Introduction to PDT

Photodynamic therapy (PDT) is an emerging non-invasive cancer treatment [1] that uses photosensitizers, light, and endogenous molecular oxygen to kill cancer cells and microorganisms [2]. Hermann von Tappeiner was the first to propose the “photodynamic effect,” where certain dyes make microorganisms sensitive to light, and exposure to sunlight leads to rapid cell death [3]. As medicine continues to evolve, cancer treatment strategies evolve also. PDT is a safer cancer treatment with fewer side effects [4]. Clinical studies have shown that PDT has been increasingly used in the treatment of solid tumors, including those of the brain, head and neck, skin, esophagus, lung, gastrointestinal tract, pancreas, bladder, prostate, breast, cervix, ovary, and basal cell carcinoma [5-7].

The anti-tumor mechanism of PDT consists of two main phases (Fig. 1). A photosensitizer (PS) accumulates at the tumor site after intravenous injection. It irradiates the tumor tissue at a specific wavelength. In the first stage, the PS changes from the ground state (singlet state (S0)) to the excited singlet state (S1) after irradiation (nanosecond range). The excited state of a photosensitizer is precarious and loses excess energy through non-radiative (thermal emission) or radiative (fluorescence emission) pathways [8, 9]. The excited singlet state produces a more stable excited triplet state (T1) with parallel spins (microseconds to milliseconds) via inter-system crossover. At T1, PS undergoes two types of reactions (type I and type II). In the first pathway, electron or hydrogen atom transfer occurs between the T1 photosensitizer and the cell membrane of the biomolecule. This process forms free radical ions, leading to the production of cytotoxic hydroxyl radicals (•OH), hydrogen peroxide (H2O2), and other reactive oxygen species (ROS). The second type of reaction involves the interaction between electronically excited-triplet state photosensitizers and ground-state triplet state molecular oxygen (3O2). The excited PS transfers energy to 3O2 to form the singlet oxygen (1O2). The product 1O2 reacts with various biomolecules and is a critical factor in the induction of apoptosis and tissue destruction in cancer cells [10-12]. In addition, type I and type II reactions occur simultaneously and independently, with type II reactions playing a more important role in PDT [13, 14].

Fig. 1
figure 1

Mechanism of PDT action.

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Introduction to Porphyrins

Porphyrins are macrocyclic pigments and cofactors commonly occurring in nature. They are often referred to as the “pigments of life.” Porphyrin comes from the Greek word “Porphyra” because porphyrins are usually bright purple or red [15]. The backbone of porphyrins is porphine (Fig. 2A), which is an aromatic compound containing up to 26 π-electrons, 18 of which form a continuous plane. Its role as a metal-binding cofactor in plants (Fig. 2B) and animals (Fig. 2C) to form biomolecules is crucial for many metabolic pathways. It significantly facilitates oxygen transport during cellular respiration and energy capture during photosynthesis [16]. The insertion of metal atoms into porphyrins affects the degree of electron delocalization in the conjugated system, making their properties more diverse and further broadening the idea of the development of porphyrin-based photosensitizers [17, 18].

Fig. 2
figure 2

Structure of porphine (A), chlorophyll (Chls) (B), heme (C), and sodium porphyrin (D).

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Cancer is a massive threat to human health. It causes the death of millions of patients each year and is the second leading cause of death worldwide after cardiovascular disease [8]. With the development of PDT, impressive progress has been made in the preparation of photosensitizers, especially porphyrin-based photosensitizers [13]. Most photosensitizers used for cancer therapy have a porphyrin-based macrocyclic backbone [19-21]. The main advantages of porphyrins in photodynamic studies include (1) stability of aromatic compounds, (2) effective absorption of visible light, (3) high yield of ROS, (4) easy functionalization modifications, and structural diversity, and (5) long triplet state lifetime and low dark toxicity [22]. Hematoporphyrin (HPD) was first used by Dougherty in 1978 to treat gastrointestinal cancer [23, 24]. HPD has shown excellent therapeutic effect under light irradiation (100 mW/cm2). Sodium porphyrin (Fig. 2D) is the world’s first approved photosensitizer for the treatment of cancer [13]. It is reusable, has virtually no side effects, and does not develop drug resistance.

