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Fluorescence Guided Activatable Cancer Theranostics: Its Development and Prospect
DOI: http://ITexLi.115104
current century. Theranostics represents a pioneering cornerstone in nanomedicine,
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possessing both diagnostic and therapeutic capacities that work simultaneously and
complementarily. It offers the capability to provide immediate insights into real-time
drug activation, distribution, and the scope of therapeutic interventions. Typically,
magnetic particles, mesoporous silica, carbon, and polymer nanoparticles are used to
create theranostic systems. The emergence of small molecular theranostic prodrugs
in nanomedicine, which incorporate fluorophores as optical markers, has gained
traction for monitoring the process of drug delivery and release. This is due to their
capacity to activate fluorescence signals simultaneously with drug release. In this
design approach, commonly, the reporter fluorophore and chemotherapeutic agents
need to be activated simultaneously and detached from the carrier. To achieve this,
one or two covalent bonds must be broken specifically within the cancerous region.
The aim is to prevent the premature release of the drug into the bloodstream or
normal cells. The active form of the drugs will only be attained upon encountering
specific entities that are notably abundant within the cancer cells. The most sophisticated approach involves designing a theranostic prodrug that ensures seamless
breakage of the chemical bond precisely at the tumor site. Figure
illustrates the
framework for the potential activation of both the reporter and the drug. In each
instance, there exists an activation or breaking point, which triggers the cleavage
process. Subsequently, the self-immolative linker undergoes fragmentation through
a distinct decomposition process that enables the formation of both the reporter
fluorophore and the drug, allowing them to attain their active states. In the Type
1 approach, cleavage happens at the center of the linker, leading to the production
of both the active drug and fluorophore through self-immolation. In the Type 2
approach, the triggering unit in the tumor-specific region activates a coordinated
pathway to generate both the fluorophore and drug via the elimination strategy with
the formation of unstable quinoid methide intermediates. In the Type 3 strategy, the
triggering unit within the theranostic is activated upon interaction with the cancerous
entity, subsequently producing the drug and fluorophore either simultaneously or
stepwise within a kinetically controlled timeframe. These approaches entail a comprehensive exploration of endogenous stimuli-responsive theranostic, which is further
elaborated in the following subsections.
Figure 1.
The design strategy of various self-immolative theranostics.
Smart Drug Delivery Systems – Futuristic Window in Cancer Therapy
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. Glutathione-activated theragnostic
Glutathione (GSH) is a tripeptide composed of glutamic acid, cysteine, and glycine. Functioning alongside its oxidized form glutathione disulfide (GSSG), it serves
to uphold cellular redox balance. Primarily generated in the cytosol, it predominantly
remains there, safeguarding against oxidative stress by counteracting reactive oxygen
species (ROS). Moreover, glutathione (GSH) plays a pivotal part in numerous cellular
activities, including cell differentiation, proliferation, and apoptosis.
The concentration of GSH in cancer cells is significantly higher than that found
in normal cells, with intracellular GSH levels (1–10mM) approximately 1000 times
greater than those in the extracellular compartment (2–20μM). This heightened
GSH concentration renders neoplastic tissues more resistant to chemotherapy [24].
Additionally, increased GSH levels in certain tumor cells are commonly linked to
elevated GSH-related enzyme activity, such as γ-glutamylcysteine ligase (GCL) and
γ-glutamyl-transpeptidase (GGT), along with an upregulation of GSH-transporting
export pumps [25]. Undoubtedly, GSH can serve as a significant trigger for the
targeted delivery of chemotherapy to the tumor microenvironment.
