Lung-Targeting Perylenediimide Nanocomposites for Efficient Therapy of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis, an idiopathic interstitial lung disease with high mortality, remains challenging to treat due to the lack of clinically approved lung-targeting drugs. Herein, we present PDIC-DPC, a perylenediimide derivative that exhibits superior lung-selective enrichment. PDIC-DPC forms nanocomposites with plasma proteins, including fibrinogen beta chain and vitronectin, which bind to pulmonary endothelial receptors for lung-specific accumulation. Moreover, PDIC-DPC significantly suppresses transforming growth factor beta1 and activates adenosine monophosphate-activated protein kinase. As a result, compared to existing therapeutic drugs, PDIC-DPC achieves superior therapeutic outcomes, evidenced by the lowest Ashcroft score, significantly improved pulmonary function, and an extended survival rate in a bleomycin-induced pulmonary fibrosis model. This study elucidates the lung-selective enrichment of assembled prodrug from biological perspectives and affords a platform enabling therapeutic efficiency on idiopathic pulmonary fibrosis.

Brain-Targeted Black Phosphorus-Based Nanotherapeutic Platform for Enhanced Hypericin Delivery in Depression.

Depression is a significant global health concern that remains inadequately treated due to the limited effectiveness of conventional drug therapies. One potential therapeutic agent, hypericin (HYP), is identified as an effective natural antidepressant. However, its poor water solubility, low bioavailability, and limited ability to penetrate the brain parenchyma have hindered its clinical application. To address these shortcomings and enhance the therapeutic efficacy of HYP, it is loaded onto black phosphorus nanosheets (BP) modified with the neural cell-targeting peptide RVG29 to synthesize a nanoplatform named BP-RVG29@HYP (BRH). This platform served as a nanocarrier for HYP and integrated the advantages of BP with advanced delivery methods and precise targeting strategies. Under the influence of 808 nm near-infrared irradiation (NIR), BRH effectively traversed an in vitro BBB model. In vivo experiments validated these findings, demonstrating that treatment with BRH significantly alleviated depressive-like behaviors and oxidative stress in mice. Importantly, BRH exhibited an excellent safety profile, causing minimal adverse effects, which highlighted its potential as a promising therapeutic agent. In brief, this novel nanocarrier holds great promise in the development of antidepressant drugs and can create new avenues for the treatment of depression.

Hypericin Alleviates Chronic Kidney Disease-induced Left Ventricular Hypertrophy by Regulation of FGF23-FGFR4 Signaling Pathway.

ABSTRACT: Chronic kidney disease (CKD) is a significant global health threat that imposes a substantial burden on both individuals and societies. CKD frequently correlates with cardiovascular events, particularly left ventricular hypertrophy (LVH), which contributes to the high mortality rate associated with CKD. Fibroblast growth factor 23 (FGF23), a hormone primarily involved in regulating calcium and phosphorus metabolism, has been identified as a major risk factor for LVH in CKD patients. Elevated serum FGF23 levels are known to induce LVH and myocardial fibrosis by activating the fibroblast growth factor receptor 4 (FGFR4) signal pathway. Therefore, targeting FGFR4 and its downstream signaling pathways holds potential as a treatment strategy for cardiac dysfunction in CKD. In our current study, we have discovered that Hypericin, a key component derived from Hypericum perforatum , has the ability to alleviate CKD-related LVH by targeting the FGFR4/phospholipase C gamma 1 (PLCγ1) signaling pathway. Through in vitro experiments using rat cardiac myocyte H9c2 cells, we observed that Hypericin effectively inhibits FGF23-induced hypertrophy and fibrosis by suppressing the FGFR4/PLCγ1/calcineurin/nuclear factor of activated T-cell (NFAT3) signaling pathway. In addition, our in vivo studies using mice on a high-phosphate diet and rat models of 5/6 nephrectomy demonstrated that Hypericin has therapeutic effects against CKD-induced LVH by modulating the FGFR4/PLCγ1/calcineurin/NFAT3 signaling pathway. In conclusion, our research highlights the potential of Hypericin as a candidate for the treatment of CKD-induced cardiomyopathy. By suppressing the FGFR4/PLCγ1 signaling pathway, Hypericin shows promise in attenuating LVH and myocardial fibrosis associated with CKD.

