Graphene quantum dots (GQDs) -based nanomaterials for improving [607208]

European Journal of Medicinal
Chemistry
Manuscript Draft

Manuscript Number: EJMECH -D-19-01380

Title: Graphene quantum dots (GQDs) -based nanomaterials for improving
photodynami c therapy in cancer treatment

Article Type: Review Article

Keywords: Graphene quantum dots; Photodynamic therapy; Nanomaterials;
Drug delivery; Cancer therapy

Abstract: Conventional photosensitizers (PSs) for photodynamic therapy
(PDT) are confi ned by their physicochemical and biological properties,
including poor water solubility, low photostability and few tumor
selectivity. In recent years, several nanomaterials have been developed
as novel PSs and PSs drug delivery systems. Graphene quantum d ots (GQDs)
as novel nanomaterials, have received significant interest in the field
of biomedical applications. It is worth noting that a large amount of
research is devoted to GQDs -based nanocomposites for cancer treatment,
especially for PDT, in that they can act not only as more favorable PSs
but also nanoplatforms for delivering PSs. In this review, the biological
behavior and physicochemical properties of GQDs for PDT are described in
detail, and the application of GQDs -based nanocomposites in improved PDT
and PDT-based combination therapies is analyzed. Therefore, this review
may provide a new strategy for designing efficient PDT systems for cancer
treatment.

GQDsDopingModified GQDsPhotosensitizers
Anti-cancer drugs
Cancer Cells
Vessel
NucleusLight
ROS
Graphical Abstract (for review)
Click here to download Graphical Abstract (for review): Graphical Abstract.pdf

Graphene quantum dots (GQDs) -based nanomaterials for 1
improving photodynamic therapy in cancer treatment 2
Huayang Fan1, Xianghua Yu1, Ke Wang1, Yijia Yin2, Yajie Tang3,4*, Yaling Tang5*，Xinhua Liang1* 3
4
1 State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & 5
Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan 6
University, No.14, Sec. 3, Renminnan Road, Chengdu Sichuan 610041, China 7
8
2 State Key Laboratory of Oral Diseas es & National Clinical Research Center for Oral Diseases & 9
Department of Orthodontics , West China Hospital of Stomatology, Sichuan University, No.14, Sec. 3, 10
Renminnan Road, Chengdu Sichuan 610041, China 11
12
3 State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China 13
14
4 Hubei Key Laboratory of Industrial Microbiology, Hubei Provincial Cooperative Innovation Center 15
of Industrial Fermentation, Key Laboratory of Fermentation Enginee ring (Ministry of Education), 16
Hubei University of Technology, Wuhan 430068 China 17
18
5 State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & 19
Department of Oral Pathology , West China Hospital of Stomatology, Sichuan Univ ersity, No.14, Sec. 3, 20
Renminnan Road, Chengdu Sichuan 610041, China 21
22
* Correspondence: 23
Corresponding author1: Yaling Tang 24
E-mail address: [anonimizat] 25
Corresponding author2: Yajie Tang 26
E-mail address: [anonimizat] 27
Corresponding author3: Xinh ua Liang 28
E-mail address: [anonimizat] 29
30
31
32
33
34
35
36
37
38
39
40
41
42 *Manuscript
Click here to download Manuscript: GQDs.docx Click here to view linked References