Within the small molecular theranostic approach, both the fluorophore and drug are
linked through a spacer containing a susceptible “–S–S–” linker, which can be broken
down by cellular glutathione (GSH). In addition to this responsive bis-thiol (–S–S–)
linker, a cancer-targeting ligand has been integrated to enhance the selective release of
therapeutics in specific cancer cells. As depicted in
Figure , the doxorubicin linked
with folate () and containing an –S–S– linker initially displayed inactive doxorubicin
fluorescence due to an atypical photoinduced electron transfer (PET) to the folic acid
[26]. After being taken up by folate receptor-positive (FR+) adenocarcinoma human
alveolar basal epithelial cells (A549), theragnostic prodrug exhibited red fluorescence,
indicating the activation of doxorubicin. In 24hours, Dox completely translocated to
the nucleus and integrated with topoisomerase II. In contrast, folate receptor-negative
breast cancer cells (MCF-7) subjected to the same treatment did not exhibit any
indication of doxorubicin release, as there was no observable fluorescence representing
active doxorubicin Crucially, the stability of doxorubicin in plasma has enhanced, with
a half-life (t1/2) of 47hours. Its efficacy in eradicating FR+ cancer cells was notably
heightened, with an IC50 value of 1.27μM, surpassing that of doxorubicin alone.
Figure 2.
Theranostic prodrug 1 [26] and its activation.
Fluorescence Guided Activatable Cancer Theranostics: Its Development and Prospect
DOI: http://ITexLi.115104
Figure 3.
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GSH-mediated activation of theranostics 2.
Another theranostic prodrug [27], in combination with SN-38, a topoisomerase
I inhibitor, and biotinylated rhodol, has demonstrated effective anticancer properties
in biotin receptor-positive A549 cells and HeLa cells. Both of these biotin receptorpositive cell lines exhibited strong green fluorescence attributed to the production of
active rhodol. Conversely, the normal NIH3T3 cell line did not display this activity,
despite having sufficient glutathione. This indicates that biotin in (Figure ) plays a
crucial role in the selective internalization of via receptor-mediated endocytosis in
biotin receptor-positive cell lines. Ex-vivo data indicated that the theranostic prodrug
selectively has accumulated in the tumor, resulting in the reduction of tumor volume
and weight. Different cancer cell-specific ligands, anti-cancer medications, and fluorophore/imaging instruments have been employed to monitor selective chemotherapeutic
interventions in both in vitro and in vivo settings, as outlined in Table [28–35].
Theranostics Drugs Target ligand Abs./emission
CPT RGD (Arg-Gly-Asp)
Gemcitabin D-biotin 700 (TP) /450 A549 — [29]
Gemcitabine Folate 630/700 KB KB cell mice [30]
DCM-S-CPT
(PEG-PL)
Gemcitabin D-biotin 700/7 20 A549 — [32]
HJ-inhibitor D-biotin 430/540 HepG2 — [33]
chlorambucil — 430/540 HeLa — [34]
TP- SN-38 PVA-biotin 510/550 HeLa — [35]
Table 1.
Examples of GSH-stimuli responsive theranostics.
(λ
/ λem) nm
abs
integrin
CPT EPR effect 465/665 BCap-37 BCap-37 tumor
430/535 U87 — [28]
In vitro
cells
Ex vivo/in vivo Ref.
xenograft mod
[31]
Smart Drug Delivery Systems – Futuristic Window in Cancer Therapy
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While GSH-triggered theranostics have primarily been examined, concerns about
stability in blood plasma persist due to the considerable presence of thiol entities in
the blood.
