Perylene-Mediated Cytoskeletal Dysfunction Remodels Cancer-Associated Fibroblasts to Augment Antitumor Immunotherapy.

Targeted reprogramming of cancer-associated fibroblasts (CAFs) is one of the most essential cancer therapies. However, how to reprogram active CAFs toward deactivated state still remains immense challenge. To tackle this challenge, herein, one perylene N, N'-bis(2-((dimethylammonium)ethylene)-2-(methoxylethyl))-1, 6, 7, 12-tetrachloroperylene-3, 4, 9, 10-tetracarboxylic diimide (PDIC-OC) is prepared, which can trigger endogenous reactive oxygen species (ROS) burst to result in cytoskeletal dysfunction and cell apoptosis so that suppress transforming growth factor ß (TGF-ß) production. As a result, PDIC-OC can reprogram the activated CAFs and relieve immunosuppressive tumor microenvironment by efficient polarization of M2-typed macrophages into M1-typed ones, downregulation of alpha-smooth muscle actin (α-SMA), alleviation of hypoxic state to promote infiltration of cytotoxic T lymphocytes, and ultimately realizes outstanding antitumor performance on B16F10 tumor-xenografted and lung-metastatic mouse model even at low concentration of 1 mg kg-1 body weight. This work thus presents a novel strategy that cytoskeleton dysfunction and cell apoptosis cooperatively suppress the secretion of TGF-ß to reprogram CAFs and meanwhile clarifies intrinsic mechanism for perylene-triggered chemo-immunotherapy against hypoxic tumors.

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.

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.

Hypericin supramolecular assembles: A way to increase the skin availability and photodynamic efficiency in tumor cells.

Cyclodextrins (CDs) are molecules approved by the FDA and show promise in increasing the solubility of hydrophobic molecules and making them more available to the skin. These CDs have been used to form complexes with some photosensitizers for Photodynamic Therapy (PDT), such as Hypericin (HY). HY is a lipophilic photosensitizer known for its exceptional fluorescence and singlet oxygen quantum yield generation of over 20 % under 590 nm irradiation. In this study, we found a six-fold increase in the release of HY in vitro after complexation with ß-CD. The ß-CDHY assembly also demonstrated better skin retention, which is crucial for the topical application of this photosensitizer. Furthermore, the ß-CD complexation led to a significant increase in the phototoxicity of HY at three different light doses (3, 6, and 10 J cm-2) due to its improved water solubility and higher in vitro accumulation (approximately two times compared with free HY) in HeLa and Vero cell lines.

Effective transport of aggregated hypericin encapsulated in SBA-15 nanoporous silica particles for photodynamic therapy of cancer cells.

Photodynamic therapy (PDT) represents an interesting modality for the elimination of damaged biomaterials and cells. This treatment takes advantage of the photosensitizing properties of molecules that are active only when irradiated with light. In the present work, a dual property of hypericin, a hydrophobic molecule with high performance in photodiagnostics and photodynamic therapy, was exploited. The non-fluorescent and photodynamically inactive form of hypericin aggregates was loaded into the nanopores of SBA-15 silica particles. The synthesized particles were characterized by infrared spectroscopy, thermogravimetry, differential thermal analysis, small-angle X-ray scattering and transmission electron microscopy. Hypericin aggregates were confirmed by absorption spectra typical of aggregated hypericin and by its short fluorescence lifetime. Release of hypericin from the particles was observed toward serum proteins, mimicking physiological conditions. Temperature- and time-dependent uptake of hypericin by cancer cells showed gradual release of hypericin from the particles and active cellular transport by endocytosis. A closer examination of SBA-15-hypericin uptake by fluorescence lifetime imaging showed that aggregated hypericin molecules, characterized by a short fluorescence lifetime (∼4 ns), were still present in the SBA-15 particles upon uptake by cells. However, monomerization of hypericin in cancer cells was observed by extending the hypericin fluorescence lifetime by ∼8 ns, preferentially in lipid compartments and the plasma membrane. This suggests a promising prognosis for delayed biological activity of the entire cargo, which was confirmed by effective PDT in vitro. In summary, this work presents an approach for safe, inactive delivery of hypericin that is activated at the target site in cells and tissues.

Redistribution of hydrophobic hypericin from nanoporous particles of SBA-15 silica in vitro, in cells and in vivo.

