Promotion and Detection of Cell-Cell Interactions through a Bioorthogonal Approach.

Manipulation of cell-cell interactions via cell surface modification is crucial in tissue engineering and cell-based therapy. To be able to monitor intercellular interactions, it can also provide useful information for understanding how the cells interact and communicate. We report herein a facile bioorthogonal strategy to promote and monitor cell-cell interactions. It involves the use of a maleimide-appended tetrazine-caged boron dipyrromethene (BODIPY)-based fluorescent probe and a maleimide-substituted bicyclo[6.1.0]non-4-yne (BCN) to modify the membrane of macrophage (RAW 264.7) and cancer (HT29, HeLa, and A431) cells, respectively, via maleimide-thiol conjugation. After modification, the two kinds of cells interact strongly through inverse electron-demand Diels-Alder reaction of the surface tetrazine and BCN moieties. The coupling also disrupts the tetrazine quenching unit, restoring the fluorescence emission of the BODIPY core on the cell-cell interface, and promotes phagocytosis. Hence, this approach can promote and facilitate the detection of intercellular interactions, rendering it potentially useful for macrophage-based immunotherapy.

Immobilising an acid-cleavable dimeric phthalocyanine on gold nanobipyramids for intracellular pH detection and photodynamic elimination of cancer cells.

An acetal-linked dimeric phthalocyanine has been synthesised and immobilised on the surface of gold nanobipyramids. The resulting nanocomposite serves as a highly sensitive probe for intracellular pH through its acid-responsive fluorescence and surface-enhanced Raman scattering signals. The phthalocyanine units released in the acidic intracellular environment can also effectively eliminate the cancer cells upon light irradiation, rendering this simple fabricated nanosystem a bimodal and bifunctional theranostic agent.

Enzyme-Responsive Double-Locked Photodynamic Molecular Beacon for Targeted Photodynamic Anticancer Therapy.

An advanced photodynamic molecular beacon (PMB) was designed and synthesized, in which a distyryl boron dipyrromethene (DSBDP)-based photosensitizer and a Black Hole Quencher 3 moiety were connected via two peptide segments containing the sequences PLGVR and GFLG, respectively, of a cyclic peptide. These two short peptide sequences are well-known substrates of matrix metalloproteinase-2 (MMP-2) and cathepsin B, respectively, both of which are overexpressed in a wide range of cancer cells either extracellularly (for MMP-2) or intracellularly (for cathepsin B). Owing to the efficient Förster resonance energy transfer between the two components, this PMB was fully quenched in the native form. Only upon interaction with both MMP-2 and cathepsin B, either in a buffer solution or in cancer cells, both of the segments were cleaved specifically, and the two components could be completely separated, thereby restoring the photodynamic activities of the DSBDP moiety. This PMB could also be activated in tumors, and it effectively suppressed the tumor growth in A549 tumor-bearing nude mice upon laser irradiation without causing notable side effects. In particular, it did not cause skin photosensitivity, which is a very common side effect of photodynamic therapy (PDT) using conventional "always-on" photosensitizers. The overall results showed that this "double-locked" PMB functioned as a biological AND logic gate that could only be unlocked by the coexistence of two tumor-associated enzymes, which could greatly enhance the tumor specificity in PDT.

Toward Precise Antitumoral Photodynamic Therapy Using a Dual Receptor-Mediated Bioorthogonal Activation Approach.

Targeted delivery and specific activation of photosensitizers can greatly improve the treatment outcome of photodynamic therapy. To this end, we report herein a novel dual receptor-mediated bioorthogonal activation approach to enhance the tumor specificity of the photodynamic action. It involves the targeted delivery of a biotinylated boron dipyrromethene (BODIPY)-based photosensitizer, which is quenched in the native form by the attached 1,2,4,5-tetrazine unit, and an epidermal growth factor receptor (EGFR)-targeting cyclic peptide conjugated with a bicycle[6.1.0]non-4-yne moiety. Only for cancer cells that overexpress both the biotin receptor and EGFR, the two components can be internalized preferentially where they undergo an inverse electron-demand Diels-Alder reaction, leading to restoration of the photodynamic activity of the BODIPY core. By using a range of cell lines with different expression levels of these two receptors, we have demonstrated that this stepwise "deliver-and-click" approach can confine the photodynamic action on a specific type of cancer cells.

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.

A Tumor-Targeting Dual-Stimuli-Activatable Photodynamic Molecular Beacon for Precise Photodynamic Therapy.