. Hydrogen peroxide mediated theranostics
Hydrogen peroxide is a widely recognized reactive oxygen species formed
from superoxide in mitochondria. It plays a critical role in cell signaling, proliferation, and maintaining redox balance. However, it is continuously produced in
cancer cells (at a rate of 0.5nmol per 1×104 cells per hour) [36]. The cancer cells
utilize hydrogen peroxide as a mechanism to acquire nutrients from neighboring
fibroblasts through stromal induction of autophagy and mitophagy. Moreover, it
leads to DNA damage, cellular metabolism, and inflammation by activating NFκB
[37]. Consequently, endogenous H
can serve as an excellent stimulant for the
2O2
cancer cell-specific activation of theranostic prodrugs. The theranostic prodrug
(Figure ) operates based on a type 2 strategy, activated in the presence of
either endogenous or exogenous H2O2. It involves a two-step process to release the
topoisomerase I inhibitor SN-38. The initial step, considered to be the slowest, is
regarded as the rate-determining step where the boronate group transforms into
OH, subsequently undergoing a 1,8-elimination reaction to generate SN-38. The
unstable quinone methide form of coumarin spontaneously transforms, leading
to the manifestation of blue fluorescence in metastatic murine melanoma B16F10
cells and human cervical cancer cell lines (HeLa cells) [38]. It yielded promising
outcomes in a murine mouse model of lung metastatic cancer, exhibiting a notably
higher survival rate compared to the untreated group. Kim and colleagues noted
that the combination of 5-fluorouracil (5-FU) with the self-monitoring intrinsic
(mitochondrial) apoptosis marker ethidium bromide demonstrated significant
antitumor activity in the A549-xenografted mice model. The theranostic prodrug
resulted in a notable reduction in tumor size compared to both the control group
and only the drug alone [39]. Shabat et al. observed that their prodrug, quinone
cyanine 7-CPT, was activated in human glioblastoma multiform (GBM) U-87 cells
in the presence of exogenous H2O2 (5 equivalents) [40]. The complete release of
camptothecin (CPT) occurred within 90minutes, monitored by the emission of
quinone cyanine 7 (QCy7) at 720nm. Administering the theranostic prodrug intratumorally and through intravenous tail vein injection in U-87 MG tumor-bearing
Figure 4.
H2O2 mediated SN-38 activation.
Fluorescence Guided Activatable Cancer Theranostics: Its Development and Prospect
DOI: http://ITexLi.115104
mice generated a strong fluorescence signal in the tumor region, attributed to the
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activation of the drug.
Utilizing ROS-mediated theranostic, particularly the activation of prodrugs
induced by H2O
could represent a futuristic strategy to enhance the therapeutic
2
potential of anticancer drugs.
.
Hydrogen sulfide stimuli-responsive theranostic
Hydrogen sulfide, a gasotransmitter, plays a crucial role in neurotransmission.
Enzymes like cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) are
overexpressed in certain cancer cells to generate endogenous H2S [41]. This compound aids in cellular bioenergetics, supporting tumor growth and proliferation,
and also facilitates angiogenesis and vasorelaxation, contributing to the blood supply
and nutrient provision for the tumor [42, 43]. Other than colon cancer, Bhuniya et
al.
noted that cervical cancer cells (HeLa cells), breast cancer cells (MDA-MB-231 cells),
and prostate cancer cells (DU145 cells) generate a larger amount of H2S in the mitochondria compared to normal cells [44]. The same research group later employed a
distinctive strategy to specifically decrease the mitochondrial inner membrane potential (Ψ) in cancer cells by releasing the 2,4-dinitrophenol protonophore (Figure
,
prodrug ) [45]. The blue emission of the coumarin fluorophore tracked the degree of
protonophore activation. It was noted that prodrug (Figure
) led to elevated ROS
production and ATP depletion in both colon cancer cells (HCT116 cells) and cervical
cancer cells (HeLa cells), along with a decrease in the mitochondrial inner membrane potential (Ψ). These effects selectively halted the proliferation of cancer cells
(HCT116 cells and HeLa cells) while maintaining the proportional growth of normal
cells (3T3L1). Another theranostic agent, TP-HS, released the topoisomerase I inhibitor SN-38 in colon cancer cells (HCT116 cells) and lung cancer cells (A549 cells),
which was monitored using the rhodol fluorophore [46]. In contrast, TP-HS did not
exhibit any indication of SN-38 release in normal human fibroblast cells, WI-38 cells.
It demonstrated cancer cell-specific antiproliferative activity, while SN-38 resulted
in nonspecific growth arrest in both normal and cancer cells. Furthermore, SN-38
led to undesired necrosis pathway cancer cell death (18%), whereas the theranostic
prodrug TP-HS exclusively followed the apoptosis pathway, arresting cancer growth.
The H2S-Gem theranostic prodrug exhibited dose and time-dependent labeling of
Figure 5.
Endogenous H2S-mediated protonophore activation.
