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Despite untiring efforts to develop therapies for pancreatic ductal adenocarcinoma (PDAC), survival statistics remain dismal, necessitating distinct approaches. Photodynamic priming (PDP), which improves drug delivery and combination regimens, as well as tumor photodestruction are key attributes of photodynamic therapy (PDT), making it a distinctive clinical option for PDAC. Localized, high-payload nanomedicine-assisted delivery of photosensitizers (PSs), with molecular specificity and controlled photoactivation, thus becomes critical in order to reduce collateral toxicity during more expansive photodynamic activation procedures with curative intent. As such, targeted photoactivable lipid-based nanomedicines are an ideal candidate but have failed to provide greater than two-fold cancer cell selectivity, if at all, due to their extensive multivariant physical, optical, and chemical complexity. Here, we report (1) a systematic multivariant tuning approach to engineer (Cet, anti-EGFR mAb) photoimmunonanoconjugates (PINs), and (2) stroma-rich heterotypic PDAC in vitro and in vivo models incorporating patient-derived pancreatic cancer-associated fibroblasts (PCAFs) that recapitulate the desmoplasia observed in the clinic. These offer a comprehensive, disease-specific framework for the development of Cet-PINs. Specificity-tuning of the PINs, in terms of PS lipid anchoring, electrostatic modulation, Cet orientation, and Cet surface densities, achieved ∼16-fold binding specificities and rapid penetration of the heterotypic organoids within 1 h, thereby providing a ∼16-fold enhancement in molecular targeted NIR photodestruction. As a demonstration of their inherent amenability for multifunctionality, encapsulation of high payloads of gemcitabine hydrochloride, 5-fluorouracil, and oxaliplatin within the Cet-PINs further improved their antitumor efficacy in the heterotypic organoids. In heterotypic desmoplastic tumors, the Cet-PINs efficiently penetrated up to 470 μm away from blood vessels, and photodynamic activation resulted in substantial tumor necrosis, which was not elicited in T47D tumors (low EGFR) or when using untargeted constructs in both tumor types. Photodynamic activation of the Cet-PINs in the heterotypic desmoplastic tumors resulted in collagen photomodulation, with a 1.5-fold reduction in collagen density, suggesting that PDP may also hold potential for conquering desmoplasia. The in vivo safety profile of photodynamic activation of the Cet-PINs was also substantially improved, as compared to the untargeted constructs. While treatment using the Cet-PINs did not cause any detriment to the mice's health or to healthy proximal tissue, photodynamic activation of untargeted constructs induced severe acute cachexia and weight loss in all treated mice, with substantial peripheral skin necrosis, muscle necrosis, and bowel perforation. This study is the first report demonstrating the true value of molecular targeting for NIR-activable PINs. These constructs integrate high payload delivery, efficient photodestruction, molecular precision, and collagen photomodulation in desmoplastic PDAC tumors in a single treatment using a single construct. Such combined PIN platforms and heterocellular models open up an array of further multiplexed combination therapies to synergistically control desmoplastic tumor progression and extend PDAC patient survival.

