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Although nearly 30 years have passed since the introduction of the first clinically approved photosensitizer for photodynamic therapy, progress in developing new pharmaceutical formulations remains unsatisfactory. This review highlights that despite years of research, many recurring challenges and issues remain unresolved. The paper includes an analysis of selected essential studies involving aminolevulinic acid and its derivatives, as well as other photosensitizers with potential for development as medical products. Among various possible vehicles, special attention is given to gelatin, alginates, poly(ethylene oxide), polyacrylic acid, and chitosan. The focus is particularly on infectious and cancerous diseases. Key aspects of developing new semi-solid drug forms should prioritize the creation of easily manufacturable and biocompatible preparations for clinical use. At the same time, new formulations should preserve the primary function of photosensitizers, which is the generation of reactive oxygen species capable of destroying pathogenic cells or tumors. Additionally, the use of adjuvant properties of carriers, which can enhance the effectiveness of macrocycles, is emphasized, especially in chitosan-based antibacterial formulations. Current research indicates that many promising dyes and macrocyclic compounds with high potential as photosensitizers in photodynamic therapy remain unexplored in formulation and development work. This review outlines potential new and previously explored pathways for advancing photosensitizers as active pharmaceutical ingredients (APIs).

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In photosynthetic light-harvesting complexes, strong interaction between chromophores enables efficient absorption of solar radiation and has been suggested to enable ultrafast energy funneling to the reaction center. To examine whether similar effects can be realized in synthetic systems, and to determine the mechanisms of energy transfer, we synthesized and characterized a series of bioinspired arrays containing strongly-coupled BODIPY dimers as energy donors and chlorin derivatives as energy acceptors. The BODIPY dimers feature broad absorption in the range of 500-600 nm, complementing the chlorin absorption to provide absorption across the entire visible spectrum. Ultrafast (~10 ps) energy transfer was observed from photoexcited BODIPY dyads to chlorin subunits. Surprisingly, the energy-transfer rate is nearly independent of the position where the BODIPY dimer is attached to the chlorin and of the type of connecting linker. In addition, the energy-transfer rate from BODIPY dimers to chlorin is slower than the corresponding rate in arrays containing BODIPY monomers. The lower rate, corresponding to less efficient through-bond transfer, is most likely due to weaker electronic coupling between the ground state of the chlorin acceptor and the delocalized electronic state of the BODIPY dimer, compared to the localized state of a BODIPY monomer.

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Aim: Evaluation of lymphatic drainage can be challenging to differentiate between separate drainage basins because only one 'color' is typically employed in sentinel node studies. This study aimed to test the feasibility of multicolor in vivo lymphangiography using newly developed organic polymer dots. Materials & methods: Biocompatible, purely organic, hydroporphyrin-doped near-infrared-emitting polymer dots were developed and evaluated for in vivo multicolor imaging in mouse lymph nodes. Results & conclusion: The authors demonstrated successful multicolor in vivo fluorescence lymphangiography using polymer dots, each tuned to a different emission spectrum. This allows minimally invasive visualization of at least four separate lymphatic drainage basins using fluorescent nanoparticles, which have the potential for clinical translation.

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Strong excitonic coupling in photosynthetic systems is believed to enable efficient light absorption and quantitative charge separation, motivating the development of artificial multi-chromophore arrays with equally strong or even stronger excitonic coupling. However, large excitonic coupling strengths have typically been accompanied by fast non-radiative recombination, limiting the potential of the arrays for solar energy conversion as well as other applications such as fluorescent labeling. Here, we report giant excitonic coupling leading to broad optical absorption in bioinspired BODIPY dyads that have high photostability, excited-state lifetimes at the nanosecond scale, and fluorescence quantum yields of nearly 50%. Through the synthesis, spectroscopic characterization, and computational modeling of a series of dyads with different linking moieties, we show that the strongest coupling is obtained with diethynylmaleimide linkers, for which the coupling occurs through space between BODIPY units with small separations and slipped co-facial orientations. Other linkers allow for broad tuning of both the relative through-bond and through-space coupling contributions and the overall strength of interpigment coupling, with a tradeoff observed in general between the strength of the two coupling mechanisms. These findings open the door to the synthesis of molecular systems that function effectively as light-harvesting antennas and as electron donors or acceptors for solar energy conversion.

