meso-Tetrahexyl-7,8-dihydroxychlorin and Its Conversion to ß-Modified Derivatives.

meso-Tetrahexylporphyrin was converted to its corresponding 7,8-dihydroxychlorin using an osmium tetroxide-mediated dihydroxylation strategy. Its diol moiety was shown to be able to undergo a number of subsequent oxidation reactions to form a chlorin dione and porpholactone, the first meso-alkylporphyrin-based porphyrinoid containing a non-pyrrolic building block. Further, the diol chlorin was shown to be susceptible to dehydration, forming the porphyrin enol that is in equilibrium with its keto-chlorin form. The meso-hexylchlorin dione could be reduced and it underwent mono- and bis-methylation reactions using methyl-Grignard reagents, and trifluoromethylation using the Ruppert-Prakash reagent. The optical and spectroscopic properties of the products are discussed and contrasted to their corresponding meso-aryl derivatives (where known). This contribution establishes meso-tetrahexyl-7,8-dihydroxychlorins as a new and versatile class of chlorins that is susceptible to a broad range of conversions to generate functionalized chlorins and a pyrrole-modified chlorin analogue.

Bromo- and glycosyl-substituted BODIPYs for application in photodynamic therapy and imaging.

The introduction of heavy atoms into the BODIPY-core structure has proven to be a straightforward strategy for optimizing the design of such dyes towards enhanced generation of singlet oxygen rendering them suitable as photosensitizers for photodynamic therapy (PDT). In this work, BODIPYs are presented by combining the concept of bromination with nucleophilic aromatic substitution (SNAr) of a pentafluorophenyl or a 4-fluoro-3-nitrophenyl moiety to introduce functional groups, thus improving the phototoxic effect of the BODIPYs as well as their solubility in the biological environment. The nucleophilic substitution enabled functionalization with various amines and alcohols as well as unprotected thiocarbohydrates. The phototoxic activity of these more than 50 BODIPYs has been assessed in cellular assays against four cancer cell lines in order to more broadly evaluate their PDT potential, thus accounting for the known variability between cell lines with respect to PDT activity. In these investigations, dibrominated polar-substituted BODIPYs, particularly dibrominated glyco-substituted compounds, showed promising potential as photomedicine candidates. Furthermore, the cellular uptake of the glycosylated BODIPYs has been confirmed via fluorescence microscopy.

The Photosensitizer Temoporfin (mTHPC) - Chemical, Pre-clinical and Clinical Developments in the Last Decade.

This review follows the research, development and clinical applications of the photosensitizer 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (mTHPC, temoporfin) in photodynamic (cancer) therapy (PDT) and other medical applications. Temoporfin is the active substance in the medicinal product Foscan® authorized in the EU for the palliative treatment of head and neck cancer. Chemistry, biochemistry and pharmacology, as well as clinical and other applications of temoporfin are addressed, including the extensive work that has been done on formulation development including liposomal formulations. The literature has been covered from 2009 to early 2022, thereby connecting it to the previous extensive review on this photosensitizer published in this journal [Senge, M. O. and J. C. Brandt (2011) Photochem. Photobiol. 87, 1240-1296] which followed its way from initial development to approval and clinical application.

A HET-CAM based vascularized intestine tumor model as a screening platform for nano-formulated photosensitizers.

The development of new tumor models for anticancer drug screening is a challenge for preclinical research. Conventional cell-based in vitro models such as 2D monolayer cell cultures or 3D spheroids allow an initial assessment of the efficacy of drugs but they have a limited prediction to the in vivo effectiveness. In contrast, in vivo animal models capture the complexity of systemic distribution, accumulation, and degradation of drugs, but visualization of the individual steps is challenging and extracting quantitative data is usually very difficult. Furthermore, there are a variety of ethical concerns related to animal tests. In accordance with the 3Rs principles of Replacement, Reduction and Refinement, alternative test systems should therefore be developed and applied in preclinical research. The Hen's egg test on chorioallantoic membrane (HET-CAM) model provides the generation of vascularized tumor spheroids and therefore, is an ideal test platform which can be used as an intermediate step between in vitro analysis and preclinical evaluation in vivo. We developed a HET-CAM based intestine tumor model to investigate the accumulation and efficacy of nano-formulated photosensitizers. Irradiation is necessary to activate the phototoxic effect. Due to the good accessibility of the vascularized tumor on the CAM, we have developed a laser irradiation setup to simulate an in vivo endoscopic irradiation. The study presents quantitative as well as qualitative data on the accumulation and efficacy of the nano-formulated photosensitizers in a vascularized intestine tumor model.

