions with 1.2%.
– Urticaria; histamine release from mast cells in dermis causing skin to itch during
treatment but it has only been reported in ALA and MAL (Methyl aminolevulinate)
photodynamic therapies.
– Innate and adaptive immune suppression.
– Other trivial side effects like miscellanea, liver parameters changes, allergic reac-
tions, and swelling [30, 34]
Late on set side effects have been reported as well, nonetheless they aren’tascommon. They consist of pigmentary changes (1%), mild to moderate scaring (0.8%), bullous pemphigoid, and most concerning, carcinogenicity [30].
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Epidermis
Dermis
Nerves
Subcutaneous
layer
Figure 9.3: Pain-inducing mechanisms of PDR.
O
Hypoxia
Sensory Nerve stimulation
ROS
ROS Increase
9.4 Some studies about carbon dots
in photodynamic therapy
Many CDs-based structures have been synthesized and their effects on the cell and animal model have been studied. We will explain their results and methods as following.
9.4.1 CCOF-1 and CCOF-2
We will briefly discuss a valuable study by Shuang Chen et al. about covalent organic
frameworks and published in advanced functional materials journal [39]. CCOF-1 and
CCOF-2 are useful structures in photodynamic therapy made of CDs and P-phenylenediamine/BODIP [39]. These structures are modified with PEG and became CCOF-1PEG and
CCOF-2PEG (Figure 9.4). CCOF-1PEG has absorption of 300–900 nm. This information
was obtained from UV–vis absorption and PL spectra. TEM image illustrates average
diameter of CCOF-1PEG to be 430 nm. DFT methods show that the size of CCOF-1PEG is
2.6 nm. Moreover, CCOF-2PEG has an absorption of 200–900 nm. The average diameter
of CCOF-2PEG is 430 nm, while alone CDs have absorption of 200–800 nm, and the average size is 5.37 nm [39].
CCOF-2PEG demonstrates high capability in photodynamic therapy because of inducing hydroxyl radicals (·OH); in order to reveal this, HeLa cell line is employed.
Cells incubate with CCOF-2PEG for 0.5 to 2 h, after cell irradiation by laser, utilizing
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Figure 9.4: Schematic indication of CCOF-1@PEG and CCOF-2PEG synthesis [39].
Figure 9.5: These charts illustrate the low cytotoxicity of CCOF-2PEG in two cell lines, irradiation and
without irradiation [39].
2,7-dichlorofluorescein di acetate (DCFH-DA). In photodynamic therapy, low cytotoxicity is essential. In order to check the cytotoxicity of CCOF-2PEG tumour cells (cancer
cell lines like HeLa cells) and normal cells (e.g., L929 cells), incubate with CCOF-2PEG
without laser irradiating. In this situation, the methyl thiazolyltetrazolium (MTT)
assay on these cells indicates that cell death is shallow (Figure 9.5) [39]. In contrast,
CCOF-2PEG has an excellent ability to induce ROS and doesn’t have cytotoxicity [39].
The performance of CCOF-2PEG in anti-t umour acti vity was studied and results
show high anti-tumour activity. This study used U14 tumour-bearing mice as an animal model. Mice were divided into four groups; the first and second group were injected
with PBS and the third and fourth group were injected with CCOF-2PEG; however, groups
two and four were illuminated by laser and tumours were weighted. The third group
was also irradiated by laser and measured but tumour’sweightwasreduced[39].
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