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.

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.

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.

Near-Infrared Hypocrellin Derivatives for Synergistic Photodynamic and Photothermal Therapy.

Hypocrellin B (HB) derived from naturally produced hypocrellins has attracted considerable attention in photodynamic therapy (PDT) because of its excellent photosensitive properties. However, the weak absorption within a "phototherapy window" (600-900 nm) and poor water solubility of HB have limited its clinical application. In this study, two HB derivatives (i. e., HE and HF) were designed and synthesized for the first time by introducing two different substituent groups into the HB structure. The obtained derivatives showed a broad absorption band covering the near-infrared (NIR) region, NIR emission (peaked at 805 nm), and singlet oxygen quantum yields of 0.27/0.31. HE-PEG-NPs were also prepared using 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-mPEG2000) to achieve excellent dispersion in water and further explored their practical applications. HE-PEG-NPs not only retained their 1 O2 -generating ability, but also exhibited a photothermal conversion efficiency of 25.9%. In vitro and in vivo therapeutic results revealed that the synergetic effect of HE-PEG-NPs on PDT and photothermal therapy (PTT) could achieve a good performance. Therefore, HE-PEG-NPs could be regarded as a promising phototheranostic agent.

Regioisomer-manipulating thio-perylenediimide nanoagents for photothermal/photodynamic theranostics.

Thionated perylenediimides (PDIs) can potentially generate thermal and reactive oxygen species and thus can be used as theranostic agents for photothermal/photodynamic therapy. Herein, thionated cis-/trans-isomer PDI-CS and PDI-TS were designed and prepared to investigate thionation engineering on therapeutic performance. The results revealed that the photodynamic performance is less associated with the positon of sulfur atoms. By contrast, trans-isomer PDI-TS showed a photothermal conversion efficiency of up to 58.4%, which was 40% higher than that of PDI-CS (∼41.6%). An in vitro half-maximal inhibitory concentration of ∼7.78 µg mL-1 was achieved for PDI-TS, which was 1.7-fold smaller than that of PDI-CS, strongly reasserting the regioisomer-modulated phototheranostic performance. Notably, the strong π-π and CS interactions in PDI-TS nanoagents are essential factors attributed to their excellent photothermal performance, indicating that the optimization of non-bonding interactions is an ingenious way to improve phototheranostic performance. This work provides a facile means of creating thio-perylenediimides that possess excellent antitumor properties and a novel proof of concept to improve therapeutic performance through the optimization of non-bonding interactions.

Hypericin photodynamic activity in DPPC liposomes - part II: stability and application in melanoma B16-F10 cancer cells.

Hypericin (Hyp) is considered a promising photosensitizer for Photodynamic Therapy (PDT), due to its high hydrophobicity, affinity for cell membranes, low toxicity and high photooxidation activity. In this study, Hyp photophysical properties and photodynamic activity against melanoma B16-F10 cells were optimized using DPPC liposomes (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) as a drug delivery system. This nanoparticle is used as a cell membrane biomimetic model and solubilizes hydrophobic drugs. Hyp oxygen singlet lifetime (τ) in DPPC was approximately two-fold larger than that in P-123 micelles (Pluronic™ surfactants), reflecting a more hydrophobic environment provided by the DPPC liposome. On the other hand, singlet oxygen quantum yield values (ΦΔ1O2) in DPPC and P-123 were similar; Hyp molecules were preserved as monomers. The Hyp/DPPC liposome aqueous dispersion was stable during fluorescence emission and the liposome diameter remained stable for at least five days at 30 °C. However, the liposomes collapsed after the lyophilization/rehydration process, which was resolved by adding the lyoprotectant Trehalose to the liposome dispersion before lyophilization. Cell viability of the Hyp/DPPC formulation was assessed against healthy HaCat cells and high-metastatic melanoma B16-F10 cells. Hyp incorporated into the DPPC carrier presented a higher selectivity index than the Hyp sample previously solubilized in ethanol under the illumination effect. Moreover, the IC50 was lower for Hyp in DPPC than for Hyp pre-solubilized in ethanol. These results indicate the potential of the formulation of Hyp/DPPC for future biomedical applications in PDT treatment.

Near-IR oxime-based solvatochromic perylene diimide probe as a chemosensor for Pd species and Cu2+ ions in water and live cells.

