Photodynamic therapy (PDT), performed with low-fluence rates, can improve antitumor responses and prevent adverse effects. However, photosensitizers (PSs) for low-fluence PDT treatment are rarely reported. Herein, we exploited an amphiphilic chlorin-based PS, named DYSP-C34, which has a variety of beneficial biological properties, such as improved water solubility, better cellular permeability, specific localization and enhanced phototoxicity under low light dose irradiation. In addition, DYSP-C34 could effectively accumulate in a mouse subcutaneous xenograft tumor and exhibit substantial tumor regression after irradiation with an extremely low light fluence (6 J/cm2). Meanwhile, the excellent phototoxicity could stimulate the host immune system and lead to a strong inhibition of tumor growth synergistically. These results indicated the potential value of DYSP-C34 as a chlorin-type PS for low-fluence PDT application.

Exploring facile and effective therapeutic modalities for synergistically controlling primary tumor and metastasis remains a pressing clinical need. Sonodynamic therapy (SDT) offers the possibility of noninvasively eradicating local solid tumors, but lacks antimetastatic activity because of its limited ability in generating systemic antitumor effect. Here, we exploited a previously unidentified ultrasound-driven “molecular machine,” DYSP-C34 (C34 for short), with multiple attractive features, emerging from preferential tumor accumulation, potent ultrasound-triggered cytotoxicity, and intrinsic immune-boosting capacity. Driven by the ultrasound, C34 functioned not only as a tumor cell killing reagent but also as an immune booster that could potentiate robust adaptive antitumor immunity by directly stimulating dendritic cells, resulting in the eradication of the primary solid tumor along with the inhibition of metastasis. This molecular machine, C34, rendered great promise to achieve systemic treatment against cancer via unimolecule-mediated SDT.

Photodynamic therapy (PDT) has garnered immense research interest. PDT can directly kill the cells via a combination of photosensitizer, light, and molecular oxygen. It has emerged as a promising therapeutic option for cancer treatment owing to its advantages such as minimized systemic toxicity, minimal invasiveness, high therapeutic efficacy, and potential for developing antitumor immunity. The novel photosensitizer 32-(4-methoxyphenyl)-152-aspartyl-chlorin e6 (DYSP-C34) was synthesized by introducing a 32-aryl substitution and amino acid substituent of the Chenghai chlorin (CHC). Briefly, 32-(4-methoxyphenyl) substitution was achieved via olefin metathesis reactions. The aspartic acid side chain was introduced regioselectively at C-152, followed by hydrolysis to yield the target DYSP-C34. CHC with the same chemical structure as chlorin e6 was prepared from chlorophyll a, which was extracted from Spirulina powders derived from Chenghai Lake in the Yunnan province of China. This strategy successfully endowed the resultant photosensitizer with better cellular permeability and increased water solubility. In addition, the photodynamic antitumor effects of PDT largely depend on the dose of photosensitizer used, time between photosensitizer administration and light exposure, and possibly other still poorly known variables. Determination of optimal conditions for PDT requires a coordinated interdisciplinary effort. Therefore, the pharmacokinetics and tissue distribution of DYSP-C34 in vivo are critical for the efficacy and safety of PDT. Herein, a high performance liquid chromatography-ultraviolet (HPLC-UV) detection method was established for the determination of the new photosensitizer DYSP-C34 in rat plasma. The sample preparation involved a protein-precipitation and liquid-liquid extraction method. Methanol was used to precipitate proteins and chloroform was used to extract chlorins. Then, DYSP-C34 was separated on a Unitary C18 column (250 mm×4.6 mm, 5 μm) with a mobile phase comprising methanol and 5 mmol/L tetrabutylammonium phosphate buffer solution (70∶30, v/v). The flow rate was 1.0 mL/min with UV detection using a wavelength of 400 nm at 40 ℃. Results showed that DYSP-C34 and chlorin e6 trimethyl ester (IS) were well separated under these conditions. The method was sensitive and sufficiently precise with a good linear relationship (determination coefficient (r2)=0.9941) over the range of 1-200 μg/mL in rat plasma. At three spiked levels (8, 40, and 120 μg/mL), the average recoveries were 74.39%, 69.71%, and 65.89%, respectively. The intra-day and inter-day relative standard deviations (RSDs) were lower than 5%. The precision met the requirements of biological sample determination. Furthermore, DYSP-C34 was stable in rat plasma under various storage conditions at room temperature, three freeze-thaw cycles, and long-term cryopreservation. The validated method was successfully applied to the pharmacokinetic study of DYSP-C34 after intravenous injection of a single dose in rat plasma. The pharmacokinetic parameters after intravenous injection of DYSP-C34 (16 mg/kg) were calculated. The plasma half-life (t1/2z) was 6.98 h, the area under the plasma concentration-time curve AUC(0-∞) was 1025.01 h·mg/L and the mean retention time MRT(0-∞) was 9.19 h. In addition, the results of DYSP-C34 distribution in tumor-bearing mice showed that DYSP-C34 could accumulate in tumor tissues, with higher concentrations in liver and kidney tissues, and lower concentrations in heart, spleen, and lung tissues. In summary, a specific, simple, and accurate HPLC-UV method was developed and validated for the determination of DYSP-C34 in rat plasma and tumor-bearing mouse tissues. The pharmacokinetics of DYSP-C34 after intravenous administration in rats and the tissue distribution characteristics of tumor-bearing mice were clarified for the first time. It is significant for clinical rational drug use and pharmacodynamic research. Therefore, choosing an appropriate time for light treatment time can achieve the best photodynamic effect. The results of pharmacokinetics and tissue distribution of DYSP-C34 provide vital guidance for subsequent pharmacodynamic research and further clinical trials in terms of dosage, light time, light toxicity and side effects.