Nanoporous silica is nowadays used in various fields of nano- and micro-materials research. The advantage of nanoporous material is that it can be filled with various hydrophilic and hydrophobic molecules, which are then delivered to the target cells and tissues. In the present study, we have studied the interaction of nanoporous silica with hydrophobic and photodynamically active molecule - hypericin. Hypericin was adsorbed on/in SBA-15 silica, which led to the disappearance of its fluorescence due to hypericin aggregate formation. However, it was observed here that hypericin can be easily redistributed from these particles towards proteins and lipids in serum and cells in vitro and in vivo. Moreover, the charged surface character of SBA-15 pores forced the creation of protein/lipid corona on particles. Such complex enabled monomerization of hypericin on the surface of particles presented by fluorescence in the corona and singlet oxygen production suitable for photodynamic therapy (PDT). The PDT efficacy achieved by introducing the new construct into the PDT protocol was comparable to the efficacy of hypericin PDT. In conclusion, this study demonstrates a promising approach for the delivery of hydrophobic photosensitizers to cancer cells by nanoporous silica using fluorescence techniques.

Anti-Epstein-Barr Viral Agents from the Medicinal Herb-Derived Fungus Alternaria alstroemeriae Km2286.

Chromatographic separation on the liquid-state fermented products produced by the fungal strain Alternaria alstroemeriae Km2286 isolated from the littoral medicinal herb Atriplex maximowicziana Makino resulted in the isolation of compounds 1-9. Structures were determined by spectroscopic analysis as four undescribed perylenequinones, altertromins A-D (1-4), along with altertoxin IV (5), altertoxin VIII (6), stemphyperylenol (7), tenuazonic acid (8), and allo-tenuazonic acid (9). Compounds 1-6 exhibited antiviral activities against Epstein-Barr virus (EBV) with EC50 values ranging from 0.17 ± 0.07 to 3.13 ± 0.31 µM and selectivity indices higher than 10. In an anti-neuroinflammatory assay, compounds 1-4, 6, and 7 showed inhibitory activity of nitric oxide production in lipopolysaccharide-induced microglial BV-2 cells, with IC50 values ranging from 0.33 ± 0.04 to 4.08 ± 0.53 µM without significant cytotoxicity. This is the first report to describe perylenequinone-type compounds with potent anti-EBV and anti-neuroinflammatory activities.

Comprehensive Thione-Derived Perylene Diimides and Their Bio-Conjugation for Simultaneous Imaging, Tracking, and Targeted Photodynamic Therapy.

In this study, the chromophore 3,4,9,10-perylenetetracarboxylic diimide (PDI) is anchored with phenyl substituents at the imide N site, followed by thionation, yielding a series of thione products 1S-PDI-D, 2S-cis-PDI-D, 2S-trans-PDI-D, 3S-PDI-D, and 4S-PDI-D, respectively, with n = 1, 2, 3, and 4 thione. The photophysical properties are dependent on the number of anchored thiones, where the observed prominent lower-lying absorption is assigned to the S0 → S2(ππ*) transition and is red-shifted upon increasing the number of thiones; the lowest-lying excited state is ascribed to a transition-forbidden S1(nπ*) configuration. All nS-PDIs are non-emissive in solution but reveal an excellent two-photon absorption cross-section of >800 GM. Supported by the femtosecond transient absorption study, the S1(nπ*) → T1(ππ*) intersystem crossing (ISC) rate is > 1012 s-1, resulting in ∼100% triplet population. The lowest-lying T1(ππ*) energy is calculated to be in the order of 1S-PDI-D > 2S-cis-PDI-D ∼ 2S-trans-PDI-D > 3S-PDI-D > 4S-PDI-D, where the T1 energy of 1S-PDI-D (1.10 eV) is higher than that (0.97 eV) of the 1O2 1Δg state. 1S-PDI-D is further modified by either conjugation with peptide FC131 on the two terminal sides, forming 1S-FC131, or linkage with peptide FC131 and cyanine5 dye on each terminal, yielding Cy5-1S-FC131. In vitro experiments show power of 1S-FC131 and Cy5-1S-FC131 in recognizing A549 cells out of other three lung normal cells and effective photodynamic therapy. In vivo, both molecular composites demonstrate outstanding antitumor ability in A549 xenografted tumor mice, where Cy5-1S-FC131 shows superiority of simultaneous fluorescence tracking and targeted photodynamic therapy.