A near-IR perylene diimide probe (OPR-PDI) containing an oxime-propargyl hybrid moiety at the bay position, was designed and synthesized for detection of Pd species and Cu2+ ions in 90% water, the solid state and MG-63 live cells. The aggregation tendency of OPR-PDI in different polarity solvents transmits solvatochromic and fluorochromic properties to differentiate certain organic solvents. Supramolecular aggregates of OPR-PDI in 90% water act as a dual chemosensor for palladium (Pd) species via de-propargylation or hydrolysis of the Schiff-base and Cu2+ ions via complexation with the O/N binding site with a low limit of detection (LOD) of the order of 7.9 × 10-8 M and 3.4 × 10-7 M respectively. TLC strips coated with OPR-PDI can be applied for sensing of Pd0 and Cu2+ ions in the solid state at levels as low as 34.6 ng cm-2 and 10.5 ng cm-2. OPR-PDI imprinted TLC strips could be used as paper sheets for writing coloured alphabets using Pd0 and Cu2+ ions as ink. Moreover, MTT assay showed that OPR-PDI has very low cytotoxicity (IC50 = 230 µM), good permeability, biocompatibility and can be applied for bio-imaging of Pd species and Cu2+ ions in MG-63 cells. DFT calculations, and cyclic voltammetric (CV) and NMR titration studies have also been discussed.

Hypocrellin Derivative-Loaded Calcium Phosphate Nanorods as NIR Light-Triggered Phototheranostic Agents with Enhanced Tumor Accumulation for Cancer Therapy.

Dopamine modified hypocrellin B (DAHB) derivative-loaded calcium phosphate nanorods (DAHB@CaP NRs) were prepared as a novel phototheranostic agent for effective tumor imaging and therapy. DAHB@CaP NRs were obtained through microwave treatment using DAHB, CaCl2 , NH3 ⋅H2 O, and H3 PO4 as precursors. The DAHB@CaP NRs possessed the following advantages: 1) efficient absorption in the near-infrared (NIR) region from 650 nm to 800 nm; 2) maximum NIR emission at approximately 735 nm; 3) enhanced cellular uptake efficiency in vitro and in vivo; and 4) efficient inhibition of tumor growth and low biotoxicity. These properties indicate the high capability of DAHB@CaP NRs for NIR fluorescence (FL) imaging-guided photodynamic therapy of cancer, thus offering promising new prospects for clinical applications.

Molecular size, shape, and electric charges: essential for perylene bisimide-based DNA intercalator to localize in cell nuclei and inhibit cancer cell growth.

The molecular properties concerning size, shape, and electric charges of the planar aromatic DNA intercalators are still poorly understood. Herein, a series of water-soluble perylene bisimide (PBI) derivatives containing a rigid and planar aromatic nanoscaffold with different size, shape, and electric charges were synthesized. Using histochemistry and cell viability assays on animal tissues and cancer cells, we revealed the molecular properties required for successful DNA intercalators to localize in cell nuclei and inhibit cancer cells. Small molecular size and the strong polarity of hydrophilic substituents are prerequisites for PBI-based DNA intercalators. A large number of charges facilitate the nucleic accumulation of these DNA intercalators, while fewer charges and planar aromatic nanoscaffold more efficiently inhibit cancer cell growth.

Hypericin: chemical synthesis and biosynthesis.

Hypericin is one of the most important phenanthoperylene quinones extracted mainly from plants of the genus Hypericum belonging to the sections Euhypericum and Campylosporus of Keller's classification. Widespread attention to the antiviral and anti-tumor properties of hypericin has spurred investigations of the chemical synthesis and biosynthesis of this unique compound. However, the synthetic strategies are challenging for organic and biological chemists. In this review, specific significant advances in total synthesis, semi-synthesis, and biosynthesis in the past decades are summarized.

Synthesis of biocompatible glycodendrimer based on fluorescent perylene bisimides and its bioimaging.

A novel water-soluble fluorescent glycodendrimer based on perylene bisimides is synthesized, which exhibits high fluorescence quantum yield of 54%. While the binding interactions of PBI-Man with Concanavalin A (Con A) are studied by fluorescence spectra and CD spectra, which show strong binding affinity for Con A with the binding constant of 3.8 × 10(7) m(-1) for monomeric mannose, nearly four orders of magnitude higher affinity than the monovalent mannose ligand. Furthermore, the fluorescence imaging of macrophage cell with PBI-Man is investigated, and shows selectively binding interaction with the mannose receptor-medicated cell entry. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) activities of PBI-Man show that PBI-Man as a biocompatible agent is noncytotoxic to living cells.