Photodynamic therapy (PDT) has garnered immense research interest. PDT can directly kill the cells via a combination of photosensitizer, light, and molecular oxygen. It has emerged as a promising therapeutic option for cancer treatment owing to its advantages such as minimized systemic toxicity, minimal invasiveness, high therapeutic efficacy, and potential for developing antitumor immunity. The novel photosensitizer 32-(4-methoxyphenyl)-152-aspartyl-chlorin e6 (DYSP-C34) was synthesized by introducing a 32-aryl substitution and amino acid substituent of the Chenghai chlorin (CHC). Briefly, 32-(4-methoxyphenyl) substitution was achieved via olefin metathesis reactions. The aspartic acid side chain was introduced regioselectively at C-152, followed by hydrolysis to yield the target DYSP-C34. CHC with the same chemical structure as chlorin e6 was prepared from chlorophyll a, which was extracted from Spirulina powders derived from Chenghai Lake in the Yunnan province of China. This strategy successfully endowed the resultant photosensitizer with better cellular permeability and increased water solubility. In addition, the photodynamic antitumor effects of PDT largely depend on the dose of photosensitizer used, time between photosensitizer administration and light exposure, and possibly other still poorly known variables. Determination of optimal conditions for PDT requires a coordinated interdisciplinary effort. Therefore, the pharmacokinetics and tissue distribution of DYSP-C34 in vivo are critical for the efficacy and safety of PDT. Herein, a high performance liquid chromatography-ultraviolet (HPLC-UV) detection method was established for the determination of the new photosensitizer DYSP-C34 in rat plasma. The sample preparation involved a protein-precipitation and liquid-liquid extraction method. Methanol was used to precipitate proteins and chloroform was used to extract chlorins. Then, DYSP-C34 was separated on a Unitary C18 column (250 mm×4.6 mm, 5 μm) with a mobile phase comprising methanol and 5 mmol/L tetrabutylammonium phosphate buffer solution (70∶30, v/v). The flow rate was 1.0 mL/min with UV detection using a wavelength of 400 nm at 40 ℃. Results showed that DYSP-C34 and chlorin e6 trimethyl ester (IS) were well separated under these conditions. The method was sensitive and sufficiently precise with a good linear relationship (determination coefficient (r2)=0.9941) over the range of 1-200 μg/mL in rat plasma. At three spiked levels (8, 40, and 120 μg/mL), the average recoveries were 74.39%, 69.71%, and 65.89%, respectively. The intra-day and inter-day relative standard deviations (RSDs) were lower than 5%. The precision met the requirements of biological sample determination. Furthermore, DYSP-C34 was stable in rat plasma under various storage conditions at room temperature, three freeze-thaw cycles, and long-term cryopreservation. The validated method was successfully applied to the pharmacokinetic study of DYSP-C34 after intravenous injection of a single dose in rat plasma. The pharmacokinetic parameters after intravenous injection of DYSP-C34 (16 mg/kg) were calculated. The plasma half-life (t1/2z) was 6.98 h, the area under the plasma concentration-time curve AUC(0-∞) was 1025.01 h·mg/L and the mean retention time MRT(0-∞) was 9.19 h. In addition, the results of DYSP-C34 distribution in tumor-bearing mice showed that DYSP-C34 could accumulate in tumor tissues, with higher concentrations in liver and kidney tissues, and lower concentrations in heart, spleen, and lung tissues. In summary, a specific, simple, and accurate HPLC-UV method was developed and validated for the determination of DYSP-C34 in rat plasma and tumor-bearing mouse tissues. The pharmacokinetics of DYSP-C34 after intravenous administration in rats and the tissue distribution characteristics of tumor-bearing mice were clarified for the first time. It is significant for clinical rational drug use and pharmacodynamic research. Therefore, choosing an appropriate time for light treatment time can achieve the best photodynamic effect. The results of pharmacokinetics and tissue distribution of DYSP-C34 provide vital guidance for subsequent pharmacodynamic research and further clinical trials in terms of dosage, light time, light toxicity and side effects.