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cancer cells, including HeLa cells and A549 cells, while remaining inactive in normal
human fibroblast cells WI38 [47]. It displayed the highest toxicity to both cancer
cells (HeLa and A549) at concentrations below 5.0μM of H2S-Gem. However, it did
not demonstrate any toxicity in WI38 cells even after 72hours of incubation. In this
scenario, the coumarin fluorophore acted as a reporter, providing temporal information on the activation of gemcitabine. Shi and colleagues created a photo-controlled
camptothecin release theranostic tailored for endogenous H2S-rich cancer cells [48].
The nanoplatform, known as NPs@BOD/CPT, was composed of borondipyrromethene (InTBOC-Cl) functioning as an H2S-triggered near-infrared (NIR) photothermal
agent, with camptothecin-11 (CPT-11) enclosed within thermosensitive nanoparticles. In the absence of H2S, it did not exhibit any hyperthermic effects, and there was
no leakage of CPT-11 when exposed to NIR laser. However, in the presence of 576nm
NIR light, the theranostic induced significant damage to cancer cells, owing to the
combined action of hyperthermia and the release of CPT-11 in the presence of H
2
In an in vivo HCT116 tumor-bearing mouse model, treatment with nano-theranostics
and NIR irradiation reduced tumor size and volume after 14days.
Utilizing endogenous H2S for theranostic activation in different cancer types is a
distinctive strategy. It eliminates the need for a cancer-specific ligand, simplifying the
synthesis of theranostic prodrugs, and making the process more straightforward and
highly valuable for cancer treatment.
. pH-dependent theranostics activation
S.
The characteristic feature of cancer is an extracellular acidic environment. This
arises from the production of lactic acid through anaerobic glycolysis and increased
activity of the pentose pathway, resulting in the generation of carbonic acid within
cancer cells. While the extracellular pHe in normal cells typically remains at 7.4,
the extracellular pHe in cancer cells, on the other hand, ranges between 6.3 and 7.1.
Therefore, the hydronium ion (H3O+) may serve as a potential trigger for the activation of chemotherapeutics from their theranostic prodrug systems. Zhang et al.
introduced a theranostic compound called Mal-hyd-Dox, which utilized an integrinspecific Gly-Arg-Gly-Asp-Ser (GRDS)-oligopeptide as a cancer cell-specific ligand
[49]. Dox was linked to the compound via an acid-sensitive hydrazone linker, and it
featured a coumarin reporter. The fluorescence emitted by coumarin in Mal-hyd-Dox
was initially faint; however, it became luminescent upon internalization in U87 cells
within an acidic endosomal environment. The compound exhibited an IC50 value of
0.19μgmL−1, and both Dox and coumarin acted as reporters, providing red and blue
channel images, respectively. In the same category, Zhang et al. developed a double
FRET-based theragnostic that both caspase (Asp-Glu-Val-Asp (DEVD) peptide
sequence) and pH act as stimulators for releasing doxorubicin in U87 cells [50]. The
cleavage of the DEVD peptide sequence regenerated a green fluorescence based on
fluorescein, demonstrating a pronounced anticancer effect (IC50=4.3×10−6M).
Sessler and colleagues introduced a bimodal multifunctional theranostic conjugate
(as depicted in
Figure ) with T1-weighted MRI. This conjugate consisted of a paramagnetic motexafin gadolinium (MGd) texaphyrin unit connected to two doxorubicin subunits via an acid-labile hydrazone linker [51]. Initially, the fluorescence signal
of doxorubicin was quenched by the strong paramagnetic signal of gadolinium ions.
However, upon internalization in A549 (human lung) cancer cells and CT26 (murine
colon carcinoma) cancer cells, the labile hydrazone bond encountered the acidic pH
of cellular lysosomes (approximately pH4.5), resulting in the release of doxorubicin.
Fluorescence Guided Activatable Cancer Theranostics: Its Development and Prospect
DOI: http://ITexLi.115104
Figure 6.
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pH-responsive paramagnetic theranostic 5.