The contradictory role of febuxostat in ABCG2 expression and potentiating hypericin-mediated photodynamic therapy in colorectal cancers.

Photodynamic Therapy (PDT) is an emerging method to treat colorectal cancers (CRC). Hypericin (HYP) is an effective mediator of PDT and the ABCG2 inhibitor, Febuxostat (FBX) could augment PDT. HT29 and HEK293 cells showed light dependant cytotoxic response to PDT in both 2D and 3D cell models. FBX co-treatment was not found to improve PDT cytotoxicity. Next, ABCG2 protein expression was observed in HT29 but not in HEK293 cells. However, ABCG2 gene expression analysis did not support protein expression results as ABCG2 gene expression results were found to be higher in HEK293 cells. Although HYP treatment was found to significantly reduce ABCG2 gene expression levels in both cell lines, FBX treatment partially restored ABCG2 gene expression. Our findings indicate that FBX co-treatment may not be suitable for augmenting HYP-mediated PDT in CRC but could potentially be useful for other applications.

Hypericin-mediated photodynamic therapy inhibits metastasis and EMT of colorectal cancer cells by regulating RhoA-ROCK1 signaling pathway.

Colorectal cancer (CRC) is significantly contributed to global cancer mortality rates. Treating CRC is particularly challenging due to metastasis and drug resistance. There is a pressing need for new treatment strategies against metastatic CRC. Photodynamic therapy (PDT) offers a well-established, minimally invasive treatment option for cancer with limited side effects. Hypericin (HYP), a potent photosensitizer for PDT, has been documented to induce cytotoxicity and apoptosis in various types of cancers. However, there are few reports on the inhibitory effects of HYP-mediated PDT on the metastatic ability of CRC cells. Here, we evaluate the inhibitory effects of HYP-mediated PDT against metastatic CRC cells and define its underlying mechanisms. Wound-healing and Transwell assays show that HYP-mediated PDT suppresses migration and invasion of CRC cells. F-actin visualization assays indicate HYP-mediated PDT decreases F-actin formation in CRC cells. TEM assays reveal HYP-mediated PDT disrupts pseudopodia formation of CRC cells. Mechanistically, immunofluorescence and western blotting results show that HYP-mediated PDT upregulates E-cadherin and downregulates N-cadherin and Vimentin. HYP-mediated PDT also suppresses key EMT regulators, including Snail, MMP9, ZEB1 and α-SMA. Additionally, the expressions of RhoA and ROCK1 are downregulated by HYP-mediated PDT. Together, these findings suggest that HYP-mediated PDT inhibits the migration and invasion of HCT116 and SW620 cells by modulating EMT and RhoA-ROCK1 signaling pathway. Thus, HYP-mediated PDT presents a potential therapeutic option for CRC.

A novel photosensitizer based on hypocrellin B activated by cysteine over-expressed in cancer cells.

L-Cysteine (Cys)-activatable photosensitizer 3 was designed and synthesized based on hypocrellin B (1). Cys is a novel tumor-associated biomarker. 3 exhibited negligible photosensitizing ability without Cys. However, when 1 was released from 3 by reaction with Cys, the photosensitizing activity was restored. Furthermore, 3 showed selective and effective photo-cytotoxicity against only cancer cells such as HeLa and A549 cells that highly express Cys when irradiated with 660 nm light, which is inside the phototherapeutic window.

Insights on the Mechanical Properties of SARS-CoV-2 Particles and the Effects of the Photosensitizer Hypericin.

SARS-CoV-2 is a highly pathogenic virus responsible for the COVID-19 disease. It belongs to the Coronaviridae family, characterized by a phospholipid envelope, which is crucial for viral entry and replication in host cells. Hypericin, a lipophilic, naturally occurring photosensitizer, was reported to effectively inactivate enveloped viruses, including SARS-CoV-2, upon light irradiation. In addition to its photodynamic activity, Hyp was found to exert an antiviral action also in the dark. This study explores the mechanical properties of heat-inactivated SARS-CoV-2 viral particles using Atomic Force Microscopy (AFM). Results reveal a flexible structure under external stress, potentially contributing to the virus pathogenicity. Although the fixation protocol causes damage to some particles, correlation with fluorescence demonstrates colocalization of partially degraded virions with their genome. The impact of hypericin on the mechanical properties of the virus was assessed and found particularly relevant in dark conditions. These preliminary results suggest that hypericin can affect the mechanical properties of the viral envelope, an effect that warrants further investigation in the context of antiviral therapies.