Abstract : Conventional photosensitizer s (PSs) for photodynamic therapy (PDT ) are 1
confined by their p hysicochemical and biological properties, including poor water 2
solubility, low photostability and few tumor selectivity . In recent years, several 3
nanomaterials have been developed as novel PSs and PSs drug delivery systems . 4
Graphene quantum dots (GQD s) as novel nanomaterials , have received significant 5
interest in the field of biomedical applications . It is worth noting that a large amount 6
of research is devoted to GQDs -based nanocomposites for cancer treatment, 7
especially for PDT , in that they can act not only as more favorable PSs but also 8
nanoplatforms for delivering PSs. In this review, the biological behavior and 9
physicochemical properties of GQDs for PDT are described in detail, and the 10
application of GQDs -based nanocomposites in improved PDT and PDT -based 11
combination therapies is analyzed. Therefore, this review may provide a new strategy 12
for designing efficient PDT systems for cancer treatment. 13
Key words: Graphene quantum dots ; Photo dynamic therapy ; Nanomaterials ; 14
Drug delivery ; Cancer therapy . 15
1. Introduction 16
Cancer has gained constant and extensive research interest as the world’s second 17
leading cause of death [1]. In clinical, each type of cancer demand s a specific 18
treatment which usually involves one or more modalities , such as surgery, 19
chemotherapy and radiation . However, these conventional cancer treatments have 20
plenty disadvantages , thus limited therapeutic efficacy can be achieved. Hence, it is 21
essential to develop less invasive and more effectively treatment s. 22
Rece ntly, photodynamic therapy (PDT) has arouse d widely attention and 23
achieve d a great development as an anticancer strategy , and may be an alternative to 24
conventional cancer therapies [2, 3] . This approach is characterized by minimally 25
invasion in nature, lower systemic toxicity, fewer side effects as well as negligible 26
drug resistance [4]. PDT demands the collaboration of light, oxygen and PSs [5]. 27
Localized PSs after activated by light can convert oxygen into cytotoxic reactive 28
oxygen species (ROS), such as singlet oxygen (1O2), causing photocytotoxicity and 29
cell death via apoptosis, necrosis or autophagy [6]. In addition , PSs play a key role in 30
influencing the efficacy and side effect s of PDT. 31
There are three generation photosensitizer s (PSs) developed for achieving 32
improved PDT [7]. In the 1970s, t he first -generation PSs came o ut, which were a 33
complex mixture of porphyrin s, and the main limitations of them were poor light 34
penetration, low clearance rate (cause skin hypersensitivity) and poor selectivity [8]. 35
The second -generation PSs began to develop since the 1980s, such as 36
5-aminolevulinic acid (ALA), chlorin and hypocrellins . They exhibit ed high singlet 37
oxygen generation, better penetration depth owing to their strong absorption from 650 38
to 800 nm and higher selectivity , yet still poor [9]. However, the sec ond-generation 39
PSs showed poor performance in water solubility and photostability, which 40
significantly limited the use for PDT [10]. Hence , new emerging method s for the 41
third -generation PSs like PSs drug delivery systems were developed [11, 12] . With the 42
development of nanomedicine, several nanomaterials including carbon -based 43

nanoparticle s (NPs) [13, 14] , gold NPs [15], quantum dots [16], polymeric 1
nanoparticles (PNPs) [17], hydrogels [18], dendrimers [19], hollow mesoporous silica 2
nanoparticles (HMSNPs) [20] and up-conversion nanoparticles (UCNPs) [21] were 3
used as PSs nanocarriers, thus enabling precise drug delivery and improving the 4
efficacy of PD T. 5
Graphene quantum dots (GQD s), as novel zero-dimensional ( 0D) graphene 6
nano material s, have many advantages over conventional organic PSs, such as good 7
biocompatibility, high water solubility and photostability , particularly with superior 8
optical properties and facile surface functionalization property [22-25]. Comparing to 9
two-dimensional ( 2D) graphene nanomaterials (such as graphene oxide and reduced 10
graphene oxide) , GQD s exhibit some unique merits, such as excellent 11
photoluminescence property due to the quantum confinement [26]. Most importantly, 12
owing to the above excellent properties of GQDs, they are used as novel PDT agent s 13
and can be modified with other biomolecules so that well-defined characteristics 14
could be reached to perform selectively and more efficient PDT, which have been 15
verified by lots of experiments . 16
So far , considerable efforts have been devoted into GQD s-based nanomaterials in 17
PDT and PDT-based combin ation therapy for cancer (Table 1 ). In this review, we 18
focus on the current studies for achiev ing enhanced PDT and PDT-based combin ation 19
therapy based on GQDs -based nanomaterial s through different surface modifications 20
and design of GQDs (fig. 1) . It will provide a comprehensive understanding of the 21
physicochemical and biological properties of GQDs for PDT and how they are 22
modified and designed as drug delivery nanoplatforms for PDT -based cancer 23
treatment. 24

