Photoactive immunoconjugates for targeted photodynamic therapy of cancer.

Photodynamic therapy (PDT) has been used as an alternative or as a complement of conventional approaches for cancer treatment. In PDT, the reactive oxygen species (ROS) produced from the interaction between the photosensitizer (PS), visible light and molecular oxygen, kill malignant cells by triggering a cascade of cytotoxic reactions. In this process, the PS plays an extremely important role in the effectiveness of the therapy. In the present work, a new photoimmunoconjugate (PIC), based on cetuximab and the known third generation PS-glycophthalocyanine ZnPcGal4, was synthesized via reductive amination. The rationale behind this was the simultaneous cancer-associated specific targeting of PIC and photosensitization of targeted receptor positive cells. Varied reaction parameters and photodynamic conditions, such as PS concentrations and both type and intensities of light, were optimized. ZnPcGal4 showed significant photoactivity against EGFR expressing A431, EGFR-transfected HCT116 and HT29 cells when irradiated with white light of stronger intensity (38 mW/cm2). Similarly, the synthesized PICs-T1 and T2 also demonstrated photoactivity with high intensity white light. The best optimized PIC: sample 28 showed no precipitation and aggregation when inspected visually and analyzed through SE-HPLC. Fluorescence excitation of sample 28 and 125I-sample 28 radioconjugate (125I-PIC, 125I-radiolabeling yield ≥95%, determined with ITLC) at 660 nm showed presence of appended ZnPcGal4. In addition, simultaneous fluorescence and radioactivity detection of the 125I-PIC in serum and PBS (pH 7.4) for the longest incubated time point of 72 h, respectively, and superimposed signals thereof demonstrated ≥99% of loading and/or labeling yield, assuring overall stability of the PIC and corresponding PIC-radioconjugate w.r.t. both the appended ZnPcGal4 and bound-125I. Moreover, real-time binding analyses on EGFR-transfected HCT116 cells showed specific binding of 125I-PIC, suggesting no alternation in the binding kinetics of the mAb after appending it with ZnPcGal4. These results suggest dual potential applications of synthesized PICs both for PDT and radio-immunotherapy of cancer.

Editorial: Photodynamic Therapy as an Important Tool for Biological Breakthroughs-Photoactive Photosensitizers Applied from Cancer to Microbial Targets.

Photodynamic therapy (PDT) stands as an approved clinical treatment for both oncologic and nononcologic disorders [...].

Porphyrin NanoMetal-Organic Frameworks as Cancer Theranostic Agents.

Metal-Organic Frameworks (MOFs) are hybrid multifunctional platforms that have found remarkable applications in cancer treatment and diagnostics. Independently, these materials can be employed in cancer treatment as intelligent drug carriers in chemotherapy, photothermal therapy, and photodynamic therapy; conversely, MOFs can further be used as diagnostic tools in fluorescence imaging, magnetic resonance imaging, computed tomography imaging, and photoacoustic imaging. One essential property of these materials is their great ability to fine-tune their composition toward a specific application by way of a judicious choice of the starting building materials (metal nodes and organic ligands). Moreover, many advancements were made concerning the preparation of these materials, including the ability to downsize the crystallites yielding nanoporous porphyrin MOFs (NMOFs) which are of great interest for clinical treatment and diagnostic theranostic tools. The usage of porphyrins as ligands allows a high degree of multifunctionality. Historically these molecules are well known for their reactive oxygen species formation and strong fluorescence characteristics, and both have proved helpful in cancer treatment and diagnostic tools. The anticipation that porphyrins in MOFs could prompt the resulting materials to multifunctional theranostic platforms is a reality nowadays with a series of remarkable and ground-breaking reports available in the literature. This is particularly remarkable in the last five years, when the scientific community witnessed rapid development in porphyrin MOFs theranostic agents through the development of imaging technologies and treatment strategies for cancer. This manuscript reviews the most relevant recent results and achievements in this particular area of interest in MOF chemistry and application.

The Surprisingly Positive Effect of Zinc-Phthalocyanines With High Photodynamic Therapy Efficacy of Melanoma Cancer.

