Two-photon excitation nanoparticles for photodynamic therapy†

Two-photon excitation (TPE) nanoparticle-based photosensitizers (PSs) that combine the advantages of TPE and nanotechnology
have emerged as attractive therapeutic agents for near-infrared red (NIR) light excited photodynamic therapy (PDT) for cancer
treatment. TPE PDT is characterized by nonlinear absorption of two relatively low-energy photons of NIR light with the resulting
emission of high-energy visible light. This high-energy light can sensitize oxygen to produce cytotoxic reactive oxygen species
(ROS) and singlet oxygen (1O2) which can kill cancer cells. The long-wavelength light used to excite TPE NPs allows for deeper
tissue penetration to achieve efficient PDT of deep-seated tumors. Moreover, TPE nanoparticles normally have large two-photon
absorption (TPA) cross-sections, which hold great potential as efficient two-photon donors in PDT. In this review, we will
summarize the recent advances made in the development of TPE nanoparticles for cancer PDT. Five different TPE nanoparticles,
including quantum dots (QDs), carbon nanomaterials, silica nanoparticles, gold nanomaterials, and polymer nanoparticles, are
summarized in detail, and the existing challenges as well as the future perspectives are also discussed.

Introduction
Over the past few decades, photodynamic therapy (PDT) has served as an effective cancer-therapy modality with
high spatiotemporal precision both in clinical research and practice. 1–4 PDT involves the application of a non-toxic
and tumor-localizing photosensitizer (PS) that can be activated by focused light to generate cytotoxic reactive oxygen
species (ROS) through both type I and type II mechanisms (Fig. 1a). 5–7 Type I mechanisms involve an activated PS
(known as excited triplet state PS) directly reacting with neighboring biological substrates in cancer cells to form
radicals and radical ions. These radicals can then subsequently interact with oxygen and give rise to ROS, including
superoxide anion radicals, hydroxyl radicals, or hydrogen peroxide. In type II processes, the excited triplet state PS
interacts directly with molecular oxygen to form singlet oxygen ( 1O2)—a highly cytotoxic ROS. It is recognized that
1O2 is the major ROS during PDT, which can induce cancer cell death through multifactorial mechanisms, ultimately
resulting in necrosis and/or apoptosis,8 the destruction of tumor microvasculature,9 and the stimulation of the
inflammatory and immune responses that can destroy the remaining cancer cells. 10,11 Compared to conventional
cancer therapies (i.e., surgery, chemotherapy, radiotherapy), PDT has benefits of being a non-invasive, localized
therapy promoting fast healing of heathy tissue to reduce long-term morbidity and improve the patients’ quality of
life.12–14 Typically, the application of PDT requires a PS molecule, light, and O 2 in order to generate the toxic ROS
that mediate cancer cell killing. To achieve effective PDT in cancer, it is crucial to develop PSs which can be
preferentially taken up by tumor cells and generate 1O2 in high quantum yield upon, ideally, NIR light excitation. 15–20
As such, various small molecule organic PSs such as porphyrins, methylene blue, and BODIPY have been
extensively made.21–26 However, most small molecule PSs are subject to relatively low PDT outcomes due to low
extinction coefficients and emission quantumyields, absorption wavelengths in the visible spectrum, and poor
solubility in aqueous media resulting in aggregation. These characteristics yield reduced 1O2 generation in tumor
mtissues and ineffective therapy. 27–31 Importantly, the selectivity of these PSs for tumor tissue over healthy tissue is
poor and often results in significant off-target tissue damage. 32,33 Alternatively, nanoparticle-based PSs represent the
next generation of PDT PSs that can overcome the limitations of smallmolecule dyes to improve the anti-tumor
efficiency of PDT improve the performance of PSs. Compared to small molecule PSs, the application of
nanoparticles to PDT shows the following benefits: (1) large extinction coefficients and emission quantum yields to
provide more efficient photosensitization; (2) the ability to encapsulate hydrophobic PSs and maintain them in their
monomeric form, preserving their photosensitizing activity, improving their solubility, and maximizing their 1O2
quantum yield under physiological conditions; 28 (3) the facile control of nanoparticle size and structure to maximize
surface-to-volume ratios and optimize the loading efficiency of PS molecules for PDT; (4) the ability to increase
accumulation in solid tumors through passive targeting by the enhanced permeability and retention effect (EPR); 37,38
(5) the well-established surface modification chemistries to attach targeting ligands and functional groups (e.g.
poly(ethylene)glycol) to improve pharmacokinetics, biodistribution, specificity and cell uptake in order to enhance
the efficacy of in vivo PDT; (6) the ability to rationally design multifunctional nanocomposites by integrating various
sensitizers with chemo-therapeutic drugs and multimodality molecular imaging agents to achieve imaging-guided
PDT. As such, a variety of nanoparticle PSs, including polymeric, metallic, magnetic, carbon material-based,
semiconductor-based and silica-based nanoparticles have been developed, and some excellent reviews covering some
of them have been published. In spite of the aforementioned benefits of nanoparticle PSs for PDT, their translation to
the clinic remains a challenge. One of the major obstacles in using these nanoparticle PSs for efficient cancer therapy
is that their activation wavelengths are generally in the UV-visible range. This falls outside of the first biological
transparency window (650–950 nm), resulting in poor tissue penetration of the excitation light. Therefore, it is rather
difficult to activate the PS molecules to generate sufficient cytotoxic 1O2 to kill cancer cells located in deep tissue.
