A Self-Assembly-Induced Exciton Delocalization Strategy for Converting a Perylene Diimide Derivative from a Type-II to Type-I Photosensitizer.

Type-I photosensitizers have shown advantages in addressing the shortcomings of traditional oxygen-dependent type-II photosensitizers for the photodynamic therapy (PDT) of hypoxic tumors. However, developing type-I photosensitizers is yet a huge challenge because the type-II energy transfer process is much faster than the type-I electron transfer process. Herein, from the fundamental point of view, an effective approach is proposed to improve the electron transfer efficiency of the photosensitizer by lowering the internal reorganization energy and exciton binding energy via self-assembly-induced exciton delocalization. An example proof is presented by the design of a perylene diimide (PDI)-based photosensitizer (PDIMp) that can generate singlet oxygen (1O2) via a type-II energy transfer process in the monomeric state, but induce the generation of superoxide anion (O2˙-) via a type-I electron transfer process in the aggregated state. Significantly, with the addition ofcucurbit[6]uril (CB[6]), the self-assembled PDIMp can convert back to the monomeric state via host-guest complexation and consequently recover the generation of 1O2. The biological evaluations reveal that supramolecular nanoparticles (PDIMp-NPs) derived from PDIMp show superior phototherapeutic performance via synergistic type-I PDT and mild photothermal therapy (PTT) against cancer under either normoxia or hypoxia conditions.

Perylene-Based Reactive Oxygen Species Supergenerator for Immunogenic Photochemotherapy against Hypoxic Tumors.

Reactive oxygen species (ROS) can act as cytotoxic radicals to directly kill tumor cells and concurrently trigger immunogenic cell death (ICD) to efficiently achieve tumor therapy. Thus motivated, we herein present one perylene monoamide-based ROS supergenerator (PMIC-NC) that not only induces hypoxia-enhanced Type-I ROS burst aided by proton transients but also triggers Type-I/II ROS production by electron or energy transfer under near-infrared (NIR) light irradiation and also elicits a strong ICD effect. More interesting, the mitochondria- and lung-specific distribution of PMIC-NC also boosts the tumor therapeutic efficiency. As a result, PMIC-NC was employed for NIR-triggered photodynamic therapy, hypoxia-enhanced chemotherapy and also displayed robust immunogenicity for systemic tumor eradication. This work thus contributes one proof-of-concept demonstration of perylene as an integrated therapeutic platform for efficient immunogenic photochemotherapy against hypoxic tumors.

Simultaneous Photodiagnosis and Photodynamic Treatment of Metastatic Melanoma.

Metastatic melanoma (MM) has a poor prognosis and is attributed to late diagnoses only when metastases has already occurred. Thus, early diagnosis is crucial to improve its overall treatment efficacy. The standard diagnostic tools for MM are incisional biopsies and/or fine needle aspiration biopsies, while standard treatments involve surgery, chemotherapy, or irradiation therapy. The combination of photodynamic diagnosis (PDD) and therapy (PDT) utilizes a photosensitizer (PS) that, when excited by light of a low wavelength, can be used for fluorescent non-destructive diagnosis. However, when the same PS is activated at a higher wavelength of light, it can be cytotoxic and induce tumor destruction. This paper focuses on PS drugs that have been used for PDD as well as PDT treatment of MM. Furthermore, it emphasizes the need for continued investigation into enhanced PS delivery via active biomarkers and passive nanoparticle systems. This should improve PS drug absorption in MM cells and increase effectiveness of combinative photodynamic methods for the enhanced diagnosis and treatment of MM can become a reality.

Yield enhancement strategies of rare pharmaceutical metabolites from endophytes.

Endophytes are barely untapped as vital sources in the medicine. They are microorganisms which mostly exist in plants. As they are exploited, it is accepted that endophytes can produce active metabolites that possess same function as their hosts such as taxol, podophyllotoxin, hypericin, and azadirachtin. These metabolites have been promising potential usefulness in safety and human health concerns. We are supposed to adopt measures to raise production for the low yield of metabolites. This paper summarizes the latest advances in various bioprocess optimization strategies. These techniques can overcome the limitations associated with rare pharmaceutical metabolite-producing endophytic fungi. These strategies include strain improvement, genome shuffling, medium optimization, fermentation conditions optimization, addition of specific factor, addition of solid sorbent, and co-culturing. It will enable endophytes to produce high and sustainable production of rare pharmaceutical metabolites.