Phthalocyanine (Pc) dyes are photoactive molecules that can absorb and emit light in the visible spectrum, especially in the red region of the spectrum, with great potential for biological scopes. For this target, it is important to guarantee a high Pc solubility, and the use of suitable pyridinium units on their structure can be a good strategy to use effective photosensitizers (PSs) for photodynamic therapy (PDT) against cancer cells. Zn(II) phthalocyanines (ZnPcs) conjugated with thiopyridinium units (1-3) were evaluated as PS drugs against B16F10 melanoma cells, and their photophysical, photochemical, and in vitro photobiological properties were determined. The photodynamic efficiency of the tetra- and octa-cationic ZnPcs 1-3 was studied and compared at 1, 2, 5, 10, and 20 µM. The different number of charge units, and the presence/absence of a-F atoms on the Pc structure, contributes for their PDT efficacy. The 3-(4',5'-dimethylthiazol-2'-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays on B16F10 melanoma cells show a moderate to high capacity to be photoinactivated by ZnPcs 1-3 (ZnPc 1 > ZnPc 2 > ZnPc 3). The best PDT conditions were found at a Pc concentration of 20 µM, under red light (λ = 660 ± 20 nm) at an irradiance of 4.5 mW/cm2 for 667 s (light dose of 3 J/cm2). In these conditions, it is noteworthy that the cationic ZnPc 1 shows a promising photoinactivation ratio, reaching the detection limit of the MTT method. Moreover, these results are comparable to the better ones in the literature.

Spherical and rod shaped mesoporous silica nanoparticles for cancer-targeted and photosensitizer delivery in photodynamic therapy.

Mesoporous silica nanoparticles (MSNPs) have attracted much attention in many biomedical applications. One of the fields in which smart functional nanosystems have found wide application is cancer treatment. Here, we present new silica nanoparticle-based systems which have been explored as efficient vehicles to transport and deliver photosensitizers (PSs) into tumor tissues during photodynamic therapy (PDT). In this work, we report the preparation, characterization, and in vitro studies of distinct shaped MSNPs grafted with S-glycoside porphyrins (Pors). The ensuing nanomaterials were fully characterized, and their properties as third-generation PSs for PDT against two bladder cancer cell lines, HT-1376 and UM-UC-3, were examined. The best uptake results were obtained for MSNP-PS2, while MSNP-PS1 showed the lowest cellular uptake among the nanocarriers tested, but revealed the best phototoxicity in both cancer cells. Overall, the phototoxicity was higher with MSNPs than with mesoporous silica nanorods (MSNRs) and higher uptake and phototoxicity were consistently observed in UM-UC-3 rather than in HT-1376 cancer cells.

Complex cellular environments imaged by SERS nanoprobes using sugars as an all-in-one vector.

Raman spectroscopy coupled with confocal microscopy offers an alternative bioimaging technique overcoming limitations associated with sensitivity, tissue penetration and image resolution. Allied to the surface-enhanced Raman scattering (SERS) properties of gold nanoparticles (AuNP), we designed SERS nanoprobes with enhanced properties and straightforward application as bio-labelling agents for gliomas. The ensuing nanoprobes coated with simple sugar units (galactose or glucose) allowed assessing information about their intracellular localization (vesicular structures), with impressive sensitivity towards complex environments and proved the ability to overcome biological auto-fluorescence and high penetration in tissues. We validate the use of sugars as an all-in-one vector (Raman reporter, conferring high stability, biocompatibility and affinity to glioma cells) as imaging agents using an impressive technique.

Unsymmetrical cationic porphyrin-cyclodextrin bioconjugates for photoinactivation of Escherichia coli.

