A promising anticancer drug: a photosensitizer based on the porphyrin skeleton

This article reviews the research status of porphyrin photosensitizers; future perspectives and current challenges are discussed.

Abstract

Photodynamic therapy (PDT) is a minimally invasive combination of treatments that treat tumors and other diseases by using photosensitizers, light and oxygen to produce cytotoxic reactive oxygen species (ROS) inducing tumor cell apoptosis. Photosensitizers are the key part of PDT for clinical application and experimental research, and most of them are porphyrin compounds at present. Due to their unique affinity for tumor tissues, porphyrins are not only excellent photosensitizers, but also good carriers to transport other active drugs into tumor tissues, which can exert synergistic anticancer effects of PDT and chemotherapy. This article reviews the clinical development of porphyrin photosensitizers and the research status of porphyrin containing bioactive groups. Finally, future perspectives and the current challenges of photosensitizers based on the porphyrin skeleton are discussed.

1. Introduction

Cancer remains a great threat to human health worldwide. Early detection, accurate diagnosis and scientific treatment are the key to improve cancer survival rates. Currently, chemotherapy is still commonly used in cancer treatment. Drugs for chemotherapy can be administered in various ways and usually produce therapeutic effects after distribution throughout the body via blood circulation. However, due to the poor targeting and lack of selectivity of this treatment method, it would cause great damage to normal cells and have considerable toxic and side effects. Thus, it has become an urgent need to develop new anti-tumor drugs with good targeting and low side effects.1–3

Porphyrins are compounds with many important functions (Fig. 1a). They are widely used in biocatalysis,4 medicine,5 bionics and environmental protection.6–8 One of the prominent advantages of most porphyrins is that they have special affinity for cancer cells, which enables them to selectively remain in cancer cells, providing feasibility for the research of anti-tumor targeted drugs.9–13 Another important use of porphyrins is as photosensitizers for PDT of tumors and other diseases.

Fig. 1. (a and b) Basic structure of porphyrin and hematoporphyrin.

Compared with traditional treatment methods, PDT seems to be a promising approach for anticancer treatment.14–16 PDT is a minimally invasive combinatorial therapy with advantages of not damaging normal tissue, low toxic side effects and relatively little pain, and it has been clinically proved to have early disease diagnosis, especially for cancer. PDT is a photo-treatment of tumors and other diseases using photosensitizing agents, light, and oxygen, generating cytotoxic ROS that induce tumour apoptosis.17–19 The main mechanism of action is shown in Fig. 2. First, the patient is given a local or systemic injection of a drug containing a photosensitizer. Then, the photosensitizer is selectively enriched around tumor tissues but not normal ones to produce a photodynamic killing effect after irradiation with light of a specific wavelength.20–22 It should be noted that the photodynamic therapeutic effect of photosensitizers is not limited to the excitation of an external light source. Researchers found that X-rays can also be used to stimulate the photodynamic effect of the photosensitizers.23 Single-molecule chemiluminescent photosensitizers can be used to induce PDT through an internal chemiluminescence reaction without irradiation by an external light source.24–27 This can better solve the problem of low penetration of ultraviolet/visible light to biological tissues and is conducive to the treatment of deep malignant tumors.

Fig. 2. Schematic diagram of photodynamic therapy.

Photosensitizers are key to the biological efficacy, and the study of porphyrins as photosensitizers has been ongoing for a long time. As early as 1942, Auler et al.28 found that hematoporphyrins selectively concentrate on tumor tissues (Fig. 1b). Since then, studies have confirmed that porphyrins have a certain targeting effect on tumor cells.29,30 ROS produced by light has a cleavage effect on biomolecules such as DNA, and has a killing effect on tumor cells, opening up a new way for early diagnosis and treatment of cancer.