Efforts to design NIR light sources have been made in order to ensure efficient delivery of light into deeper tissues
for PDT. One of the recently explored techniques to enhance tissue penetration is through lanthanide-doped
upconversion nanoparticle (UCNP)-based PSs. These UCNPs can be excited by low energy NIR light and emit high
energy photons at shorter wavelengths to activate encapsulated PS molecules. Compared to the traditional down-
conversion luminescent nanomaterials, UCNPs have better tissue penetration depth, higher photochemical stability,
and lower auto-fluorescence background, which have made UNCPs successful for preclinical in vivo imaging
applications Another class of nanoparticle-based PS depends on two-photon absorption. This class of nanoparticle
PSs integrates the advantages of two-photon excitation (TPE) with those of nanotechnology, and have recently
emerged as an attractive alternative to the NIRexcitable PDT technique. 52–55 While traditional one-photon excitation
of PSs requires the absorption of a single photon equal to the band-gap energy of the PSs, TPE occurs when two
lower energy photons of infrared light are absorbed by the PS, where the sum of the photon energies are equal to the
band-gap of energy, thereby allowing for deeper light penetration and lower photo-bleaching of PS molecules in
tissues (Fig. 1a).46–58 A key feature of TPE is the nonlinearity of photon absorption that makes it possible to activate
PSs at the focal point of the laser beam. This allows for better spatial control of PS activation in three-dimensions
during PDT, reducing off-target damage to surrounding healthy tissues. For example, the use ofTPEPDTto
selectively close off a single bloodvessel located at the focus of the laser beam was successfully demonstrated by
Anderson and co-workers in 2008, 59 suggesting highly precise spatial treatment in vivo. Since then, great progress
has been made in advancing TPE PDT, including the development of various TPE nanoparticles with enhanced
tumor targeting abilities and enhanced 1O2 quantum yield upon TPE, which could provide a potential to achieve
precise cancer PDT at deeper tissue penetration. In this article, we will summarize the recent advance in the
development of TPE nanoparticles for cancer PDT pre-clinically. The currently reported TPE nanoparticles for PDT
mainly include semiconductor quantum dots (QDs), carbon nanomaterials, silica-based nanoparticles, gold
nanoparticles and polymeric nanomaterials (Fig. 1b). These nanoparticles were either designed as two-photon
absorption (TPA) materials (e.g. QDs) to sensitize encapsulated PS molecules or used as tumor-targeting
nanocarriers (e.g. silica-based nanoparticles) to load two-photon absorption dyes and PSs. Table S1 in the ESI †
summarizes various TPE nanoparticles reported to date for PDT applications, which we will separately describe in
this review. Moreover, the current challenges and perspective of TPE nanoparticle-based PDT as a feasible
theranostic technology will be discussed as well. Although there have been many recently published reviews
regarding the applications of nanoparticles in PDT, 12,39–45 none of them have discussed TPE nanoparticle-based PDT
in detail. Therefore, this review will focus on the TPE field in efforts to assimilate the state-of-the-art and inform
future research on the design of TPE nanoparticles for precise cancer PDT. TPE QD-based PSs
Semiconductor nanoparticles (QDs) have been a topic of considerable interest in biomedical applications due to their
prominent optical properties, including high emission quantum yield, high photostability, broad absorption spectra,
and narrow and symmetrical emission wavelengths. 60–63 QDs also have large extinction coefficients (e = 104–107 M_1
cm_1), providing the ability to serve as strong light absorbers. 64,65 Moreover, their optical properties can be precisely
tuned from the UV to NIR region through facile modification of their size and composition. 66–69 These unique
advantages offer great promise in molecular imaging and PDT applications. 70–73 In addition to their large extinction
coefficients, large TPA cross-sections (s2 = 103–105 Goeppert–Mayer, or GM, units. The methods for measuring TPA
cross-sections are briefly discussed in the ESI†53,74) were also found for certain well-designed QDs. 75–77 Together,
these properties make QDs outstanding two-photon sensitizers for PDT. In order to trigger 1O2 generation during TPE
PDT, QDs could either act as PSs on their own to directly sensitize O 2, or act as energy donors that can transfer the
energy to attached PSs to indirectly sensitize O 2. However, the efficacy of QDs themselves to directly sensitize O 2 for
the generation of 1O2 is rather low (B5%).78 Therefore, current research on QD-based TPE PDT is mainly focused on
the design of different QD–PS nanocomposites, with the goal of enhancing the energy transfer efficacy and
improving the 1O2 quantum yield. The first report of using QDs for TPE PDT applications was demonstrated by
Dayal et al. in 2008.79 In their study, uniform CdSe QDs with an average diameter of 5 nm were synthesized, and
silicon phthalocyanine 4 (Pc 4) was then covalently linked to the surface of QDs to prepare QD-Pc 4
nanocomposites. The QDs were chosen to have a maximum fluorescence at 635 nm, which overlapped well with the
absorption of Pc 4 to ensure efficient energy transfer. Following TPE of the QDs at 1100 nm, the two-photon
sensitization of Pc 4 molecules occurred via a fluorescence resonance energy transfer (FRET) process between QD
and Pc 4, with an energy transfer efficiency of B70%. Encouraged by this, Wen et al. used water-soluble CdTe QDs
as TPE energy donors to sensitize the electrostatically absorbed meso-tetra(4-sulfonatophenyl)porphyrin
dihydrochloride (TSPP) molecules under 800 nm laser excitation, and an energy transfer efficiency as high as
82.6%was observed.80 In 2013, Skripka et al. exploited a QD-chlorin e6 (Ce6) complex formed through hydrophobic
interactions between Ce6 molecules and lipid-coated QDs, which were effectively excited via two-photon irradiation
at 1030 nm. When multiple Ce6 molecules were bound to the surface of QD, the energy transfer efficiency upon TPE
reached about 80%.81 Though it is encouraging that two-photon triggered energy transfer between the QDs and PS
molecules was observed, it has to be mentioned that these preliminary studies were merely conducted in solution, and
the 1O2 production efficiency remains unknown. In 2011, Qi et al. determined the 1O2 generation efficiency of a
biocompatible CdSe/ZnS QD-based PS under TPE (Fig. 2). Core shell CdSe/ZnS QDs protected by tri-n-
octylphosphine oxide (TOPO) were encapsulated with an amphiphilic polymer to improve their water solubility and
biocompatibility. The TPA cross-section values for amphiphile-coated QDs were found to range from 800 to 2400
GM, which are much larger than those of porphyrin itself. To ensure efficient energy transfer, a water soluble
