Selective photodynamic eradication of senescent cells with a ß-galactosidase-activated photosensitiser.

A ß-galactosidase-responsive photosensitiser has been designed and synthesised. It contains a galactosyl substrate, a boron dipyrromethene-based photosensitising unit and a black hole quencher 2 connected via an AB2-type self-immolative linker. This novel photosensitiser can be selectively activated by the senescence-associated ß-galactosidase in senescent cells, leading to restoration in fluorescence emission and effective killing of the cells via photodynamic action.

Specific Activation of Photosensitizer with Extrinsic Enzyme for Precisive Photodynamic Therapy.

Delivery of functional proteins into the intracellular space has been a challenging task that could lead to a myriad of therapeutic applications. We report herein a novel bioconjugation strategy for enzyme modification and selective delivery into cancer cells for lock-and-key-type activation of photosensitizers. Using a bifunctional linker containing a bis(bromomethyl)phenyl group and an o-phthalaldehyde moiety, it could induce cyclization of the peptide sequence Ac-NH-CRGDfC-CONH2 through site-specific dibenzylation with the two cysteine residues and further coupling with ß-galactosidase via the phthalaldehyde-amine capture reaction. This facile two-step one-pot procedure enabled the preparation of cyclic RGD-modified ß-galactosidase readily, which could be internalized selectively into αvß3 integrin-overexpressed cancer cells. Upon encountering an intrinsically quenched distyryl boron dipyrromethene-based photosensitizer conjugated with a galactose moiety through a self-immolative linker inside the cells, the extrinsic enzyme induced specific cleavage of the ß-galactosidic bond followed by self-immolation to release an activated derivative, thereby restoring the photodynamic activities and causing cell death effectively. The high specificity of this extrinsic enzyme-activated photosensitizing system was also demonstrated in vivo using nude mice bearing an αvß3 integrin-positive U87-MG tumor. The specific activation at the tumor site resulted in lighting up and complete eradication of the tumor upon laser irradiation, while by using the native ß-galactosidase, the effects were largely reduced. In contrast to the conventional activation using intrinsic enzymes, this extrinsic enzyme activatable approach can further minimize the nonspecific activation toward precisive photodynamic therapy.

Site-Specific Displacement-Driven Activation of Supramolecular Photosensitizing Nanoassemblies for Antitumoral Photodynamic Therapy.

The delivery and activation of photosensitizers in a specific manner is crucial in photodynamic therapy. For an antitumoral application, it can confine the photodynamic action on the cancer cells, thereby enhancing the treatment efficacy and reducing the side effects. We report herein a novel supramolecular photosensitizing nanosystem that can be specifically activated in cancer cells and tumors that overexpress epidermal growth factor receptor (EGFR). It involves the self-assembly of the amphiphilic host-guest complex of a ß-cyclodextrin-conjugated phthalocyanine-based photosensitizer (Pc-CD) and a ferrocene-substituted poly(ethylene glycol) (Mn = 2000) (Fc-PEG) in aqueous media. The resulting nanosystem Pc-CD@Fc-PEG with a hydrodynamic diameter of 124-147 nm could not emit fluorescence and generate reactive oxygen species due to the self-quenching effect and the ferrocene-based quencher. Upon interactions with molecules of adamantane substituted with an EGFR-targeting peptide (Ad-QRH*) in water and in EGFR-positive HT29 and A431 cells, the ferrocene guest species were displaced, resulting in disassembly of the nanoparticles and restoration of these photoactivities. The half-maximal inhibitory concentration values were down to 1.24 µM (for HT29 cells). The nanosystem Pc-CD@Fc-PEG could also be activated in an Ad-QRH*-treated HT29 tumor in nude mice, leading to increased intratumoral fluorescence intensity and effective eradication of the tumor upon laser irradiation. The results showed that this two-step supramolecular approach can actualize site-specific photosensitization and minimize nonspecific phototoxicity in a general photodynamic treatment.

Dual Cathepsin B and Glutathione-Activated Dimeric and Trimeric Phthalocyanine-Based Photodynamic Molecular Beacons for Targeted Photodynamic Therapy.