1
Table 1 2
Recent examples of GQDs -based nanocomposites for PDT -based therapy 3
Structure Synthesis of GQDs Binding force Highlights Ref
GQD s Electrochemical oxidation of graphite – First demonstrated GQD -mediated photodynamic
cytotoxicity. [27]
GQD s Hydrothermal method –
The highest 1O2-generating efficiency ever reported [28]
GQDs Exfoliating and disintegrating graphite flakes – GQDs have an impressive enhancement of ROS
generation over conventional photosensitizers [25]
γ-irradiated GQD s Electrochemical oxidation of graphite rods – Using γ-irradiation to modulate and develop GQD optical
properties [29]
OH‐GQDs – – OH‐GQDs lead to G0‐G1 arrest and cells senescence [30]
UCNP -GQD/TRITC Hydrothermal method Covalent bond (between GQDs and UCNP )
Covalent bond (between TRITC and UCNP -GQD ) Mitochondrial specific photodynamic therapy upon NIR
light [31]
N-GQD -RBs Solvothermal method (synthesis of N-GQDs ) Covalent bond (between N-GQD s and RBs) Two-photon induced precise and deep PDT [32]
GQD@MnO2 Hydrothermal method Electrostatic force (between GQDs and MnO2 ) Detect ed GSH in a living system and enhance PDT
efficacy [33]
HA-GQD -SiO2 – π-π stacking (between GQDs and HA) Superior 1O2 generation abilit y [34]
GQD -SS-Ce6 Improved Hummers method Disulfide bon d (between GQD sand Ce6) Redox -responsive photodynamic nanosystem cause
effective suppression of tumor growth [35]
Ag-GQDs/DOX Bottom -up method π-π stacking (between Ag -GQDs and DOX) Ag-GQDs as a general platform incorporat ed PDT and
chemotherapy [36]
GQDs@hMSN(DOX) -PEG Hydrothermal treatment Covalent bond (between GQD s and h MNS ) Combined enhanced drug delivery platfor m and PDT [37]
N-GQD -DOX -APTES Hydrothermal method (s ynthesis of N-GQD ) Covalent bond (between GQDs and APTES ) Nucleus -targeted drug delivery and controllable PTT [38]
PC@GCpD(Gd) – – Combined i mmunotherapy and PDT [39]
Lu-TP&Gd -TP/GQD -RGD Hydrothermal method A bridge of poly (ethylene glycol) (between GQDs and RGD)
π-π stacking and hydrophobic interactions (between GQD -RGD and Lu-TP&Gd -TP) Multiple combined therapies including BRT, PDT and
PTT [40]
HMNS/SiO2/GQDs -DOX Bottom -up method Amino bond (between GQD s and HMNS/SiO2 )
π-π stacking, hydrogen bond and electrostatic interaction (between GQD s and Ce6) Multiple combined therapies including PTT, PDT and
chemotherapy [41]

Fig. 1. Schematics of GQDs -based nanomaterials for enhanced PDT and combined therapy .
2. Physicochemical and biological properties of GQD s for PDT
Graphene quantum dot s (GQD s) as 0D graphene material s with good
biocompatibility, their lateral dimensions typically less than 10 nm [42]. The synthesis
methods of GQD s are two completely different methods , top-down and bottom -up,
which have been systemically discussed [42]. It has small size, excellent solubility, as
well as other physical and chemical properties, including highly tunable
photoluminescence (PL) [43, 44] , electrochemiluminescence, excellent photostability,
multi -photon excitation and facile surface functionaliz ation . The collective properties
make GQD s promising in sensors [45-48], bioimaging [49-53], drug delivery system
[54, 55] . In addition, combined with its fluorescence imaging and drug delivery
properties, GQDs and GQDs -based nanocomposites can simultaneously achieve
tumor diagnosis and target PDT, which can be used as a new strategy for future cancer
treatment.
GQDs as novel PSs have a high ROS generation property, and several recent
studies have evaluate d the photodynamic cytotoxicity of GQD s in vivo and in vitro .
Markovic et al. [27] firstly demonstrat ed the photodynamic cytotoxicity of GQD s in
vitro . Under blue light illumination , GQD s generate d ROS which was successfully
detected by DHR and Electron Paramagnetic Resonance (EPR) spectroscopy to kill
human glioma cells (U251 ). Through immunoblotting and RNA interference, results
show that photoexcited GQD s can easily access to target cancer cells, and initiat e
autophagy contribut ing to the photodynamic cytotoxicity (Fig. 2) . The cell death
displayed both apoptosis and autophagy. Ge et al. [28] show ed high capability of
GQDs to generate 1O2, and the generation yield was greater than 1 .3. Also, GQDs
exhibited great PDT efficacy in vitro experiment , the cell viability of HeLa cells was
less than 20% after incubated HeLa cells with the 1.8 µM GQDs solution . In vivo
experiment, GQDs significantly inhibited the growth of subcutaneous breast cancer
xenografts in female mice after 17 days, and no tumor regrowth was observed over 50
days. Furthermore, Jovanovic et al. [29] found that structural change of GQDs can
cause direct impact on their physicochemical properties. After irradiat ion at lower
doses of γ -irradiation, the diameter and structure of GQDs were revised , as well as
their photoluminescence intensity, singlet oxygen production and so on were