A multifunctional photodynamic molecular beacon (PMB) has been designed and synthesized which contains an epidermal growth factor receptor (EGFR)-targeting cyclic peptide and a trimeric phthalocyanine skeleton in which the three zinc(II) phthalocyanine units are each substituted with a glutathione (GSH)-responsive 2,4-dinitrobenzenesulfonate (DNBS) quencher and are linked via two cathepsin B-cleavable GFLG peptide chains. This tailor-made conjugate is fully quenched in the native form due to the photoinduced electron transfer effect of the DNBS moieties and the self-quenching of the phthalocyanine units. It can target the EGFR overexpressed in cancer cells, and after receptor-mediated endocytosis, it can be activated selectively by the co-existence of intracellular GSH and cathepsin B, both of which are also overproduced in cancer cells, in terms of fluorescence emission and singlet oxygen generation. The cell-selective behavior of this PMB has been demonstrated using a range of cancer cells with different expression levels of EGFR, while the stimuli-responsive properties have been studied both in vitro and in various aqueous media. The overall results show that this advanced PMB, which exhibits several levels of control of the tumor specificity, is a promising photosensitizer for precise antitumoral photodynamic therapy.

[3 + 1] Mixed Cyclization: A Synthetic Route to Prepare Low-Symmetry Phthalocyanines.

A novel synthetic strategy for low-symmetry phthalocyanines has been developed, which involves the base-promoted cyclization of a preconnected trisphthalonitrile and a free phthalonitrile in the presence of a metal template. By using this [3 + 1] mixed cyclization approach, a series of zinc(II) phthalocyanine derivatives have been synthesized in up to 12% yields, including a very rare ABCD-type phthalocyanine and an amphiphilic ABAC-type analogue that can self-assemble in aqueous media, forming stable spherical nanoparticles.

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.

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.

C=C Bond Oxidative Cleavage of BODIPY Photocages by Visible Light.

Photocages for protection and the controlled release of bioactive compounds have been widely investigated. However, the vast majority of these photocages employ the cleavage of single bonds and high-energy ultraviolet light. The construction of a photoactivation system that uses visible light to cleave unsaturated bonds still remains a challenge. Herein, we report a regioselective oxidative cleavage of C=C bonds from a boron-dipyrrolemethene (BODIPY)-based photocage by illumination at 630 nm, resulting in a free aldehyde and a thiol fluorescent probe. This strategy was demonstrated in live HeLa cells, and the generated α-formyl-BODIPY allowed real-time monitoring of aldehyde release in the cells. In particular, it is shown that a mannose-functionalized photocage can target HepG2 cells.

Nanoparticles for Triple Drug Release for Combined Chemo- and Photodynamic Therapy.

A pH-responsive drug delivery system (DDS) based on mesoporous silica nanoparticles (MSNs) has been prepared for the delivery of three anticancer drugs with different modes of action. The novelty of this system is its ability to combine synergistic chemotherapy and photodynamic therapy. A photoactive conjugate of a phthalocyanine (Pc) and a topoisomerase I inhibitor (topo-I), namely camptothecin (CPT), linked by a poly(ethylene glycol) (PEG) chain has been synthesized and then loaded into the mesopores of MSNs. Doxorubicin (DOX), which is a topoisomerase II inhibitor (topo-II), has also been covalently anchored to the outer surface of the MSNs through a dihydrazide PEG linker. In the acidic environment of tumor cells, selective release of the three drugs takes place. In vitro studies have demonstrated the endocytosis of the system into HeLa and HepG2 cells, and the subsequent release of the three drugs into the cytoplasm and nucleus. Furthermore, the cytotoxic effect of DOX, CPT and Pc has been assessed in vitro before and upon light irradiation.

The unique features and promises of phthalocyanines as advanced photosensitisers for photodynamic therapy of cancer.

Phthalocyanines exhibit superior photoproperties that make them a surely attractive class of photosensitisers for photodynamic therapy of cancer. Several derivatives are at various phases of clinical trials, and efforts have been put continuously to improve their photodynamic efficacy. To this end, various strategies have been applied to develop advanced phthalocyanines with optimised photoproperties, dual therapeutic actions, tumour-targeting properties and/or specific activation at tumour sites. The advantageous properties and potential of phthalocyanines as advanced photosensitisers for photodynamic therapy of cancer are highlighted in this tutorial review.

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.

Ferric Ion Driven Assembly of Catalase-like Supramolecular Photosensitizing Nanozymes for Combating Hypoxic Tumors.