Two dual stimuli-activated photosensitizers were developed, in which two or three glutathione (GSH)-responsive 2,4-dinitrobenzenesulfonate (DNBS)-substituted zinc(II) phthalocyanine units were connected via one or two cathepsin B-cleavable Gly-Phe-Leu-Gly peptide linker(s). These dimeric and trimeric phthalocyanines were fully quenched in the native form due to the photoinduced electron transfer to the DNBS substituents and the self-quenching of the phthalocyanine units. In the presence of GSH and cathepsin B, or upon internalization into A549 and HepG2 cancer cells, these probes were activated through the release of free phthalocyanine units. The intracellular fluorescence intensity was increased upon post-incubation with GSH ester or reduced upon pre-treatment with a cathepsin B inhibitor. Upon light irradiation, these photosensitizers became highly cytotoxic with IC50 values of 0.21-0.39 µM. The photocytotoxicity was also dependent on the intracellular GSH and cathepsin B levels. The results showed that these conjugates could serve as smart photosensitizers for targeted photodynamic therapy.

Glutathione-degradable polydopamine nanoparticles as a versatile platform for fabrication of advanced photosensitisers for anticancer therapy.

A series of glutathione (GSH)-responsive polydopamine (PDA) nanoparticles (NPs) were prepared using a disulfide-linked dopamine dimer as starting material, of which the size could be tuned systematically by adjusting the amount of ammonia solution used. Molecules of a phthalocyanine (Pc)-based photosensitiser and an epidermal growth factor receptor (EGFR)-targeting peptide were then sequentially immobilised on the surface of the NPs through coupling with the surface functionalities of PDA. The immobilised Pc molecules in the resulting nanosystem were photodynamically inactive due to the strong self-quenching effect and the quenching by the PDA core. Upon exposure to GSH in phosphate-buffered saline or EGFR-positive cancer cells, namely A549 and A431 cells, the NPs were disassembled through cleavage of the disulfide linkages to release the Pc molecules, thereby restoring their fluorescence emission and singlet oxygen generation. The NPs with the smallest size (ca. 200 nm in diameter) exhibited the highest cellular uptake and high photocytotoxicity with IC50 values as low as 0.05 µM based on Pc. These NPs could also accumulate and be activated in the tumour of A431 tumour-bearing nude mice, lighting up the tumour with fluorescence over a period of 72 h and completely eradicating the tumour through laser irradiation for 10 min (675 nm, 20 J cm-2). The results suggest that these biodegradable and versatile PDA-based NPs can serve as a promising nanoplatform for fabrication of advanced photosensitisers for targeted photodynamic therapy.

One-pot peptide cyclisation and surface modification of photosensitiser-loaded red blood cells for targeted photodynamic therapy.

We report herein a one-pot approach to cyclise a tumour-targeting peptide and conjugate it on the surface of red blood cells loaded with a boron dipyrromethene-based photosensitiser using a bifunctional linker consisting of a bis(bromomethyl)phenyl unit and an ortho-phthalaldehyde unit. This cell-based photosensitiser with surface modification with cyclic RGD peptide moieties can selectively bind against the αvß3 integrin-overexpressed cancer cells, leading to enhanced photocytotoxicity. The results demonstrate that this facile strategy is effective for live-cell surface modification for a wide range of applications.

Targeted Delivery and Site-Specific Activation of ß-Cyclodextrin-Conjugated Photosensitizers for Photodynamic Therapy through a Supramolecular Bio-orthogonal Approach.

Targeted delivery of photosensitizers using hydrophilic and tumor-directing carriers and site-specific activation of their photocytotoxicity are two common strategies to enhance the specificity of anticancer photodynamic therapy. We report herein a novel supramolecular bio-orthogonal approach to integrate these two functions. A ß-cyclodextrin-substituted aza-boron-dipyrromethene-based photosensitizer was first complexed with a ferrocene-substituted black-hole quencher to inhibit its photosensitizing ability. Upon encountering the adamantane moieties that had been delivered to target cancer cells through specific binding of the conjugated peptide to the overexpressed epidermal growth factor receptor, the ferrocene-based guest species were displaced due to the stronger binding interactions between ß-cyclodextrin and adamantane, thereby restoring the photodynamic activity of the photosensitizer. Hence, this two-step process enabled targeted delivery and site-specific activation of the photosensitizer, as demonstrated through a series of experiments in aqueous media, in a range of cancer cell lines and in tumor-bearing nude mice.

Enhancement of innate and adaptive anti-tumor immunity by serum obtained from vascular photodynamic therapy-cured BALB/c mouse.