2. Double action mechanism of porphyrins

2.1. Mechanism of aggregation in tumor cells

At present, the relatively mature explanation of the aggregation mechanisms of porphyrins in tumor cells are the theory of low-density lipoprotein receptors and pH difference. Studies have shown that low-density lipoprotein receptors are more active in rapidly proliferating tumor cells,31–33 which contribute to the binding of porphyrins to tumor cells. Lipophilic porphyrins bind to the center of the low-density lipoprotein receptor and enter tumor cells through a specific membrane receptor that interacts with the low-density lipoprotein receptor apolipoprotein B. Therefore, porphyrins can selectively accumulate in tumor cells. In addition, Pottier et al.34 pointed out that tumor tissues have lower pH values than normal tissues because of their faster metabolism. Generally, the pH of normal tissues is 7.0–8.0, and the pH values of tumor tissues are 5.85–7.68. Porphyrins enter cells by passive diffusion, and the diffusion would efficiently increase with the decrease of pH values. The larger the tumor tissue, the lower the pH values.34 This facilitates preferential aggregation of porphyrins in tumor tissues. Porphyrins, such as protoporphyrin, hematoporphyrin and deuteroporphyrin, include four pyrrole nitrogen atoms inside the porphyrin system and two carboxylic acid groups on their periphery. Thus, there is a balance between protonation and deprotonation among them (Fig. 3). In tumor tissues with a typical pH value of 6.5, porphyrins exist in four forms in the equilibrium system, and 44% of porphyrins were neutral. Meanwhile, in the equilibrium system of normal tissues at pH 7.4, porphyrins exist in three forms, and only 3% of the porphyrins were neutral. Neutral molecules are more likely to penetrate cell membranes and enter cells than charged ions.35 Therefore, the phenomenon that porphyrins preferentially aggregate in tumor tissues can be explained. What's more, researchers36 demonstrated that pH is a key factor affecting the retention of porphyrin protein conjugates in cells by the affinity experiments of phosphate porphyrins and bovine serum albumin (BSA), which also provides strong evidence for the pH theory to promote the aggregation of porphyrins in tumor tissues.

Fig. 3. Acid–base equilibria of typical dicarboxylic acid porphyrin (P(CO₂H)₂) in healthy and malignant tissues. P is the parent skeleton of porphyrin. HP⁺ indicates that the porphyrin imino nitrogen is protonated to form a cationic porphyrin and COO^– indicates that the carboxyl group is deprotonated to generate a local negative charge in the peripheral group.

2.2. Photodynamic killing effect on tumor cells

Photosensitizers, light and oxygen are the three elements of PDT. The photoactive reaction of photosensitizers produces ROS such as singlet oxygen, which is the main cause of tumor tissue damage.37 When the porphyrin photosensitizer is excited by appropriate wavelength illumination, the photosensitizer that absorbs photon energy transitions from the ground state to the excited singlet state. The lifetime of the excited singlet state is very short, and the energy of the excited singlet state can be emitted by heat dissipation decay or fluorescence emission, or by intersystem intersection to an excited triplet state with a relatively low energy and a long lifetime. In the triplet state, the photosensitizer can generate ROS by two mechanisms, as shown in Fig. 4.38,39 In the presence of molecular oxygen, superoxide radicals and hydroxyl radicals are formed by type I reaction, and singlet oxygen radicals are generated by type II reaction.40,41 These ROS can damage most types of biomolecules such as amino acids, lipids, nucleic acids, and the like.

Fig. 4. Photosensitizer molecules produce reactive oxygen species.

3. Anticancer drugs based on the porphyrin skeleton

The core idea of drug design includes the design and assembly principle of the target, to find a substance with a similar structure to the reported pharmaceutically active compound, and to combine several pharmaceutically active fragments to produce a synergistic effect. Porphyrins are favored by more and more scientists because of their unique photosensitivity and tumor aggregation. Research based on porphyrins mainly includes the following two types: porphyrin photodynamic anticancer drugs and porphyrin-bonded anticancer drugs.