porphyrin (TrisMPyP-COOH) as the FRET acceptor was then covalently conjugated to the surface of the polymer-
coated QDs, resulting in a final ratio of porphyrin to QD of 18 : 1. Upon TPE of QDs at 800 nm, an efficient FRET
from QD to porphyrin occurred and the 1O2 generation efficiency was found to be two-fold greater than that of the
porphyrin solution alone. This work provided the groundwork from which TPE QD-based PSs could be applied for
PDT in biological systems.82 Fowley et al. later developed a CdSe/ZnS QDs–Rose Bengal (RB) conjugate that
effectively produced 1O2 and was capable of killing tumor cells upon TPE at 800 nm (Fig. 3). 83 In their study, a water
soluble mercaptopropionic acid (MPA) coated CdSe/ZnS QD with a maximum emission of 525 nm was synthesized,
and subsequently conjugated to RB via an amide linkage. A high loading capacity of 64 RB molecules was achieved
on the surface of each QD, which improved the aqueous solubility of RB and resulted in a FRET efficiency as high
as 99.5%between the QD and RB. The 1O2 generation efficiency of the QD–RB nanocomposites in cultured HeLa
cells upon TPE at 800 nm was evaluated using a fluorescent 1O2 indicator 20,70-dichlorodihydrofluorescein diacetate
(DHFA), which showed a bright green intracellular fluorescence. In contrast, only negligible fluorescence was
observed in HeLa cells incubated with both QD–RB and DHFA without TPE. These results suggested that the QD–
RB conjugate was able to produce cytotoxic 1O2 in living human tumor cells in vitro upon TPE, which could
ultimately induce tumor cell death. In 2013, Chou et al.84 used a core–shell CdSe/CdS/ZnS QD as the TPE energy
donor to sensitize electrostatically absorbed sulfonated aluminum phthalocyanine (AlPcS) molecules upon two-
photon irradiation with an unfocused 800 nm femtosecond (fs) pulsed laser beam (Fig. 4). The FRET efficiency of
the QD–AlPcS nanocomposite in water was found to be as high as 90%, and the 1O2 generation under TPE at 800 nm
was tested in aqueous solution with an 1O2 sensitive substrate, 1,3-diphenylisobenzofuran (DPBF). The aqueous
solution of QD–AlPcS and DPBF was irradiated at 800 nm with the fs laser at a low power density of 92 mW mm _2,
resulting in a remarkable reduction of the DPBF fluorescence that indicated a large amount of 1O2 generation. The
FRET mediated 1O2 generation and PDT was subsequently studied in vitro in cultured human HeLa and KB cancer
cells incubated with QD–AlPcS (0.1–5 mM) for 1 h. Upon TPE at 800 nm, both HeLa and KB cells were efficiently
killed, with the degree of cell kill proportional to the irradiation dose. More than 90% of the HeLa cells were killed
following 60 min irradiation with the 800 nm fs pulsed laser. Importantly, there was no significant difference in the
PDT killing efficiency between one-photon excitation and TPE paradigms, suggesting that QD–AlPcS are feasible
for TPE PDT. This is the first report showing that TPE of a QD–PS conjugate could compete with the traditional
one-photon excitation to achieve PDT in killing efficiency against cancer cells. Despite the progress that has
beenmade toward building TPE QD–PS nanocomposites, their suitability for in vivo PDT still needs to be verified.
Very recently, Lemon et al. have employed micelle-encapsulated QD–palladium( II) porphyrin (PdPS) complexes to
construct water soluble and biocompatible QD–PdPS assemblies as two-photon oxygen sensors. 85 Due to the
improved biocompatibility and unique optical properties of the nanocomposite, the application of QD–PdPS
assemblies to noninvasively image and report on vascular oxygen levels in living mice was successfully
demonstrated under two-photon irradiation using a window chamber model. Though their application for in vivo
PDT was constrained due to negligible 1O2 production, this work demonstrated the potential to build a more efficient
TPE QD–PS system, replacing PdPS by other PS moieties with higher 1O2 generation efficiency (e.g. Pc 4). This
system would have the potential to enable the generation of large amounts of 1O2 under TPE to initiate precise in vivo
PDT for cancer treatment.

3. TPE carbon nanomaterial-based PSs

Even with the initial PDT success demonstrating their in vivo potential, the QD-based PSs inherently rely upon
toxic metal ions (e.g. Cd2+) for their synthesis. As a result, their safety in vivo remains a major concern. Recently,
carbon-based nanomaterials including carbon dots, carbon nanotubes and graphene have emerged as relatively
benign nanomaterials for optical imaging and related biomedical applications. 86–89 Carbon-based nanomaterials
havemanymerits over QDs and traditional fluorescence dyes, such as robust chemical inertness, easy
functionalization, high resistance to photobleaching, good biocompatibility and large TPA cross-sections. 90–93 These
carbon-based nanomaterials hold great promise to develop novel carbon nanomaterialbased PSs enabling efficient
PDT upon two-photon irradiation. Currently, various carbon-based nanomaterials for TPE PDT have been explored,
which will be discussed in detail in the following section. In 2013, Fowley et al. reported the first example of using
carbon dots (CQDs) in combination with traditional PSs for TPE PDT. In this work, hydrophilic CQDs with a
quantum yield of 13% were prepared, and a hydrophobic PS protoporphyrin IX (PTIX) was then covalently linked to
their surface via an amide bond to make water soluble CQD–PTIX nanocomposites (Fig. 5). 94 The CQDs have a
maximum emission at 430 nm, which overlapped well with the absorption of PTIX, ensuring efficient FRET under
TPE at 800 nm. The 1O2 generation efficiency of the CQD–PTIX through this FRET process was determined by
DPBF, showing a 30% reduction in DPBF absorbance. This suggested that a significant quantity of 1O2 was
generated, which was able to induce an 82% reduction in HeLa cell viability following treatment with both CQD–
PTIX (1 mM) and two-photon laser excitation at 800 nm. Moreover, the TPE-induced therapeutic efficacy of the
CQD–PTIX nanocomposites in vivo was also evaluated in a syngeneic radiation-induced fibrosarcoma (RIF-1)
mouse tumor model. In this model, mice treated with an intratumoral injection of CQD–PTIX (30 mM) showed a
60% shrinkage in tumor size 4 days after two-photon irradiation at 800 nm. In contrast, the control mice that were
treated with either CQD–PTIX or a 800 nm laser alone showed a significant 65% increase in tumor size. Encouraged
by this result,Wang et al. also used water-soluble CQDs as the TPE energy donors to sensitize the electrostatically
absorbed PS 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP) to yield CQD–
TMPyP conjugates for TPE PDT (Fig. 6). 95 The CQDs they used had a high TPA cross-section with a maximum
value of 15000 GM at 700 nm, about two orders of magnitude higher than that of TMPyP alone. Therefore, the 1O2
generation efficiency of the CQD– TMPyP conjugates was enhanced over the small molecule PS alone, which