Enhancing PDT by Improving the Light Energy Capture Efficiency of Porphyrin Photosensitizers

Porphyrins are widely used in PDT owing to their unique photosensitive properties. However, porphyrins have disadvantages in cancer therapy. For example, porphyrins have low water solubility, causing them to readily aggregate by stacking. This quenches the electronically excited states, reduces the quantum yield of 1O2, and weakens the effectiveness of PDT. Additionally, the maximum absorption wavelength of porphyrins is not in the red-light region; however, longer wavelengths of red light are generally chosen in PDT because the transmission rate to human tissues is higher at this wavelength with efficient irradiation deeper into tumor tissues (same type of light source), enabling the photosensitizers to produce photodynamic therapeutic effects. Therefore, these deficiencies need to be addressed in porphyrin photosensitizers to enhance the light energy capture efficiency. Previous reports have focused on PDT mechanisms and discussion of nanoparticles [8, 25, 26]. There is no systematic discussion on improving the efficiency of porphyrin’s light energy capture. This paper includes discussion about the structural modifications that enhance the absorption of porphyrin photosensitizers in the red-light region for better penetration and improved light energy capture efficiency. Ultimately, improving the efficiency of 1O2 generation in tumor tissues enhances the photodynamic therapeutic effect of the photosensitizer; indirect excitation of porphyrin photosensitizers using fluorescent donors (fluorescence resonance energy transfer) has been successfully used to address these problems. It is expected that this paper will be valuable in PDT.

Structural Modification Induces Red-shift in the Spectral Absorption Band of Porphyrin Photosensitizers

Most recent studies have focused on enhancing the photophysical properties of PS through various structural modifications. These include binding with other molecules, metallization, and nanotechnological applications [13, 27]. Hilmey et al. synthesized a series of dithioporphyrin-based photosensitizers and evaluated photodynamic properties [28]. The results showed that the different combinations of heteroatoms in the center of the porphyrin ring resulted in I-band absorption peaks of these compounds with longer wavelengths than that of Photofrin. Moreover, the new porphyrin coordination compounds synthesized in this study efficiently generated 1O2 under I-band irradiation. The redshift of the I-band absorption peak increased the light penetration depth, which is of great significance for the clinical application of porphyrin-based photosensitizers (Fig. 3). Cheng et al. fabricated a composite photosensitizer [29] by mixing DNA G-quadruplexes with hydrophilic porphyrin (TMPipEOPP)4+•4I−. This photosensitizer exhibited a new absorption band at approximately 700 nm. Interestingly, the absorption intensity of the new photosensitizer in the Q-band was much higher than that of free TMPipEOPP. For example, the molar absorption coefficient of the complex formed by TMPipEOPP with the G-tetramer AS1411 is approximately 47,000 L/mol−1 cm−1 at 700 nm, which is 7.4 times higher than that of free TMPipEOPP at 650 nm. Compared with the conventional porphyrin photosensitizer, the excitation wavelength of the composite photosensitizer is redshifted by approximately 50 nm (650–700 nm), which is favorable for light penetration. Additionally, the light absorption efficiency of the composite photosensitizer increased by approximately 7.4 times, which significantly improved the 1O2 generation capacity and enhanced the PDT effect.

Fig. 3
figure 3

Porphyrin photosensitizers structure modification to enhance PDT. The redshift of the absorption spectral region of the photosensitizer enhances the penetration of deep tissues.