3.1. Porphyrin photodynamic anticancer drugs

Photosensitizers in PDT were developed in the 1970s, mainly hematoporphyrin derivatives (HPDs). In the 1960s, Lipson42 prepared HPDs and found that they were localized in tumor tissues. In the early 1970s, Kelly and Snell43 combined HPDs with light and successfully applied them to the treatment of bladder cancer, demonstrating the potential application of porphyrins in destroying tumor tissues. Since then, porphyrin photosensitizers have received more and more attention, becoming the most important photosensitizer for clinical PDT (Table 1).44,45 Photofrin® derived from HPDs is the first photosensitizer approved by the FDA. It is the most frequently used photosensitizer and is clinically used for the treatment of esophageal cancer, bladder cancer, gastric cancer, etc. (Fig. 5a). Overholt and Panjehpour46 used Photofrin® to treat patients with esophageal cancer and found that over 80% of patients eliminated foreign body hyperplasia and achieved good therapeutic results. Nseyo et al.47 have reported the effectiveness of PDT in the management of patients with recurrent superficial bladder cancer. Of the 58 patients, 90% did not relapse after receiving PDT. C. Xiao-Jun et al.48 applied photosensitive PDT to 30 patients with intractable bronchial lung cancer, and the total effective inhibition rate reached 86.7%. Meanwhile, the bronchial blocking rate dropped from 90.0% (27/30) before PDT to 16.7% (4/30) after PDT. Photofrin® has the strongest absorption peak at 630 nm. 24 to 48 hours after intravenous injection into the bloodstream, the target site was subjected to photodynamic irradiation treatment. However, it takes 4–8 weeks for the photosensitizer to be excluded from the body after the end of treatment, during which patients must be protected from light in order to avoid skin photosensitivity.44,49 As the first-generation porphyrin photosensitizer, Photofrin® has many areas for improvement, such as cumbersome synthesis steps, long residence time in the body, severe photosensitivity of the skin, poor absorption at 630 nm and low tissue penetration, which limit its application in medicine.50–52

Table 1. Some porphyrins and their analogue photosensitizers in clinical trials.

Macrocyclic type platform	Photosensitizer	Trade name	Excitation wavelength	Manufacturer	Potential indications	Ref.
Porphyrin	Hematoporphyrin derivatives	Photofrin®	630 nm	Axcan Pharma, Canada	Esophageal cancer, bladder cancer, gastric cancer, bronchial cancer	45–48
	Hematoporphyrin derivatives	Hemoporfin®	630 nm	Fudan Zj, China	Port wine stains, early gastric cancer and rectal cancer	57–60
Chlorin	Temoporfin	Foscan®	652 nm	Biolitec, Germany	Prostate cancer, head and neck cancer, pancreatic cancer	61, 62
	Verteporfin	Visudyne®	690 nm	Novartis, Switzerland	Age related macular degeneration, vertebral metastases	66–69
	2-(1-Hexyloxyethyl)-2-devinyl pyropheophorbide-alpha	Photochlor®	665 nm	Roswell Park, USA	Basal cell carcinoma, esophageal, cancers, head and neck tumors	53
	Rostaporfin	Purlytin®	659 nm	Mrvt, USA	Kaposi's sarcoma, cutaneous metastatic adenocarcinomas, prostate, brain, lung cancers, basal cell carcinomas	54
	Talaporfin sodium	Laserphyrin®	664 nm	Meiji Seika, Japan	Early lung cancer	55
Bacteriochlorin	Padeliporfin	Tookad®	763 nm	Steba Biotech, UK	Prostate cancer	70–74
Phthalocyanine	Suftalanzinc	Photocyanine®	676 nm	Longhua Pharm, China	Gastrointestinal cancer, colon cancer, esophageal and nasopharyngeal cancer solid tumors	77, 78
	Silicon phthalocyanine	Pc4®	675 nm	Case Western Res., USA	Cutaneous and subcutaneous lesions from diverse solid tumor origins	56

Fig. 5. (a–f) Structural formula of photosensitizers commonly used in the clinic.