improved , and all these changes ma de GQDs better PDT agent s. In addition , GQDs
could be modified with other molecules (such as hydroxyl [30] and polyethylene
glycol [56]), through revisi ng the properties of GQDs and GQDs -based materials , and
make GQDs more suitable for PDT.

Fig. 2. Autophagy of GQDs to produce photodynamic cytotoxicity . Reproduced with permission [27].
Copyright 2012, Elsevier.
3. GQDs -based nanocomposites for enhanced PDT
PSs and photoexcitation are the key to improve efficacy of PDT, since the PDT
major process involves cytotoxic intracellular ROS produced by excited PSs upon
photoexcitation. Owing to the facile surface functionalization property of GQDs, the y
can readily be modified with other biocompatible molecules to perform enhanced
PDT. In addition, s mall-sized GQDs could be highly influenced by the surface
functional groups and structural change. Precisely decorat ing GQDs with specific
characteristics plays a key role to get ideal PDT agents . Herein, we summarize the
current GQDs -based nanocomposites developed for enhanced P DT, which based on

two-photon excitation (TPE) as a laser source and modified GQDs -based
nanomaterials as P DT agents .
3.1 Two-photon excitation induced GQDs -based nanocomposites for PDT
Recently, TPE has gained great attention in the field of biomedical imaging and
detection [57]. Compared with one -photon excitation, TPE exhibits several
advantages such as fine spatial resolution, strong penetrating ability and reduced
photodamage in biosystems [58, 59] . More importantly, TPE has been applied to
improve treatment efficiency in PDT [6, 60] . Owing to the TPE property of GQDs,
several TPE induced GQDs -based nanomaterials have been developed for improved
PDT.
Meng and co -workers [33] synthesized a novel multifunctional two -photo
nanoprobe based on GQDs, GQD@MnO2, which was designed for imaging of
glutathione (GSH) in biosystems and improved PDT (Fig. 3). GSH, as a ubiquitous
tripeptide and the most prevalent intracellular thiol, was reported to consume 1O2,
thus tremendously decreasing the effectiveness of PDT , and the effect of GSH on
PDT efficiency was further confirmed in vitro. The GQD@MnO2 nanoprobe showed
high sensitivity and high selectivity toward GSH, and intracellular GSH can reduce
MnO2 nanosheets , leading to a lower GSH level , thus the efficacy of GQDs on PDT
was significantly improved [61]. In vitro experiment , after incubating HeLa cells with
GQDs or GQD@MnO2 with 560 nm irradiation for 30 min, results showed that the
PDT efficiency of GQD@MnO2 was significantly better than that of GQDs. In
addition , nitrogen (N) doped GQD s also exhibit good two-photon propert y and have
been developed for enhanced P DT.

Fig. 3. Schematic illustration of GQD@MnO2 and GQD -SS-Ce6 with GSH regulated photoactivity for PDT.
Reproduced with permission [33]. Copyright 201 8, The Royal Society of Chemistry .
Reproduced with permission [35]. Copyright 2016, American Chemical Society.

3.2 Modified GQDs -based nanocomposites for PDT
3.2.1 Nitrogen Doped GQD s for PDT
Doping GQD s with heteroatoms is an effective method for modif ying their
electron density and optical properties for well-defined characteristics [62]. Currently,
great efforts have been given to nitrogen ( N) doped GQD s (N-GQDs) for PDT , and
various facile approaches for synthesizing N-GQDs are reported including
hydrothermal method, electrochemiluminescenc e, solvothermal method , solvent free
synthesis , thermal treatment and t hermal pyrolysis method [63]. Doping GQDs with

