Photodynamic therapy (PDT) relies on the administration of photosensitizers (PSs) that are activated by light at a specific wavelength to produce cytotoxic reactive oxygen species (ROS) to kill cancer cells and other pathogens. As a result, photosensitizer, light source, and molecular oxygen are three critical components for PDT. When irradiated by light, good photosensitizers should exhibit strong light absorption, high photo-stability, and efficient ROS generation. To ensure effective light energy delivery to deep-seated diseases, the light source should be capable of penetrating biological tissues. Porphyrin derivatives are currently the majority of clinically approved photosensitizers by the Food and Drug Administration (FDA). Although they are activated by red light (~630 nm) in clinical applications to improve light penetration, this is not the wavelength at which they absorb the most. To improve the light penetration, photosensitizers with absorption in the red and near-infrared (NIR) regions, such as methylene blue and indocyanine green (ICG), were developed. ICG exhibits strong NIR absorption, but it suffers from low ROS generation efficiency, fast body clearance, and poor photo-stability. Furthermore, due to the energy threshold of the PS required to ensure efficient energy transfer from the triplet state to oxygen molecules, developing photosensitizers with long-wavelength absorption is extremely difficult. In comparison to the UV and visible light commonly used in traditional PDT, NIR light penetrates biological tissues much better due to the lower scattering effect. Two-photon excitation is one effective strategy of using NIR light as the light source for PDT in which the PSs are activated by the simultaneous absorption of two NIR photons to produce ROS [Fig. 1(a)]. Because the probability of two-photon absorption is very low for a photosensitizer, light with high peak intensity, such as an ultrafast femtosecond pulsed laser, is used for two-photon excitation. Furthermore, the laser is usually focused to increase the efficiency of two-photon excitation. When compared to one-photon excitation, two-photon absorption occurs only at the focal plane, and the out-of-focus signals are suppressed [Fig. 1(b)], which is due to the square dependence of the two-photon fluorescence intensity on the excitation light power. Two-photon excited PDT (2P-PDT) has considerable promise for high-precision deep tissue theranostics thanks to NIR light excitation and high-order nonlinear optical effects.

The efficiency of ROS generation and the two-photon absorption cross-section are two main factors that determine the therapeutic efficiency of 2P-PDT. Traditional photosensitizers, such as porphyrin derivatives and chlorophyll derivatives, have been adopted in a variety of 2P-PDT applications (Fig. 2). Despite efficient ROS generation, their poor water solubility, small two-photon absorption cross-section, and low photo-stability have limited their performance in practical applications. Over the last two decades, new generation PSs have been developed to improve cancer targeting. PSs are linked with antibodies, proteins, and other molecules that are overexpressed on cancer cells to improve selectivity and therapeutic performance. Furthermore, PSs are loaded onto a variety of nanoparticles, including quantum dots, gold-based nanoparticles, and polymer micelles, to build nano-platforms for efficient drug delivery. The intersystem crossing (ISC) process from the singlet excited state to the triplet excited state is critical in the generation of ROS. By incorporating heavy atoms into photosensitizers, such as iridium, ruthenium, iodine, and bromine, the spin-orbit coupling (SOC) can be enhanced, which favors the improvement of the ISC process. Furthermore, reducing the singlet and triplet energy gaps (ΔE_ST) can help to improve the ISC process (Fig. 3). Photosensitizers with donor-acceptor (D-A) structures have been developed in recent years to reduce the ΔE_ST value by separating the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO). Moreover, the D-A structured molecules also favor a large two-photon absorption cross-section. By adjusting the donor and acceptor moieties, and introducing π-conjugated linkers, their two-photon absorption can be improved due to the enhanced strength of the donor and acceptor, and the extended conjugation length (Fig. 4). Most organic photosensitizers are highly hydrophobic and show reduced ROS generation and fluorescence reduction in an aqueous environment due to an aggregation-caused quenching effect. To address this issue, novel photosensitizers with aggregation-induced emission (AIE) properties have been developed (Fig. 6). The AIE nanoparticle-based PSs performed excellently well in two-photon imaging and 2P-PDT, owing to their enhanced ROS generation and fluorescence intensity in aggregate states as nanoparticle formulations. Due to its specific advantages for deep tissue penetration, 2P-PDT has been used in image-guided precise blood vessel closure in vivo (Fig. 7). To improve ROS generation and two-photon absorption in a single photosensitizer molecule, a new concept called polymerization-enhanced two-photon photosensitization was proposed (Fig. 8). Recently, an NIR-II light (1000-1700 nm) activable photosensitizer with greater therapeutic depth than the NIR-I (700-950 nm) excitation was developed (Fig. 9). Even though the action section for 2P-PDT is relatively small, its applications for bulky solid tumor therapy have shown some promise with the advancement of ROS generation and light source (Fig. 10).

2P-PDT is promising for precise cancer therapy in deep tissues due to the advantages of NIR light excitation and high-order nonlinear optical process. Efforts have been made to improve the ROS generation and two-photon absorption of PSs. Traditional PSs suffer from aggregation-caused quenching problems which are addressed by novel AIE PSs. Compared with one-photon PDT, 2P-PDT should not be focused on the therapy of large solid tumors due to its small action section. 2P-PDT has a lot of potential in basic research applications that require high resolution and precision, such as building a stroke model in mouse brain, precise intracellular ROS activation, selective cancer cell killing in vivo, etc. NIR-II excitation should be considered to improve the therapeutic depth of 2P-PDT. However, shifting the absorption of PSs to long-wavelength while maintaining ROS generation efficiency is a challenge. Therefore, future development for 2P-PDT should concentrate on extremely efficient PSs with long-wavelength absorption, NIR-II light excitation, increased two-photon excitation efficiency with a large field, and a single PS molecule capable of both good ROS generation and two-photon absorption capability.