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Mono-L-aspartyl chlorin e6 (NPe6) is a hydrophilic chlorin derived from chlorophyll a with good photosensitivity. Chan et al. used NPe6 for photodynamic therapy [30]. Compared to Photofrin, NPe6 with increased absorption in the near-infrared spectrum is thought to improve photon utilization and the depth of light penetration within the cancer tissue. In phase I trials, NPe6 has been shown to exhibit potent tumor-killing effects when the timing of photoactivation is associated with or near-peak plasma levels. The data show a 95% either complete or partial response to NPe6-based phototherapy if the cutaneous lesions were basal cell carcinoma. Other malignant cutaneous lesions demonstrated an overall 67% complete or partial response rate. Moreover, Bellnier et al. showed that [31] chlorophyll elicits considerably less potential for cutaneous phototoxicity than in patients receiving Photofrin.

Metal-modified Porphyrin Photosensitizers Enhance Light Energy Capture Efficiency to Enhance PDT

Metal porphyrins are widely present in nature and their ability to cleave DNA nucleases has attracted significant attention in the last few years. Thus, combining porphyrins with metals provides additional antitumor activity and tumor selectivity. The biodistribution of the metals inside and outside tumor cells can also be tracked [32]. Owing to paramagnetic effects, the photocatalytic activity of metal complexes is heavily dependent on the central metal [33]. Therefore, many researchers have inserted metals into the porphyrin rings to maintain their stability and photophysical properties. Moreover, the structure of β-substituted porphyrin is similar to that of natural porphyrins and has been widely used in biological research. These studies have shown that some transition metal compounds such as iridium, ruthenium, rhenium, and osmium exert good antitumor activity, especially in photodynamics [34-37]. Particularly, Ru is less toxic to normal cells, making it a good option for PDT application [38]. Schmitt et al. synthesized four aromatic Ru(II) derivatives (C6H5Me or p-PriC6H4Me) and evaluated compounds as potential dual photosensitizers or chemotherapeutic agents against human melanoma cells Me300 [39]. It showed that the uptake and high photosensitizing activity of metal Ru in human melanoma cell models were promoted under red light (652 nm) irradiation at only 5 J/cm2. In another study, Zhang et al. synthesized porphyrin derivatives containing Ru(II) polypyridyl porphyrins or zinc(II) porphyrin structures and evaluated their cytotoxicity against human nasopharyngeal carcinoma HK-1 and cervical cancer HeLa cells [40]. They showed that porphyrins containing Ru had high 1O2 quantum yields, rapid cellular uptake, low dark cytotoxicity, and potent photocytotoxicity (80% of Ru-L-containing incubated HK-1 cells were killed at a concentration of 1 μM and yellow light dose of 3 J/cm2).

Self-assembling Porphyrin Nanoparticles Improve Light Energy Capture Efficiency and Enhance PDT

A significant disadvantage of porphyrin-based photosensitizers is that they readily aggregate through stacking, leading to the quenching of electronically excited states, thereby reducing the quantum yield of 1O2 and diminishing the effectiveness of PDT [41-44]. Self-assembly is a natural phenomenon and a powerful method for applying multifunctional nanomaterials in biological applications [45-47]. Recent advances in polymer synthesis have led to the development of various controlled polymerization techniques, such as anionic or reactive radical polymerization. Polymers with different structures can self-assemble into aggregates with different morphologies under certain conditions, such as linear block copolymers (BCP), graft copolymers, and dendritic polymers [48].

For instance, BCP has the same basic structure as lipids (hydrophobic head and hydrophilic tail) but is composed of chemically different polymeric chains, which are covalently linked in a series of two or more segments. Amphiphilic BCP self-assembles into nanoparticles in aqueous solution via thermodynamic or kinetic variation. The shell is a hydrophilic polymer, and the hydrophobic region in the center can be used as an excellent carrier for the porphyrin photosensitizer to minimize the adverse interactions between the hydrophobic photosensitizer and water [49]. Self-assembly into nanoparticles enables porphyrins to effectively address their tendency to aggregate during PDT, efficiently capture light energy, and generate 1O2 (Fig. 4). Studies of meso-tetra-4-hydroxyphenylporphyrin (mTHPP) with polyethylene glycol molecules (PEG) have shown that the complexes formed improve the solubility of porphyrin photosensitizers and reduce their aggregation in aqueous environments, thus allowing efficient capture of light energy to generate 1O2 [50, 51].