Consequently, the release of doxorubicin was monitored using T1-weighted MRI and
fluorescence imaging (Dox). This integration of MRI and optical imaging modalities allows for the visualization of tumors using MRI and the analysis of histological
samples through optical imaging. Additionally, it enables the delineation of tumors
via both MRI and intra-operative optical imaging.
Later Wang et al. incorporated PEGylated biotin as a targeting ligand to improve
the selectivity for precise anticancer activity in biotin receptor-positive human
colorectal cancer cell lines (HT-29 and LS180 cells) [52]. The theranostic prodrug
(Figure ) combined labile hydrazone derivatives of doxorubicin and conjugation of PEGylated biotin. The prodrug was applied to colorectal cancer cell lines
beforehand, resulting in a halt in the cell cycle with percentages of 69.54 and 76.76%
at 12h and 24h, respectively. In contrast, colorectal cancer cells that were not treated
showed a G1 phase percentage of 67.45%. On the contrary, prodrug cannot release
payload to receptor-negative human embryonic kidney (HEK 293) cells. Prodrug
readily enters cells through receptor-mediated endocytosis and is then localized
in the nucleoli following the breakdown of the hydrazone bond in the lysosome.
Figure 7.
pH-responsive PEGylated biotin theranostic 6.
Smart Drug Delivery Systems – Futuristic Window in Cancer Therapy
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Figure 8.
pH driven photodynamic therapeutic NIR theranostic 7.
Duringinvivo biodistribution studies, free doxorubicin displayed notable accumulation in heart tissue, while prodrug primarily accumulated in tumor tissue. Prodrug
exhibited a considerable tumor volume and weight loss in LS180 cells inoculated
mice without causing overall body weight reduction. Conversely, mice with tumors,
injected with doxorubicin, experienced significant weight loss posttreatment. The
interaction between the ligand and receptor promotes increased uptake of prodrug
by the tumor tissues, and cellular acidosis facilitates the controlled release of the
anticancer agent doxorubicin within the tumor site, thereby minimizing its adverse
effects on overall health.
In general, these strategies revolve concept of prodrugs, wherein established
commercial drugs are repurposed as part of a novel formulation system following
chemical alterations in anticancer medications. Occasionally, these modifications can
impact the drug’s metabolic properties. Kamkaew et al. devised a novel pH-triggered
photodynamic therapy approach using theranostic (Figure ) as its foundation [53].
In an acidic environment, it demonstrated a significant increase in reactive oxygen
species generation compared to physiological pH levels (around pH7.4). Essentially,
within an acidic environment, there is a decrease in the energy gap between orbitals
and an enhancement in intramolecular charge transfer, promoting the formation of
singlet oxygen under NIR irradiation at 850nm. Consequently, theranostic molecule
exhibited robust photodynamic efficacy in HepG2 hepatic carcinoma cells, while
remaining inert in normal human embryonic kidney cells. Additionally, it exhibited
efficacy in penetrating deep-seated tumor tissues, suggesting potential for enhanced
in vivo outcomes.
While the acid labile activation of theranostics demonstrated promising outcomes, a substantial portion of the payload may be released in the extracellular matrix
due to its acidic nature. Consequently, this conventional strategy might not suffice as
a robust approach.
. β-Galactosidase stimulated theranostics
β-galactosidase exhibits notable potency as a cancer biomarker in specific cancer
subtypes, including colon cancers, lung cancers, liver cancers, and ovarian cancers
[54]. Bhuniya and colleagues observed that a coumarin-conjugated theranostic
(Figure ) released the nucleoside drug gemcitabine to hepatic carcinoma cells
(HepG2) upon encountering cellular galactosidase, as compared to cervical cancer
cells and normal human foreskin (HFF-1) cells [55]. The coumarin fluorophore
monitored the entire cellular uptake and drug release process. Similarly, Kolemen
and Gunbas et al. utilized a β-galactosidase activatable iodo-resorufin derivative
Fluorescence Guided Activatable Cancer Theranostics: Its Development and Prospect
DOI: http://ITexLi.115104
Figure 9.
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β-Galactosidase activated cancer theranostics.