Molecular dynamics simulation on water/oil interface with model asphaltene subjected to electric field.

HYPOTHESIS: The droplet-medium interfaces of petroleum emulsions are often stabilized by the indigenous surface-active compounds (e.g., asphaltenes), causing undesired issues. While demulsification by electric field is a promising technique, fundamental study on the droplet-medium interface influenced by electric field is limited. Molecular dynamics (MD) simulations are expected to provide microscopic insights into the nano-scaled water/oil interface. METHODS: MD simulations are conducted to study the adsorption of model asphaltene molecules (represented by N-(1-hexylheptyl)-N'-(5-carboxylicpentyl) perylene-3,4,9,10-tetracarboxylic bisimide (C5Pe)) on a water-toluene interface under various strengths of electric field. The adsorption amount and structural feature of C5Pe molecules at water-toluene interface are investigated, and the effects of electric field and salt are discussed. FINDINGS: C5Pe molecules tend to adsorb on the water-oil interface. As the electric field strength increases, the adsorption amount first slightly increases (or remains constant) and then decreases. The electric field disrupts the compact π-π stacking between C5Pe molecules and increases their mobility, causing a dispersed distribution of the molecules with a wide range of orientations relative to the interface. Within the studied range, the addition of salt ions appears to stabilize the interface at high electric field. These results provide useful insights into the mechanism and feasibility of demulsification under electric field.

Spectroscopy studies of interaction hypericin with an anti-cancer therapy drug doxorubicin.

The presented study was designed to estimate the ability of hypericin to interact with the anticancer drug doxorubicin. The hetero-association of hypericin and doxorubicin was investigated with absorption and fluorescence spectroscopy methods in aqueous solution of DMSO in two-component mixtures: doxorubicin-hypericin and three component mixtures: DNA-doxorubicin-hypericin. The data indicate that hypericin forms complexes with doxorubicin and that the association constants are on the order of 300,000 M-1 in a buffer with 30% DMSO content. The absorption spectra of the hypericin - doxorubicin complexes were examined as well. Owing to its ability to interact with flat aromatic compounds, hypericin may potentially be used as an interceptor molecule to detoxification of patients after chemotherapy.

Biocompatible hypocrellin A-Fe(III) nanoparticles exhibiting efficient photo-activated CDT in vitro and in vivo.

Chemodynamic therapy (CDT) is novel and promising for cancer treatment, however, the potential systematic toxicity of the used nanoparticles is still a big concern. In this work the biocompatible hypocrellin A-Fe(III) nanoparticles (HA-Fe(III) NPs) were synthesized and studied. The CDT effect of HA-Fe(III) NPs in the dark is negligible but can be photo-activated upon red light irradiation, which is meaningful to realize precise CDT treatment by selective light irradiation. Moreover, HA-Fe(III) NPs can also generate O2˙-, which may be converted into H2O2 to further enhance the CDT effect. As a result, HA-Fe(III) NPs had little cytotoxicity in the dark at the concentration up to 200 µg ml-1, but exhibited efficient CDT activity upon red light irradiation under both normoxic and hypoxic conditions. The in vivo results also showed that HA-Fe(III) NPs can efficiently inhibit tumor growth upon 628 nm light irradiation.

In Situ Hypoxia-Induced Supramolecular Perylene Diimide Radical Anions in Tumors for Photothermal Therapy with Improved Specificity.

Considering that hypoxia is closely associated with tumor proliferation, invasion, metastasis, and drug resistance, it is of great significance to overcome hypoxia in tumor treatment. Herein, we report a hypoxia-induced specific photothermal therapy (PTT) based on the photothermal agent of supramolecular perylene diimide radical anions. Hypoxic regions in various tumors display strong reductive ability, and in such environments the supramolecular complex of a perylene diimide derivative and cucurbit[7]uril could be reduced to supramolecular perylene diimide radical anions. Benefiting from the strong NIR absorption and good photothermal conversion performance of the in situ generated supramolecular perylene diimide radical anions, the hypoxia-induced PTT strategy exhibits excellent photothermal therapeutic efficiency as well as good specificity and biological safety. Moreover, hypoxia inducible factor expression of tumors decreases to the normal level after PTT treatment. It is anticipated that such a hypoxia-induced specific PTT strategy opens new horizons for photothermal therapy against hypoxic tumors with improved specificity and safety.