N is an effectively method to modify their optical and electrical propertie s (widens the
light adsorption and prolongs the PL lifetime ) [42]. Particularly, N -GQDs exhibited
good TPE propert y.
Kuo and colleagues [64] developed N -GQDs by functionalized them with amino
molecules (amino -N-GQDs ). N-GQDs exhibited improved photochemical and
electrochemical properties and after being modified with the amino -group the
electronic properties of N -GQD s were further enhanced . Human cervical carcinoma
cells (KB-50) were co -cultured with amino -N-GQDs. T he amino -N-GQDs displayed
good biocompatibility to KB -50 as well as high photodynamic cytotoxicity to KB-50
under two-photo excitation. In addition, N -GQDs can act as not only a good PS but
also a nanocarrier for efficiently delivering and activat ing other conventional PSs. Sun
et al. [32] synthesized stable photosensitizer coupled N-GQDs , N-GQD -RB,
involving a conventional photosensitizer Rose Bengal (RB) . N-GQDs exhibited l arge
two-photon absorption cross -section (TPAC) , which ensure d two-photon induced
fluorescence resonance energy transfer (FRET ) efficiency. Under the FRET process ,
RB c ould be activated by N -GQD s with two-photon irradiation and efficiently
perform ed its photodynamic cytotoxicity . MTT assay was applied to evaluate the
photocytotoxicity of N -GQD -RB. After treated MCF -7 cells with N -GQD -RB for
with 480 nm laser irradiat ion for 10 minutes , the cell viability was dose -dependent
declining from 100% to 35% wh ile the N -GQD -RB dose was increased from 0 to 60
μL. In vivo experiment, results showed that, the target vessel is clearer and deeper in
the two -photon image s than in the one-photon image s. In addition, in blood vessel
closure experiment, the closure was reached after irradiation for about 8 minutes.
Above results indicated N -GQD -RB have huge potential for precise and deep PDT.
Thanks to the superior physicochemical properties of GQDs as well as N -doping
strategy further amend ing optical and electrical properties of GQDs, N -GQDs have
great potentials as PDT agents, and as nanoplatform s involving more therapeutic
agents so that could achieve multiple combination therap ies, which would be
mentio ned later.
3.2.2 PS Doped GQD s for PDT
Most of conventional organic photosensitizers (PSs) such as chlorin e6 (Ce6) and
hypocrellin A (HA) suffer from poor water solubility . GQD s can be a suitable
nanocarrier to optimize the efficacy of PSs and improve the water solubility of PSs
for improved PDT therapeutic outcome .
Du and co-workers [35] develop ed GQD s-based nanocomposite s with redox
responsive photo -activity by integrat ing Ce6 onto GQD s via disulfide bond (Fig. 3).
Additionally, Pluronic F -127 was coated on the GQD -SS-Ce6 as a stabilizer. When
GQD -SS-Ce6 reaches local tumor tissue, GSH in the tumor cells induces disulfide
bond cleavage. Ce6 c ould then be released from the GQDs nanoplatform and its
phototoxicity is restored. Even under light irradiation conditions, the nanocomposite
exhibit ed fluorescence quenching characteristics and slight phototoxicity, and the
photo -activity of PSs can be selectively restored only in the presence of a reducing
agent. In vitro and in vivo photodynamic experiment , the redox -responsive GQD s
nanocomposite exhibited an effective suppression of HeLa cells growth . In addition,

HA as an efficient second -generation PS has great photodynamic activity upon the
phototherapeutic window area of 480 -600 nm [65, 66] . Zhou et al. [34] prepared a
nano composite HA -GQD in which HA and GQDs interact through p -p stacking,
which simultaneously has imaging and PDT capabilities. Then HA -GQD was
encapsulated in porous silica nanoparticles to prepare HA -GQD -SiO2 , in order to
avoid the separation of the nanocomp osites during drug delivery and it further
increased the fluorescence signal intensity and 1O2 production capacity of HA -GQD.
And the porous matrix properties as well as silica shell protection of SiO2 ensure d the
imaging and PDT properties of HA -GQD . In vitro experiments, HA -GQD -SiO2
showed better PDT effect on HeLa cells than HA and HA -GQD, indicating that
HA-GQD -SiO2 can be used as a better PDT agent . As a result, GQDs can act as a PS
nano carrier not only improve the properties of conventional organic PSs but also used
for delivery PSs precisely, making GQDs -based nanocomposites more favorable for
PDT.
3.2.3 Other Modified GQDs for PDT
In addition, modified GQDs can exhibit targeted PDT property. Zhang et al. [31]
proposed a strategy by covalently modif ying GQDs on upconversion nanoparticles
(UCNP ) via amide linkages for highly efficacious PDT (Fig. 4). Then,
tetramethylrhodamine -5-isothiocyanate (TRITC) [67, 68] , a mitochondria -targeting
fluorescent probe , was used to modif ied UCNP -GQD to obtain UCNP -GQD /TRITC ,
and their mitochondria -targeting ability was confirmed by the line scan analysis .
Mitochondria, an important organelle that produces energy, its direct damage and
destruction can rapidly promote programmed cell apoptosis [69]. In addition,
UCNP -GQD/TRITC c ould precisely produce a large amount of 1O2 in mitochondria
under near-infrared (NIR) laser irradiation. Therefore, mitochondria -targeted PDT
strategy is expected to be used to accurately treat cancer.