Photodynamic inactivation (PDI) of microorganisms has been used for the treatment of bacterial infection. PDI is based on the combination of three non-toxic elements: a photosensitizer (PS), light and molecular oxygen, which lead to the formation of reactive oxygen species (ROS) that cause lethal oxidative damage into the target pathogenic bacteria. For that, clinical approved tetrapyrrolic macrocycles, with particular emphasis on photoactive porphyrin (Por) dyes, have been used as PS in PDI for different biomedical applications. Two novel unsymmetrical free-base thiopyridyl Pors conjugated with α- or γ-CD units (Pors 2 and 3) were prepared and the corresponding cationic ones (Pors 2a and 3a) were assessed as water-soluble photosensitizer (PS) agents by photophysical, photochemical and E. coli photobiological studies. The presence of the CD unit and the positive charges on the Por periphery (2a and 3a) enhance their solubility in aqueous media. The photoactivity of the two cationic Pors 2a and 3a ensures their potential as PDI drugs against Gram-negative bacteria model, a bioluminescent E. coli, which the best PDI efficiency was determined for Por 3a that achieved the highest bacterial reduction of 4.0 log10 (ANOVA, p < 0.0001), reaching the detection limit of the method after 15 min.

Phthalocyanines for G-quadruplex aptamers binding.

The G-quadruplex (G4)-forming sequence within the AS1411 derivatives with alternative nucleobases and backbones can improve the chemical and biological properties of AS1411. Zn(II) phthalocyanine (ZnPc) derivatives have potential as high-affinity G4 ligands because they have similar size and shape to the G-quartets. The interactions of four Zn(II) phthalocyanines with the G4 AS1411 aptamer and its derivatives were determined by biophysical techniques, molecular docking and gel electrophoresis. Cell viability assay was carried out to evaluate the antiproliferative effects of Zn(II) phthalocyanines and complexes. CD experiments showed structural changes after addition of ZnPc 4, consistent with multiple binding modes and conformations shown by NMR and gel electrophoresis. CD melting confirmed that ZnPc 2 and ZnPc 4, both containing eight positive charges, are able to stabilize the AT11 G4 structure (ΔTm > 30 °C and 18.5 °C, respectively). Molecular docking studies of ZnPc 3 and ZnPc 4 suggested a preferential binding to the 3'- and 5'-end, respectively, of the AT11 G4. ZnPc 3 and its AT11 and AT11-L0 complexes revealed pronounced cytotoxic effect against cervical cancer cells and no cytotoxicity to normal human cells. Zn(II) phthalocyanines provide the basis for the development of effective therapeutic agents as G4 ligands.

Synthesis, Characterization and Photodynamic Activity against Bladder Cancer Cells of Novel Triazole-Porphyrin Derivatives.

Novel triazole-porphyrin derivatives (TZ-PORs) were synthesized through the Heck reaction and then incorporated into polyvinylpyrrolidone (PVP) micelles. After verifying that this incorporation did not compromise the photophysical and chemical features of TZ-PORs as photosensitizers, the phototoxicity of the formulations towards cancer cells was screened. Biological studies show high photodynamic activity of all PVP-TZ-POR formulations against a bladder cancer cell line with a particular highlight to PVP-TZ-POR 7e and 7f that are able to significantly reduce HT-1376 cell viability, while they had no effect on control ARPE-19 cells.

Caveolin-1 Modulation Increases Efficacy of a Galacto-Conjugated Phthalocyanine in Bladder Cancer Cells Resistant to Photodynamic Therapy.

Photodynamic therapy (PDT) has demonstrated encouraging anticancer therapeutic results, but the current clinically approved photosensitizers (PSs) are not ideal in the treatment of bladder cancer. Conventional PSs have low selectivity to the bladder tumor tissue and induce toxicity or bystander effects on nontumor urothelium. Previous studies demonstrated that the use of galactose-photosensitizer (PS) conjugates is a more selective method of delivering PDT-mediated toxicity due to their ability to recognize carbohydrate-binding domains overexpressed in bladder tumors. Using patient-derived bladder tumor specimens cultured ex vivo and bladder cancer cell lines with different PDT sensitivity, we find that a galactose-phthalocyanine (PcGal16) accumulates in bladder tumors expressing galactose-binding proteins and internalizes through an endocytic process. The endocytosis mechanism is cell line-dependent. In HT-1376 bladder cancer lines resistant to PDT, depletion of caveolin-1-the main structural protein of caveolae structures-increased the amount of sugar-binding proteins, i.e. GLUT1, at the cell membrane resulting in an improved PcGal16 uptake and PDT efficacy. These data show the potential of ex vivo cultures of bladder cancer, that ideally could mimic the original microenvironment, in screening galacto-PDT agents. Additionally, our studies demonstrate that PDT efficacy in bladder cancer depends on the endocytic mechanisms that regulate PS accumulation and internalization in cancer cells.