(Figure ) for photodynamic therapy against cancer, showing selective activation
in malignant U-87MG cells and the production of reactive oxygen species (ROS)
[56]. Kim et al. demonstrated β-galactosidase stimulated release of doxorubicin from
Gal-Dox () (Figure ) in receptor-positive HT29 and HepG2 cells compared to the
receptor-negative cervical cancer cells (HeLa cells) [57]. In an in vivo HT29-bearing
mice model, the administration of Gal-Dox exhibited significant inhibition of tumor
growth (53.1%) compared to free Dox treatment (34.9%). Yet, it remains unclear in
nearly every instance whether alterations have occurred in the pharmacokinetics of
the parent drug.
Recently identified as a cancer biomarker, β-galactosidase is poised to find
diverse applications in activating theranostic agents for targeted cancer treatments.
However, till now, a limited number of theranostics have been reported. Most of the
theranostics cases introduce anticancer agents with reporter fluorophore through
a covalent linker, which may perturb the chemotherapeutic activity of the parent
drugs, and sometimes alter metabolic ingredients which introduce unwanted toxicity.
Additionally, it is crucial to focus on the design approach and gather thorough pharmacokinetic data on theranostic prodrugs. It is even more advantageous to consider
a novel strategy centered around theranostics to circumvent reliance on currently
available drugs.
. Theranostics for tumor hypoxia
Unrestrained proliferation of blood vessels leads to the restriction of blood supply
in the tumor area, creating a harsh hypoxic microenvironment. This condition is
exacerbated by the anaerobic glycolysis in tumor tissues, triggering hypoxia through
an excessive expression of hypoxia-inducible factor (HIF1α) [58]. Consequently,
the HIF1α suppressor, prolyl hydroxylase, fails to regulate the expression of HIF1α.
The hypoxic tumor is located at a significant distance from the closest bloodstream
(≥150μm) (Figure ), thereby preventing the diffusion of oxygen to the cancer
cells, resulting in a decreased partial oxygen pressure in this zone (pO2≤20mmHg).
Consequently, hypoxia diffuses heterogeneously within the solid tumor, leading to
the development of metastatic tumor masses.
The diminished oxygen pressure during hypoxia significantly impacts the effectiveness of current anticancer treatments. In the context of radiotherapy, the inadequate generation of reactive oxygen species in hypoxic areas often leads to radiation
resistance, thereby impeding the creation of lethal damage to DNA in cancer cells.
Furthermore, the hypoxia-induced HIF-1α/β factor and free oxygen radicals may
Smart Drug Delivery Systems – Futuristic Window in Cancer Therapy
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Figure 10.
Schematic representation of hypoxic tumor formation.
stimulate the production of angiogenesis factors, counteracting the damage intended
to be inflicted by radiation therapy. Likewise, the efficiency of chemotherapeutic
agents is generally compromised in hypoxic tumor regions. These agents may face
obstacles in reaching the hypoxic tumor area. Moreover, elevated extracellular acidosis in hypoxia plays a pivotal role in impeding the cellular uptake of chemotherapeutic
Theranostics Activating
group
NO
AzP NN SN-38 Green FL HeLa 4T1-cells
PDU-DB -NO NO
NO
Azo-M NN melphalan analog NIR FL HeLa 4T1 Tumor-
PANO() NH
AzCDF NO
Active drug Imaging
SN-38 Green FL HeLa, A549 HeLa cells
2
Gemcitabine Green FL MGC-803
2
SN-38 Green FL HeLa – [62]
2
OH KDAC inhibitor Pano – HCT116 cells OE21
2
3,4-difluorobenzylidene
2
curcumin
modality
Green FL MDA-MB-231 MDA-MB-231
In vitro cells In vivo /
cells
ex-vivo
xenograft
mouse model
inoculated
xenograft
murine mouse
model
MCF-7-cellinoculated
xenograft
mice
bearing
xenograft
mouse
xenografts
xenografts
Table 2.
Information about various hypoxia-induced theranostic activation system.
Ref.
[59]
[60]
[61]
[63]
[64]
[65]
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