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Semiconducting polymer dots (Pdots) are rapidly becoming one of the most studied nanoparticles in fluorescence bioimaging and sensing. Their small size, high brightness, and resistance to photobleaching make them one of the most attractive fluorophores for fluorescence imaging and sensing applications. This paper highlights our recent advances in fluorescence bioimaging and sensing with nanoscale luminescent Pdots, specifically the use of organic dyes as dopant molecules to modify the optical properties of Pdots to enable deep red and near infrared fluorescence bioimaging applications and to impart sensitivity of dye doped Pdots towards selected analytes. Building on our earlier work, we report the formation of secondary antibody-conjugated Pdots and provide Cryo-TEM evidence for their formation. We demonstrate the selective targeting of the antibody-conjugated Pdots to FLAG-tagged FLS2 membrane receptors in genetically engineered plant leaf cells. We also report the formation of a new class of luminescent Pdots with emission wavelengths of around 1000 nm. Finally, we demonstrate the formation and utility of oxygen sensing Pdots in aqueous media.

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Electronic interactions between tetrapyrroles are utilized in natural photosynthetic systems to tune the light-harvesting and energy-/charge-transfer processes in these assemblies. Such interactions also can be employed to tailor the electronic properties of tetrapyrrolic dyads and larger arrays for use in materials science and biomedical research. Here, we have utilized static and time-resolved optical spectroscopy to characterize the optical absorption and emission properties of a set of chlorin and bacteriochlorin dyads with varying degrees of through-bond (TB) and through-space (TS) interactions between the constituent macrocycles. The dyads consist of two chlorins or two bacteriochlorins joined by a linker that utilizes a triple-double-triple-bond (enediyne) motif in which the double-bond portion is an ester-substituted ethylene or o-phenylene unit. The photophysical studies are coupled with density functional theory (DFT) calculations to probe the ground-state molecular orbital (MO) characteristics of the dyads and time-dependent DFT calculations (TDDFT) to elucidate excited-state properties. The latter include electronic characteristics of the singlet excited-state manifold and the absorption transitions to these states from the electronic ground state. A comparison of the MO and calculated spectral properties of each dyad with the linker present versus disrupted (by eliminating the double-bond portion) gives insight into the relative contributions of TB versus TS interactions to the electronic properties of the dyads. The results show that the TB and TS contributions are additive (constructively interfere), which is not always the case for molecular dyads. Most of the dyads have shorter lifetimes of the lowest singlet excited state compared to the parent monomer, which derives from increased S1 → S0 internal conversion. The enhancement is greater for the dyads in benzonitrile than in toluene. The studies provide insights into the nature of the electronic interactions between the constituents in the tetrapyrrole arrays and how these interactions dictate the spectral properties and excited-state decay characteristics.

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Near-infrared (NIR) fluorescent semiconductor polymer dots (Pdots) have shown great potential for fluorescence imaging due to their exceptional chemical and photophysical properties. This paper describes the synthesis of NIR-emitting Pdots with great control and tunability of emission peak wavelength. The Pdots were prepared by doping poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-(2,1',3)-thiadiazole)] (PFBT), a semiconducting polymer commonly used as a host polymer in luminescent Pdots, with a series of chlorins and bacteriochlorins with varying functional groups. Chlorins and bacteriochlorins are ideal dopants due to their high hydrophobicity, which precludes their use as molecular probes in aqueous biological media but on the other hand prevents their leakage when doped into Pdots. Additionally, chlorins and bacteriochlorins have narrow deep red to NIR-emission bands and the wide array of synthetic modifications available for modifying their molecular structure enables tuning their emission predictably and systematically. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements show the chlorin- and bacteriochlorin-doped Pdots to be nearly spherical with an average diameter of 46 ± 12 nm. Efficient energy transfer between PFBT and the doped chlorins or bacteriochlorins decreases the PFBT donor emission to near baseline level and increases the emission of the doped dyes that serve as acceptors. The chlorin- and bacteriochlorin-doped Pdots show narrow emission bands ranging from 640 to 820 nm depending on the doped dye. The paper demonstrates the utility of the systematic chlorin and bacteriochlorin synthesis approach by preparing Pdots of varying emission peak wavelength, utilizing them to visualize multiple targets using wide-field fluorescence microscopy, binding them to secondary antibodies, and determining the binding of secondary antibody-conjugated Pdots to primary antibody-labeled receptors in plant cells. Additionally, the chlorin- and bacteriochlorin-doped Pdots show a blinking behavior that could enable their use in super-resolution imaging methods like STORM.

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A series of 3,5-bis(hetero)arylethenyl-substituted BODIPY derivatives have been prepared by Knoevenagel-type condensation of alkyl-substituted BODIPY with the corresponding aldehydes. 2-Pyrrolylethenyl-substituted derivatives feature near-IR emission (λem > 700 nm) with a high fluorescence quantum yield. Both the emission maxima and fluorescence quantum yields are relatively insensitive to solvent polarity, contrary to the corresponding near-IR-emitting 4-(N,N-dimethylaminophenyl)ethenyl derivatives. Alkylation at the N-pyrrolic position of the ethenyl substituent allows for the installation of the hydrophilic PEG group and afforded amphiphilic BODIPY derivatives. Overall, 2-pyrrolylethenyl-substituted BODIPY derivatives appear to be versatile fluorophores with potential applications in near-IR imaging.