Investigating Alkylated Prodigiosenes and Their Cu(II)-Dependent Biological Activity: Interactions with DNA, Antimicrobial and Photoinduced Anticancer Activity.

Prodigiosenes are a family of red pigments with versatile biological activity. Their tripyrrolic core structure has been modified many times in order to manipulate the spectrum of activity. We have been looking systematically at prodigiosenes substituted at the C ring with alkyl chains of different lengths, in order to assess the relevance of this substituent in a context that has not been investigated before for these derivatives: Cu(II) complexation, DNA binding, self-activated DNA cleavage, photoinduced cytotoxicity and antimicrobial activity. Our results indicate that the hydrophobic substituent has a clear influence on the different aspects of their biological activity. The cytotoxicity study of the Cu(II) complexes of these prodigiosenes shows that they exhibit a strong cytotoxic effect towards the tested tumor cell lines. The Cu(II) complex of a prodigiosene lacking any alkyl chain excelled in its photoinduced anticancer activity, thus demonstrating the potential of prodigiosenes and their metal complexes for an application in photodynamic therapy (PDT). Two derivatives along with their Cu(II) complexes showed also antimicrobial activity against Staphylococcus aureus strains.

Dipyrrinato-Iridium(III) Complexes for Application in Photodynamic Therapy and Antimicrobial Photodynamic Inactivation.

The generation of bio-targetable photosensitizers is of utmost importance to the emerging field of photodynamic therapy and antimicrobial (photo-)therapy. A synthetic strategy is presented in which chelating dipyrrin moieties are used to enhance the known photoactivity of iridium(III) metal complexes. Formed complexes can thus be functionalized in a facile manner with a range of targeting groups at their chemically active reaction sites. Dipyrrins with N- and O-substituents afforded (dipy)iridium(III) complexes via complexation with the respective Cp*-iridium(III) and ppy-iridium(III) precursors (dipy=dipyrrinato, Cp*=pentamethyl-η5 -cyclopentadienyl, ppy=2-phenylpyridyl). Similarly, electron-deficient [IrIII (dipy)(ppy)2 ] complexes could be used for post-functionalization, forming alkenyl, alkynyl and glyco-appended iridium(III) complexes. The phototoxic activity of these complexes has been assessed in cellular and bacterial assays with and without light; the [IrIII (Cl)(Cp*)(dipy)] complexes and the glyco-substituted iridium(III) complexes showing particular promise as photomedicine candidates. Representative crystal structures of the complexes are also presented.

Exploring the relationship between structure and activity in BODIPYs designed for antimicrobial phototherapy.

A synthetic strategy to BODIPY dyes is presented giving access to a range of new compounds relevant in the context of antimicrobial photodynamic therapy (aPDT). BODIPYs with the 8-(4-fluoro-3-nitrophenyl) and the 8-pentafluorophenyl substituents were used for the synthesis of new mono- and dibrominated BODIPYs. The para-fluorine atoms in these electron-withdrawing groups facilitate functional modification via nucleophilic aromatic substitution (SNAr) with a number of amines and thio-carbohydrates. Subsequently, the antibacterial phototoxic activity of these BODIPYs has been assessed in bacterial assays against the Gram-positive germ S. aureus and also against the Gram-negative germ P. aeruginosa. The bacterial assays allowed to identify substitution patterns which ensured antibacterial activity not only in phosphate-buffered saline (PBS) but also in the presence of serum, hereby more realistically modelling the complex biological environment that is present in clinical applications.

Matryoshka-Type Liposomes Offer the Improved Delivery of Temoporfin to Tumor Spheroids.