A facile approach to assemble catalase-like photosensitizing nanozymes with a self-oxygen-supplying ability was developed. The process involved Fe3+ -driven self-assembly of fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids. By adding a zinc(II) phthalocyanine-based photosensitizer (ZnPc) and the hypoxia-inducible factor 1 (HIF-1) inhibitor acriflavine (ACF) during the Fe3+ -promoted self-assembly of Fmoc-protected cysteine (Fmoc-Cys), the nanovesicles Fmoc-Cys/Fe@Pc and Fmoc-Cys/Fe@Pc/ACF were prepared, which could be disassembled intracellularly. The released Fe3+ could catalyze the transformation of H2 O2 enriched in cancer cells to oxygen efficiently, thereby ameliorating the hypoxic condition and promoting the photosensitizing activity of the released ZnPc. With an additional therapeutic component, Fmoc-Cys/Fe@Pc/ACF exhibited higher in vitro and in vivo photodynamic activities than Fmoc-Cys/Fe@Pc, demonstrating the synergistic effect of ZnPc and ACF.

Boron(III) Carbazosubphthalocyanines: Core-Expanded Antiaromatic Boron(III) Subphthalocyanine Analogues.

Condensation of 1,8-diamino-3,6-dichlorocarbazole with a series of disubstituted 1,3-diiminoisoindolines, followed by treatment with BF3 ⋅OEt2 led to the formation of the corresponding core-expanded boron(III) subphthalocyanine analogues. These air-stable π-conjugated boron(III) carbazosubphthalocyanines possess two boron-containing seven-membered-ring units and a 16 π-electron skeleton, and represent the first examples of antiaromatic boron(III) subphthalocyanine analogues as supported by spectroscopic and theoretical studies. The molecular structure of one of these compounds was unambiguously determined by single-crystal X-ray diffraction analysis. In contrast to typical boron(III) subphthalocyanines, which adopt a cone-shaped structure, the π skeleton of this compound is almost planar.

Assemblies of Boron Dipyrromethene/Porphyrin, Phthalocyanine, and C60 Moieties as Artificial Models of Photosynthesis: Synthesis, Supramolecular Interactions, and Photophysical Studies.

A series of light-harvesting conjugates based on a zinc(II) phthalocyanine core with either two or four boron dipyrromethene (BODIPY) or porphyrin units have been synthesized and characterized. The conjugation of BODIPY/porphyrin units can extend the absorptions of the phthalocyanine core to cover most of the visible region. Upon addition of an imidazole-substituted C60 (C60 Im), it can axially bind to the zinc(II) center of the phthalocyanine core through metal-ligand interactions. The resulting complexes form photosynthetic antenna-reaction center mimics in which the BODIPY/porphyrin units serve as the antennas to capture the light and transfer the energy to the phthalocyanine core by efficient excitation energy transfer. The excited phthalocyanine is then quenched by the axially bound C60 Im moiety by electron transfer, which has been supported by computational studies. The photoinduced processes of the assemblies have been studied in detail by various steady-state and time-resolved spectroscopic methods. By femtosecond transient absorption spectroscopic studies, the lifetimes of the charge-separated state of the bis(BODIPY) and bis(porphyrin) systems have been determined to be 3.2 and 4.0 ns, respectively.

Disulfide-Linked Dendritic Oligomeric Phthalocyanines as Glutathione-Responsive Photosensitizers for Photodynamic Therapy.

A series of disulfide-linked dendritic phthalocyanines were synthesized by using the CuI -catalyzed alkyne-azide cycloaddition reaction as the key step. Whereas these compounds were essentially nonaggregated in N,N-dimethylformamide, they were stacked in citrate solution (pH 7.4, with 1 % Cremophor EL), as shown by the broad appearance of their Q-band absorption. Having two-to-six zinc(II) phthalocyanine units in a molecule, these compounds were significantly self-quenched, particularly in citrate solution. Both the fluorescence intensity and singlet-oxygen generation efficiency were significantly lower than those of the monomeric counterparts, and the self-quenching efficiency increased as the number of phthalocyanine units increased. Upon interaction with 5 mm glutathione (GSH) in citrate solution, the fluorescence intensity of these compounds increased as a result of cleavage of the disulfide linkages and separation of the phthalocyanine units, which thereby reduced the self-quenching effect. The "on/off" ratios were found to be 7, 18, 23, and 21 for the dimeric (PC2), trimeric (PC3), tetrameric (PC4), and hexameric (PC6) systems, respectively. GSH also enhanced the fluorescence emission inside human colon adenocarcinoma HT29 cells and promoted the formation of singlet oxygen of these compounds. Upon irradiation, their half maximal inhibitory concentration (IC50 ) values were found to be in the range of 0.18 to 0.38 µm. Finally, the biodistribution and activation of PC2 and PC6 were also examined in HT29 tumor-bearing nude mice. For both compounds, the fluorescence intensity per unit area at the tumor was found to grow gradually during the first 24 h. Whereas the intensity then dropped for PC2, the intensity for PC6 remained steady over the following 6 d, which might have been a result of the enhanced permeability and retention effect arising from the larger molecular mass of the hexameric system.