Fig. 4
figure 4

Self-assembled porphyrin nanoparticles restrict porphyrin aggregation to enhance PDT. Advantages: (a) Limited tetraphenylporphyrin precipitates. (b) High 1O2 generation efficiency.

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On this basis, Jin et al. prepared alternative copolymers (P(MIPOSS-alt-VBTPP)-b-POEGMA) using 4-vinylbenzyl terminal tetraphenylporphyrin (VBTPP) and maleimide isobutyl polyhedral oligomeric sesquioxane (MIPOSS) as starting monomers using alternating reversible addition-rupture chain transfer (RAFT) polymerization [52]. Porphyrins and polyhedral oligomeric sesquisiloxanes (POSS) were alternately mounted on the backbone and self-assembled into nanoparticles in water. In vitro cytotoxicity assay revealed that the PM nanoparticles without POSS units (control group) were significantly less phototoxic than those of the experimental group. In vivo efficacy evaluation experiments showed that the tumors of both nanoparticle-treated mice were suppressed, and the tumors in one group were almost completely eradicated. The spatial cage structure and alternating structure of the POSS units in the block copolymer can effectively reduce aggregation-induced quenching (AIQ) among the porphyrin units. Therefore, the light energy capture efficiency of the photosensitizer was improved, and the effect of PDT was enhanced.

Self-assembling porphyrin nanoparticles have attracted considerable attention because of their excellent water dispersibility. In addition, meso-substitution is a simple synthetic route to introduce flexible chains that can improve the biocompatibility and light energy capture efficiency of the photosensitizers [53]. Pan et al. synthesized two novel meso-A2B2-porphyrins (P1 and P2) with symmetrical phenylethynyl groups by introducing amphiphilic triethylene glycol groups on porphyrins [54]. The corresponding hydrophilic nanoparticles were prepared using a solvent exchange-based self-assembly method. The meso-capping structure of the central porphyrins was modified with amphiphilic triethylene glycol groups, which showed good biocompatibility compared with that of the other photosensitizers [55, 56]. Moreover, P1 and P2 exhibit strong near-infrared absorption in the typical therapeutic window (600–800 nm) and effectively generate 1O2. The nanoparticles showed high phototoxicity to Hela cells under 650-nm laser irradiation. Wu et al. [57] designed and synthesized two novel porphyrin photosensitizers (NI-Por and NI-ZnPor). Subsequently, the NI-Por and NI-ZnPor nanoparticles, which showed good hydrophilicity and moderate particle size (approximately 60 nm), were prepared via a self-assembly process. They showed high efficiency of ROS generation in detection experiments with 2′,7′-dichloro-fluorescein (DCFH) as a probe. The feasibility of nanoparticles as photosensitizers for PDT was further evaluated using the CCK-8 assay. Under light irradiation, NI-Por and NI-ZnPor nanoparticles showed significant therapeutic effects on HeLa cells. In this study, the introduction of PEG chains on porphyrins enhanced the hydrophilicity of the photosensitizers [58]. The bandgap of the molecule was narrowed owing to the strong electron-absorbing ability of the 1,8-naphthalimide moiety, which resulted in the redshift of the absorption peak and enhanced near-infrared (NIR) absorption [59, 60]. Moreover, nanoparticles accumulated at the tumor site through enhanced permeability and retention (EPR) effects and exhibit significant cytotoxicity in PDT.