Objectives Numerous studies employ mathematical methods, such as Monte Carlo simulation, to predict the tumor killing effects of photodynamic therapy (PDT) by simulating optical propagation, photosensitizer distribution, and oxygen distribution. Whether these models faithfully reflect tumor killing is unknown, and model validation using tumor cross sections in these studies is usually insufficient to answer this question. To fill this gap in our knowledge, we employed a mouse model of breast cancer to determine the spatiotemporal effects of PDT using direct histopathological and biochemical analyses of whole tumors. Methods We prepared approximately 700 5-μm-thick serial sections of breast tumors of syngeneic mice treated with PDT employing the photosensitizer photocarcinorin (PsD-007, a second-generation photosensitizer developed in China). Three adjoining sections were subjected to hematoxylin and eosin staining to assess necrosis, the TUNEL assay to evaluate apoptosis, and CD31 staining to detect angiogenesis, respectively. We then generated a three-dimensional (3D) reconstruction of the tumor to evaluate these processes. We simultaneously used the Monte Carlo method to develop a model of light distribution throughout the tumor to evaluate the actual and simulated tumor killing effects induced by PDT. Results Tumor necrosis decreased exponentially as a function of distance from the source of illumination, while the distributions of apoptosis and neovascularization were independent of light distribution. Most apoptosis occurred in the lower layers (3000–4000 μm) of the tumor where the light intensity was too low to excite the photosensitizer. Neovascularization occurred at depths ranging from 2500 to 3500 μm. These analyses provided a 3D view of how a tumor is destroyed using PDT. Conclusions Although the optical distribution model predicted tumor necrosis caused by PDT, it was ineffective in predicting the sites of apoptosis and vascular destruction. Mathematical modeling is limited in its capabilities required to gain a comprehensive understanding of the spatiotemporal events associated with PDT. The mouse model developed here will serve as a platform for detailed direct histopathological, biochemical, and molecular genetic analyses of the effects of PDT, which will facilitate the development of optimized treatment strategies.

With the rapidly emerging designs and applications of light-activated, photodynamic therapy (PDT)-based nanoconstructs, photonanomedicines (PNMs), an unmet need exists to establish whether conventional methods of photochemical and photophysical characterization of photosensitizers are relevant for evaluating new PNMs in order to intelligently guide their design. As a model system, we build on the clinical formulation of benzoporphyrin derivative (BPD), Visudyne(®), by developing a panel of nanolipid formulations entrapping new lipidated chemical variants of BPD with differing chemical, photochemical and photophysical properties. These are 16:0 and 20:0 lysophosphocholine-BPD (16:0/20:0 BPD-PC), DSPE-PEG-BPD and BPD-cholesterol. We show that Visudyne(®) was the most phototoxic formulation to OVCAR-5 cells, and the least effective was liposomal DSPE-PEG-BPD. However, these differences did not match their optical, photophysical and photochemical properties, as the static BPD quenching was highest in Visudyne, which also exhibited the lowest generation of singlet oxygen production. Furthermore, we establish that OVCAR-5 cell phototoxicity also does not correlate with rates of photosensitizer photobleaching and fluorescence quantum yields in any nanolipid formulations. These findings warrant critical future studies into subcellular targets and molecular mechanisms of phototoxicity of photodynamic nanoconstructs, as more reliable prognostic surrogates for predicting efficacy in order to appropriately and intelligently guide their design.

With increasing knowledge of the relevance of vascular cell adhesion molecule 1 (VCAM-1) for tumor growth, metastasis, angiogenesis, and related processes, it has become an attractive anti-tumor strategy to target VCAM-1 expression on the tumor vasculature. We designed a new targeted nanodrug, denoted PVQ, based on a photosensitizer (for the photodynamic effect), VCAM-1 target and quantum dot (QD) carrier, using conjugated water-dispersible colloidal CdSe–CdS/ZnS QDs, protoporphyrin IX (PpIX) photosensitizers, and VCAM-1 binding peptides. Its targeting ability and photodynamic therapy (PDT) efficiency against VCAM-1 expression in human umbilical vein endothelial cells (HUVECs) were then investigated. Conjugates of QD–VCAM-1 binding peptide (VQ), PpIX–VCAM-1 binding peptide (PV), and PVQ prepared using amide coupling were verified by agarose gel electrophoresis, Fourier transform infrared spectroscopy, and fluorescence spectrometry. VCAM-1 expression in HUVECs was induced by TNF-α treatment. PVQ conjugates were co-cultured with VCAM-1 expressing (VCAM-1(+)) and non-expressing (VCAM-1(−)) HUVECs, and target imaging, ROS generation, cell death, and apoptosis were analyzed using confocal fluorescence microscopy. VCAM-1 target imaging could not distinguish between VCAM-1(+) and VCAM-1(−) HUVECs after only 6 h of incubation; however it could distinguish between the cells after incubation for 24 h. After incubation for ca. 30 min, PVQ generated a significantly higher yield of ROS (3.6 fold) in VCAM-1(+) HUVECs compared with VCAM-1(−) cells, during 10 min of irradiation at a wavelength of 405 nm, and this was followed by a second rise in ROS at 30 min after irradiation. Moreover, cell destruction was observed clearly in VCAM-1(+) cells treated with PVQ and almost all cells became round after 30 min of irradiation at 405 nm. PVQ-induced PDT effects caused a significant apoptosis (onset and late apoptosis) in VCAM-1(+) HUVECs at 6 h after PDT treatment. In conclusion, PVQ shows a great potential for targeted PDT in cancer therapy.