To solve this problem, researchers developed the second-generation porphyrin and porphyrin analogue photosensitizers (chlorin, bacteriochlorin and phthalocyanine),53–56 such as Hemoporfin® (porphyrin), Foscan®, Visudyne® (chlorin), Tookad® (bacteriochlorin) and Photocyanine® (phthalocyanine). (Fig. 5b–f). Hemoporfin® is a relatively new PDT photosensitizer synthesized by De-Yu Xu et al.57 and has been clinically tested in China. It is used as a vascular-targeted drug to treat port wine stains (PWS) and achieve satisfactory clinical efficacy in adherence to operational specifications.58 However, in practical treatment, it has lower efficacy for purple and thickened type PWS than pink PWS because of the limited ability of the light source to penetrate biological tissues, which is also a problem worth considering.59 The researchers60 conducted a preliminary study on the clinical study of Hemoporfin® used in PDT of digestive tract tumors. Patients with early gastric cancer and rectal cancer can achieve complete remission, while patients with advanced esophageal cancer can only mildly be relieved. At present, there are relatively few relevant clinical reports, and further standardization operations and optimization of various test conditions are required. As a typical chlorin sensitizer, Foscan® is widely used to treat prostate cancer, head and neck cancer, etc. Clinical studies were performed on 14 patients with prostate cancer using temoporfin. After PDT, the level of prostate-specific antigen had decreased in 9 patients, 5 patients had complete tumor disappearance, and necrosis was found to involve up to 91% of the prostate cross section.61 In addition, after 35 patients with head and neck cancer received PDT, 60% of the patients achieved good local inhibition. All the patients had a recurrence-free survival rate of more than 50% within 1 year.62 Although Foscan® is promising for clinical application, it easily forms aggregates after injection and causes serious inflammation because of poor solubility. Hence, how to reduce its toxic side effects and ease the suffering of patients remains to be further explored.63–65 Visudyne® is indicated for the treatment of patients with age-related macular degeneration, pathological myopia, suspected ocular histoplasmosis or typical systolic choroidal neovascularization.66,67 It also efficiently induced tumour necrosis even in advanced pancreatic cancer.68 In addition, clinical Phase I trials have initially shown that Visudyne® is safe to treat vertebral metastases, both from a pharmacological and neurological perspective.69 Tookad® is a derivative of Pd(ii) bacteriopheophytin, which is an amphiphilic photosensitizer and is commonly used as a vascular targeting drug.70–72 It can be activated at longer wavelengths, giving it the ability to kill deep malignant tumor tissues. Clinical studies have shown that it has a good effect on prostate cancer, and there are no obvious side effects.73,74 The treatment advantage is that the time interval between administration and light treatment is not long, and it can be removed from the blood in about 20 minutes, so the light treatment can be carried out immediately after administration. High doses can cause skin sensitivity, but can greatly reduce sensitivity for its rapid elimination rate.75,76 Photocyanine® is a disubstituted amphiphilic phthalocyanine compound. Anti-tumor in vivo and in vitro experiments have shown that it could significantly inhibit the growth of tumor tissues such as gastrointestinal cancer, colon cancer, solid tumor and other tumor tissues. The clinical Phase I results initially indicate that it is safe, effective and quality controllable, and its efficacy needs to be further verified in Phase II and III clinical trials and further optimization of treatment options is also required.77,78

Compared with the first-generation porphyrin photosensitizers, the second-generation porphyrin photosensitizers have higher absorption in the near-infrared spectral region, better ROS production efficiency, faster tumor aggregation, easier removal from normal tissues, and lower skin photosensitivity. After years of development, they have been relatively mature, partially overcoming the shortcomings of the first-generation porphyrin photosensitizers, and become more in line with the characteristics of ideal photosensitizers.

As drug delivery systems, researchers have designed third-generation porphyrin photosensitizers with molecular recognition function by connecting second-generation porphyrin photosensitizers with bioactive molecules such as peptides, anti-tumor monoclonal antibodies, liposomes, folic acid, and sugars.79 These so-called Nano photosensitizers (Nanops) include a porphyrin phospholipid self-assembled nano-preparation,80 porphyrin-polypeptide self-assembled nano-preparation,81,82 and porphyrin-metallic organic framework self-assembled nano-preparation.83–86 All of them have high loading capacity of photosensitizers, maintain the monomer form of photosensitizers and prevent self-quenching, further improve the selective aggregation of drugs in focus sites or tumor tissues, reduce toxic and side effects and enhance the anticancer effect.79,87 However, the third-generation porphyrin photosensitizers are currently in the research and development stage and have not been used in clinical trials.