Fig. 4. Schematic illustration of UCNP -GQD/TRITC synthesis and mitochondria -targeted PDT . Reproduced

with permission [31]. Copyright 201 8, Elsevier .
4. GQDs -based nanocomposites for PDT -based combination therapy
Monotherapy such as chemotherapy is often not as effective as we anticipated ,
combination therapy with multiple treatment modalities often shows better efficacy in
cancer treatment. Herein, we summarize d the recent designed GQDs -based
nanocomposites performing for PDT with one or more therap ies, including
chemotherapy, photothermal therapy, i mmunotherapy and biological redox therapy .
4.1 PDT with Chemo therapy
Chemotherapy as the one of the most common clinical treatment s for cancer s has
several limitations , including low efficacy, high drug resistance, and amounts of side
effects [70, 71] . Integrating chemotherapy with PDT, which has good synergistic
effect s, will be a promising cancer treatment strategy. GQDs, as a novel drug
nanocarrier, has a small volume, high surface -to-volume ratio, and rich active site s on
the surface, which can be utilized to efficiently load and release chemotherapeutic
drugs [54]. In addition, GQDs are currently used to deliver doxorubicin (DOX), and it
significantly enhances the cytotoxicity of DOX.
Ju et al. [38] designed and prepared N -GQD -DOX -APTES, which can be used
for nuclear targeted delivery of anticancer drugs . N-GQD s were prepared by the
hydrothermal method and then DOX was loaded (N-GQD -DOX) . 3-(aminopropyl)
triethoxysilane (APTES) as a charge reversal agent were grafted onto the
N-GQD -DOX to enhance the stability of DOX [72]. APTES exhibited pH dependent
charge -reversal characteristic and could convert the drug delivery system to positively
charged state during the acidic tumor microenvironment , in that it increase d the
ability of the tumor cells to internalize the nanocomposites. Furthermore, enhanced
nuclear targeting ability of N -GQD -DOX -APTES was confirmed by fluorescence
imaging, and accumulation of DOX in the nucleus was observed. Compared to
N-GQD -DOX, N -GQD -DOX -APTES limits the release rate of DOX and has a
protective effect on the release of DOX. The combination of chemotherapy and PDT
based on N -GQD -DOX -APTES signific antly improved the treatment efficiency and
showed higher anticancer effectiveness than free DOX and N -GQD -DOX -APTES
without laser irradiation (fig. 5). Silver nanoparticles (Ag -NPs) as anticancer agents ,
the antitumor activity has bene reported [73, 74] . Habiba et al. [36] employ ed Ag-NPs
to decorate GQDs and coupl ed them with DOX to form Ag -GQD /DOX as a new
anti-cancer nanocomplex. The Ag-GQD displayed high efficiency in delivering DOX
better than that of GQDs as well as low cytotoxicity in normal Vero cells. In SOSG
detection, Ag -GQDs produce d higher singlet oxygen than GQDs, indicating that they
have high PDT potential . In vitro experiment, Ag-GQD /DOX showed higher
chemotherapy efficiencies for DU145 and HeLa cells and the combination of
chemotherapy and PDT based on Ag-GQD /DOX was superior to monotherapy in the
treatment of DU145 and HeLa cells.
Furthermore , hollow mesoporous silica nanoparticles (hMSN) with porous and
hollow structures can be used as support systems for GQDs, which not only regulate
their retention time in the body, but also have extra space to accommodate more

therapeutic molecules [75, 76] . Yang et al. [37] prepared GQDs@hMSN -PEG NPs by
loading polyethylene glycol modified GQDs into the cavity of hMSN, which has good
biocompatibility , excellent stability and strong 1O2 generation capacity . And th is drug
delivery system can efficiently load ed DOX, about 0.780 mg DOX /mg
nanocomposites. The released behavior was pH -dependent . In acidic condition (pH
5.0), the amount of released DOX could be around 57.6% after 4 days. In vivo
experiments, successful accumulation of GQDs@hMSN and efficient delivery of
DOX to breast tumor tissue were observed in tumor -bearing mice . These GQDs -based
nanomaterials can be used to improve drug loading and delivery efficiency and are
powerful candidates for combination therapy with PDT and chemotherapy.