Development of a novel formulation with hypericin to treat cutaneous leishmaniasis based on photodynamic therapy in in vitro and in vivo studies.

An evaluation of the leishmanicidal activity in vitro and in vivo of hypericin, an expanded-spectrum photosensitizer found in Hypericum perforatum, is presented. Hypericin was evaluated against intracellular amastigotes in vitro of Leishmania (Viannia) panamensis. A topical formulation containing 0.5% hypericin was developed and assayed in vivo in a hamster model of cutaneous leishmaniasis. Results demonstrate that hypericin induces a significant antiamastigote effect in vitro against L. panamensis by decreasing the number of parasites inside infected cells. The topical formulation of 0.5% hypericin allows healing of L. panamensis-induced lesions upon a topical application of 40 mg/day plus visible-light irradiation (5 J/cm(2), 15 min), twice a week for 3 weeks.

Successful in vivo tumor visualization using fluorescence laparoscopy in a mouse model of disseminated alveolar rhabdomyosarcoma.

BACKGROUND: Surgery for rhabdomyosarcoma is challenging due to a lack of clear delineation between tumor and surrounding tissue. Mutilating surgery can be necessary in difficult tumor localizations. Therefore, novel diagnostic and therapeutic modalities are required. The aim of this study was to evaluate the in vivo tumor detection of RMS using fluorescence laparoscopy and to analyze the efficacy of hypericin-induced photodynamic therapy in a mouse model. METHODS: Seventeen NOD/LtSz-scid IL2Rγnull-mice were divided into four groups. In group 1, mCherry-expressing tumor cells and in group 2-4 non-transfected tumor cells were xenotransplanted. Three weeks later, one fluorochrome per group (ICG, ICG-cetuximab, hypericin) was injected. Fluorescence laparoscopy was carried out and tumors were resected using fluorescence guidance. In the hypericin group, photodynamic therapy was performed using blue light and apoptosis was evaluated by TUNEL test. RESULTS: A clear discrimination between healthy and tumor tissue was feasible by fluorescending properties with mCherry expressing tumor cells and after injection of hypericin. No fluorescence was detected in mice injected with ICG and ICG-labeled cetuximab. Hypericin photodynamic therapy induced apoptosis of tumor cells after exposure to blue light. CONCLUSIONS: Intraoperative photodynamic diagnosis was feasible using mCherry-transfected tumor cells or hypericin. Additionally, intraoperative photodynamic therapy was possible and effective.

ER stress, autophagy and immunogenic cell death in photodynamic therapy-induced anti-cancer immune responses.

Tumours are a form of pseudo-organs with their own microenvironment where the cancer cells nurture a dysfunctional immune environment incapable of inciting anti-tumour immunity. It had been proposed that the only way to counteract such an immune system dysfunction in tumours is by eliciting, therapeutically, a cancer cell death pathway that is accompanied by high immunogenicity and possibly inhibits or reduces the influence of the pro-tumourigenic cytokine signalling. Subsequently, a small and a large-scale screening study as well as several targeted studies found that few, selected anticancer therapeutic regimens are able to induce a promising kind of cancer cell demise called immunogenic cell death (ICD), which can activate the immune system owing to the spatiotemporally defined emission of danger signals. Recently, photodynamic therapy (PDT) utilizing the photosensitiser, hypericin (Hyp), became the first PDT paradigm characterized to be capable of inducing bona fide ICD. In the present perspective, we discuss the various technical, conceptual, and molecular advancements and unprecedented results revealed by Hyp-PDT that have influenced the fields of ICD, ER stress biology, cancer cell death, anti-cancer immune responses, photoimmunology and PDT.

Dual-modal imaging and photodynamic therapy using upconversion nanoparticles for tumor cells.

Here we synthesized silica-coated NaYF4:Yb,Tm@NaGdF4 nanoparticles with hypocrellin photosensitizers covalently incorporated inside the silica shells, combining dual modal imaging and photodynamic therapy (PDT) functions together. Under excitation at 980 nm, the tumor-targeting specificity of the as-prepared nanomaterials efficiently enhanced as folic acid (FA) was conjugated. The internalization of UCNPs@SiO2@hypocrellin A-FA in HeLa cells and HEK-293 cells was observed by confocal microscopy and in vitro magnetic resonance imaging (MRI), which demonstrated that the as-prepared nanocomposites have the ability to target folate receptor (FR) (+) cells. Moreover, magnetic resonance (MR) measurements also demonstrated that the as-prepared nanocomposites could be used as a contrast agent for MRI. All these results showed the feasibility and potential of the as-prepared nanocomposites for simultaneous imaging and PDT application.