3.2. Porphyrin-bonded bioactive drugs

The low penetration of light to tissues is one of the main weaknesses of conventional photosensitizers, which enormously limits the clinical application of photosensitizers, so the development of new photosensitizers is extra significant. Porphyrins were bonded to anticancer active substances to form complexes, which were selectively transported to tumor tissues and the substances were released by the targeting function of porphyrins, reducing the toxic and side effects on normal tissues and realizing the comprehensive effect of chemotherapy and PDT. To a certain extent, the conjugates overcome the low penetration depth of light and improve the efficacy of porphyrin photosensitizers. The anticancer drugs of these porphyrin-bonded bioactive groups are described below (Table 2).

Table 2. Collected data of porphyrin-bonded biological groups.

Porphyrin conjugate a	ROS generation	λ ex	Radiation conditions	Yield of 1O2	Activity data	Potential indications	Ref.
Porphyrin–platinum 6a and 6b	—	401/421 nm	IR lamp, 60 J cm–2, 20 min	—	In vitro: T/C corr: 5 μM (J82/MDA-MB-231)	Bladder cancer, breast cancer	88–90
6c	—	—	—	—	In vitro: ED50: 2.4 μg mL–1 (L1210) In vivo: T/C: 30 mg kg–1, 206% (L1210)	Leukemia, lung adenocarcinoma	91
6d	1O2	420 nm	420 nm, 6.95 J cm–2, 15 min	0.54	In vitro: IC50: 0.019 μM (CP70)	Cervical cancer, ovarian cancer	92
6e	1O2	427 nm	50 W LED, 30 min	0.76	In vitro: IC50: 0.12 μM (Colon 26)	Colon cancer, sarcoma	93
					In vitro: IC50: 0.08 μM (Sarcoma 180)
Porphyrin–5-fluorouracil 7a (M = Zn)	—	424 nm	—	—	Inhibition rate: in vitro (1 μM, SMMC7721, 13.88%)	Liver cancer, colon cancer	99
					Inhibition rate: in vivo (50 mg kg–1, LoVo tumor, 70%)
Porphyrin–nitrogen mustard 8a	1O2	400 nm	—	—	Inhibition rate: in vitro (5 μg mL–1, SMMC7721 40%)	Liver cancer	104
8b (R = H)	1O2	400 nm	—	—	In vitro: IC50: 6 μg mL–1 (Bel-7404)	Liver cancer	106
Porphyrin–piperazine 9a	—	430 nm	—	—	In vitro: ID50: 8 mg mL–1 (HNE1)	Liver cancer, stomach cancer	113
Porphyrin–anthraquinone 10a	—	420 nm	500 W tungsten halogen white lamp, 2 h	—	In vitro: IC50: 2 μM (HCT116)	Colon cancer	115
10b (M = Zn)	—	418 nm	Ultraviolet light, 6 h	—	In vitro: IC50: 8.83 μM (HeLa)	Cervical cancer	116
Porphyrin–ferrocene 11a	1O2	415 nm	620 nm, 12 mW cm–2, 30 min	—	In vitro: IC50: 25 μM (MCF-7)	Breast cancer	119

^aThe conjugate number represents the corresponding conjugate in the figures.