PET/CT Imaging with an 18F-Labeled Galactodendritic Unit in a Galectin-1-Overexpressing Orthotopic Bladder Cancer Model.

Galectins are carbohydrate-binding proteins overexpressed in bladder cancer (BCa) cells. Dendritic galactose moieties have a high affinity for galectin-expressing tumor cells. We radiolabeled a dendritic galactose carbohydrate with 18F (18F-labeled galactodendritic unit 4) and examined its potential in imaging urothelial malignancies. Methods: The 18F-labeled first-generation galactodendritic unit 4 was obtained from its tosylate precursor. We conducted in vivo studies in a galectin-expressing UMUC3 orthotopic BCa model to determine the ability of 18F-labeled galactodendritic unit 4 to image BCa. Results: Intravesical administration of 18F-labeled galactodendritic unit 4 allowed specific accumulation of the carbohydrate radiotracer in galectin-1-overexpressing UMUC3 orthotopic tumors when imaged with PET. The 18F-labeled galactodendritic unit 4 was not found to accumulate in nontumor murine bladders. Conclusion: The 18F-labeled galactodendritic unit 4 and similar analogs may be clinically relevant and exploitable for PET imaging of galectin-1-overexpressing bladder tumors.

Highly Efficient Singlet Oxygen Generators Based on Ruthenium Phthalocyanines: Synthesis, Characterization and in vitro Evaluation for Photodynamic Therapy.

Invited for the cover of this issue is the group of Torres at the University of Madrid. The image of the cover of this issue depicts cancer cells being destroyed by reactive singlet oxygen produced by ruthenium phthalocyanine glycoconjugates under red light. The work, developed at the Universities of Madrid, Aveiro, Lisbon and Coimbra, describes ruthenium phthalocyanines as powerful bladder cancer PDT agents. Read the full text of the article at 10.1002/chem.201903546.

Highly Efficient Singlet Oxygen Generators Based on Ruthenium Phthalocyanines: Synthesis, Characterization and in vitro Evaluation for Photodynamic Therapy.

The synthesis of ruthenium(II) phthalocyanines (RuPcs) endowed with one carbohydrate unit-that is, glucose, galactose and mannose-and a dimethylsulfoxide (DMSO) ligand at the two axial coordination sites, respectively, is described. Two series of compounds, one unsubstituted at the periphery, and the other one bearing eight PEG chains at the isoindole meta-positions, have been prepared. The presence of the axial DMSO unit significantly increases the phthalocyanine singlet oxygen quantum yields, related to other comparable RuPcs. The compounds have been evaluated for PDT treatment in bladder cancer cells. In vitro studies have revealed high phototoxicity for RuPcs unsubstituted at their periphery. The phototoxicity of PEG-substituted RuPcs has been considerably improved by repeated light irradiation. The choice of the axial carbohydrate introduced little differences in the cellular uptake for both series of photosensitizers, but the phototoxic effects were considerably higher for compounds bearing mannose units.

Multicharged Phthalocyanines as Selective Ligands for G-Quadruplex DNA Structures.

The stabilization of G-Quadruplex DNA structures by ligands is a promising strategy for telomerase inhibition in cancer therapy since this enzyme is responsible for the unlimited proliferation of cancer cells. To assess the potential of a compound as a telomerase inhibitor, selectivity for quadruplex over duplex DNA is a fundamental attribute, as the drug must be able to recognize quadruplex DNA in the presence of a large amount of duplex DNA, in the cellular nucleus. By using different spectroscopic techniques, such as ultraviolet-visible, fluorescence and circular dichroism, this work evaluates the potential of a series of multicharged phthalocyanines, bearing four or eight positive charges, as G-Quadruplex stabilizing ligands. This work led us to conclude that the existence of a balance between the number and position of the positive charges in the phthalocyanine structure is a fundamental attribute for its selectivity for G-Quadruplex structures over duplex DNA structures. Two of the studied phthalocyanines, one with four peripheral positive charges (ZnPc1) and the other with less exposed eight positive charges (ZnPc4) showed high selectivity and affinity for G-Quadruplex over duplex DNA structures and were able to accumulate in the nucleus of UM-UC-3 bladder cancer cells.