Although several approaches have been developed to reduce AIQ, such as using POSS units on polymer side chains to isolate tetraphenylporphyrin (TPP) or developing tree-like macromolecules around TPP, the drug loading capacity (LC) of PS is relatively low because of the introduction of redundant non-therapeutic groups [52]. Therefore, enhancing the LC of porphyrin photosensitizers is necessary to improve their light-energy capture efficiency. Zheng et al. developed the first poly-TPP nanoparticles by cross-linking degradable reactive oxygen clusters, thiometallic linkers, and tetraphenyl-porphyrin derivatives followed by co-precipitation [61]. These nanoparticles had high quantitative loading efficiency (> 99%), homogeneous nanoparticles (no aggregation), and increased quantum yield of 1O2 (ΦΔ = 0.79 in dimethyl sulfoxide compared with 0.52 of the original TPP). In vivo antitumor effect study of the nanoparticles showed that TPP nanoparticles exerted significant inhibitory effect on tumor growth, and the tumor volume was approximately 1/10 that of the PBS group after 10 days. In contrast, poly-TPP nanoparticles showed the best PDT therapeutic effect because of their efficient drug-release ability.

Nanomaterial Modification of Porphyrin Photosensitizers to Enhance Light Energy Capture Efficiency for PDT

Nanotechnology has significantly developed over the last decade. The use of nanomaterial platforms for diagnostics and therapeutics has enabled precise drug delivery to target tissues and increased the effectiveness of anticancer treatments [62, 63]. The enhancement of photodynamic photosensitizer activity by metal nanoparticles through increased generation of 1O2 has garnered significant interest [64, 65]. Under light excitation, the surface electrons of metal nanoparticles exhibit collective oscillations (fixed-domain surface plasmon excitations) that excite and enhance a series of optical processes near the surface of metal nanoparticles, such as metal-enhanced singlet oxygen generation, surface-enhanced Raman scattering (SERS), absorption, and fluorescence and phosphorescence emission intensities [66]. Gold nanorods (Au NRs) can enhance ROS generation in PDT applications because of their high biocompatibility, stability, and tunable plasmonic resonance bands [67-69] (Fig. 5).

Fig. 5
figure 5

Nanomaterials modified porphyrin photosensitizers to enhance PDT. Enhanced light capture efficiency increases 1O2 generation.

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Ferreira et al. synthesized two different shapes of gold nanostructures (spherical and rod-like) and formed colloidal hybrid systems with 5,10,15,20-tetrakis(N-methylpyridinium-4-yl) porphyrin tosylate salt (H2TM4PyP(OTs)4) (POR), which can be used for PDT in the visible light range [67]. The ROS generation assay showed that the hybrid system consisting of Au NRs and POR showed a higher efficiency of 1O2 generation relative to the other components by one order of magnitude. This effect was attributed to the enhanced local electric field around the Au NRs, which led to increased light absorption by the photosensitizer. The resulting effective energy transfer to the oxygen molecules formed 1O2. Duman et al. reported nanocomposites of 5,10,15,20-tetrakis(1-methyl 4-pyridinio) porphyrin tetra(p-toluenesulfonate) (TMPyP) with Au NRs as nanocarrier systems for PDT and fluorescence imaging [70•]. To improve biocompatibility and promote cellular uptake, the NRs were wrapped with polyacrylic acid (PAA) and effectively loaded with cationic porphyrins via electrostatic interactions. In vitro cytotoxicity assays showed that TMPyP-loaded NRs exhibited higher phototoxicity than the same concentration of free photosensitizer. This is because the loading of TMPyP onto the Au NRs increases the absorption and emission intensity of the photosensitizer, which promotes the generation of 1O2 under light irradiation, ultimately enhancing the PDT effect.