Despite untiring efforts to develop therapies for pancreatic ductal adenocarcinoma (PDAC), survival statistics remain dismal, necessitating distinct approaches. Photodynamic priming (PDP), which improves drug delivery and combination regimens, as well as tumor photodestruction are key attributes of photodynamic therapy (PDT), making it a distinctive clinical option for PDAC. Localized, high-payload nanomedicine-assisted delivery of photosensitizers (PSs), with molecular specificity and controlled photoactivation, thus becomes critical in order to reduce collateral toxicity during more expansive photodynamic activation procedures with curative intent. As such, targeted photoactivable lipid-based nanomedicines are an ideal candidate but have failed to provide greater than two-fold cancer cell selectivity, if at all, due to their extensive multivariant physical, optical, and chemical complexity. Here, we report (1) a systematic multivariant tuning approach to engineer (Cet, anti-EGFR mAb) photoimmunonanoconjugates (PINs), and (2) stroma-rich heterotypic PDAC in vitro and in vivo models incorporating patient-derived pancreatic cancer-associated fibroblasts (PCAFs) that recapitulate the desmoplasia observed in the clinic. These offer a comprehensive, disease-specific framework for the development of Cet-PINs. Specificity-tuning of the PINs, in terms of PS lipid anchoring, electrostatic modulation, Cet orientation, and Cet surface densities, achieved ~16-fold binding specificities and rapid penetration of the heterotypic organoids within 1 h, thereby providing a ~16-fold enhancement in molecular targeted NIR photodestruction. As a demonstration of their inherent amenability for multifunctionality, encapsulation of high payloads of gemcitabine hydrochloride, 5-fluorouracil, and oxaliplatin within the Cet-PINs further improved their antitumor efficacy in the heterotypic organoids. In heterotypic desmoplastic tumors, the Cet-PINs efficiently penetrated up to 470 μm away from blood vessels, and photodynamic activation resulted in substantial tumor necrosis, which was not elicited in T47D tumors (low EGFR) or when using untargeted constructs in both tumor types. Photodynamic activation of the Cet-PINs in the heterotypic desmoplastic tumors resulted in collagen photomodulation, with a 1.5-fold reduction in collagen density, suggesting that PDP may also hold potential for conquering desmoplasia. The in vivo safety profile of photodynamic activation of the Cet-PINs was also substantially improved, as compared to the untargeted constructs. While treatment using the Cet-PINs did not cause any detriment to the mice’s health or to healthy proximal tissue, photodynamic activation of untargeted constructs induced severe acute cachexia and weight loss in all treated mice, with substantial peripheral skin necrosis, muscle necrosis, and bowel perforation. This study is the first report demonstrating the true value of molecular targeting for NIR-activable PINs. These constructs integrate high payload delivery, efficient photodestruction, molecular precision, and collagen photomodulation in desmoplastic PDAC tumors in a single treatment using a single construct. Such combined PIN platforms and heterocellular models open up an array of further multiplexed combination therapies to synergistically control desmoplastic tumor progression and extend PDAC patient survival.