resulted in significant damage to HeLa cells under TPE at 700 nm As alternative carbon-based nanomaterials to the
CQDs that served as energy donors for TPE PDT, Zhang et al. have recently taken single-walled carbon nanotubes
(SWCNTs) as nanocarriers to load Ru(II) complexes to build TPE nanocomposite Ru@SWCNTs for bimodal photo-
thermal (PTT) and two-photon excited photodynamic therapy (PTT-TPE PDT). 96 In their design (Fig. 7), the Ru(II)
complex was employed as an effective TPE agent with a TPA cross-section of 494 GM at 808 nm, and was loaded on
the surface of SWCNTs through noncovalent p–p interactions. Initially, the fluorescence of the Ru( II) complex was
quenched by the SWCNTs, leading to low 1O2 production. Upon TPE at 808 nm, the SWCNTs absorbed light and
generated heat to achieve a PTT effect. This increasing temperature also triggered the release of the Ru( II) complexes
from the SWCNTs, leading to the recovery of the 1O2 production. This was verified both in vitro and in cultured
HeLa cells. The PTT-TPE PDT combined therapy using the Ru@SWCNTs was demonstrated in both two-
dimensional (2D) cancer cell and three-dimensional (3D) multicellular tumor spheroid (MCTS) models, showing
superior anticancer efficacies over either PDT with the Ru( II) complexes or PTT with the SWCNTs alone. Moreover,
in vivo studies were also conducted utilizing the intratumoral injection of 100 mL of a 1 mgmL_1 aqueous dispersion
of Ru@SWCNTs. In this study, nearly complete ablation of tumors under twophoton irradiation at 808 nm was
observed. These results highlighted that the bimodal PTT and TPE PDT from Ru@SWCNTs were efficacious for
cancer therapy.

4. TPE silica nanomaterial-based PSs

Silica-based nanoparticles are another class of attractive nanomaterials that has been widely used in biomedicine due
to their unique characteristics amongst nanomaterials, including high biocompatibility, ease of preparation, well-
defined and tunable nanostructures, and large surface areas. 97–99 Moreover, mesoporous silica nanoparticles (MSNs)
are endowed with large pore volumes and tunable pore sizes, which hold a great capacity to load various imaging and
therapeutic agents for biomedical applications. Encouraged by these properties, the use of silicabased nanoparticles
to build PSs for effective TPE PDT has also been successfully demonstrated both in vitro and in vivo, which is
discussed in detail in the following section. In 2007, Kim et al. took organically modified silica (ORMOSIL)
nanoparticles as vectors to co-encapsulate aggregation-enhanced two-photon absorbing fluorescent dye aggregates of
9,10-bis(40- (400-amino-styryl)styryl)anthracene (BDSA) as a donor and the PS 2-devinyl-2-(1-
hexyloxyethyl)pyropheophorbide (HPPH) as an acceptor to build nanoassemblies for TPE PDT (Fig. 8). 100 The
system was found to provide several advantages: (1) the enhanced fluorescence of the BDSA in the aggregated state
can greatly overcome the notorious issue of aggregation induced fluorescence quenching of most organic dyes, which
is beneficial to maintain energy transfer to the PS necessary for PDT; (2) the encapsulation of the hydrophobic HPPH
PS in the aqueous soluble ORMOSIL nanoparticles can dramatically improve the water solubility of the HPPH,
ensuring a strong fluorescence emission following the acceptance of energy from BDSA upon TPE; (3) the porosity
of the ORMOSIL nanoparticles allowed O 2 molecules to diffuse in and out of the particle matrix and facilitated their
sensitization to form 1O2. As such, a FRET efficiency as high as 73.0% was observed upon TPE at 850 nm, which
displayed a high capacity to generate 1O2 in aqueous solution. Moreover, the nanoassemblies were readily taken up
by HeLa cells and produced an intense two-photon excited intracellular fluorescence. An intracellular FRET
efficiency up to 36% was estimated by using an ‘‘acceptor bleaching’’ method, suggesting high nanocomposite
stability in the intracellular environment. Upon TPE, obvious HeLa cell necrosis was observed after laser irradiation
at 850 nm. These results suggested that co-encapsulation of TPA dyes and PSs in porous silica nanoparticles is a
feasible approach to develop drug-carrier nanoassemblies enabling TPE PDT. This was also demonstrated by Cheng
et al., in which MSN co-encapsulating the TPA dye fluorescein isothiocyanate (FITC) and the PS (Pd-meso-tetra(4-
carboxyphenyl)porphyrin, PdTPP) resulted in high 1O2 production and efficient TPE PDT both in vitro and in vivo
(Fig. 9).101 In spite of the promising results from the indirect TPE of PSs using FRET from co-encapsulated TPA
dyes, the inevitable loss of light power during the energy transfer process has prompted the development of silica
nanoparticles directly encapsulating two-photon excitable PS molecules. As such, the two-photon light would
directly excite the PSs for 1O2 production. For example, Qian et al. developed protoporphyrin IX (PpIX) doped
ORMOSIL nanoparticles with an average diameter of about 25 nm as TPE PDT agents (Fig. 10). 102 The doped PpIX
could be directly excited by an 800 nm fs laser to emit strong fluorescence in HeLa cells as observed by confocal
microscopy. This same 800 nm fs laser in the confocal microscope was used as a direct irradiation source to trigger
PDT in HeLa cells, resulting in an obvious change in morphology indicative of cell death following 2 min of
irradiation. In contrast, no obvious cytotoxicity was observed in non-treated HeLa cells after irradiation. These
results indicated that the direct excitation of the PpIX in the ORMOSIL nanoparticles through its efficient TPA was
able to induce 1O2 generation within tumor cells as a demonstration of anticancer therapy. Velusamy et al. have
reported another example of using porous silica nanoparticles to encapsulate a new PS molecule 11,12-
dibutoxydibenzo[a,c]-phenazine bridged amine for TPE PDT. 103 Because of the considerably high TPA cross-section
(B7000 GM excited at 800 nm) of the encapsulated PS molecules, the nanoparticles can be directly excited under 800
nm light to generate 1O2 with a quantum yield of 0.51 in D 2O. These doped nanoparticles were able to induce a
significant 40% mortality in macrophage cells in vitro (Raw 264.7) upon 800 nm fs pulsed laser irradiation at 2.6 W
cm_2 for 3 min. Recently, Gary-Bobo et al. prepared mannose-functionalized MSNs covalently encapsulating
approximately 6850 units of banana-shaped biphotonic quadrupolar chromophores as effective TPA PSs (Fig. 11). 104
The TPA properties of the PS molecule (1200 GM) were retained in the MSNs, leading to extremely large TPA
cross-sections (up to 8 MGM) for a single MSN that improved 1O2 production upon TPE. The subsequent TPE PDT
experiments in cultured cells showed that the mannosefunctionalized MSNs (MSN1–mannose) had clearly higher
PDT efficacy than that of non-functionalized nanoparticles (MSN1). After incubation with 20 mg mL_1 MSN1–
mannose for 24 h, nearly 100% of MCF-7 breast cancer cells were killed following TPE at 760 nm for 3 s.