The balance between the amount of drug delivered to tumor tissue and the homogeneity of its distribution is a challenge in the efficient delivery of photosensitizers (PSs) in photodynamic therapy (PDT) of cancer. To date, many efforts have been made using various nanomaterials to efficiently deliver temoporfin (mTHPC), one of the most potent photosensitizers. The present study aimed to develop double-loaded matryoshka-type hybrid nanoparticles encapsulating mTHPC/cyclodextrin inclusion complexes in mTHPC-loaded liposomes. This system was expected to improve the transport of mTHPC to target tissues and to strengthen its accumulation in the tumor tissue. Double-loaded hybrid nanoparticles (DL-DCL) were prepared, characterized, and tested in 2D and 3D in vitro models and in xenografted mice in vivo. Our studies indicated that DL-DCL provided deep penetration of mTHPC into the multicellular tumor spheroids via cyclodextrin nanoshuttles once the liposomes had been destabilized by serum proteins. Unexpectedly, we observed similar PDT efficiency in xenografted HT29 tumors for liposomal mTHPC formulation (Foslip®) and DL-DCL.

Trends and targets in antiviral phototherapy.

Photodynamic therapy (PDT) is a well-established treatment option in the treatment of certain cancerous and pre-cancerous lesions. Though best-known for its application in tumor therapy, historically the photodynamic effect was first demonstrated against bacteria at the beginning of the 20th century. Today, in light of spreading antibiotic resistance and the rise of new infections, this photodynamic inactivation (PDI) of microbes, such as bacteria, fungi, and viruses, is gaining considerable attention. This review focuses on the PDI of viruses as an alternative treatment in antiviral therapy, but also as a means of viral decontamination, covering mainly the literature of the last decade. The PDI of viruses shares the general action mechanism of photodynamic applications: the irradiation of a dye with light and the subsequent generation of reactive oxygen species (ROS) which are the effective phototoxic agents damaging virus targets by reacting with viral nucleic acids, lipids and proteins. Interestingly, a light-independent antiviral activity has also been found for some of these dyes. This review covers the compound classes employed in the PDI of viruses and their various areas of use. In the medical area, currently two fields stand out in which the PDI of viruses has found broader application: the purification of blood products and the treatment of human papilloma virus manifestations. However, the PDI of viruses has also found interest in such diverse areas as water and surface decontamination, and biosafety.

A dialysis-based in vitro drug release assay to study dynamics of the drug-protein transfer of temoporfin liposomes.

Today, a growing number of nanotherapeutics is utilized to deliver poorly soluble compounds using the intravenous route of administration. The drug release and the direct transfer of the active pharmaceutical ingredient to serum proteins plays an important role in bioavailability and accumulation of the drug at the target site. It is closely related to the formation of a protein corona as well as the plasma protein binding of the compound. In the present study, two in vitro drug release methods, the flow-through cell and the dispersion releaser technology, were evaluated with regards to their capability to measure a time-resolved profile of the serum protein binding. In this context, the photosensitizer temoporfin and temoporfin-loaded liposomes were tested. While in the fine capillaries of the flow-through cell a rapid agglomeration of proteins occurred, the dispersion releaser technology in combination with the four-step model enabled the measurement of the transfer of drugs from liposomes to proteins. In presence of 10% of fetal calf serum approximately 20% of the model compound temoporfin were bound to serum proteins within the first 3 h. At higher serum concentration this binding remained stable for approximately 10 h.

Nucleophilic Thioglycosylation of Pentafluorophenyl-Substituted Porphyrinoids: Synthesis of Glycosylated Calix[n]phyrin and [28]Hexaphyrin Systems.

The use of carbohydrate thiolates for facile, high-yielding, regio- and stereoselective nucleophilic substitution reactions of complex pentafluorophenyl-substituted porphyrinoids is reported. The title reaction has successfully been applied to calix[4]phyrin, calix[6]phyrin, and [28]hexaphyrin substrates. The novel glycoporphyrinoid products with their extraordinary structures and unique photophysical properties are soluble in aqueous solutions and can serve as platforms for applications in biomedicine, catalysis, coordination, or redox chemistry.