3.2.1. Porphyrin–platinum conjugates

Platinum anticancer drugs are a class of metal anticancer drugs that are widely used in clinical practice. For example, cisplatin has excellent anticancer effects on various cancers such as bladder cancer, head and neck cancer and testicular cancer. However, it also has some shortcomings, such as poor water solubility, low oral efficacy, and serious side effects from continuous use. By improving the targeting of cancer cells and reducing its concentration in normal tissues, the toxic and side effects of cisplatin can be effectively reduced. Therefore, researchers modified the structure of cisplatin and developed a new class of porphyrin–cisplatin active compounds. Brunner et al.88–90 conducted a systematic study on porphyrin–cisplatin and designed and synthesized a series of ester porphyrin–cisplatin conjugates (Fig. 6a and b). Through anti-tumor experiments in vitro, it was found that some conjugates have good inhibitory activity against bladder cancer cells TCC-SUP/J82 and breast cancer cells MDA-MB-231, and their effect was better than that of cisplatin. Under light irradiation, the conjugates have a good synergistic anticancer effect by exerting the efficacy of both PDT and chemotherapy. Kim et al.91 prepared a series of polyethylene glycol porphyrin and cisplatin conjugates, and found that the water solubility of these dimers was improved due to the introduction of polyethylene glycol (Fig. 6c). Anti-tumor experiments in vitro showed that the conjugate could inhibit the proliferation of various cancer cells such as the leukemia L1210 cell line, and the anticancer activity was significantly stronger than that of cisplatin. Meanwhile, the cisplatin concentration in cancer cells was increased, indicating that the targeting ability of cisplatin modified by porphyrin was significantly improved. N. Anu et al.92 reported the synthesis of novel platinum–porphyrin conjugates as well as their photophysical characterization and in vitro light-induced anticancer properties (Fig. 6d). These conjugates showed only minimal cytotoxicity in the dark, but were highly cytotoxic under 420 nm light exposure. After incubation with HeLa cells, the nuclear Pt concentration was 30 times higher than that of cisplatin, indicating that these conjugates have a good affinity for tumor cells, and that such compounds can bind to DNA and induce photocleavage of DNA, indicating that DNA may be the main target of these conjugates. All of these favorable characteristics imply that porphyrin–platinum conjugates are worth exploring as novel PDT anticancer agents in vivo. Hu et al.93 combined the porphyrin skeleton containing gallium(iii) with the platinum(ii) group to obtain two porphyrin–cisplatinum ion compounds (Fig. 6e and f). Both conjugates exhibit high singlet oxygen quantum yields and remarkable photocytotoxicity. In particular, the compound (Fig. 6e) showed a lower IC₅₀ value under illumination (Colon 26: 0.12 μM; Sarcoma 180: 0.08 μM), and in in vivo PDT assay, it almost completely inhibited tumor growth. The results indicate that the compound (Fig. 6e) is a promising PDT anticancer drug. Lim et al.94 assembled porphyrin with oxaliplatin and evaluated its biological activity. As compared with monotherapy, the conjugate exhibited 3-fold enhanced cytotoxicity and 2-fold increase in the apoptosis. Experiments in vivo confirmed that the conjugate effectively inhibited tumor growth without causing systemic toxicity in mice. The above studies show that porphyrin–platinum conjugates have excellent performance in targeted localization, exhibit synergistic effects of PDT and chemotherapy, and have the potential to become highly effective and low-toxicity new antitumor drugs.

Fig. 6. (a–f) Structure of the porphyrin–platinum conjugates.

3.2.2. Porphyrin–5-fluorouracil conjugates

5-Fluorouracil is a kind of broad-spectrum anticancer drug, which blocks the replication of DNA and RNA by interfering with the synthesis of nucleic acid, so as to block the growth of cancer cells. Although it has a good therapeutic effect on many solid tumors, such as colon cancer,95 liver cancer,96 breast cancer,97 and rectal cancer,98 its toxic and side effects on the gastrointestinal tract and blood hinder its clinical application. Li et al.99 designed and synthesized a novel type of potential targeting anticancer drug based on porphyrin and 5-fluorouracil (Fig. 7). The anticancer activity of the porphyrin–5-fluorouracil compound was evaluated by MTT assay. As the results showed, the conjugate inhibited the growth of liver cancer cell SMCC-7721 by more than twice as much as 5-fluorouracil in the absence of light. Since no light treatment was used, only 5-fluorouracil was used for anticancer action, indicating that the porphyrin in the porphyrin–fluorouracil duplex actually enhances the targeting of fluorouracil and enhances the anticancer activity of 5-fluorouracil. Two metalloporphyrin–5-fluorouracil amino acid conjugates were synthesized by W. Shi,100 and the in vitro PDT effect of conjugates on human esophageal cancer Ec9706 cells were evaluated by standard cytotoxicity assay. The results indicated that 5-fluorouracil and l-phenylalanine could significantly improve the photocytotoxicity of porphyrins, and the transition metals Co(ii) and Mn(ii) also had significant effects on the photoactivity of porphyrins. Not only that, researchers found that 5-fluorouracil could better assist the porphyrin photodynamic efficacy. After pre-treatment of squamous cell carcinoma with 5-fluorouracil, porphyrin was added. It was found that the addition of 5-fluorouracil increased the selectivity of porphyrin to tumors by 4 times and significantly improved the photodynamic efficacy.101 This means that 5-fluorouracil combined with porphyrin is a potential treatment for skin cancer. Therefore, we can conclude that porphyrin combined with 5-fluorouracil has good biological activity and can significantly inhibit the proliferation of a variety of cancer cells, and has a great development potential in the treatment of malignant tumors.