Fig. 5. (A) Results of in vitro experiments with N -GQD -DOX -APTES (B) Drug release capacity
of different drug delivery systems Reproduced with permission [38]. Copyright 2019, Wiley -VCH.
4.2 PDT with PTT
Photothermal therapy (PTT) is an effective cancer therapy strategy . During the
PTT process, the PSs converts the absorbed light energy into heat under light
illumination, causing the temperature of the local tissue to rapidly rise to an effective
treatment temperature for a period of time to ablate the cancer cells without damaging
the normal tissue [77]. Particularly , GQD s exhibit excellent photothermal conversion
efficiency because of its good near-infrared light absorbance, excellent thermal
conductivity, and low toxicity and ha ve been s uccessfully designed for PTT [78-84].
Combin ing PDT with PTT could enhanc e the conversion of light to ener gy in that
improv e the anticancer efficiency [85]. Cao et al. [86] synthesized a multifunctional
nano platform encompass ing diagnostic and therapeutic functions, GQD -PEG -P. In
this nanocomposite, P is one of the second -generation PSs, porphyrin derivative, with
strong 1O2 productivity and low toxicity [87]. GQD -PEG -P exhibit ed excellent
physicochemical properties such as good physiological stability , fluorescent imaging
ability , and can be used as an effective drug delivery system. In addition, it can be
used for intracellular cancer -associated miRNA detect ion after modification by
molecular beacon (MB) gene probe. After being taken up by human lung cancer cells
(A549) , the MB dissociate d from the surface of GQD -PEG -P and emit ted a
fluorescent signal to effectively locate cancer cells, which means it can be used as a
diagnostic tool for cancer. Importantly, it also achieved good PTT conversion
efficiency (calculated to 28.58% ) with 980 nm laser irradiation , as well as high singlet

oxygen generation ability with 635 nm laser irradiation , which was evaluated by
DPBF chemical probe and fluorescent SOSG chemical probe . The PTT and PDT
properties of GQD -PEG -P were respectively tested for non -small cell lung cancer
(A549) cells, and significant cell membrane damage was observed. Furthermore,
MTT assay was used to investigate the PTT/PDT synergic treatment. When irradiated
with 635 nm and 980 nm lasers, the cell death rate of A549 cells which treated by
GQD -PEG -P was significantly increased to 86.0%, while the death rates under the
635 nm single laser and 980 nm single laser irradiation were 38.7% and 69.5%. In
vivo experiments, multicellular tumor spheres (MCTS) of MCF -7 cells were used to
evaluate GQD -PEG -P, and the results showed that nearly all MCF -7 cells were
destro yed. The proposed GQD -PEG -P offers great promise for cancer diagnosis and
treatment.
4.3 PDT with Immunotherapy
Cancer immunotherapy as revolutionizing oncology refers to the treatment by
activatin g or suppressing the immune system . Combination therapy based on PDT
and immunotherapy offers the potential to increase the anti -tumor immune response
and increase the sensitivity of tumors to immunotherapy in a safer and more efficient
manner [88].
Wu et al. [39] designed a novel multifunctional nanocomposite PC@GCpD (Gd)
by combin ing polydopamine -stabilized GQDs nanocomposites (GCpD) with
immunostimulatory polycationic polymers (PC) for magnetic resonance and
fluorescence imaging guide d photo immunotherapy . The nanocomposite produce d a
synergistic effect of photothermal and photodynami c therapy by GCpD to effectively
kill tumor cells . And it can deliver PC to endosome Toll-like receptor 9 (TLR9),
resulting in activation and infiltration of T lymphocytes by sustained stimulation of
secretion of pro -inflammatory cytokines and maturation of dendritic cells. In vivo
experiments, under laser irradiation, Gd almost completely inhibited the growth of
tumor tissue in the breast cancer model of EMT6 mice, which means that combined
phototherapy and immunotherapy have excellent synergistic effects . In addition, in
vivo drug delivery and biodistribution of Gd can be tracked using dual imaging,
indicating that the nanocom posite has the potential for precise cancer treatment.
4.4 Multiple combined therapies
Yang et al. [40] fabricated Lu-TP&Gd -TP/GQD -RGD , which provided a
synergistic therapy combining PDT, PTT and biological redox therapy (BRT). In this
nanoconstruct, a cyclic peptide (RGD peptide) was conjugated to GQDs, and the
selectivit y of GQD -RGD was improved [89]. GQD -RGD as drug delivery
nanovehicles , was loaded with a PDT agent lutetium (III) texaphyrin (Lu -TP) [90] and
a redox active drug Gadolinium (III) texaphyrin (Gd -TP) [91] via π−π stacking. In
vitro experiment, more than 62% of the A549 cells exhibited apoptosis or necrosis
after treated by Lu -TP&Gd -TP/GQD -RGD with irradiation . In vivo experiment , it
showed quiet low toxicity to A549 tumor -bearing mice. And the therapeutic effect of
the nanoconstruct was evaluated exhibiting a superior synergistic PDT/PTT/BRT
effect. Wo et al. [41] synthesize d a multimodal system LP -HMNS/SiO2/GQDs , which
encompas sed four synergistic therapeutic effects, including magneto -mechanical,