Temoporfin-in-Cyclodextrin-in-Liposome-A New Approach for Anticancer Drug Delivery: The Optimization of Composition.

The main goal of this study was to use hybrid delivery system for effective transportation of temoporfin (meta-tetrakis(3-hydroxyphenyl)chlorin, mTHPC) to target tissue. We suggested to couple two independent delivery systems (liposomes and inclusion complexes) to achieve drug-in-cyclodextrin-in-liposome (DCL) nanoconstructs. We further optimized the composition of DCLs, aiming to alter in a more favorable way a distribution of temoporfin in tumor tissue. We have prepared DCLs with different compositions varying the concentration of mTHPC and the type of ß-cyclodextrin (ß-CD) derivatives (Hydroxypropyl-, Methyl- and Trimethyl-ß-CD). DCLs were prepared by thin-hydration technique and mTHPC/ß-CD complexes were added at hydration step. The size was about 135 nm with the surface charge of (-38 mV). We have demonstrated that DCLs are stable and almost all mTHPC is bound to ß-CDs in the inner aqueous liposome core. Among all tested DCLs, trimethyl-ß-CD-based DCL demonstrated a homogenous accumulation of mTHPC across tumor spheroid volume, thus supposing optimal mTHPC distribution.

Pre-/post-functionalization in dipyrrin metal complexes - antitumor and antibacterial activity of their glycosylated derivatives.

A post-functionalization route to tris(dipyrrinato) metal complexes is presented giving access to a range of new complexes relevant in the context of medicinal inorganic chemistry. A pentafluorophenyl group in the meso-position of the dipyrrin ligand serves as an anchor for the connection with alcohols and thiocarbohydrates. The photochemotherapeutic activity of the complexes has been assessed in cellular assays with tumor cell lines and against the Gram-positive bacterium S. aureus. Finally, it is shown that this post-functionalization is also applicable to other dipyrrinato metal complexes.

Photodynamic activity of Temoporfin nanoparticles induces a shift to the M1-like phenotype in M2-polarized macrophages.

The monocyte/macrophage cell lineage reveals an enormous plasticity, which is required for tissue homeostasis, but is also undermined in various disease states, leading to a functional involvement of macrophages in major human diseases such as atherosclerosis and cancer. We recently generated in vivo evidence that crystalline, nonfluorescent nanoparticles of the hydrophobic porphyrin-related photosensitizer Aluminum phthalocyanine are selectively dissolved and thus may be used for specific fluorescent labelling of rejected, but not of accepted xenotransplants. This led us to hypothesize that nanoparticles made of planar photosensitizers such as porphyrins and chlorins were preferentially taken up and dissolved by macrophages, which was verified by in vitro studies. Here, using an in vitro system for macrophage differentiation/polarization of the human monocyte THP-1 cell line, we demonstrate differential uptake/dissolution of Temoporfin-derived nanoparticles in polarized macrophages, which resulted in differential photosensitivity. More importantly, low dose photodynamic sensitization using Temoporfin nanoparticles can be used to trigger M1 re-polarization of THP-1 cells previously polarized to the M2 state. Thus, sublethal photodynamic treatment using Temoporfin nanoparticles might be applied to induce a phenotypic shift of tumor-associated macrophages for the correction of an immunosuppressive microenvironment in the treatment of cancer, which may synergize with immune checkpoint inhibition.

Chlorin Nanoparticles for Tissue Diagnostics and Photodynamic Therapy.