Encapsulating pH-Responsive Doxorubicin-Phthalocyanine Conjugates in Mesoporous Silica Nanoparticles for Combined Photodynamic Therapy and Controlled Chemotherapy.

Doxorubicin (Dox) was conjugated to a zinc(II) phthalocyanine (ZnPc) through an acid-cleavable hydrazone linker. This azido-containing conjugate was then anchored to the nanochannels of an alkyne-modified mesoporous silica nanoparticle (MSN) system via copper(I)-catalyzed azide-alkyne cycloaddition. An analogous nanosystem was also prepared by immobilization of a hydrazine-substituted ZnPc to the MSN followed by coupling with Dox. The release of Dox under acidic conditions was studied in phosphate-buffered saline. After internalization into human hepatocellular carcinoma HepG2 cells, these nanoparticles showed fluorescence not only for ZnPc, but also for Dox, suggesting that release of Dox was triggered by the acidic intracellular environment. The chemocytotoxic Dox together with singlet oxygen generated upon irradiation on the encapsulated ZnPc in these MSNs could kill the cells effectively. A synergistic cytotoxicity was suggested by a less-than-unity combination index. These nanoparticles function as both nanophotosensitizers for photodynamic therapy and as nanoplatforms for pH-controlled drug release.

Aminophthalocyanine-Mediated Photodynamic Inactivation of Leishmania tropica.

Photodynamic inactivation ofLeishmaniaspp. requires the cellular uptake of photosensitizers, e.g., endocytosis of silicon(IV)-phthalocyanines (PC) axially substituted with bulky ligands. We report here that when substituted with amino-containing ligands, the PCs (PC1 and PC2) were endocytosed and displayed improved potency againstLeishmania tropicapromastigotes and axenic amastigotesin vitro The uptake of these PCs by bothLeishmaniastages followed saturation kinetics, as expected. Sensitive assays were developed for assessing the photodynamic inactivation ofLeishmaniaspp. by rendering them fluorescent in two ways: transfecting promastigotes to express green fluorescent protein (GFP) and loading them with carboxyfluorescein succinimidyl ester (CFSE). PC-sensitizedLeishmania tropicastrains were seen microscopically to lose their motility, structural integrity, and GFP/CFSE fluorescence after exposure to red light (wavelength, ∼650 nm) at a fluence of 1 to 2 J cm(-2) Quantitative fluorescence assays based on the loss of GFP/CFSE from liveLeishmania tropicashowed that PC1 and PC2 dose dependently sensitized both stages for photoinactivation, consistent with the results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay.Leishmania tropicastrains are >100 times more sensitive than their host cells or macrophages to PC1- and PC2-mediated photoinactivation, judging from the estimated 50% effective concentrations (EC50s) of these cells. Axial substitution of the PC with amino groups instead of other ligands appears to increase its leishmanial photolytic activity by up to 40-fold. PC1 and PC2 are thus potentially useful for photodynamic therapy of leishmaniasis and for oxidative photoinactivation ofLeishmaniaspp. for use as vaccines or vaccine carriers.

pH- and Thiol-Responsive BODIPY-Based Photosensitizers for Targeted Photodynamic Therapy.

A diiodo distyryl boron dipyrromethene (BODIPY) core was conjugated to two ferrocenyl quenchers through acid-labile ketal and/or thiol-cleavable disulfide linkers, of which the fluorescence and photosensitizing properties were significantly quenched through a photoinduced electron-transfer process. The two symmetrical analogues that contained either the ketal or disulfide linkers could only be activated by a single stimulus, whereas the unsymmetrical analogue was responsive to dual stimuli. Upon interaction with acid and/or dithiothreitol (DTT), these linkers were cleaved selectively. The separation of the BODIPY core and the ferrocenyl moieties restored the photoactivities of the former in phosphate buffered saline and inside the MCF-7 breast cancer cells, rendering these compounds as potential activable photosensitizers for targeted photodynamic therapy. The dual activable analogue exhibited the greatest enhancement in intracellular fluorescence intensity in both an acidic environment (pH 5) and the presence of DTT (4 mm). Its photocytotoxicity against MCF-7 cells also increased by about twofold upon preincubation with 4 mm of DTT. The activation of this compound was also demonstrated in nude mice bearing a HT29 human colorectal carcinoma. A significant increase in fluorescence intensity in the tumor was observed over 9 h after intratumoral injection.