Photodynamic therapy (PDT) is a clinically approved treatment for various types of cancer. Besides killing the tumor cells directly, PDT has also been reported to trigger anti-tumor immunity. In our previous study, BAM-SiPc-based PDT was shown to induce immunogenic cell death on CT26 murine colon tumor cells in vitro. Using the BALB/c mouse animal model and a vascular-PDT (VPDT) approach, it could also eradicate tumor in ∼ 70% of tumor-bearing mice and elicit an anti-tumor immune response. In the present study, the serum obtained from the VPDT-cured mice was studied and found to possess various immunomodulatory properties. In in vitro studies, it stimulated cytokine secretions of IL-6 and C-X-C motif chemokine ligands 1-3 in CT26 cells through the NF-κB and MAPK pathways. The complement protein C5a boosted in the serum was shown to be involved in the process. The serum also induced calreticulin exposure on CT26 cells and activated dendritic cells. It contained CT26-targeting antibodies which, through the Fc region, induced macrophage engulfment of the tumor cells. In in vivo studies, inoculation of the serum-treated CT26 cells to mice demonstrated a retarded tumor growth with leukocytes, particularly T cells, attracted to the tumor site. In addition, the VPDT-cured mice showed different degrees of resistance against challenge of other types of murine tumor cells, for example, the breast tumor 4T1 and EMT6 cells.

Facile Synthesis of Cyclic Peptide-Phthalocyanine Conjugates for Epidermal Growth Factor Receptor-Targeted Photodynamic Therapy.

A facile procedure for in situ peptide cyclization and phthalocyanine conjugation was developed by utilizing a bifunctional linker incorporated with a bis(bromomethyl)benzene unit and a cyclopentadiene moiety. These functional groups facilitated the nucleophilic substitution with the two cysteine residues of the linear peptides followed by the Diels-Alder reaction with the maleimide moiety attached to a zinc(II) phthalocyanine. With this approach, three cyclic peptide-phthalocyanine conjugates were prepared in 20-26% isolated yield via a one-pot procedure. One of the conjugates containing a cyclic form of the epidermal growth factor receptor (EGFR)-binding peptide sequence CMYIEALDKYAC displayed superior features as an advanced photosensitizer. It showed preferential uptake by two EGFR-positive cancer cell lines (HT29 and HCT116) compared with two EGFR-negative counterparts (HeLa and HEK293), resulting in significantly higher photocytotoxicity. Intravenous administration of this conjugate into HT29 tumor-bearing nude mice resulted in selective localization in tumor and effective inhibition of tumor growth upon photodynamic treatment.

Immunogenic necroptosis in the anti-tumor photodynamic action of BAM-SiPc, a silicon(IV) phthalocyanine-based photosensitizer.

Photodynamic therapy (PDT) is an anti-tumor modality which employs three individually non-toxic substances, including photosensitizer, light and oxygen, to produce a toxic effect. Besides causing damage to blood vessels that supply oxygen and nutrients to the tumor and killing the tumor by a direct cytotoxic effect, PDT has also been known to trigger an anti-tumor immune response. For instance, our previous study showed that PDT with BAM-SiPc, a silicon(IV) phthalocyanine based-photosensitizer, can not only eradicate the mouse CT26 tumor cells in a Balb/c mouse model, but also protect the mice against further re-challenge of the tumor cells through an immunomodulatory mechanism. To understand more about the immune effect, the biochemical actions of BAM-SiPc-PDT on CT26 cells were studied in the in vitro system. It was confirmed that the PDT treatment could induce immunogenic necroptosis in the tumor cells. Upon treatment, different damage-associated molecular patterns were exposed onto the cell surface or released from the cells. Among them, calreticulin was found to translocate to the cell membrane through a pathway similar to that in chemotherapy. The activation of immune response was also demonstrated by an increase in the expression of different chemokines.

Facile one-pot synthesis of cyclic peptide-conjugated photosensitisers for targeted photodynamic therapy.

A novel synthetic strategy for in situ cyclisation of peptides and conjugation with functional boron dipyrromethenes (BODIPYs) has been developed. Linear peptides with up to 16 amino acid residues can be cyclised effectively and the resulting conjugates can be isolated in higher than 20% yield. One of the conjugates having a cyclic RGD moiety has been studied both in vitro and in vivo. It exhibits high and selective affinity towards the αvß3-positive cell lines and induces high photocytotoxicity. The conjugate can also selectively localise in and effectively inhibit the growth of αvß3-overexpressed tumour in vivo.

Cadherin-17 Targeted Near-Infrared Photoimmunotherapy for Treatment of Gastrointestinal Cancer.