Synthetic Biology-based Construction of Unnatural Perylenequinones with Improved Photodynamic Anticancer Activities.

The construction of structural complexity and diversity of natural products is crucial for drug discovery and development. To overcome high dark toxicity and poor photostability of natural photosensitizer perylenequinones (PQs) for photodynamic therapy, herein, we aim to introduce the structural complexity and diversity to biosynthesize the desired unnatural PQs in fungus Cercospora through synthetic biology-based strategy. Thus, we first elucidate the intricate biosynthetic pathways of class B PQs and reveal how the branching enzymes create their structural complexity and diversity from a common ancestor. This enables the rational reprogramming of cercosporin biosynthetic pathway in Cercospora to generate diverse unnatural PQs without chemical modification. Among them, unnatural cercosporin A displays remarkably low dark toxicity and high photostability with retention of great photodynamic anticancer and antimicrobial activities. Moreover, it is found that, unlike cercosporin, unnatural cercosporin A could be selectively accumulated in cancer cells, providing potential targets for drug development. Therefore, this work provides a comprehensive foundation for preparing unnatural products with customized functions through synthetic biology-based strategies, thus facilitating drug discovery pipelines from nature.

Hypericin as a promising natural bioactive naphthodianthrone: A review of its pharmacology, pharmacokinetics, toxicity, and safety.

Hypericin can be derived from St. John's wort, which is widely spread around the world. As a natural product, it has been put into clinical practice such as wound healing and depression for a long time. In this article, we review the pharmacology, pharmacokinetics, and safety of hypericin, aiming to introduce the research advances and provide a full evaluation of it. Turns out hypericin, as a natural photosensitizer, exhibits an excellent capacity for anticancer, neuroprotection, and elimination of microorganisms, especially when activated by light, potent anticancer and antimicrobial effects are obtained after photodynamic therapy. The mechanisms of its therapeutic effects involve the induction of cell death, inhibition of cell cycle progression, inhibition of the reuptake of amines, and inhibition of virus replication. The pharmacokinetics properties indicate that hypericin has poor water solubility and bioavailability. The distribution and excretion are fast, and it is metabolized in bile. The toxicity of hypericin is rarely reported and the conventional use of it rarely causes adverse effects except for photosensitization. Therefore, we may conclude that hypericin can be used safely and effectively against a variety of diseases. We hope to provide researchers with detailed guidance and enlighten the development of it.

Alkyl Derivatives of Perylene Photosensitizing Antivirals: Towards Understanding the Influence of Lipophilicity.

Amphipathic perylene derivatives are broad-spectrum antivirals against enveloped viruses that act as fusion inhibitors in a light-dependent manner. The compounds target the lipid bilayer of the viral envelope using the lipophilic perylene moiety and photogenerating singlet oxygen, thereby causing damage to unsaturated lipids. Previous studies show that variation of the polar part of the molecule is important for antiviral activity. Here, we report modification of the lipophilic part of the molecule, perylene, by the introduction of 4-, 8-, and 12-carbon alkyls into position 9(10) of the perylene residue. Using Friedel-Crafts acylation and Wolff-Kishner reduction, three 3-acetyl-9(10)-alkylperylenes were synthesized from perylene and used to prepare 9 nucleoside and 12 non-nucleoside amphipathic derivatives. These compounds were characterized as fluorophores and singlet oxygen generators, as well as tested as antivirals against herpes virus-1 (HSV-1) and vesicular stomatitis virus (VSV), both known for causing superficial skin/mucosa lesions and thus serving as suitable candidates for photodynamic therapy. The results suggest that derivatives with a short alkyl chain (butyl) have strong antiviral activity, whereas the introduction of longer alkyl substituents (n = 8 and 12) to the perylenyethynyl scaffold results in a dramatic reduction of antiviral activity. This phenomenon is likely attributable to the increased lipophilicity of the compounds and their ability to form insoluble aggregates. Moreover, molecular dynamic studies revealed that alkylated perylene derivatives are predominately located closer to the middle of the bilayer compared to non-alkylated derivatives. The predicted probability of superficial positioning correlated with antiviral activity, suggesting that singlet oxygen generation is achieved in the subsurface layer of the membrane, where the perylene group is more accessible to dissolved oxygen.