A perylene derivative regulates HIF-1α and Stat3 signaling pathways.

It is becoming increasingly evident that improving the cure rate of many cancers will require treatment regimens hit more than one validated tumor targets. Developing an anti-cancer agent that targets two oncoproteins simultaneously is a promising strategy for accomplishing this goal. It would be expected to promote drug efficacy, reduce therapy-resistant without introducing additional toxic side effects. HIF-1α is a key regulator of the cellular response to hypoxia and is involved in tumor angiogenesis and cancer cell survival, glucose metabolism, and invasion. Stat3 has several oncogenic functions, including suppression of anti-tumor immune responses and promotion of inflammation. Recently, we have identified the perylene derivative, TEL03, as a dual inhibitor that targets both HIF-1α and Stat3. TEL03 blocks the expression of both HIF-1α and Stat3, regulated oncogenes (e.g., Bcl-2, VEGF, Glut1, and others) in cancer cells, and induces cancer cell apoptosis. The results demonstrated that: (i) TEL03 blocks Stat3 phosphorylation, and inhibits Stat3 transcriptional activity; and (ii) interferes the binding of HIF-1α to p300/CBP inducing its degradation by proteasomes under hypoxic conditions. Our in vivo tests showed that as a dual inhibitor, TEL03 dramatically inhibited tumor growth, and provided the evidence that targeting both HIF-1α and Stat3 simultaneously could be a promising strategy for breast and pancreatic cancer therapies.

Retinal injury thresholds for 532, 578, and 630 nm lasers in connection to photodynamic therapy for choroidal neovascularization.

BACKGROUND AND OBJECTIVES: The purpose of this study was to explore the retinal injury thresholds in rabbits and evaluate the influence of retinal pigmentation on threshold irradiance at laser wavelengths of 532, 578, and 630 nm which might be involved in hypocrellin B (HB) and hematoporphyrin monomethyl ether (HMME) photodynamic therapy (PDT) for choroidal neovascularization (CNV). MATERIALS AND METHODS: The eyes of pigmented and non-pigmented rabbits were exposed to 532, 578, and 630 nm lasers coupled to a slit lamp biological microscope. The exposure duration was 100 seconds and the retinal spot size was 2 mm throughout the experiment. The minimum visible lesions were detected by funduscopy at 1 and 24 hours post exposure. Bliss probit analysis was performed to determine the ED50 thresholds, fiducial limits and probit slope. RESULTS: In pigmented rabbits, the 24-hour retinal threshold irradiances at 532, 578, and 630 nm were 1,003, 1,475, and 1,720 mW/cm(2) , respectively. In non-pigmented rabbits, the 24-hour threshold irradiances were 1,657, 1,865, and 15,360 mW/cm(2) , respectively. The ED50 for 24-hour observation differed very little from the ED50 for 1-hour observation. The non-pigmented rabbits required a ninefold increase in threshold irradiance at 630 nm comparing to the pigmented rabbits. CONCLUSION: This study will contribute to the knowledge base for the limits of laser irradiance in application of HB or HMME PDT for CNV.

Toward Type I/II ROS Generation Photoimmunotherapy by Molecular Engineering of Semiconducting Perylene Diimide.

As prospective phototheranostic agents for cancer imaging and therapy, semiconducting organic molecule-based nanomedicines are developed. However, near-infrared (NIR) emission, and tunable type I (O2 • -) and type II (1O2) photoinduced reactive oxygen species (ROS) generation to boost cancer photoimmunotherapy remains a big challenge. Herein, a series of D-π-A structures, NIR absorbing perylene diimides (PDIs) with heavy atom bromide modification at the bay position of PDIs are prepared for investigating the optimal photoinduced type I/II ROS generation. The heavy atom effect has demonstrated a reduction of molecular ∆EST and promotion of the intersystem crossing processes of PDIs, enhancing the photodynamic therapy (PDT) efficacy. The modification of three bromides and one pyrrolidine at the bay position of PDI (TBDT) has demonstrated the best type I/II PDT performance by batch experiments and theoretical calculations. TBDT based nanoplatforms (TBDT NPs) enable type I/II PDT in the hypoxic tumor microenvironment as a strong immunogenic cell death (ICD) inducer. Moreover, TBDT NPs showing NIR emission allow in vivo bioimaging guided phototherapy of tumor. This work uses novel PDIs with adjustable type I/II ROS production to promote antitumor immune response and accomplish effective tumor eradication, consequently offering molecular guidelines for building high-efficiency ICD inducers.