BACKGROUND: Organic crystalline nanoparticles (NPs) are not fluorescent due to the crystalline structure of the flat molecules organized in layers. In earlier experiments with Aluminum Phthalocyanine (AlPc)-derived NPs, the preferential uptake and dissolution by macrophages was demonstrated [3]. Therefore, inflamed tissue or cancer tissue with accumulated macrophages may exhibit specific fluorescence in contrast to healthy tissue which does not fluoresce. The present study addresses the photobiological effects of NP generated from Temoporfin (mTHPC), a clinically utilized photosensitizer belonging to the chlorin family. METHODS: In-vitro investigations addressing uptake, dissolution and phototoxicity of mTHPC NP vs. the liposomal mTHPC formulation Foslip were performed using J774A.1 macrophages and L929 fibroblasts. For total NP uptake analysis, the cells were lysed, the nanoparticles dissolved and the fluorescence quantified. The intracellular molecular dissolution was measured by flow cytometry. Fluorescence microscopy served for controlling intracellular localization of the dissolved fluorescing molecules. Reaction mechanisms after PDT (mitochondrial activity, apoptosis) were analyzed using fluorescent markers in cell-based assays and flow cytometry. RESULTS: Organic crystalline NP of different size were produced from mTHPC raw material. NP were internalized more efficiently in J774A.1 macrophages when compared to L929 fibroblasts, whereas uptake and fluorescence of Foslip was similar between the cell lines. NP dissolution correlated with internalization levels for larger particles in the range of 200-500 nm. Smaller particles (45 nm in diameter) were taken up at high levels in macrophages, but were not dissolved efficiently, resulting in comparatively low intracellular fluorescence. Whereas Foslip was predominantly localized in membranes, NP-mediated fluorescence also co-localized with acidic vesicles, suggesting endocytosis/phagocytosis as a major uptake mechanism. In macrophages, phototoxicity of NPs was stronger than in fibroblasts, even exceeding Foslip when administered in identical amounts. In both cell lines, phototoxicity correlated with mitochondrial depolarization and enhanced activation of caspase 3. CONCLUSIONS: Due to their preferential uptake/dissolution in macrophages, mTHPC NP may have potential for the diagnosis and photodynamic treatment of macrophage-associated disorders such as inflammation and cancer.

Lipid-DNAs as Solubilizers of mTHPC.

Hydrophobic drug candidates require innovative formulation agents. We designed and synthesized lipid-DNA polymers containing varying numbers of hydrophobic alkyl chains. The hydrophobicity of these amphiphiles is easily tunable by introducing a defined number of alkyl chain-modified nucleotides during standard solid-phase synthesis of DNA using an automated DNA synthesizer. We observed that the resulting self-assembled micelles solubilize the poorly water-soluble drug, meta-tetra-hydroxyphenyl-chlorin (mTHPC) used in photodynamic therapy (PDT) with high loading concentrations and loading capacities. A cell viability study showed that mTHPC-loaded micelles exhibit good biocompatibility without irradiation, and high PDT efficacy upon irradiation. Lipid-DNAs provide a novel class of drug-delivery vehicle, and hybridization of DNA offers a potentially facile route for further functionalization of the drug-delivery system with, for instance, targeting or imaging moieties.

Hyperbranched Polyglycerol Loaded with (Zinc-)Porphyrins: Photosensitizer Release Under Reductive and Acidic Conditions for Improved Photodynamic Therapy.

An adaptable approach toward cleavable nanoparticle carrier systems for photodynamic therapy (PDT) is presented, comprising a biocompatible carrier loaded with multiple photosensitizer (PS) molecules related to the clinically employed PS Temoporfin, two linkers cleavable under different triggers and glyco-targeting with mannose. A synthetic pathway to stimuli responsive hyperbranched polyglycerol (hPG) porphyrin conjugates via the copper(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC) or the strain-promoted alkyne-azide cycloaddition (SPAAC) has been developed. The PS 10,15,20-tris(3-hydroxyphenyl)-5-(2,3,4,5,6-pentafluorophenyl)porphyrin was functionalized with disulfide containing cystamine and acid-labile benzacetal linkers. Conjugates with reductively and pH labile linkers were thus obtained. Cleavage of the active PS agents from the polymer carrier is shown in several different release studies. The uptake of the conjugates into the cells is demonstrated via confocal laser scanning microscopy (CLSM) and flow cytometry. Finally, the antitumor and antibacterial phototoxicity of selected conjugates has been assessed in four different tumor cell lines and in cultures of the bacterium Staphylococcus aureus. The conjugates exhibited phototoxicity in several tumor cell lines in which conjugates with reductively cleavable linkers were more efficient compared to conjugates with acid-cleavable linkers. For S. aureus, strong phototoxicity was observed for a combination of the reductively cleavable and the pH labile linker and likewise for the cleavable conjugate with mannose targeting groups. The results thus suggest that the conjugates have potential for antitumor as well as antibacterial PDT.