Hypericin-Based Photodynamic Therapy Displays Higher Selectivity and Phototoxicity towards Melanoma and Squamous Cell Cancer Compared to Normal Keratinocytes In Vitro.

The aim of this study was to explore the potential of hypericin, a naturally occurring photosensi-tizer, for photodynamic therapy (PDT) in skin cancer, investigating its phototoxic effects and mechanisms of action in cancer cells compared to normal skin keratinocytes, squamous cell cancer (SCC-25) cells and melanoma (MUG-Mel2) cells. Hypericin was applied at concentrations ranging from 0.1-40 µM to HaCaT, SCC-25, and MUG-Mel2 cells. After 24 h of incubation, the cells were exposed to orange light at 3.6 J/cm2 or 7.2 J/cm2. Phototoxicity was assessed using MTT and SRB tests. Cellular uptake was measured by flow cytometry. Apoptosis-positive cells were estimated through TUNEL for apoptotic bodies' visualization. Hypericin exhibited a higher phototoxic reaction in cancer cells compared to normal keratinocytes after irradiation. Cancer cells demonstrated increased and selective uptake of hypericin. Apoptosis was observed in SCC-25 and MUG-Mel2 cells following PDT. Our findings suggest that hypericin-based PDT is a promising and less invasive approach for treating skin cancer. The higher phototoxic reaction, selective uptake by cancer cells, and observed proapoptotic properties support the promising role of hypericin-based PDT in skin cancer treatment.

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.

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.

Naturally Occurring Partially Reduced Perylenequinones from Fungi.

This Review provides a critical analysis of the literature covering the naturally occurring partially reduced perylenequinones (PQs) from fungi without carbon substituents (which can be named class A perylenequinones) and discusses their structures, stereochemistry, biosynthesis, and biological activities as appropriate. Perylenequinones are natural pigments with a perylene skeleton produced by certain fungi, aphids, some plants, and animal species. These compounds display several biological activities, e.g., antimicrobial, anti-HIV, photosensitizers, cytotoxic, and phytotoxic. It describes 36 fungal PQs and cites 81 references, covering from 1956 to August 2022.

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.

Nucleoside Analogs and Perylene Derivatives Modulate Phase Separation of SARS-CoV-2 N Protein and Genomic RNA In Vitro.

The life cycle of severe acute respiratory syndrome coronavirus 2 includes several steps that are supposedly mediated by liquid-liquid phase separation (LLPS) of the viral nucleocapsid protein (N) and genomic RNA. To facilitate the rational design of LLPS-targeting therapeutics, we modeled N-RNA biomolecular condensates in vitro and analyzed their sensitivity to several small-molecule antivirals. The model condensates were obtained and visualized under physiological conditions using an optimized RNA sequence enriched with N-binding motifs. The antivirals were selected based on their presumed ability to compete with RNA for specific N sites or interfere with non-specific pi-pi/cation-pi interactions. The set of antivirals included fleximers, 5'-norcarbocyclic nucleoside analogs, and perylene-harboring nucleoside analogs as well as non-nucleoside amphiphilic and hydrophobic perylene derivatives. Most of these antivirals enhanced the formation of N-RNA condensates. Hydrophobic perylene derivatives and 5'-norcarbocyclic derivatives caused up to 50-fold and 15-fold enhancement, respectively. Molecular modeling data argue that hydrophobic compounds do not hamper specific N-RNA interactions and may promote non-specific ones. These findings shed light on the determinants of potent small-molecule modulators of viral LLPS.

Antifungal Activity of Amphiphilic Perylene Bisimides.