Regeneration of Phytochemicals by Structure-Driven Organization of Microbial Biosynthetic Steps.

Medicinal phytochemicals, such as artemisinin and taxol, have impacted the world, and hypericin might do so if its availability issue could be addressed. Hypericin is the hallmark component of Saint John's wort (Hypericum perforatum L.), an approved depression alleviator documented in the US, European, and British pharmacopoeias with its additional effectiveness against diverse cancers and viruses. However, the academia-to-industry transition of hypericin remain hampered by its low in planta abundance, unfeasible bulk chemical synthesis, and unclear biosynthetic mechanism. Here, we present a strategy consisting of the hypericin-structure-centered modification and reorganization of microbial biosynthetic steps in the repurposed cells that have been tamed to enable the designed consecutive reactions to afford hypericin (43.1 mg L-1 ), without acquiring its biosynthetic knowledge in native plants. The study provides a synthetic biology route to hypericin and establishes a platform for biosustainable access to medicinal phytochemicals.

A perylene diimide-based nanoring architecture for exogenous and endogenous ATP detection: biochemical assay for monitoring phosphorylation of glucose.

Positively charged amphiphiles hold great significance in supramolecular chemistry due to their good solubility, and physiochemical and molecular recognition properties. Herein, we report the synthesis, characterization and molecular recognition properties of the dicationic amphiphile based on perylene diimide-tyrosine alkyl amide amine (PDI 3). PDI 3 showed the formation of a nanoring architecture in the self-assembled aggregated state (90% H2O-DMSO mixture) as observed by SEM and TEM studies. The diameter of the nanoring is around 30-50 nm with a height varying from 1 to 2 nm. The self-assembled aggregates of PDI 3 are very sensitive towards nucleoside triphosphates. Upon addition of ATP, PDI 3 showed a decrease in the absorbance and emission intensity at 535 and 580 nm (due to the monomer state), respectively. The lowest detection limit for ATP is 10.8 nM (UV) and 3.06 nM (FI). Upon interaction of ATP with PDI 3, the nanoring morphology transformed into a spherical structure. These changes could be attributed to the formation of ionic self-assembled aggregates between dicationic PDI 3 and negatively charged ATP via electrostatic and H-bonding interactions. The complexation mechanism of PDI 3 and ATP was confirmed by optical, NMR, Job's plot, DLS, SEM and AFM studies. PDI 3 displays low cytotoxicity toward MG-63 cells and can be successfully used for the detection of exogenous and endogenous ATP. The resulting PDI 3 + ATP complex is successfully used as a 'turn-on' biochemical assay for monitoring phosphorylation of glucose.

Hypocrellin B-based activatable photosensitizers for specific photodynamic effects against high H2O2-expressing cancer cells.

A novel tumor-related biomarker, a H2O2-activatable photosensitizer 4 based on the 1,3-dicarbonyl enol moieties of hypocrellin B (3), was designed and synthesized. The photosensitizer 4 showed a blue-shifted absorption band compared with 3, and showed negligible photosensitizing ability without H2O2. However, the release of 3 from 4 by the reaction with H2O2 regenerated the photosensitizing ability. Furthermore, 4 exhibited selective and effective photo-cytotoxicity against high H2O2-expressing cancer cells upon photo-irradiation with 660 nm light, which is inside the phototherapeutic window.

A novel hypocrellin-based assembly for sonodynamic therapy against glioblastoma.

The non-invasive treatment of glioblastoma (GBM) is of great significance and can greatly reduce the complications of craniotomy. Sonodynamic therapy (SDT) is an emerging tumor therapeutic strategy that overcomes some fatal flaws of photodynamic therapy (PDT). Different from PDT, SDT has deep tissue penetration and can be applied in the non-invasive treatment of deep-seated tumors. However, effective sonosensitizers that can be used for SDT of GBM are still very rare. Herein, we have prepared a suitable assembly based on a hypocrellin derivative (CTHB) with good biocompatibility. Excitedly, the hypocrellin-based assembly (CTHB NPs) can effectively produce reactive oxygen species under ultrasound stimulation. The inherent fluorescence and photoacoustic imaging characteristics of the CTHB NPs are conducive to the precise positioning of the tumors. It has been proved both in subcutaneous and in intracranial tumor models that CTHB NPs can be used as an effective sonosensitizer to inhibit tumor growth under ultrasound irradiation. This hypocrellin-based assembly has a good clinical prospect in the non-invasive treatment of GBM.