In vitro and in vivo antitumor activity of a novel hypocrellin B derivative for photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity toward tumor cells by irradiating photosensitizers with light exposure to produce reactive oxygen species (ROS). An ideal photosensitizer is a crucial element to PDT. In the current study, we evaluated the photodynamic activity of a novel photosensitizer, the derivative of hypocrellin B (HB), 17-(3-amino-1-pentanesulfonic acid)-substituted hypocrellin B Schiff-base (PENSHB), both in vitro and in vivo. METHODS: Physicochemical characteristics of the novel photosensitizer were compared with that of its parent HB. The intracellular distribution of photosensitizers and mitochondrial membrane potential were detected with laser scanning confocal microscopy. The pathway of cell death was analyzed by flow cytometry. The release of proapoptotic proteins was evaluated by Western blot. S180 tumor model was used to evaluate the antitumor effects of PENHB-mediated PDT. RESULTS: Compared with its parent HB, water solubility of the derivative was improved enormously (6.6 mg/ml vs. 4.6 µg/ml), rendering its intravenous injection feasible without auxiliary solvent. The derivative had better PDT effect than HB in vitro under similar dark cytotoxicity. Moreover, PENSHB-mediated PDT was able to induce mitochondrial inner membrane permeabilisation, cytochrome c release, caspase-3 activation and subsequent apoptotic death. In vivo study showed that more than half of tumor bearing mice were cured by PENSHB-mediated PDT. CONCLUSIONS: In vitro and in vivo studies suggest that PENSHB is an effective photosensitizer for PDT to tumors. Therefore, PENSHB as a novel photosensitizer has a good prospect of clinical application.

Simultaneous enhanced diagnosis and photodynamic therapy of photosensitizer-doped perylene nanoparticles via doping, fluorescence resonance energy transfer, and antenna effect.

We demonstrate a new strategy for preparing photosensitizer-doped perylene nanoparticles via doping, fluorescence resonance energy transfer, and antenna effect for simultaneous enhanced diagnosis and photodynamic therapy.

Photodynamic mechanisms induced by a combination of hypericin and a chlorin based-photosensitizer in head and neck squamous cell carcinoma cells.

The aim of this study was to elucidate photodynamic therapy (PDT) effects mediated by hypericin and a liposomal meso-tetrahydroxyphenyl chlorin (mTHPC) derivative, with focus on their 1:1 mixture, on head and neck squamous cell carcinoma cell lines. Absorption, excitation and photobleaching were monitored using fluorescence spectrometry, showing the same spectral patterns for the mixture as measured for single photosensitizers. In the mixture mTHPC showed a prolonged photo-stability. Singlet oxygen yield for light-activated mTHPC was Φ(Δ) = 0.66, for hypericin Φ(Δ) = 0.25 and for the mixture Φ(Δ) = ~0.4. A linear increase of singlet oxygen yield for mTHPC and the mixture was found, whereas hypericin achieved saturation after 35 min. Reactive oxygen species fluorescence was only visible after hypericin and mixture-induced PDT. Cell viability was also more affected with these two treatment options under the selected conditions. Examination of death pathways showed that hypericin-mediated cell death was apoptotic, with mTHPC necrotic and the 1:1 mixture showed features of both. Changes in gene expression after PDT indicated strong up-regulation of selected heat-shock proteins. The application of photosensitizer mixtures with the features of reduced dark toxicity and combined apoptotic and necrotic cell death may be beneficial in clinical PDT. This will be the focus of our future investigations.

Controlled aggregation of peptide-substituted perylene-bisimides.

Controlled aggregation of perylene bisimides in multiple modes has been achieved via symmetric substitution with peptides. Using optical probes of aggregation, the balance of hydrophobic and electrostatic forces are found to play a key role in directing self assembly and are exploited via solvent, pH, and specific extrinsic ion effects.

Synthesis of vanadyl-hypocrellin A complex and its photodynamic properties research.

Hypocrellin A is an efficient photodynamic agent against many tumor cells and viruses. However, it was found that the preparation of injectable formula for HA was highly hampered by the poor water solubility of these compounds. So, here, a new water-soluble vanadyl-hypocrellin A complex was first synthesized and the complex forming process was studied using spectral and thermal dynamics methods. The results indicated that VO(2+)-HA can stable in aqueous solutions and exhibit increased photostability, affinity and photocleavage ability toward ctDNA under anaerobic condition. Moreover, in vitro studies illustrated that VO(2+)-HA also had strong anti-cancer activity.

Quantitative and site-directed chemical modification of hypocrellins toward direct drug delivery and effective photodynamic activity.

For photodynamic therapy (PDT) treatment of microvascular diseases, drugs are delivered via blood circulation and the targets are vasculature endothelial cells, for which the contradictory requirements of hydrophilicity and lipophilicity of the drugs have been achieved by liposome preparations. Herein, it is demonstrated that the drug delivery and target affinity are achieved by a single chemical compound, hypocrellin B (HB) derivative 6 selected from three novel aminoalkanesulfonic acid HB derivatives, 5-7. 6 exhibits a much higher PDT activity (IC(50) = 22 nM) on human gastric carcinoma BGC823 cells than HB, while it has no cellular toxicity in the dark. On the basis of estimation of the clinically required concentration according to relative PDT activity and clinical criteria, it can be predicted that 6 is directly deliverable to and PDT effective on target cells. The enhanced red absorption and superhigh photoactivity suggest that 6 is more powerful for PDT of tumors than HB.

A phase 1/2 study of orally administered synthetic hypericin for treatment of recurrent malignant gliomas.