Importantly, the observed cytotoxicity was highly specific to excitation by light in the NIR region. No cell death was
observed in the MSN1–mannose treated cells either without irradiation or exposure to daylight for 4 h. These results
support the importance of NIR-sensitive nanoparticles to improve PDT outcomes since TPE in the NIR enables
higher tissue penetration and high spatial precision of cell death while avoiding unwelcomed side effects caused by
daylight (i.e. non-specific and non-therapeutic) exposure. The subsequent examination of TPE PDT in vivo was
carried out on nude mice bearing HCT-116 xenografts, and a strong reduction in tumor weight ( ca. 70%) for mice
treated with both MSN1–mannose and 760 nm laser irradiation was observed in comparison to tumors treated with
saline solution alone or treated with MSN1– mannose but no NIR irradiation. Moreover, TPE PDT with MSN1–
mannose in mice also prevented the formation of liver and colon macrometastases. These results suggest that focal
and targeted TPE PDT using multifunctionalized MSNs hold great promise to achieve non-invasive treatment of
small localized solid tumors. Inspired by this, a further application of a similar silica nanoparticle PS system was also
reported for the in vitro treatment of retinoblastoma.105 In contrast to the above examples in which the silica
nanoparticles were merely used as nanocarriers, Secret et al. directly used porous silicon nanoparticles (pSiNPs) as
TPE energy transfer donors to sensitize the covalently linked PS molecule 5-(4-iso-thiocyanatophenyl)- 10,15,20-
tris(4-N-methylpyridiniumyl)porphyrin trichloride (Porph) under 800 nm laser excitation. 106 The pSiNPs had a
relatively large porous volume (1.6 mL g_1) and surface area (242 m2 g_1), allowing for grafting multiple components
including PSs and targeting ligands. Moreover, the pSiNPs were biodegradable and can be excited by a two-photon
NIR laser, making pSiNPs efficient as energy donors for TPE PDT. To increase the uptake in tumor cells, a tumor
targeting ligand of mannose was attached to the surface of the pSiNPs using either phenyl squarate- or ketone-based
linking chemistry. The TPA cross-section of the mannose and Porph functionalized silica nanoparticles (pSiNP–
Porph–Man) at 800 nm was found to be 360 GM, which is more than 6-fold over Porph itself (55 GM). The
measurement of the 1O2 generation of pSiNP–Porph– Man upon irradiation at 800 nm in aqueous solution showed
significant production of 1O2 and/or ROS, which was able to cause significant death of MCF-7 cells in vitro.
However, a comparable amount of free Porph was not able to induce cell death under the same conditions, suggesting
that the energy transfer from the two-photon excited pSiNPs to Porph contributed greatly to improve the TPE-PDT
efficiency.

5. TPE gold nanomaterial-based PSs

Gold nanomaterials, including gold nanorods, nanocages, nanoshells, and nanostars, have found widespread
applications for cancer diagnosis and therapy on account of their Good biocompatibility, surface versatility, tunable
size and unique optical properties.107–110 In addition to these properties, it has been demonstrated that spherical gold
nanoparticles can be directly excited to yield cytotoxic 1O2. Since gold nanoparticles generally have superior
photostability and high resistance to oxidative decomposition by ROS oxidation, they are able to act as PSs for
PDT.111 Recently, it has also been demonstrated that single gold nanoparticles with different shapes displayed
dramatically different TPA cross-sections, with B83 GM for nanospheres, B500 GM for nanocubes, B1.5 _ 103 GM
for nanotriangles, B4.2 _ 104 GM for nanorods, and B4.0 _ 106 GM for nanostars at 820 nm.112 These characteristics
suggest a high potential for the gold nanoplatform applied to TPE PDT. Furthermore, gold nanoparticles are known
to have a strong plasmon resonance band, allowing them to effectively absorb NIR light and efficiently convert the
photons into thermal energy, thus causing irreversible hyperthermic ablation of cancer cells. Therefore, upon TPE
with an NIR laser, a synergistic effect of PDT and PTT could be realized, which would enhance the photo-induced
cancer cell death as demonstrated previously by Ru@SWCNTs. The following section will summarize the recent
progress in the development of gold nanoparticles for cancer therapy via the TPE process. In 2012, Zhao et al.