The alteration of temoporfin distribution in multicellular tumor spheroids by ß-cyclodextrins.

To be effective anticancer drugs must penetrate tissue efficiently, reaching all target population of cancer cells in a concentration sufficient to exert a therapeutic effect. This study aimed to investigate the ability of methyl-ß-cyclodextrin (Me-ß-CD) and 2-hydroxypropyl-ß-cyclodextrin (Hp-ß-CD) to alter the penetration and diffusion of temoporfin (mTHPC) in HT29 multicellular tumor spheroids. mTHPC had а nonhomogenous distribution only on the periphery of spheroids. The presence of ß-CDs significantly altered the distribution of mTHPC consisting in the increase of both the depth of photosensitizer penetration and accumulation in HT29 spheroids. We suggest that this improvement is related to the nanoshuttle mechanism of ß-CD action, when ß-CDs facilitate mTHPC transportation to the cells in the inner layers of spheroids. As a result of mTHPC distribution improvement, ß-CDs enhance mTHPC photosensitizing activity towards HT29 multicellular tumor spheroids. The observed effects strongly depend on the type of ß-CD. Thus, varying the type of ß-CD we can finely tune the possibility of using mTHPC for diagnostic (delimitation of tumor margins) or therapeutic purposes.

Mannose-Functionalized Hyperbranched Polyglycerol Loaded with Zinc Porphyrin: Investigation of the Multivalency Effect in Antibacterial Photodynamic Therapy.

The antibacterial photodynamic activity of hyperbranched polyglycerol (hPG) loaded with zinc porphyrin photosensitizers and mannose units was investigated. hPG, with a MW of 19.5 kDa, was functionalized with about 15 molecules of the photosensitizer {5,10,15-tris(3-hydroxyphenyl)-20-[4-(prop-2-yn-1-ylamino)tetrafluorophenyl]porphyrinato}-zinc(II) by using copper(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC). These nanoparticle conjugates were functionalized systematically with increasing loadings of mannose in the range of approximately 20 to 110 groups. With higher mannose loadings (ca. 58-110 groups) the water-insoluble zinc porphyrin photosensitizer could thus be transferred into a water-soluble form. Targeting of the conjugates was proven in binding studies to the mannose-specific lectin concanavalin A (Con A) by using surface plasmon resonance (SPR). The antibacterial phototoxicity of the conjugates on Staphylococcus aureus (as a typical Gram-positive germ) was investigated in phosphate-buffered saline (PBS). It was shown that conjugates with approximately 70-110 mannose units exhibit significant antibacterial activity, whereas conjugates with approximately 20-60 units did not induce bacterial killing at all. These results give an insight into the multivalency effect in combination with photodynamic therapy (PDT). On addition of serum to the bacterial cultures, a quenching of this antibacterial phototoxicity was observed. In fluorescence studies with the conjugates in the presence of increasing bovine serum albumin (BSA) concentrations, protein-conjugate associations could be identified as a plausible cause for this quenching.

Quantitative bioimaging of platinum group elements in tumor spheroids.

Limited drug penetration into tumor tissue is a significant factor to the effectiveness of cancer therapy. Tumor spheroids, a 3D cell culture model system, can be used to study drug penetration for pharmaceutical development. In this study, a method for quantitative bioimaging of platinum group elements by laser ablation (LA) coupled to inductively coupled plasma mass spectrometry (ICP-MS) is presented. Different matrix-matched standards were used to develop a quantitative LA-ICP-MS method with high spatial resolution. To investigate drug penetration, tumor spheroids were incubated with platinum complexes (Pt(II)acetylacetonate, cisplatin) and the palladium tagged photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). Distribution and accumulation of the pharmaceuticals were determined with the developed method.