photothermal, photodynamic and chemo therapies . In this nanocomposite, a silica
shell containing amino groups was coated with hollow magnetic nanospheres
(HMNSs) of Fe3O4 and then GQDs were conjugated with them via amino bond , as
well as liposomes was used as a stabilizer . Fe 3O4 nanoparticles have been used in
magnetic induction hypertherm ia therapy , for example, with NIR laser irradiation
Fe3O4 nanoparticles could rapidly generating heat then inhibiting tumor growth by the
photothermal effect [92, 93] . LP-HMNS/SiO2/GQDs as a nanocarrier was further
loaded with DOX (LP-HMNS/SiO2/GQDs -DOX ). GQD s with huge specific areas,
superior photothermal conversion efficiency and capacity of generating ROS
contribute s to the multimodal system in drug lo ading efficiency, photothermal
conversion and intracellular ROS generation. In vivo, Eca-109 cells were exposed
under laser irradiati on or magnetic field stimulation, and results showing that
synergistic therapeutic effects can be achieved, as well as which were significantly
higher than Eca-109 cells with one stimulus.
5. Conclusion and perspective
In conclusion, GQDs stand out with excellent p hysicochemical properties , high
ROS generation capacity and imaging property , reported as great theranostics agent s
for PDT . And further combin ed with surface modification GQDs can act as drug
delivery nanocarriers, and various functional molecules have been utilized to adjust
GQDs -based nanocomposites with more favorable properties , which have been
extensively studied in vivo and in vitro for enhanced PDT and PDT-based
combination therapy . Together with bioimaging and selectively drug deliver
properties, GQDs may serve as the third -generation PS s, exhibiting great potential for
improved PDT.
However, owing to the large heterogeneity , GQDs prepared by different methods
make them exhibit a vast array of physicochemical properties . And the potential
toxicities of GQDs limit their practical use. It was reported that GQDs could induce
inflammatory response, and cause acute inflammation [94, 95] , and DNA damage
towards fibroblast cell lines (NIH-3T3 cells ) [96], which must be taken into careful
consideration when GQDs are used for future clinical studies . In addition, pure GQD s
exhibits limited PDT efficiency so that synthesize of GQDs with sharp characteristics
should be paid attention to . How to improve the PDT performance of GQD s by
precisely engineer their properties is still a significant and challenging task. Besides ,
similar to the triggered -release mechanism (pH, redox , GSH) mentioned above ,
GQDs-based nanocomposites as PDT agent s could be selectively activate d upon
enzymes , which has gained much attention in other carriers such as hydrogels .
Overall, G QDs are novel and potential nanomaterials for improved PDT in cancer
therapy . With the help of surface functionalization strategies and property control , it is
expected to see the clinical application of GQD s in the near future.

Acknowledgments
This work was supported by National Natural Science Foundation of China grants
(Nos. 81672672, 81572650, and 81772891) and by State Key Laboratory of Oral

Diseases Special Funded Projects.

Conflict of interest statement
The authors declare no conflicts of interest.

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