Photodynamic therapy (PDT) is a non-invasive and innovative therapeutic approach which has been increasingly applied in clinical cancer therapy. As the central element of PDT, the development of novel photosensitizers (PSs) with longer absorption wavelength, proper lipophilic/hydrophilic profiles, target tissue selectivity, and higher photo−/lowest dark-cytotoxicity is a challenging task. Previously, we designed and synthesized a series of novel long-wavelength chlorin e6 (Ce6)-based PSs via introducing aromatic groups to the vinyl of Ce6 skeleton. The new formed compounds with π-extension system exhibited improved photodynamic effects and spectral characteristics. Among these π-conjugated chlorin PSs, (E)-32-(4-methoxyphenyl)-chlorin e6, named A15, was expected to be a potent antitumor candidate as a PDT agent due to its good photobiological properties. Herein, in this work, we evaluated the effectiveness of A15 in cancer PDT. In vitro, a novel rare earth probe, ATTA-Eu3+ was applied to detect the singlet oxygen (1O2) production of A15 in solution and human hepatoma HepG2 cells, respectively. Moreover, A15 exhibited strong phototoxicity and weak dark cytotoxity to HepG2 cells. In H22 tumor bearing mice, A15 showed excellent tumor accumulation ability via i.v. administration and induced tumor regression, followed by laser treatment. These results indicated that A15 is a potential novel π–extension chlorin-type PS for PDT applications.

Targeting tumor acidic microenvironment, a cis-aconitate linked pyropheophorbide a dimer 3 was designed and prepared The observed fluorescence quenching of 3 verified our FRET based molecular design. Acid-dependent cleavage in aqueous solution, singlet oxygen generation, and cytotoxiciry against HepG2 cell lines of dimer 3 were investigated. The dimer demonstrated different levels of fluorescence recovery when incubated in acidic aqueous environment as well as effective phototoxicity against HepG2 cells.

In order to increase the photodynamic effect of Chlorin e6, four Chlorin e6-artesunate conjugates were designed and synthesized. Among them, three conjugates (3, 6, 9) contained single artesunate side chain at 152, 173 and 131 of Chlorin e6, respectively, and one conjugate (11) contained three artesunate side chains. In the in vitro evaluation of photodynamic effect, the four conjugates showed more potent phototoxicity against HepG2 cells than Chlorin e6. The introduction of artesunate side chain significantly increased the intracellular ROS production, although the production of singlet oxygen was not improved. Compound 11 exhibited much more potent phototoxicity than the other conjugates because the three artesunate side chains greatly enhanced the ROS production and cellular uptake. The results demonstrated that the conjugation of Chlorin e6 and artesunate could accomplish synergistic effects of chemo-phototherapy, and finally enhanced their antiproliferative effects.

32-Aryl substitution generated the maximum wavelength redshift and increased the in vitro phototoxicity against HepG2 cells simultaneously.