For biological applications, Au NRs must be highly stable and biocompatible. However, severe cytotoxicity and low stability are two issues that hinder the practical application of Au NRs. Au NRs are usually coated with cetyl trimethyl ammonium bromide (CTAB) bilayers, which are essential structural directing agents in the most popular Au NRs synthesis methods. CTAB-coated Au NRs exert significant cytotoxicity in human cells [71, 72]. Silicon dioxide is well-suited as a coating material for Au NRs. Silicate systems have been used to synthesize bulk, film, and granular silicate mesoporous structures for a wide range of applications [73, 74]. Zhang et al. [75] synthesized 4-carboxyphenyl porphyrin-conjugated silica coated gold nanorods (AuNR@SiO2-TCPP). To eliminate the toxicity of residual CTAB, the mesoporous silica with a high drug LC and good biocompatibility was coated onto the surface of the Au NRs [76]. In an MTT assay, the survival rate of A549 cells treated with AuNR@SiO2-TCPP (100 μg/mL) was 21%. In animal experiments, AuNR@SiO2-TCPP exhibited significant tumor suppressive effects. Additionally, in vivo biosafety assays in tumor-bearing mice showed no apparent organ damage or inflammatory response in the AuNR@SiO2-TCPP-treated group. In another study, Lebepe et al. [77••] reported a photosensitizer (GO@Au NRs-TMePyP) consisting of water-soluble cationic porphyrin (5,10,15,20-tetrakis(3-methyl pyridyl)porphyrin, TMePyP) and Au NRs anchored on a graphene-oxide (GO) sheet. The photodynamic efficiency of graphene-based Au NRs was improved along with reduced cytotoxicity [78]. In the 1O2 detection experiment, GO@Au NRs-TMePyP exhibited efficient 1O2 generation. Moreover, the MBT-2 cytotoxicity assay showed that GO@Au NRs-TMePyP has excellent biocompatibility and great potential for biological applications.

The Au NRs deform under NIR laser irradiation and eventually become spheres that cannot effectively absorb NIR light. Therefore, photostable nanomaterials appear to be attractive options for future applications. Fullerene (C60) is a carbon nanomaterial with unique photochemical and electrochemical properties, physical properties, and low systemic toxicity; hence, it is a promising candidate for in vivo targeted drug delivery [79, 80]. Moreover, C60 absorbs visible light and enters the long-lived triplet state through effective intersystem crossing to generate ROS, which has great potential in PDT [81, 82]. However, carbon nanotubes and graphene oxide do not have this function. Shi et al. [83] modified iron oxide nanoparticles (IONPs) on the surface of C60 and conjugated them with hematoporphyrin monomethyl ether (HMME) after PEG modification to obtain multifunctional C60-IONP-PEG/HMME nanocomposites. The cytotoxicity assays showed that C60-IONP-PEG/HMME was not cytotoxic without light irradiation. Moreover, the ROS generation efficiency of the photosensitizer was effectively increased by laser irradiation. Compared with that of free HMME, C60-IONP-PEG/HMME showed a significant photodynamic killing effect on B16-F10 cells, and PDT was significantly enhanced in mouse tumor models in vivo.

Fluorescence Resonance Energy Transfer System Enhances Light Energy Capture Efficiency to Enhance PDT

Fluorescence resonance energy transfer (FRET)–based drug delivery systems for indirect activation of PS drugs via donor fluorophores have been extensively investigated in PDT. The transfer of excitation energy from the donor to the PS significantly improve its light capture efficiency and expand the range of light sources. Thus, the efficiency of 1O2 generation by PS is greatly improved, which ultimately improves the PDT effect of photosensitizers [84, 85] (Fig. 6).

Fig. 6
figure 6

FRET system enhances PDT by improving the efficiency of light energy capture.

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Single-photon Excitation Donor Type