reported polyvinylpyrrolidone (PVP) coated gold nanorods (AuNRs) which displayed high biocompatibility and
could be employed as PSs for TPE PDT (Fig. 12). 113 AuNRs were prepared using different aspect ratios, all of which
showed high TPA cross-sections (from 2.2 _ 104 to 3 _ 104 GM) and the ability to generate 1O2 under TPE as
monitored by chemical oxidation of aqueous solutions of 9,10-anthracenediyl-bis(methylene)dimalonic acid
(ABDA). The TPE-induced 1O2 production efficiency of Au NRs was found to be much higher than that of
commonly used PSs such as RB and Indocyanine Green (ICG). The cellular studies showed that the PVP coated
AuNRs had high uptake efficiency in HeLa cells and were able to kill the cells upon 808 nm fs pulsed laser
excitation in an irradiation time-dependent manner. After 15 min irradiation, the cell viability declined to 18%,
indicating the induction of significant cell death. This was also confirmed by monitoring intracellular fluorescence
directly, showing extensive ethidium bromide (EB) staining that indicated a late stage of cell death. Based on this,
Zhao et al. further demonstrated that the two-photon induced 1O2 generation capacities were further enhanced when
Au nanospheres and short AuNRs were present in aggregated form. 114 Because Au nanoparticles normally exist in
aggregated form when present in biological environments, it will be very attractive to use the aggregated Au
nanoparticles for TPE PDT. In 2014, the same group reported another AuNR-based TPE PDT nanocomposite
AuNR/SiO2–T790 consisting of an AuNR core with strong TPE fluorescence, a silica shell with controllable
thickness, and a layer of covalently conjugated porphyrin molecules (T790) with high 1O2 production quantum yield
(0.47) (Fig. 13).115 Upon two-photon irradiation with fs laser pulses at 800 nm, the strong plasmon resonance of the
AuNR core amplified the local electric field, resulting in respective 11.8-fold and 32.8-fold enhancement in two-
photon fluorescence emission and TPE efficiency of the attached T790. The enhancement of the TPE 1O2 generation
capacities was demonstrated in cultured HepG2 cancer cells with two-photon fluorescence imaging and PDT
efficacy. Following 800 nm laser irradiation for 8 min, up to 50% of cells were killed with AuNR/SiO 2–T790,
significantly higher than the 20% killing achieved in cells treated with AuNR/SiO 2 under the same conditions. This
suggested that AuNR/SiO2–T790 had higher 1O2 generation than that of AuNR/SiO 2, and the combination of AuNRs
with organic PSs showed promise to improve TPE PDT efficiency. In efforts to exploit the efficient PTT of Au
nanoparticles, Gao et al. have constructed hypocrellin B (HB)-incorporated mixed lipid-coated gold nanocages (lipid-
HB–AuNCs) as twophoton PTT/PDT agents for cancer therapy. 116 In their design, HB was employed as a two-photon
PDT agent that was reconstituted into mixed-lipid vesicles composed of hydrogenated soybean phosphatidylcholine,
cholesterol, and mPEG2000– distearylphosphatidylethanolamine (mPEG2000–DSPE). These vesicles were then
spread on the outer surface of AuNCs to form lipid-HB–AuNCs. With NIR two-photon irradiation at 790 nm, the
AuNCs absorbed part of the light to generate heat, while the remaining light induced the encapsulated HB to generate
1O2. Therefore, TPE PDT and PTT were simultaneously achieved, which caused a remarkable reduction in cell
viability (17.4%) as compared to that observed for either PDT (54.5%) or PTT (64.6%) alone. This study further
supports the synergistic anticancer efficiency achievable with two-photon excitable PDT and PTT. To demonstrate
the capacity of Au nanoparticles for TPE PDT in vivo, Chen et al. have recently developed mesoporous silica-coated
gold nanorods (MS-AuNRs) incorporating organic PSs (Pd-meso-tetra(4-carboxyphenyl) porphyrin, PdTPP) for two-
photon activated PDT (Fig. 14).117 In their design, the mesoporous SiO 2 shell surrounding the AuNRs significantly
improved the two-photon luminescence stability of AuNRs, with B260% better resistance to physical deformation
than that of uncoated AuNRs. Moreover, the encapsulating mesoporous SiO 2 matrix provided a large surface area for
loading high concentrations of PdTPP. This PS was activated through intra-particle plasmonic resonance energy
transfer from the two-photon excited AuNRs. Both outcomes of the design of these nanoparticles enhanced 1O2
production upon two-photon irradiation, which was verified both in vitro and in vivo in a breast cancer mouse model.
Mice treated with combined MS-AuNR–PdTPP and 800 fs pulsed laser irradiation showed inhibited tumor growth,
suggesting effective in vivo PDT. Therefore, the rational design of these PS-encapsulating Au nanoparticles could
serve as a promising nanosystem for the efficient TPE PDT both in vitro and in vivo.

6. TPE polymeric nanomaterial-based PSs

Polymeric nanomaterials have emerged as another attractive material option for the development of TPE PDT
nanosystems with unique and well-suited characteristics, such as high biocompatibility, good biodegradability and
large drug loading capacity. 118–120 They can be easily modified to have properties that are responsive to various
external or internal stimuli in cells to afford the specific and controlled release of encapsulated PSs for enhanced
PDT efficacy at the target site. 40 Moreover, some semiconducting polymeric materials exhibit encouraging optical
properties including large extinction coefficients, high fluorescence quantum yields, and excellent
photostability.45,121,122 In particular, these organic semiconducting materials also have much larger TPA cross-
sections than that of their small-molecule counterparts, providing the ability to act as efficient two-photon energy
donors. This property has the potential to enhance twophoton- induced energy transfer to nearby PS molecules,
improving two-photon induced 1O2 production, and cause cancer cell death. 52,123–129 Currently, several TPE polymeric
nanomaterial-based PSs have been developed which will be discussed in the following section. In 2007, Chen et al.