Photoimmunoconjugates: novel synthetic strategies to target and treat cancer by photodynamic therapy.

Photodynamic therapy (PDT) combines a photosensitizer (PS) with the physical energy of non-ionizing light to trigger cell death pathways. PDT has potential as a therapeutic modality to be used in alternative or in combination with other conventional cancer treatment protocols (e.g. surgery, chemotherapy and radiotherapy). Still, due to the lack of specificity of the current PSs to target the tumor cells, several studies have exploited their conjugation with targeting moieties. PSs conjugated with antibodies (Abs) or their fragments, able to bind antigens overexpressed in the tumors, have demonstrated potential in PDT of tumors. This review provides an overview of the most recent advances on photoimmunoconjugates (PICs) for cancer PDT, which involve the first and second-generation PSs conjugated to Abs. This is an update of our previous review "Antibodies armed with photosensitizers: from chemical synthesis to photobiological applications", published in 2015 in Org. Biomol. Chem.

Compromising the plasma membrane as a secondary target in photodynamic therapy-induced necrosis.

Photodynamic therapy (PDT) is a non-invasive treatment widely applied to different cancers. The goal of PDT is the photo-induced destruction of cancer cells by the activation of different cell death mechanisms, including apoptosis and/or necrosis. Recent efforts focusing on understanding the mechanisms of cell death activated by PDT find that it depends on the type of photosensitizer (PS), targeted organelles, and nature of the light used. It is generally accepted that very short incubation times are required to direct the PS to the plasma membrane (PM), while longer periods result in the accumulation of the PS in internal compartments such as the endoplasmic reticulum or mitochondria. Glycosylation of the PS targets cancer via saccharide receptors on the cell surface, and is generally assumed that these compounds rapidly internalize and accumulate, e.g. in the endoplasmic reticulum. Herein we demonstrate that a minor fraction of a glycosylated chlorin compound residing at the PM of cancer cells can activate necrosis upon illumination by compromising the PM independently of the length of the incubation period. The results presented here show that the PM can also be targeted by glycosylated PS designed to accumulate in internal organelles. PS activation to induce necrosis by compromising the plasma membrane has the benefits of fast cell death and shorter irradiation times. The findings described here expand our understanding of the cellular damage induced by phototherapies, presenting the possibility of activating another cell death mechanism based on the incubation time and type of light used.

Metal-organic framework assembled from erbium and a tetrapodal polyphosphonic acid organic linker.

A three-dimensional metal-organic framework (MOF), poly[[µ6-5'-pentahydrogen [1,1'-biphenyl]-3,3',5,5'-tetrayltetrakis(phosphonato)]erbium(III)] 2.5-hydrate], formulated as [Er(C12H11O12P4)]·2.5H2O or [Er(H5btp)]·2.5H2O (I) and isotypical with a Y3+-based MOF reported previously by our research group [Firmino et al. (2017b). Inorg. Chem. 56, 1193-1208], was constructed based solely on Er3+ and on the polyphosphonic organic linker [1,1'-biphenyl]-3,3',5,5'-tetrakis(phosphonic acid) (H8btp). The present work describes our efforts to introduce lanthanide cations into the flexible network, demonstrating that, on the one hand, the compound can be obtained using three distinct experimental methods, i.e. hydro(solvo)thermal (Hy), microwave-assisted (MW) and one-pot (Op), and, on the other hand, that crystallite size can be approximately fine-tuned according to the method employed. MOF I contains hexacoordinated Er3+ cations which are distributed in a zigzag inorganic chain running parallel to the [100] direction of the unit cell. The chains are, in turn, bridged by the anionic organic linker to form a three-dimensional 6,6-connected binodal network. This connectivity leads to the existence of one-dimensional channels (also running parallel to the [100] direction) filled with disordered and partially occupied water molecules of crystalization which are engaged in O-H...O hydrogen-bonding interactions with the [Er(H5btp)] framework. Additional weak π-π interactions [intercentroid distance = 3.957 (7) Å] exist between aromatic rings, which help to maintain the structural integrity of the network.