Recently, several fluorescent materials have been developed as donors to transfer energy to the PS via photonic excitation of donors to enhance the generation of 1O2. Owing to the excellent light capturing ability of conjugated polymers, the transfer of excitation energy to the acceptor PS along its backbone can lead to high amplification of the PS signal [86-88]. Chang et al. developed an efficient polymer-dot (Pdots) photosensitizer [89] by covalently doping porphyrins on the polymer backbone, in which the PS of tetraphenylporphyrin is covalently bound to the p-conjugated backbone of (9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-2,1,3-thiadiazole) (PFBT). The resulting Pdots have excellent stability and solved the problem of photosensitizer leaching encountered in photosensitizer-doped Pdots. Compared with that of the pure PFBT Pdots, the fluorescent PFBT-TPPx Pdots were significantly quenched by the introduction of TPP, and the light-capturing polymer backbone mainly transferred the excitation energy to the TPP unit. Moreover, the Pdots exhibited excellent performance, including high 1O2 quantum yield (35%) and low dark toxicity. Zhou et al. designed and synthesized a poly (metallocene) hyperbranched conjugated polyelectrolyte containing a platinum (II) porphyrin complex, which was used to efficiently generate 1O2 for PDT [90]. Based on the overlap between the luminescence band of the Pdots at 420 nm and the Soret and Q absorption bands, a Förster radius of 6.6 nm was calculated, indicating that FRET from poly(furan) to platinum (II) porphyrins is effective. The FRET process was further investigated and determined to exist in the Pdots. Furthermore, the ability of Pdots to kill tumor cells, which was attributed to its high 1O2 quantum yield (80%) due to the introduction of oxygen-sensitive phosphorescent platinum (II) porphyrin complexes, was confirmed using MTT colorimetric assay, flow cytometry analysis, and real-time fluorescence imaging of photoinduced in situ cell death.

In recent years, water-soluble conjugated oligomers, including oligo-(phenylene vinylene) (OPV), oligo-(phenylene ethynylene), and oligo-(thiophene ethynylene), have attracted considerable attention because of their molecular structures and tunable optical properties [91]. In 2019, Zhao et al. designed and synthesized a novel donor–acceptor porphyrin PS in the form of covalent bonds using condensation reactions with cationically conjugated oligo-(thiophene ethynylene) as the donor and 5,10,15,20–4 (4-aminophenyl) porphyrin (TPP) as the acceptor [92]. The positively charged OPV acts as an “antenna” because of its strong light capturing ability. Under white-light irradiation, intense spectral overlap occurs between OPV and the porphyrins at very short distances, resulting in excellent FRET (99%), which significantly improves the 1O2 yield of the porphyrins. The cytotoxic effects of OPV-C3-TPP, OPV-C6-TPP, and TPP on MCF-7 cells were studied using an MTT colorimetric assay. Under light irradiation, cell viability decreased with increasing concentrations of OPV-modified porphyrins after incubation with the cells. In fact, cell mortality exceeded 98% at a concentration of 5 μM.

Two-photon Excitation Donor Type

Two-photon absorbing fluorescent dyes can also be used to construct energy transfer systems for PDT. In a two-photon excited FRET system, existing photosensitizers are combined with two-photon absorption (TPA) dyes. Here, the photosensitizer unit (energy acceptor) is indirectly excited by the FRET of the two-photon absorbing dye unit (energy donor). Energy capture by the TPA donor strongly enhances the two-photon excitation efficiency of the photosensitizer, which generates 1O2 more efficiently [93, 94]. Since the two-photon absorbing donor can be excited by NIR light during this process, deeper tissue penetration can be obtained compared with that in conventional PDT [95].

Kim et al. prepared organically modified silica nanoparticles with 2-desethylene-2-(1-hexyloxyethyl) pyromellitic chlorophyll acid (HPPH) as an energy acceptor and 9,10-bis(4′-(4′′-aminostyryl)styryl)anthracene dye with severely distorted geometry (BDSA) as a two-photon energy donor [96]. The partial flattening of the aggregation geometry and the resulting loose stacking of molecules in the aggregated state enhanced two-photon absorption. At an excitation wavelength of 425 nm, the fluorescence intensity of the co-wrapped nanoparticles was quenched by approximately 70% for BDSA emission and amplified by approximately 5 times for HPPH emission compared with the fluorescence intensity of nanoparticles containing equal amounts of dye. This indicates that FRET occurred between BDSA and HPPH, which enhanced the generation of 1O2. Hammerer et al. attached triethylene glycol (PTEGTP) and diethylene glycol-α-mannosyl groups (PManTP) to the meso-phenyl moieties of porphyrins to obtain a series of porphyrin-triphenylamine-hybridized photosensitizers [97]. These new photosensitizers have a cationic charge and are highly water-soluble, thereby improving cell penetration. Laser irradiation at 500 nm induced the energy transfer process from TP to porphyrin. Moreover, the new compounds were found to be localized in the mitochondria, the preferred target organelles for PDT. In conclusion, the improved properties of the new photosensitizer significantly increased the efficiency of two-photon activated PDT.