Fig. 7. Structure of the porphyrin–5 fluorouracil conjugates.

3.2.3. Porphyrin–nitrogen mustard conjugates

Nitrogen mustard is one of the first drugs used in clinical treatment of tumors. It can alkylate the sulfhydryl groups, amino groups, and hydroxyl groups in proteins and nucleic acids in cellular components due to the presence of reactive alkyl groups. Then, it damages the structure and function of DNA, which affects the transcriptional replication of DNA, and thus affects the cells' division and growth, achieving the purpose of killing cancer cells.102,103 However, due to poor targeting, it has a large toxic side effect on normal tissues during use. In order to improve the selectivity of nitrogen mustard to cancer cells and reduce the toxic side effects, researchers designed porphyrin–nitrogen mustard conjugates. Chen et al.104,105 synthesized a series of porphyrin–nitrogen mustard conjugates (Fig. 8a). It was found that the drug concentration in tumor tissues was 40 times higher than that in the liver after administration for a period of time, showing excellent targeting performance. At the same time, the conjugates could destroy the cell membrane of the liver cancer cells, and block the synthesis of DNA, leading to apoptosis. Guo et al.106 synthesized a series of porphyrin compounds containing nitrogen mustard by an improved Lindsey method (Fig. 8b), including one nitrogen mustard structure to four nitrogen mustard structures. The inhibition rate of these compounds on Bel-7404 cells was tested by MTT assay. The results showed that the anticancer activity of the synthesized tetrasubstituted porphyrin–nitrogen mustard compounds was significantly higher than that of the monosubstituted porphyrin–nitrogen mustard compounds. Porphyrin–nitrogen mustard conjugates can combine the anticancer effects of chemical toxicity and photodynamic effects while reducing the distribution of drugs in normal tissues, reducing toxic and side effects, which leads to the prospect of developing new anticancer drugs for photochemotherapy.

Fig. 8. (a and b) Structures of the porphyrin–nitrogen mustard conjugates.

3.2.4. Porphyrin–piperazine conjugates

Piperazine derivatives have broad-spectrum pharmacological activities, such as anti-depression,107 anticancer,108 anti-fungus,109 anti-malaria,110 and anti-convulsion,111 and are widely used in medical fields. Based on the specific affinity of porphyrin for cancer cells and the antitumor activity of piperazines, Guo et al.112–114 designed and synthesized a series of porphyrin–piperazine compounds (Fig. 9a) and tested their inhibition rates on hepatoma cells Bel-7404, nasal cancer cells MCG, and gastric cancer cells NHE1. Most of the conjugates exhibited stronger inhibition of cancer cells than the positive control drugs cisplatin and 5-fluorouracil. Among them, the anticancer activity of the tetrasubstituted porphyrin–piperazine compounds was significantly higher than that of the monosubstituted porphyrin–piperazine compounds. Subsequently, they explored the mode of action of these compounds with DNA by binding experiments with calf thymus DNA (ctDNA). The results showed that these compounds bind to DNA in an intercalating manner and their binding strength was greater than that of the porphyrin precursor, which showed that the porphyrin–piperazine conjugates could bind better to DNA and had a potential medicinal value. Therefore, the porphyrin–piperazine conjugates have a potential medicinal value and are worthy of further exploration.

Fig. 9. Structure of the porphyrin–piperazine conjugates.