BACKGROUND: Hypericin is a potent inhibitor of glioma growth in vitro. To examine whether synthetic oral hypericin can be tolerated by patients with recurrent malignant gliomas (anaplastic astrocytoma and glioblastoma) and to investigate its efficacy against these tumors, the authors undertook an open-label, sequential dose escalation/de-escalation tolerance study. METHODS: Patients with documented recurrent or progressive malignant gliomas who had received standard radiation therapy with or without chemotherapy were included. Patients were excluded for previous treatment with agents known to contain hypericin or treatment within 30 days with medications known to cause photosensitivity. Enrolled patients were given gradually increasing dosages of oral synthetic hypericin (0.05-0.50 mg/kg) for up to 3 months if no toxicity was observed, and patient response to treatment was noted. The patients were examined each month and underwent magnetic resonance imaging to evaluate tumor status at 3 months. RESULTS: Synthetic hypericin administered orally appeared to provide stabilization or a slight (<50%) decrease in tumor volume (coded as stable disease) at 3 months for 7 of 42 patients (17%) and produced a tumor reduction >50% (partial response) in 2 patients (5%). Seventeen patients (40%) survived for 3 months on daily synthetic hypericin at dose levels of 0.33 ± 0.070 mg/kg daily. The mean maximum tolerated dose was 0.40 ± 0.098 mg/kg daily. Twelve patients continued on hypericin therapy beyond 3 months. The median survival was 26 weeks (Kaplan-Meier method). CONCLUSIONS: The results of this study indicated that synthetic, oral hypericin is well tolerated in this patient group. The response results were comparable to those reported from other studies of salvage therapies for recurrent malignant brain tumors.

Small-animal PET of tumor damage induced by photothermal ablation with 64Cu-bis-DOTA-hypericin.

UNLABELLED: The purpose of this study was to investigate the potential application of small-molecular-weight (64)Cu-labeled bis-DOTA-hypericin in the noninvasive assessment of response to photothermal ablation therapy. METHODS: Bis-DOTA-hypericin was labeled with (64)Cu with high efficiency (>95% without purification). Nine mice bearing subcutaneous human mammary BT474 tumors were used. Five mice were injected intratumorally with semiconductor CuS nanoparticles, followed by near-infrared laser irradiation 24 h later (12 W/cm(2) for 3 min), and 4 mice were not treated (control group). All mice were intravenously injected with (64)Cu-bis-DOTA-hypericin (24 h after laser treatment in treated mice). Small-animal PET images were acquired at 2, 6, and 24 h after radiotracer injection. All mice were killed immediately after the imaging session for biodistribution and histology study. In vitro cell uptake and surface plasmon resonance studies were performed to validate the small-animal PET results. RESULTS: (64)Cu-bis-DOTA-hypericin uptake was significantly higher in the treatment group than in the control group. The percentage injected dose per gram of tissue in the treated and control groups was 1.72 ± 0.43 and 0.76 ± 0.19, respectively (P = 0.017), at 24 h after injection. Autoradiography and histology results were consistent with selective uptake of the radiotracer in the necrotic zone of the tumor induced by photothermal ablation therapy. In vitro results showed that treated BT474 cells had a higher uptake of (64)Cu-bis-DOTA-hypericin than nontreated cells. Surface plasmon resonance study showed that bis-DOTA-hypericin had higher binding affinity to phosphatidylserine and phosphatidylethanolamine than to phosphatidylcholine. CONCLUSION: (64)Cu-bis-DOTA-hypericin has a potential to image thermal therapy-induced tumor cell damage. The affinity of (64)Cu-bis-DOTA-hypericin for injured tissues may be attributed to the breakdown of the cell membrane and exposure of phosphatidylserine or phosphatidylethanolamine to the radiotracer, which binds selectively to these phospholipids.

N-cyclic bay-substituted perylene G-quadruplex ligands have selective antiproliferative effects on cancer cells and induce telomere damage.

A series of bay-substituted perylene derivatives is reported as a new class of G-quadruplex ligands. The synthesized compounds have differing N-cyclic substituents on the bay area and differing side chains on the perylene major axis. ESI-MS and FRET measurements highlighted the strongest quadruplex binders in this series and those showing the highest quadruplex/duplex selectivity. Several biological assays were performed on these compounds, which showed that compound 5 (PPL3C) triggered a DNA damage response in transformed cells with the formation of telomeric foci containing phosphorylated γ-H2AX and 53BP1. This effect mainly occurred in replicating cells and was consistent with Pot1 dissociation. Compound 5 does not induce telomere damage in normal cells, which are unaffected by treatment with the compound, suggesting that this agent preferentially kills cancer cells. These results reinforce the notion that G-quadruplex binding compounds can act as broad inhibitors of telomere-related processes and have potential as selective antineoplastic drugs.