Semiconductor quantum dots (QDs) are nanomaterials that hold great promise for PDT applications. The QD size imparts unique optical properties that enable precise tuning from the UV to the IR region by varying the size and composition. Due to the ability to absorb light in the NIR region of the spectrum, low-intensity light can penetrate tissues and access deep tumors. Additionally, owing to their significant transition dipole moments, QDs are excellent absorbing materials that are ideal donors for activating photosensitizers in PDT [98, 99] (Fig. 7).

Fig. 7
figure 7

Two-photon excited quantum dots FRET system enhanced PDT. Large two-photon absorption cross section and deep tissue penetration.

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Chou et al. conjugated aluminum sulfonated phthalocyanine (AlPcS) to two-photon excited (TPE) QDs to form QD AlPcS conjugates for PDT [100]. The high two-photon absorption cross section (TPACS) enables the excitation of the QDs by a low-power-density 800 nm unfocused femtosecond laser and transfer the energy to the conjugated AlPcS via FRET. The FRET efficiency of the QD AlPcS conjugates was as high as 90% in water. The FRET process of cellular QD AlPcS was also observed in KB and HeLa cells under two-photon excitation using an 800-nm femtosecond laser. It effectively generated 1O2, which finally killed the tumor cells. Fowley et al. designed a carbon QD (CQD)-protoporphyrin (IX) sensitizer conjugate [101]. Laser irradiation at 800 nm caused a sizeable two-photon absorption cross-section of the CQDs to be used for indirectly exciting protoporphyrin (IX) through FRET to generate large amounts of singlet oxygen, which resulted in an 82% decrease in HeLa cell viability. Moreover, mice treated with intratumoral injection of CQD-protoporphyrin (IX) conjugate showed 60% tumor inhibition after laser irradiation at 800 nm in a fibrosarcoma-bearing mouse model.

Conclusions and Perspectives

Research on porphyrin photosensitizers has made significant progress in overcoming the problems of insufficient water solubility and limited tissue penetration of traditional photosensitizers. Structural modification, metal porphyrins, and nanotechnology have enhanced the light energy capture efficiency of photosensitizers. Moreover, light irradiation induces fluorescent donors to activate photosensitizers indirectly via FRET. The absorption spectral region of the photosensitizer is redshifted, which can be used for the treatment of deep tumor tissue. However, there are some problems to be solved with porphyrin photosensitizers in PDT. Avoiding or minimizing toxicity to normal tissues is extremely important for PDT of tumors. Some nanoscale photosensitizers, such as Au NRs and silica nanoparticles, have high redox reactivity on their surfaces, which may generate unnecessary 1O2 that is toxic to normal cells [102]. Some nanoparticles containing heavy metals cause damage to normal cells [103]. Moreover, photosensitizing drugs can be rapidly cleared in the human body, resulting in a low drug concentration in tumor tissues and unable to exert good effects of PDT [104, 105]. Therefore, biocompatible materials need to be developed to reduce the toxicity and side effects of photosensitizers on normal cells during PDT using porphyrin photosensitizers.

In conclusion, the development of novel porphyrin photosensitizers for PDT has garnered increasing interest in recent years. Despite this, the clinical application of PDT technology still requires further research. However, the rapid advances in the field of optical technology and nanotechnology show promise for the development of safer and more effective photosensitizers in the future.