A perylene diimide probe for NIR-II fluorescence imaging guided photothermal and type I/type II photodynamic synergistic therapy.

Phototherapy has garnered significant attention in the past decade. Photothermal and photodynamic synergistic therapy combined with NIR fluorescence imaging has been one of the most attractive treatment options because of the deep tissue penetration, high selectivity and excellent therapeutic effect. Benefiting from the superb photometrics and ease of modification, perylene diimide (PDI) and its derivatives have been employed as sensing probes and therapeutic agents in the biological and biomedical research fields, and exhibiting excellent potential. Herein, we reported the development of a novel organic small-molecule phototherapeutic agent, PDI-TN. The absorption of PDI-TN extends into the NIR region, which provides feasibility for NIR phototherapy. PDI-TN overcomes the traditional Aggregation-Caused Quenching (ACQ) effect and exhibits typical characteristics of Aggregation-Induced Emission (AIE). Subsequently, PDI-TN NPs were obtained by using an amphiphilic triblock copolymer F127 to encapsulate PDI-TN. Interestingly, the PDI-TN NPs not only exhibit satisfactory photothermal effects, but also can generate O2•- and 1O2 through type I and type II pathways, respectively. Additionally, the PDI-TN NPs emit strong fluorescence in the NIR-II region, and show outstanding therapeutic potential for in vivo NIR-II fluorescence imaging. To our knowledge, PDI-TN is the first PDI derivative used for NIR-II fluorescence imaging-guided photodynamic and photothermal synergistic therapy, which suggests excellent potential for future biological/biomedical applications.

Single-Photon Deep-Red Light-Triggered Direct Release of an Anticancer Drug: An Investigative Tumor Regression Study on a Breast Cancer Spheroidal Tumor Model.

Breast adenocarcinoma ranks high among the foremost lethal cancers affecting women globally, with its triple-negative subtype posing the greatest challenge due to its aggressiveness and resistance to treatment. To enhance survivorship and patients' quality of life, exploring advanced therapeutic approaches beyond conventional chemotherapies is imperative. To address this, innovative nanoscale drug delivery systems have been developed, offering precise, localized, and stimuli-triggered release of anticancer agents. Here, we present perylenemonoimide nanoparticle-based vehicles engineered for deep-red light activation, enabling direct chlorambucil release. Synthesized via the reprecipitation technique, these nanoparticles were thoroughly characterized. Light-induced drug release was monitored via spectroscopic and reverse-phase HPLC. The efficacy of the said drug delivery system was evaluated in both two-dimensional and three-dimensional spheroidal cancer models, demonstrating significant tumor regression attributed to apoptotic cell death induced by efficient drug release within cells and spheroids. This approach holds promise for advancing targeted breast cancer therapy, enhancing treatment efficacy and minimizing adverse effects.

Internal heavy atom effect of Au(III) and Pt(IV) on hypocrellin A for enhanced in vitro photodynamic therapy of cancer.

Hypocrellin A (HA), an a natural perylene quinine photosensitizers (PSs), can chelate with heavy metal ions, including Au(III) and Pt(IV), to form a 1:2 complex, which exhibits enhanced (1)O2 generation quantum yield through the increased intersystem crossing efficiency mediated by internal heavy atom effect. Besides, the chelate process greatly improved the water solubility of HA. Comparative studies with HA and complexes have demonstrated that the heavy-atom effect on HA molecules enhances the efficiency of in vitro photodynamic (PDT) efficacy.

Perylene-Mediated Electron Leakage in Respiratory Chain to Trigger Endogenous ROS Burst for Hypoxic Cancer Chemo-Immunotherapy.