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The excited-state properties and photoinduced charge-transfer (CT) kinetics in a series of symmetrical and asymmetrical Zn- and Au-ligated meso-meso-connected bacteriochlorin (BChl) complexes are studied computationally. BChl derivatives, which are excellent near-IR absorbing chromophores, are found to play a central role in bacterial photosynthetic reaction centers but are rarely used in artificial solar energy harvesting systems. The optical properties of chemically linked BChl complexes can be tuned by varying the linking group and involving different ligated metal ions. We investigate charge transfer in BChl dyads that are either directly linked or through a phenylene ring (1,4-phenylene) and which are ligating Zn or Au ions. The directly linked dyads with a nearly perpendicular arrangement of the BChl units bear markedly different properties than phenylene linked dyads. In addition, we find that the dielectric dependence of the intramolecular CT rate is very strong in neutral Zn-ligated dyads, whereas cationic Au-ligated dyads show negligible dielectric dependence of the CT rate. Rate constants of the photo induced CT process are calculated at the semiclassical Marcus level and are compared to fully quantum mechanical Fermi's golden rule based values. The rates are calculated using a screened range separated hybrid functional that offers a consistent framework for addressing environment polarization. We study solvated systems in two solvents of a low and a high scalar dielectric constant.

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Arrays of hydroporphyrins with boron complexes of dipyrromethene (BODIPY) are a promising platform for biomedical imaging or solar energy conversion, but their photophysical properties have been relatively unexplored. In this paper, we use time-resolved fluorescence, femtosecond transient absorption spectroscopy, and density-functional-theory calculations to elucidate solvent-dependent energy and electron-transfer processes in a series of chlorin- and bacteriochlorin-BODIPY arrays. Excitation of the BODIPY moiety results in ultrafast energy transfer to the hydroporphyrin moiety, regardless of the solvent. In toluene, energy is most likely transferred via the through-space Förster mechanism from the S1 state of BODIPY to the S2 state of hydroporphyrin. In DMF, substantially faster energy transfer is observed, which implies a contribution of the through-bond Dexter mechanism. In toluene, excited hydroporphyrin components show bright fluorescence, with quantum yield and fluorescence lifetime comparable to those of the benchmark monomer, whereas in DMF, moderate to significant reduction of both quantum yield and fluorescence lifetime are observed. We attribute this quenching to photoinduced charge transfer from hydroporphyrin to BODIPY. No direct spectral signature of the charge-separated state is observed, which suggests that either (1) the charge-separated state decays very quickly to the ground state or (2) virtual charge-separated states, close in energy to S1 of hydroporphyrin, promote ultrafast internal conversion.

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Targeted near-infrared (NIR) fluorescence probes are playing a significant role in biomedical imaging because NIR penetrates deeper into tissues and is associated with reduced autofluorescence compared to visible light fluorescence probes. Long-wavelength emitting 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) is an attractive platform for synthesizing NIR fluorophores because of its high photostability, high molar absorption coefficient, and sharp absorption and emission spectra. However, its lipophilicity hampers the conjugation chemistry necessary to add targeting moieties. In this study, we synthesized a novel NIR BODIPY derivative, NMP14. Substitutions of ethylene-bridged pyrrole units at the 3- or 5-position of the parent BODIPY chromophore result in a red shift of more than 200 nm. However, NMP14 cannot be conjugated to antibodies because of its hydrophobicity. Therefore, we synthesized NMP13 by adding short poly(ethylene glycol) to NMP14 and successfully conjugated NMP13 to cetuximab and trastuzumab. In vitro microscopic studies showed that NMP13 conjugated antibodies were activated after internalization and lysosomal processing, which means that NMP13 acts as an activatable probe only turning on after cellular internalization. After the administration of NMP13 conjugated antibodies, mice tumors were detected with high tumor to background ratios for a long period. These results suggest that NMP13 has potential as an activatable fluorescence probe for further clinical applications.

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Photoisomerization of 3,4-di(methoxycarbonyl)-enediyne linker in hydroporphyrin (chlorin or bacteriochlorin) dyads leads to thermally stable cis isomers, where macrocycles adopt a slipped cofacial mutual geometry with an edge-to-edge distance of ∼3.6 Å (determined by density functional theory (DFT) calculations). Absorption spectra exhibit a significant splitting of the long-wavelength Qy band, which indicates a strong electronic coupling with a strength of V = ∼477 cm-1 that increases to 725 cm-1 upon metalation of hydroporphyrins. Each dyad features a broad, structureless emission band, with large Stokes shift, which is indicative of excimer formation. DFT calculations for dyads show both strong through-bond electronic coupling and through-space electronic interactions, due to the overlap of π-orbitals. Overall, geometry, electronic structure, strength of electronic interactions, and optical properties of reported dyads closely resemble those observed for photosynthetic special pairs. Dyads reported here represent a novel type of photoactive arrays with various modes of electronic interactions between chromophores. Combining through-bond and through-space coupling appears to be a viable strategy to engineer novel optical and photochemical properties in organic conjugated materials.