Metronomic photodynamic therapy (mPDT) was developed to improve tumor-specific responses through cell death by apoptosis. We developed an mPDT suppository kit including ALA and LED suppositories and analyzed its killing effect on rectal tumors in rabbits. Methods: The ALA (10 wt%) suppository was prepared using ALA powder, type 36 semi-synthetic fatty acid glyceride, and azone. The LED suppository was constructed by encapsulating LED units and a circuit in transparent epoxy resin. VX2 cells were injected into the rectal submucosa of rabbits to establish a carcinoma model in situ. The ALA suppository was inserted into the rectal cavity for 30 min of uptake and activated for 1 h by the LED suppository at a power density of 20 mW/cm2. The mPDT process was repeated three times once a day. MRI was used to monitor tumor growth, histopathology and TUNEL staining were performed at 14 days after mPDT. Results: The overall response rate was 60% in the mPDT group using the kit in which the tumor size was decreased up to about 50% at 7 days post-mPDT and almost eliminated at 14 days. HE staining showed that only 6.16% of the tumor tissue remained after mPDT treatment. TUNEL detection showed that the apoptosis rate was 18.9%. Conclusion: We verified the killing effect of the mPDT suppository kit on rectal tumors in rabbits based on mPDT that induced tumor cell apoptosis.

This study evaluated the effects and mechanism of action of combining irreversible electroporation (IRE) and photodynamic therapy (PDT) in breast cancer cells in vitro and in vivo. Jin’s formula was used to assess killing efficacy of different IRE+PDT dosing combinations in breast cancer MCF-7 cells. Flow cytometry, high-content imaging, and confocal laser scanning microscopy were used to detect apoptosis. qRT-PCR and western blotting were used to evaluate expression of apoptosis-related genes and proteins. IRE+PDT combination therapy was administered to BALB/C mice with breast cancer tumors in vivo; tumor size was used to assess treatment efficacy. Killing mechanisms were examined using transmission electron microscopy and immunohistochemistry. We found that IRE+PDT combination therapy produced significant synergistic killing effects in breast cancer cells (highest Jin q value of 1.32). Early apoptosis rates were significantly higher in the IRE+PDT group (16.0%) than in IRE-alone (7.6%) and PDT-alone (4.6%) groups (P

Metronomic photodynamic therapy (mPDT) has emerged as an attractive treatment for the selective destruction of tumor cells by induction of apoptosis. Here, we compared the effects of mPDT and acute photodynamic therapy (aPDT) on human SW837 colorectal cancer (CRC) cells. CRC cells were subjected to mPDT using various exposure durations, concentrations of 5-aminolevulinic acid (ALA), fluence rates and energy densities. The effects were compared with those induced by aPDT. We found that apoptosis and autophagy were earlier induced to a greater extent by mPDT than by the same dose applied as aPDT. The survival rates for mPDT vs. aPDT were 35.2%, 32.4%,27.6%,31.6% vs. 85.7%, 71.1%, 67.8%, 42.1% after 3, 6, 12, and 24 h PDT, respectively. For the same time points, the apoptotic rates for mPDT vs. aPDT were 43.2%, 47.3%, 54.7%, and 50.3% vs. 14.6%, 17.6%, 27.1%, and 53.2%, respectively. mPDT induced a peak rate of autophagy of 20.0% at 3 h, whereas aPDT induced two smaller peaks at 3 h (14.1%) and 12 h (15.8%). Advanced autophagosomes were more abundant in mPDT- than aPDT-treated cells and appeared earlier after mPDT (3 h) than after aPDT (3−12 h). Western Bloting results showed that the ratio of LC3B-II/β − actin at 3 h was higher (1.04 times) after mPDT than aPDT. Collectively, these datas indicated that ALA-mPDT was more effective than the same dose of ALA-aPDT at inducing SW837 CRC cell death via apoptosis and autophagy. Thus, mPDT may be a superior choice than aPDT for the treatment of human CRC.