Carbon-1 versus Carbon-3 Linkage of d-Galactose to Porphyrins: Synthesis, Uptake, and Photodynamic Efficiency.

The use of glycosylated compounds is actively pursued as a therapeutic strategy for cancer due to the overexpression of various types of sugar receptors and transporters on most cancer cells. Conjugation of saccharides to photosensitizers such as porphyrins provides a promising strategy to improve the selectivity and cell uptake of the photosensitizers, enhancing the overall photosensitizing efficacy. Most porphyrin-carbohydrate conjugates are linked via the carbon-1 position of the carbohydrate because this is the most synthetically accessible approach. Previous studies suggest that carbon-1 galactose derivatives show diminished binding since the hydroxyl group in the carbon-1 position of the sugar is a hydrogen bond acceptor in the galectin-1 sugar binding site. We therefore synthesized two isomeric porphyrin-galactose conjugates using click chemistry: one linked via the carbon-1 of the galactose and one linked via carbon-3. Free base and zinc analogs of both conjugates were synthesized. We assessed the uptake and photodynamic therapeutic (PDT) activity of the two conjugates in both monolayer and spheroidal cell cultures of four different cell lines. For both the monolayer and spheroid models, we observe that the uptake of both conjugates is proportional to the protein levels of galectin-1 and the uptake is suppressed after preincubation with an excess of thiogalactose, as measured by fluorescence spectroscopy. Compared to that of the carbon-1 conjugate, the uptake of the carbon-3 conjugate was greater in cell lines containing high expression levels of galectin-1. After photodynamic activation, MTT and lactate dehydrogenase assays demonstrated that the conjugates induce phototoxicity in both monolayers and spheroids of cancer cells.

A Galactose Dendritic Silicon (IV) Phthalocyanine as a Photosensitizing Agent in Cancer Photodynamic Therapy.

Two protected galacto-dendritic units have been axially coordinated to the central ion of a silicon(IV) phthalocyanine to afford SiPcPGal4 containing four units of galactose per macrocycle. These biological moieties provided better solubility in aqueous medium and a sensitizer with higher absorption peaks at 680-690 nm. The photodynamic activity of SiPcPGal4 was evaluated against UM-UC-3 human bladder cancer cell line and the results were compared with the activity of the reported SiPcPGal2 and SiPc(OH)2 . SiPcPGal4 had a better uptake and it was a better toxicity inducer than SiPcPGal2 and SiPc(OH)2 owing to its four galactose units, protected by isopropylidene groups, which can act as targeted micelles.

Porphyrin modified trastuzumab improves efficacy of HER2 targeted photodynamic therapy of gastric cancer.

Gastric cancer (GC) is the 3rd deadliest cancer worldwide, due to limited treatment options and late diagnosis. Human epidermal growth factor receptor-2 (HER2) is overexpressed in ∼20% of GC cases and anti-HER2 antibody trastuzumab in combination with conventional chemotherapy, is recognized as standard therapy for HER2-positive metastatic GC. This strategy improves GC patients' survival by 2-3 months, however its optimal results in breast cancer indicate that GC survival may be improved. A new photoimmunoconjugate was developed by conjugating a porphyrin with trastuzumab (Trast:Porph) for targeted photodynamic therapy in HER2-positive GC. Using mass spectrometry analysis, the lysine residues in the trastuzumab structure most prone for porphyrin conjugation were mapped. The in vitro data demonstrates that Trast:Porph specifically binds to HER2-positive cells, accumulates intracellularly, co-localizes with lysosomal marker LAMP1, and induces massive HER2-positive cell death upon cellular irradiation. The high selectivity and cytotoxicity of Trast:Porph based photoimmunotherapy is confirmed in vivo in comparison with trastuzumab alone, using nude mice xenografted with a HER2-positive GC cell line. In the setting of human disease, these data suggest that repetitive cycles of Trast:Porph photoimmunotherapy may be used as an improved treatment strategy in HER2-positive GC patients.