In cancer photodynamic therapy (PDT), a photosensitizer taken up by cancer cells can generate reactive oxygen species upon near-infrared light activation to induce cancer cell death. To increase PDT potency and decrease its adverse effect, one approach is to conjugate the photosensitizer with an antibody that specifically targets cancer cells. In the present study, IR700, a hydrophilic phthalocyanine photosensitizer, was conjugated to the humanized monoclonal antibody ARB102, which binds specifically cadherin-17 (CDH17 aka CA17), a cell surface marker highly expressed in gastrointestinal cancer to produce ARB102-IR700. Photoimmunotherapy (PIT) of gastrointestinal cancer cell lines was conducted by ARB102-IR700 treatment and near-infrared light irradiation. The results showed that ARB102-IR700 PIT could induce cell death in CDH17-positive cancer cells with high potency. In a co-culture model, CDH17-negative and CDH17-overexpressing SW480 cells were labeled with distinct fluorescent dyes and cultured together prior to PIT treatment. The results confirmed that ARB102-IR700 PIT could kill CDH17-positive cells specifically, while leaving the adjacent CDH17-negative cells unaffected. An in vivo efficacy study was conducted using a pancreatic adenocarcinoma AsPC-1 xenograft tumor model in nude mice. Fluorescence scanning indicated that ARB102-IR700 accumulated specifically in the tumor sites. To perform PIT, at 24 and 48 h postinjection, mice were irradiated with a 680 nm laser at the tumor site to activate the photosensitizer. It was shown that ARB102-IR700 PIT could inhibit tumor growth significantly. In summary, this study demonstrated that the novel ARB102-IR700 is a promising agent for PIT in gastrointestinal cancers.

Multifunctional Molecular Therapeutic Agent for Targeted and Controlled Dual Chemo- and Photodynamic Therapy.

A novel molecular therapeutic agent was designed and synthesized, which contains three functional components, namely, a zinc(II) phthalocyanine substituted with a 2,4-dinitrobenzenesulfonate (DNBS) group as a glutathione (GSH)-activated photosensitizer, a chemo-prodrug based on combretastatin A-4 (CA4) with a singlet oxygen-cleavable aminoacrylate linker, and a biotin moiety as a tumor-targeting ligand. The conjugate showed preferential uptake toward the biotin-receptor-positive HepG2 cells compared with the low biotin-receptor-expressed HCT-116 cells used as the negative control, resulting in the restoration of the fluorescence emission and singlet oxygen generation upon removal of the DNBS group by intracellular GSH. The singlet oxygen produced not only induced a significant photodynamic effect against HepG2 cells but also triggered the cascaded release of the chemocytotoxic CA4, leading to synergistic cytotoxicity as shown by the less-than-unity combination index.

Glutathione- and light-controlled generation of singlet oxygen for triggering drug release in mesoporous silica nanoparticles.

A combined stimulus-responsive photosensitiser and drug release system based on mesoporous silica nanoparticles was prepared. This nanoplatform encapsulated molecules of zinc(ii) phthalocyanine substituted with a glutathione-cleavable 2,4-dinitrobenzenesulfonate quencher and doxorubicin linked via a singlet-oxygen-cleavable 9,10-dialkoxyanthracene linker. In the presence of glutathione (in mM range) and upon irradiation (λ > 610 nm), the phthalocyanine units were activated by detaching from the quenching component to emit fluorescence and generate singlet oxygen. The latter subsequently cleaved the 9,10-dialkoxyanthracene linker to trigger the release of a doxorubicin derivative. The glutathione- and light-controlled activation and drug-release processes on this nanoplatform were demonstrated in phosphate buffered saline. The activation in fluorescence emission by intracellular thiols was also shown inside HepG2 human hepatocellular carcinoma cells. Upon irradiation, the nanosystem exhibited high cytotoxicity due to the photodynamic effect of the activated phthalocyanine units, but the cytotoxic effect of the released Dox moieties was not notable probably due to their reduced cytotoxicity as a result of the pendant substituent and the low drug loading in the nanoparticles.

Engineering and optimization of phosphate-responsive phytase expression in Pichia pastoris yeast for phytate hydrolysis.

Phytate is the major storage form of phosphorus in plants. It is present in cereals and raw materials of vegetable origin used in animal and human diets. However, non-ruminant animals have little phytase activity in their guts and, therefore, cannot digest phytate. As a result, almost all dietary phytate is discharged into the environment, causing phosphorus pollution. Phytate is also considered as an "antinutrient" for its ability to form insoluble and stable complexes with metal ions, thus reducing dietary absorption of essential minerals. It is a dire need to develop sustainable approaches for environmentally-friendly utilization for this valuable and abundant natural resource. To this end, we engineered Pichia pastoris to express and secrete phytase in a "made-to-order" fashion in response to external level of inorganic phosphate (Pi). Responsiveness to external Pi level was achieved by generating a Pi-responsive promoter library using directed evolution. The resultant yeast strains were proven to liberate Pi from wheat-based meal in a simulated in vitro digestion model. These yeast-based whole cell biocatalysts may serve as platform hosts with potential applications in food processing industry and animal waste treatment.