Perylene derivatives can be stimulated by the hypoxic tumor microenvironment to generate radical anion that is proposed to arouse electron exchange with oxidizing substance, and in turn, realize reactive oxygen species (ROS) burst. Here, three perylene therapeutic agents, PDI-NI, PDIB-NI, and PDIC-NI, are developed and it is found that the minimum lowest unoccupied molecular orbital (LUMO) energy level makes PDIC-NI most easily accept electrons from the oxidative respiratory chain to form lots of anions, and the resultant maximum ROS generation, establishing an unambiguous mechanism for the formation of perylene radical anions in the cell, presents solid evidence for LUMO energy level determining endogenous ROS burst. Stirringly, PDIC-NI-induced ROS generation arouses enhanced mitochondrial oxidative stress and concurrently activates immunogenic cell death (ICD), which not only efficiently kills lung tumor cells but also reprograms immunosuppressive tumor microenvironment, including the cytokine secretion, dendritic cell maturation, as well as cytotoxic T lymphocytes activation, to inhibit the growth of xenografted and metastasis tumor, presenting a proof-of-concept demonstration of perylene that acts as an integrated therapeutic agent to well realize hypoxia-activated chemotherapy with ICD-induced immunotherapy on lung cancer.

Radiolabeled iodohypericin as tumor necrosis avid tracer: diagnostic and therapeutic potential.

It is estimated that 30-80% of solid tumor mass represents necrotic tissue that consists out of a significant number of dead and dying cells. The fact that these necrotic zones are restricted to dysplastic and malignant tissue and are rarely present in normal tissue makes necrosis an interesting target both for cancer diagnosis and therapy. In this study, the avidity of hypericin, [(123) I]iodohypericin and [(131) I]iodohypericin to tumor necrosis was explored for both diagnosis and therapy of experimental malignancies. The intratumoral distribution in RIF-1 tumors was investigated by means of fluorescence microscopy (hypericin) and autoradiography ([(123) I]iodohypericin). Results show high uptake of the tracers in necrosis at 24 hr, lasting for up to 72 hr p.i. Ratios of activity of [(123) I]iodohypericin in necrotic tissue over viable tumor reached up to 19.63 ± 4.66, correlating with 9.20% ID/g in necrosis. Nude mice bearing RIF-1 tumors that received three injections of 300 µCi over a 3-week treatment period showed stabilization in tumor growth for 5 days, as measured by caliper and micro-positron emission tomography using [(18) F]fluorodeoxyglucose. Based on these results, we suggest the potentials of radiolabeled hypericin (1) in diagnostic aspects including prognosis or staging assessment of bulky necrotic cancers, monitoring of treatments and therapeutic follow-up; and (2) in cancer treatment based on tumor necrosis. In conclusion, we showed that hypericin radiolabeled with iodine is a necrosis avid tracer that can be used both as a tumor diagnostic and therapeutic.

Hypericin-PDT-induced rapid necrotic death in human squamous cell carcinoma cultures after multiple treatment.

PDT (photodynamic therapy) has been used for the treatment of NMCC (non-melanoma cutaneous cancer) particularly, human SCC (squamous cell carcinoma). However, the nature of the photosensitizer, the activation light source and the mode of cell death induced post-PDT remains elusive. We tried to optimize PDT using the light-activated (320-400 nm) St John's Wort-derived compound, Hyp (hypericin). The work highlights the potential mode of cell death and the increased efficacy of the technique associated with multiple Hyp-PDT treatment. SCC cells were exposed to different concentrations of Hyp and activated with light at 1 J/cm2 for 1 or 2 days. Thereafter with the optimum dose of Hyp proliferation, ROS (reactive oxygen species), and apoptosis were analysed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay, FACS analysis and Fluorescent/Phase contrast microscopy was carried out for morphological studies. Hyp-PDT produces more ROS after 1 day compared with 2 days and the mode of cell death is a necrotic caspase-independent mechanism. We propose a novel 'double-hit/2-day' strategy to reduce the viability in SCC using Hyp-based PDT as an adjunctive treatment modality.

A single-dose toxicity study on non-radioactive iodinated hypericin for a targeted anticancer therapy in mice.

AIM: Hypericin (Hyp) and its radio-derivatives have been investigated in animal models with ischemic heart diseases and malignancies for diagnostic and therapeutic purposes. Before radioiodinated Hyp ((123)I-Hyp or (131)I-Hyp) can be considered as a clinically useful drug, vigorous evaluations on its chemotoxicity are necessary. In the present study, we examined the toxicity of a single dose of non-radioactive (127)I-Hyp in normal mice for 24 h and 14 d. METHODS: Studies were performed on 132 normal mice. (127)I -Hyp at a clinically relevant dose of 0.1 mg/kg body weight and a 100-times higher dose of 10 mg/kg was intravenously injected into 40 mice. The safety aspects of clinical manifestations, serological biochemistry, and histopathology were assessed. In another 72 mice, (127)I-Hyp was administered intravenously at assumed values to bracket the value of LD(50). The rest 20 mice were used in the control groups. RESULTS: At 24 h and 14 d following the injection of (127)I -Hyp at either 0.1 or 10 mg/kg, all mice tolerated well without mortality or any observable treatment-related symptoms. No significant differences were found in blood biochemical parameters between the test and control groups. All organs presented normal appearances upon histopathological inspection. The value of LD(50) of (127)I-Hyp in mice through intravenous injection was 20.26 mg/kg, with the 95% confidence interval between 18.90 and 21.55 mg/kg. CONCLUSION: The current study reveals a broad safety range of (127)I-Hyp, which not only supports the use of (123)I-Hyp or (131)I-Hyp in the necrosis targeting theragnostic strategy, but also serves as a valuable reference for exploring other possible applications for iodinated Hyp.