A facile design of thio-perylenediimides with controllable fluorescent, photodynamic and photothermal effects towards cancer theranostics.

A series of thionated perylenediimides with modulating phototheranostic modalities have been synthesized by a one-pot method for multiple anti-cancer applications. Compared to the initial and 4-tert-butyl phenol-substituted fluorescent perylenediimide, the obtained monothionated perylenediimide became photodynamic. With the increase of thionation degree, tetrathionated perylenediimide changed into an optimal photothermal agent.

Perylene diimide-based treatment and diagnosis of diseases.

Integrated treatment using imaging technology to monitor biological processes for the precise treatment and diagnosis of diseases to improve treatment outcomes is becoming a hot topic. Accordingly, perylene diimide (PDI) has excellent photothermal conversion and photostability, which can be used as a good material for disease treatment and diagnosis. Herein, we review the latest research progress on the real-time diagnosis of related diseases based on perylene diimide probes in the aspects of bioimaging, detection of biomarkers and determination of the pH in living cells. Furthermore, perylene diimide-based multifunctional nano-delivery systems are particularly emphasized, showing great therapeutic potential in the field of image-guided combination therapy in tumor therapy. Finally, the great opportunities and challenges still faced by perylene diimide before entering the clinic are comprehensively analyzed.

Hypericin-loaded oil-in-water nanoemulsion synthesized by ultrasonication process enhances photodynamic therapy efficiency.

Hypericin (Hy) is a hydrophobic photosensitizer used in photodynamic therapy for cancer therapeutic. In this study, Hy-loaded oil-in-water (O/W) nanoemulsions (NEs) were produced by the ultrasonication method combing different biocompatible oils and surfactants to enhance Hy aqueous solubility and bioavailability. Experimental parameters were optimized by the characterization of droplet size, zeta potential, and physicochemical properties. In vitro studies based on the release profile, cytotoxicity, cell morphology, and Hy intracellular accumulation were assayed. Hy at 100 mg L-1 was incorporated into the low viscosity (~0.005 Pa s) NEs with spherical droplets averaging 20-40 nm in size and polydispersity index <0.02. Hy release from the NE was significantly higher (4-fold) than its suspension (p < 0.001). The NEs demonstrated good physical stability during storage at 5 °C for at least six months. The Hy-loaded NEs exhibited an IC50 value 6-fold lower than Hy suspension during PDT against breast cancer cell lines (MCF-7). Cell microscopy imaging confirmed the increased cytotoxic effects of Hy-loaded NEs, showing damaged and apoptotic cells. Confocal laser scanning microscopy evidenced greater Hy delivery through NE into MCF-7 cells followed by improved intracellular ROS generation. Our results suggest that the Hy-loaded NEs can improve hypericin efficacy and assist Hy-PDT's preclinical development as a cancer treatment.

Regulating Twisted Skeleton to Construct Organ-Specific Perylene for Intensive Cancer Chemotherapy.

The systemic use of pharmaceutical drugs for cancer patients is a compromise between desirable therapy and side effects because of the intrinsic shortage of organ-specific pharmaceutical drug. Design and construction of pharmaceutical drug to achieve the organ-specific delivery is thus desperately desirable. We herein regulate perylene skeleton to effect organ-specificity and present an example of lung-specific distribution on the basis of bay-twisted PDIC-NC. We further demonstrate that PDIC-NC can target into mitochondria to act as cellular respiration inhibitor, inducing insufficient production of adenosine triphosphate, promoting endogenous H2 O2 and . OH burst, elevating calcium overload, efficiently triggering the synergistic apoptosis, autophagy and endoplasmic reticulum stress of lung cancer cells. The antitumor performance of PDIC-NC is verified on in vivo xenografted, metastasis and orthotopic lung cancer, presenting overwhelming evidences for potentially clinical application. This study contributes a proof-of-concept demonstration of twisted perylene to well attain lung-specific distribution, and meanwhile achieves intensive lung cancer chemotherapy.