MicroRNA-200 family governs ovarian inclusion cyst formation and mode of ovarian cancer spread.

Epidemiologic and histopathologic findings and the laying hen model support the long-standing incessant ovulation hypothesis and cortical inclusion cyst involvement in sporadic ovarian cancer development. MicroRNA-200 (miR-200) family is highly expressed in ovarian cancer. Herewith, we show that ovarian surface epithelial (OSE) cells with ectopic miR-200 expression formed stabilized cysts in three-dimensional (3D) organotypic culture with E-cadherin fragment expression and steroid hormone pathway activation, whereas ovarian cancer 3D cultures with miR-200 knockdown showed elevated TGF-ß expression, mitotic spindle disorientation, increased lumenization, disruption of ROCK-mediated myosin II phosphorylation, and SRC signaling, which led to histotype-dependent loss of collective movement in tumor spread. Gene expression profiling revealed that epithelial-mesenchymal transition and hypoxia were the top enriched gene sets regulated by miR-200 in both OSE and ovarian cancer cells. The molecular changes uncovered by the in vitro studies were verified in both human and laying hen ovarian cysts and tumor specimens. As miR-200 is also essential for ovulation, our results of estrogen pathway activation in miR-200-expressing OSE cells add another intriguing link between incessant ovulation and ovarian carcinogenesis.

Synthesis and biological evaluation of an epidermal growth factor receptor-targeted peptide-conjugated phthalocyanine-based photosensitiser.

A peptide-conjugated zinc(ii) phthalocyanine containing the epidermal growth factor receptor-targeted heptapeptide QRHKPRE has been prepared. The conjugate labelled as ZnPc-QRH* can selectively bind to the cell membrane of HT29 human colorectal adenocarcinoma cells in 10 min followed by internalisation upon prolonged incubation via receptor-mediated endocytosis, leading to localisation in lysosomes eventually. By manipulating the incubation time, the subcellular localisation of the conjugate can be varied and the cell-death pathways induced upon irradiation can also be altered. It has been found that photosensitisation initiated at the cell membrane and in the lysosomes would trigger cell death mainly through necrosis and apoptosis respectively. Intravenous administration of the conjugate into HT29 tumour-bearing nude mice resulted in higher accumulation in the tumour than in most major organs. The selective binding of this conjugate to tumour has also been demonstrated by comparing the results with those of the analogue with a scrambled peptide sequence (EPRQRHK). The overall results indicate that ZnPc-QRH* is a promising EGFR-targeted photosensitiser for photodynamic therapy.

A Biotinylated and Endoplasmic Reticulum-Targeted Glutathione-Responsive Zinc(II) Phthalocyanine for Targeted Photodynamic Therapy.

A biotinylated glutathione (GSH)-responsive zinc(II) phthalocyanine has been prepared and characterized. With a 2,4-dinitrobenzenesulfonyl moiety, its fluorescence emission and singlet oxygen generation were silenced in its intact state. Upon exposure to high concentration of GSH, its photosensitizing properties were restored in phosphate buffered saline and inside tumor cells. It also showed preferential uptake on HepG2 human hepatocarcinoma cells (with higher biotin receptor expression) rather than Chinese hamster ovary (CHO) cells (with lower biotin receptor expression). Upon irradiation, it caused photocytotoxicity with an IC50 value down to 0.1 µm on HepG2 cells. Moreover, it can localize in the endoplasmic reticulum (ER), causing ER stress after light irradiation.

Endoplasmic Reticulum-Localized Two-Photon-Absorbing Boron Dipyrromethenes as Advanced Photosensitizers for Photodynamic Therapy.

Two advanced boron dipyrromethene (BODIPY) based photosensitizers have been synthesized and characterized. With a glibenclamide analogous moiety, these compounds can localize in the endoplasmic reticulum (ER) of HeLa human cervical carcinoma cells and HepG2 human hepatocarcinoma cells. The BODIPY π skeleton is conjugated with two styryl or carbazolylethenyl groups, which can substantially red-shift the Q-band absorption and fluorescence emission and impart two-photon absorption (TPA) property to the chromophores. The TPA cross section of the carbazole-containing analogue reaches a value of 453 GM at 1010 nm. These compounds also behave as singlet oxygen generators with high photostability. Upon irradiation at λ > 610 nm, these photosensitizers cause photocytotoxicity to these two cell lines with IC50 values down to 0.09 µM, for which the cell death is triggered mainly by ER stress. The two-photon photodynamic activity of the distyryl derivative upon excitation at λ = 800 nm has also been demonstrated.