3.2.5. Porphyrin–anthraquinone conjugates

Anthraquinone derivatives have various biological activities such as anti-inflammatory, antibacterial, anticancer, etc. Anticancer drugs such as doxorubicin and mitoxantrone, which are commonly used in clinical practice, are typical anthraquinone compounds. Banfi et al.115 designed and synthesized a variety of porphyrin–anthraquinone conjugates by appropriately modifying emodin with tetraphenylporphyrin, and preliminarily evaluated their activity. Anti-tumor experiments in vitro showed that the inhibitory effect of the conjugates on human colon cancer cell line HCT116 was significantly stronger than that of emodin, and the compound with an amide bond had the best anti-tumor efficiency (Fig. 10a). Yang et al.116 also combined fluoroporphyrin with anthraquinone through an amide bond to synthesize a fluoroporphyrin–anthraquinone conjugate and its metal conjugate counterparts (Fig. 10b). Compared with fluoroporphyrin, all conjugates inhibited the activity of HeLa cells under ultraviolet light irradiation conditions, indicating that all of them exhibited better synergistic anticancer effects. Zhao et al.117,118 found that porphyrin–anthraquinone conjugates could be tightly bound to ctDNA by intercalation, partial insertion, etc., and the binding strength was greater than that of free base porphyrins. Under light of an appropriate wavelength, active substances such as singlet oxygen were generated, which led to DNA photodecomposition.

Fig. 10. (a and b) Structures of the porphyrin–anthraquinone conjugates.

This means that the porphyrin–anthraquinone conjugates are a class of potential photosensitizers for PDT.

3.2.6. Porphyrin–ferrocene conjugates

Chemodynamic therapy (CDT) uses endogenous chemical energy to produce ROS, which can cause cell death in the absence of additional energy stimulation such as light. Ferrocene and its derivatives can be formed by the Fenton reaction in the presence of hydrogen peroxide (H₂O₂) to form hydroxyl radicals and highly toxic ROS, which are often used in CDT. Lei et al.119 coupled porphyrin and ferrocene through amide bonds for photodynamic and chemical dynamic therapy (Fig. 11a).

Fig. 11. (a and b) Structures of the porphyrin–ferrocene conjugates.

It was found that compared with single component drugs, the ferrocene–porphyrin conjugates have excellent ROS production ability, significantly improved antitumor activity, and still have a better tumor killing effect in the form of low dose drugs.

Lippert et al.120 found that the addition of ferrocene improved the lipophilicity of porphyrin (Fig. 11b), allowing the conjugate to exhibit higher membrane permeability and significantly inhibit the formation of Candida albicans cell membranes in the dark. It is a potential antifungal drug and offers the possibility to further develop porphyrin-based systemic antifungal agents, and the synergy between CDT and PDT has great potential in tumor therapy.

4. Conclusions

Completely eradicating various cancer cells while keeping healthy host cells unaffected remains challenging at the current state of medical development. Due to its high efficiency, low toxicity and short duration of action, PDT seems to be a promising approach to anticancer therapy, while there is still a long way to go for its development. How to enhance the selective uptake and uniform distribution of photosensitizers in tumor tissues is still an urgent conundrum to be solved, especially the specific targeting of different cells and tumor tissues, to improve the efficacy and reduce their toxic and side effects on healthy tissues and organs. This may be a promising research direction, introducing functionally active groups into porphyrin photosensitizers to improve their targeting and anti-tumor activity, and then selectively delivering them to tumor tissues with transport carriers such as liposomes, magnetic nanoparticles and metal–organic frameworks. The advantages are as follows: photosensitizers bond with bioactive groups, which can exert synergistic therapeutic effects and improve the efficacy. Also, combining with a transport carrier having a bio-recognition function is conducive to improving the ability of the photosensitizers to penetrate tissues, enhancing the targeting of tumor tissues and increasing the drug-level concentration at the target site. In addition, the combination of endogenous chemiluminescent substances and porphyrins may achieve surprising effects. They may remove the limitation of low penetrability of tumor tissues to external light and eliminate deep malignant tumors through stimulation from both internal chemiluminescence and an external light source. What's more, the clinical application of PDT should also be standardized, and relevant industry standards should be formulated in terms of drug dose, light dose, light nature, light duration, efficacy, etc., which is conducive to promoting the extensive application of PDT.

Conflicts of interest

The authors declare no conflict of interests.