Tumor Microenvironment Stimuli-Responsive Single-NIR-Laser Activated Synergistic Phototherapy for Hypoxic Cancer by Perylene Functionalized Dual-Targeted Upconversion Nanoparticles.

Although synergistic therapy has shown great promise for effective treatment of cancer, the unsatisfactory therapeutic efficacy of photothermal therapy/photodynamic therapy is resulted from the absorption wavelength mismatch, tumor hypoxia, photosensitizer leakage, and inability in intelligent on-demand activation. Herein, based on the characteristics of tumor microenvironment (TME), such as the slight acidity, hypoxia, and overexpression of H2 O2 , a TME stimuli-responsive and dual-targeted composite nanoplatform (UCTTD-PC4) is strategically explored by coating a tannic acid (TA)/Fe3+ nanofilm with good biocompatibility onto the upconversion nanoparticles in an ultrafast, green and simple way. The pH-responsive feature of UCTTD-PC4 remains stable during the blood circulation, while rapidly releases Fe3+ in the slightly acidic tumor cells, which results in catalyzing H2 O2 to produce O2 and overcoming the tumor hypoxia. Notably, the emission spectrum of the UCTTD perfectly matches the absorption spectrum of the photosensitizer (perylene probe (PC4)) to achieve the enhanced therapeutic effect triggered by a single laser. This study provides a new strategy for the rational design and development of the safe and efficient single near-infrared laser-triggered synergistic treatment platform for hypoxic cancer under the guidance of multimodal imaging.

A dual-targeting anticancer approach: soil and seed principle.

PURPOSE: To test the hypothesis that targeting the microenvironment (soil) may effectively kill cancer cells (seeds) through a small-molecular weight sequential dual-targeting theragnostic strategy, or dual-targeting approach. MATERIALS AND METHODS: With approval from the institutional animal care and use committee, 24 rats were implanted with 48 liver rhabdomyosarcomas (R1). First, the vascular-disrupting agent combretastatin A4 phosphate (CA4P) was injected at a dose of 10 mg/kg to cause tumor necrosis, which became a secondary target. Then, the necrosis-avid agent hypericin was radiolabeled with iodine 131 to form (131)I-hypericin, which was injected at 300 MBq/kg 24 hours after injection of CA4P. Both molecules have small molecular weight, are naturally or synthetically derivable, are intravenously injectable, and are of unique targetablities. The tumor response in the dual-targeting group was compared with that in vehicle-control and single-targeting (CA4P or (131)I-hypericin) groups with in vivo magnetic resonance imaging and scintigrams and ex vivo gamma counting, autoradiography, and histologic analysis. Tumor volumes, tumor doubling time (TDT), and radiobiodistribution were analyzed with statistical software. P values below .05 were considered to indicate a significant difference. RESULTS: Eight days after treatment, the tumor volume of rhabdomyosarcoma in the vehicle-control group was double that in both single-targeting groups (P < .001) and was five times that in the dual-targeting group (P < .0001), without treatment-related animal death. The TDT was significantly longer in the dual-targeting group (P < .0001). Necrosis appeared as hot spots on scintigrams, corresponding to 3.13% of the injected dose of (131)I-hypericin per gram of tissue (interquartile range, 2.92%-3.97%) and a target-to-liver ratio of 20. The dose was estimated to be 100 times the cumulative dose of 50 Gy needed for radiotherapeutic response. Thus, accumulated (131)I-hypericin from CA4P-induced necrosis killed residual cancer cells with ionizing radiation and inhibited tumor regrowth. CONCLUSION: This dual-targeting approach may be a simple and workable solution for cancer treatment and deserves further exploitation.