reported the first example of using a twophoton absorbing block copolymer as a nanocarrier to encapsulate a
hydrophobic porphyrin PS from which micelles were constructed that showed enhanced two-photon induced 1O2
generation (Fig. 15).130 In their design, amphiphilic block copolymers with good water solubility and
biocompatibility were extensively loaded with PS molecules. Importantly, the hydrophobic block of the copolymer
allowed for high concentrations of PS loading while avoiding probe aggregation in aqueous solution, improving the
fluorescence emission of the PS. This copolymer encapsulating two-photon chromophores possessed much larger
TPA cross-sections than that of the PS alone. Furthermore, the chromophore fluorescence emission overlapped well
with both the Soret and Q-band absorptions of the PS molecule to ensure efficient FRET sensitization of the PSs
under two-photon irradiation. An energy transfer efficiency as high as 96% was obtained between the polymer and
the PS in micellar aqueous solutions under TPE, enhancing PS fluorescence emission by 6.5-fold. The 1O2 generation
of the micelles under TPE at 800 nm was later verified using a water soluble 1O2 sensor (ADPA), and a 2.6-fold
faster bleaching rate was observed in the micelles than without the two-photon chromophore, indicating a much
larger amount of 1O2 generation. Shen et al. have reported another TPE polymeric nanoparticle consisting of the
semiconducting polymer poly[9,9-di-bromohexylfluorene- 2,7-ylenethylene-alt-1,4-(2,5-dimethoxy)phenylene]
(PFEMO) as the host matrix and tetraphenylporphyrin (TPP) as the PS (Fig. 16). 131 TPP molecules were doped into
the PFEMO matrix to form stable nanoparticles with a uniformsize ofB50 nm. Due to the large delocalized p-
conjugated backbones, the TPA cross-section of PFEMO was found to be 2160 GM per molecule (54 GM per repeat
unit), a value significantly larger than that of TPP (12 GM). As such, PFEMO served as a reliable twophoton energy
donor from which efficient FRET from PFEMO to TPP was achieved. Overall, this formulation resulted in a
maximum of 21-fold enhancement in two-photon emission of encapsulated TPP. The measurement of two-photon
induced 1O2 generation from the nanoparticles in aqueous solution with a 1O2 sensor (ABDA) revealed that the photo-
oxidation rate for TPP-doped PFEMO NPs was 6.5-fold faster than that of TPP alone and 15.9-fold faster than that of
pure PFEMO NPs without TPP. A similar study of using a two-photon absorbing semiconducting polymer of
poly[{9,9-dioctyl-2,7-divinylene-fluoreneylene}- alt-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFPV) as
the host matrix and TPP as the PS to form small semiconducting polymer nanoparticles for TPE PDT was also
reported by Grimland and co-authors, which showed a very high TPA cross-section (5 _ 105 GM) for 1O2
generation.132 These results clearly confirmed that the efficient intraparticle FRET from conjugated polymers to TPP
under TPE enhanced the 1O2 generation capacity of this nanosystem. Based on their initial study, Shen et al. have
further modified their PFEMO–TPP system by incorporating polyoxyethylene nonylphenylether (CO-520) into the
nanoparticles. These CO-T-P NPs were stable under cell culture conditions and showed low dark cytotoxicity. Cell
studies showed that the CO-T-P NPs were actively taken up and internalized into HepG2 cancer cells through
endocytosis, resulting in strong intracellular TPE fluorescence (Fig. 17a) accompanied by a significant reduction in
cell viability under two-photon irradiation at 800 nm with a light dose of 630 J cm _2. This is the first example of TPE
PDT activity in live cancer cells with PS-doped conjugated polymer nanoparticles. 133 Encouraged by this, folic acid
was introduced onto the surface of the PS-doped conjugated polymer NPs, which showed selective TPE induced cell
death in folate receptoroverexpressing cancer cells (e.g., KB cancer cells) (Fig. 17b).56 These results suggest that the
combination of tumor targeting ligands with PS-doped conjugated polymer NPs could act as efficient TPE nano-
photosensitizers for cancer PDT. However, it should be mentioned that the application of polymeric nanomaterials
for TPE PDT is merely demonstrated in cultured cells. There is still lack of experimental evidence showing their in
vivo PDT efficacy.

7. Conclusion and future perspectives

TPE PDT has gained significant attention for cancer therapy in recent years due to the demonstration of improved
tissue penetration and spatial selectivity associated with this technique. Though a variety of classic organic PSs have
been made and have played key roles in PDT, their direct application for TPE PDT in cancer treatment is still limited
by a variety of shortcomings, including low TPA cross-sections. TPE nanoparticlebased PDT that combines the
advantages of nanotechnology and TPE hold great promise to selectively enhance 1O2 generation efficiency in deeper
tissue to improve PDT efficacy for solid and deep-seated tumors. Until now, different kinds of TPE nanoparticles
have been actively developed, aiming to exert larger TPA cross-sections and improve selective accumulation in
tumor tissues/cells in efforts to enhance TPE PDT outcomes both in vitro and in vivo. In this review, we have
summarized recent advances in the development of these TPE nanoparticles based on five different nanomaterials
(QDs, carbon, silica, gold, polymer), and their capacity to produce 1O2 and achieve efficient cancer PDT upon two-
photon irradiation. Although promising results have been demonstrated, cancer PDT using TPE nanoparticles is in its
infancy with the following challenges awaiting resolution. Firstly, TPE PDT requires the use of a pulsed laser light
source to excite PSs, and generally, the focal area of the laser beam is small in order to ensure the delivery of
adequate energy to sensitize PSs. Therefore, the effective area of the laser spot is much smaller than that of
traditional light sources (e.g., Xe light, LED). This will necessarily require a much longer irradiation time and precise
positional adjustment of the laser beam to ensure delivery of sufficient energy to the entire tumor tissue. This small
focal area currently limits the application of TPE PDT to cancers as it is not amenable to the treatment of bulky
tumors.32 New technology is emerging that has enlarged the laser beam diameter, as well as increased the laser output
energy,134 and will surely expand the applications of TPE PDT both in vivo preclinically and in clinical practice.