Perylene-based compounds, either naturally occurring or synthetic, have shown interesting biological activities. In this study, we report on the broad-spectrum antifungal properties of two lead amphiphilic perylene bisimides, compounds 4 and 5, which were synthesized from perylene-3,4,9,10-tetracarboxylic dianhydride by condensation with spermine and an ammonium salt formation. The antifungal activity was evaluated using a collection of fungal strains and clinical isolates from patients with onychomycosis or sporotrichosis. Both molecules displayed an interesting antifungal profile with MIC values in the range of 2-25 µM, being as active as several reference drugs, even more potent in some particular strains. The ammonium trifluoroacetate salt 5 showed the highest activity with a MIC value of 2.1 µM for all tested Candida spp., two Cryptococcus spp., two Fusarium spp., and one Neoscytalidium spp. strain. Therefore, these amphiphilic molecules with the perylene moiety and cationic ammonium side chains represent important structural features for the development of novel antifungals.

Axial Chiral Binaphthoquinone and Perylenequinones from the Stromata of Hypocrella bambusae Are SARS-CoV-2 Entry Inhibitors.

A new axial chiral binaphtoquinone, hypocrellone (1), and a new perylenequinone, hypomycin F (2), were isolated from the stromata of Hypocrella bambusae, together with five known compounds, 3-7. The structures of 1 and 2 were assigned by spectroscopic and HRESIMS data analyses. The axial chirality of 1 was determined by electronic circular dichroism data analysis, and the absolute configurations of 2 and 3 were determined by X-ray crystallography. The axial chirality of 7 was determined by UV-induced photooxidation from 4. Compounds 1, 4, and 5 showed inhibitory activity against pseudotyped SARS-CoV-2 infection in 293T-ACE2 cells with IC50 values of 0.17, 0.038, and 0.12 µM. Compounds 4 and 5 were also active against live SARS-CoV-2 infection with EC50 values of 0.22 and 0.21 µM, respectively. Further cell-cell fusion assays, surface plasmon resonance assays, and molecular docking studies revealed that 4 and 5 could bind with the receptor-binding domain of SARS-CoV-2 S protein to prevent its interaction with human angiotensin-converting enzyme II receptor. Our results revealed that 4 and 5 are potential SARS-CoV-2 entry inhibitors.

Influence of core extension and side chain nature in targeting G-quadruplex structures with perylene monoimide derivatives.

A structure-activity relationship (SAR) study in terms of G-quadruplex binding ability and antiproliferative activity of six fluorescent perylenemonoimide (PMIs) derivatives is reported. A positive charge seems to be the key to target G4. This study also reveals the importance of the element substitution in the potential biological activity of PMIs, being the polyethylene glycol (PEG) chains in the peri position responsible for their antiproliferative activity. Among them, the cationic PMI6 with two PEG chains is the most promising compound since its fluorescence is enhanced in the presence of G-quadruplex structures. Moreover, PMI6 binds to the human telomeric G-quadruplex hTelo with high affinity and displays a high antiproliferative potential towards HeLa (cervical adenocarcinoma), A549 (lung adenocarcinoma) and A2780 (ovarian adenocarcinoma) cells. Its fate can be followed inside cells thanks to its fluorescent properties: the compound is found to accumulate in the mitochondria.

The Effects of Photosensitizing Dyes Fagopyrin and Hypericin on Planktonic Growth and Multicellular Life in Budding Yeast.

Naphthodianthrones such as fagopyrin and hypericin found mainly in buckwheat (Fagopyrum spp.) and St. John's wort (SJW) (Hypericum perforatum L.) are natural photosensitizers inside the cell. The effect of photosensitizers was studied under dark conditions on growth, morphogenesis and induction of death in Saccharomyces cerevisiae. Fagopyrin and hypericin induced a biphasic and triphasic dose response in cellular growth, respectively, over a 10-fold concentration change. In fagopyrin-treated cells, disruptions in the normal cell cycle progression were evident by microscopy. DAPI staining revealed several cells that underwent premature mitosis without budding, a striking morphological abnormality. Flow Cytometric (FC) analysis using a concentration of 100 µM showed reduced cell viability by 41% in fagopyrin-treated cells and by 15% in hypericin-treated cells. FC revealed the development of a secondary population of G1 cells in photosensitizer-treated cultures characterized by small size and dense structures. Further, we show that fagopyrin and the closely related hypericin altered the shape and the associated fluorescence of biofilm-like structures. Colonies grown on solid medium containing photosensitizer had restricted growth, while cell-to-cell adherence within the colony was also affected. In conclusion, the photosensitizers under dark conditions affected culture growth, caused toxicity, and disrupted multicellular growth, albeit with different efficiencies.