Secondly, similar to the conventional one-photon excitation PDT, TPE PDT is also an O 2 consuming modality, that is
highly dependent on tissue O2 levels. Since solid tumor tissues are characterized by hypoxic microenvironments, the
efficacy of PDT is seriously hindered in these hypoxic regions of solid tumors. Moreover, the continuous O 2
consumption and rapid shutdown of the vasculature which are also associated with PDT could exacerbate the
hypoxia problem and further limit the efficacy of PDT. Though several methods including a combination therapy of
PDT with antiangiogenic agents135–137 and compensation of O2 depletion by controlled light exposure using light–
dark periods138–140 or extension of irradiation with low fluence rates 141–143 have been proposed to overcome this
‘PDT-induced hypoxia’, solid tumor tissues with pre-existing hypoxia are still poorly treated. Therefore, new
methods that could promote PDT independent of tumor tissue O 2 levels will be essential for the adaption of this
therapeutic modality to the clinic. Recently, two innovative studies using one-photon excitation nanoparticles that
could create new paradigms of O 2 selfenriching PDT and O2-evolving PDT have been reported by Hu’s 144 and Guo’s
group,23 respectively. These novel selfperpetuating nanosystems could overcome tumor hypoxia and achieve
effective treatment of solid tumors. Feasibly, the integration of these methods with TPE nanoparticles would allow
precise PDT of hypoxic tumor cells located in deeper solid tissue. Thirdly, the ability to avoid undesired
phototoxicity to normal tissues is another critical concern for cancer PDT. Though TPE PDT is characterized by
nonlinear absorption of two photons of infrared light, most of the PSs employed can also be sensitized by visible
light, which will cause skin phototoxicity following systemic administration of the nanoparticle system. In
consideration of this limitation, one-photon excitation PDT with activatable PSs has been proposed as an attractive
therapeutic option.145–148 In these systems PSs are initially quenched, resulting in weak fluorescence and low 1O2
generation efficiency in order to minimize phototoxicity. However, these responsive PSs can be selectively activated
by a molecular target to generate enhanced fluorescence and 1O2 production at the intended site of therapy (e.g.
tumor), leading to targeted cell death. Therefore, PDT with activatable PSs would improve the specificity of cancer
PDT by overcoming the undesired phototoxicity to healthy tissues. Furthermore, the enhanced fluorescence upon
target interaction would also allow for molecular imaging of the tumor, enabling simultaneous diagnosis and
treatment of cancer.149,150 On the basis of the progress that has been made towards activatable PDT, it is expected that
this same strategy could also be applicable to construct activatable TPE nanoparticle-based PSs, and that these next-
generation nanosystems will further contribute to precise diagnosis and treatment of cancer with reduced
phototoxicity. Moreover, the use of tumor targeting ligands conjugated to the nanoparticle surface for active delivery
of nanoparticles to tumor tissues would also reduce phototoxicity and improve PDT therapeutic outcomes. 151 Lastly,
most of the currently reported TPE nanoparticles were only evaluated in cultured cells or in living mouse models
with intratumoral injection. For clinical applications, their biodistribution, blood circulation and potential dark
toxicity after systemic administration are still unknown. Compared to organic PSs, the nanoparticles highlighted in
this review are subject to toxicity that is unique to their size and is not yet well understood, and which importantly
has become a main concern limiting their use in biomedicine. As such, it is necessary to conduct nanotoxicological
studies to determine whether these nano-sized PSs have harmful effects both on the human body and in the
environment.152,153 Many factors such as size, inherent chemical components, and surface chemistry of nanomaterials
are known to modulate toxicity. For example, the small size of nanoparticles has shown pathological effects on the
lungs, which would cause respiratory toxicity and inflammation. 154,155 Nanoparticles also have much larger surface
area which would enhance their ability to non-specifically absorb biomolecules that could lead to protein dysfunction
and proinflammatory effects.156 In addition, some studies have shown that certain nanoparticles (e.g., AuNPs, silica
NPs) exhibit high redox reactivity at their surface, which could generate unnecessary ROS and cause oxidative stress
to normal cells.157 QDs that are made of heavy metal ions (e.g., Cd2+) have been found to exert heavy metal toxicity
on biological systems due to the liberation of free Cd 2+, which would induce ROS production, cause indirect DNA
damage, and ultimately kill cells. 158,159 However, whether these Cd2+-containing QDs are also toxic to humans
remains unclear and should be carefully examined in the future. Moreover, most nanoparticles are easily trapped by
the reticoendothelial system (RES) following intravenous injections and thus give rise to two notorious sideeffects:
(1) RES trapping would make nanoparticles clear quickly from blood, leading to lower uptake of nanoparticles in
targeted tumor tissues;160 (2) high uptake of nanoparticles in the liver and spleen which is related to RES trapping
could cause liver toxicity and blood cell deficits, and initiate inflammatory responses. 161,162 It has been known that the
in vivo properties, potential nanotoxicity and nonspecific RES uptake of nanoparticles are closely related to their
surface chemistry, therefore, highly biocompatible materials (e.g., PEG) for surface coating and better bioconjugation
strategies for surface functionalization are of significant importance. 163,164 An important consideration for the future
development of TPE PDT nanosystems is the emerging strategies of in situ formation of nanoparticles from small
molecules165–167 or microbubbles.168,169 The ability to build nanoparticles at the intended site of action in the body
allows rapid delivery and prolonged retention in tumor tissues, and has shown promise for molecular imaging as well
as drug delivery. The use of either small molecules or microbubbles could overcome the RES trapping and minimize
nanotoxicity following systemic administration, while enhancing accumulation in tumor tissues upon in situ
formation of nanoparticles. These strategies could also be applicable to the in situ building of TPE nanoparticles for
cancer PDT, which may have high clinical impact due to the ability to overcome the current concerns regarding
nanotoxicity. In conclusion, there has been growing research interest focused on developing new TPE nanoparticles
over the last two decades, with a focus on the discovery of newmaterials to provide large TPA cross-sections and
high 1O2 production quantumyields to enhance PDT efficiency. It is envisioned that, on the one hand, these
nanoparticles will have improved photochemical and pharmacokinetic properties, better tumor targeting ability and
reduced phototoxicity in normal tissue.170 On the other hand, the use of two photons of NIR light instead of a single
visible photon to sensitize PSs could provide better spatial selectivity and deeper tissue penetration than that of
onephoton excitation by visible light.171 Therefore, TPE nanoparticles with these advantages will show profound
potential for precise cancer PDT. It should also be mentioned that there are still significant challenges in the
translation of nanoparticle-based PDT technologies, such as lack of nanotoxicology studies both in vitro and in vivo,
lack of inexpensive and safe lasers approved for clinical use, and lack of efficient methods to measure light dose (or
dosimetry) and distribution in a particular disease tissue. 170,172 However, with the rapid development of both optical
technology and nanotechnology, it is expected that new TPE-specific nanoparticles, inexpensive pulsed fs lasers, 173–
176 methods allowing delivery of lasers into deep-seated tissues, 177–179 and methods for the precise measurement of
light dosimetry in vivo will be developed in the future,180,181whichwill push the field ofTPE PDT for biomedical
applications rapidly forward.