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Near infrared (NIR) fluorescent probes are attractive tools for biomedical in vivo imaging due to the relatively deeper tissue penetration and lower background autofluorescence. Activatable probes are turned on only after binding to their target, further improving target to background ratios. However, the number of available activatable NIR probes is limited. In this study, we introduce two types of activatable NIR fluorophores derived from bacteriochlorin: chlorin-bacteriochlorin energy-transfer dyads and boron-dipyrromethene (BODIPY)-bacteriochlorin energy-transfer dyads. These fluorophores are characterized by multiple narrow excitation bands with relatively strong emission in the NIR. Targeted bacteriochlorin-based antibody or peptide probes have been previously limited by aggregation after conjugation. Polyethylene glycol (PEG) chains were added to improve the hydrophilicity without altering pharmacokinetics of the targeting moieties. These PEGylated bacteriochlorin-based activatable fluorophores have potential as targeted activatable, multicolor NIR fluorescent probes for in vivo applications.

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Fluorescence-detected two-dimensional electronic spectroscopy (F-2DES) projects the third-order non-linear polarization in a system as an excited electronic state population which is incoherently detected as fluorescence. Multiple variants of F-2DES have been developed. Here, we report phase-modulated F-2DES measurements on a strongly coupled symmetric bacteriochlorin dyad, a relevant 'toy' model for photosynthetic energy and charge transfer. Coherence map analysis shows that the strongest frequency observed in the dyad is well-separated from the excited state electronic energy gap, and is consistent with a vibrational frequency readily observed in bacteriochlorin monomers. Kinetic rate maps show a picosecond relaxation timescale between the excited states of the dyad. To our knowledge this is the first demonstration of coherence and kinetic analysis using the phase-modulation approach to F-2DES.

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A series of chlorin monomers and dyads has been prepared to probe the effect of ethenyl vs ethynyl linkers on the electronic conjugation and optical properties in resulting derivatives. Styryl-substituted chlorins have been prepared either by a Heck reaction or by microwave-assisted olefin metathesis, while ß-ß ethenyl-linked dyads have been synthesized from the corresponding vinyl-substituted chlorin monomer using microwave-assisted olefin metathesis. It has been found that when an ethenyl linker is connected at the ß-position of chlorin it provides stronger electronic conjugation than an ethynyl one, which is manifested by a greater bathochromic shift of the longest wavelength absorption (Q y) and emission bands. Stronger electronic coupling is particularly evident for dyads, where ethenyl-linked dyad exhibits a strong near-IR absorption band emission (λabs = 707 nm, λem = 712 nm, Φf = 0.45), compared to the deep-red absorption and emission of a corresponding ethynyl-linked dyad (λabs = 689 nm, λem = 691 nm, Φf = 0.48). The reactivity of ethenyl-linked dyads with singlet oxygen is discussed as well. The results reported here provide further guidelines for molecular design of deep-red and near-IR absorbing and intensely emitting chlorin derivatives and chlorins with extended π-electronic conjugation for a variety of photonic applications.

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Excitonically coupled bacteriochlorin (BC) dimers constitute a primary electron donor (special pair) in bacterial photosynthesis and absorbing units in light-harvesting antenna. However, the exact nature of the excited state of these dyads is still not fully understood. Here, we report a detailed spectroscopic and computational investigation of a series of symmetrical bacteriochlorin dimers, where the bacteriochlorins are connected either directly or by a phenylene bridge of variable length. The excited state of these dyads is quenched in high-dielectric solvents, which we attribute to photoinduced charge transfer. The mixing of charge transfer with the excitonic state causes accelerated (within 41 ps) decay of the excited state for the directly linked dyad, which is reduced by orders of magnitude with each additional phenyl ring separating the bacteriochlorins. These results highlight the origins of the excited-state dynamics in symmetric BC dyads and provide a new model for studying the primary processes in photosynthesis and for the development of artificial, biomimetic systems for solar energy conversion.

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A series of a rigid meso-meso directly linked chlorin-chlorin, chlorin-bacteriochlorin, and bacteriochlorin-bacteriochlorin dyads, including free bases as well as Zn(II), Pd(II), and Cu(II) complexes, has been synthesized, and their absorption, emission, singlet oxygen (1O2) photosensitization, and electronic properties have been examined. Marked bathochromic shifts of the long-wavelength Q y absorption band and increase in fluorescence quantum yields in dyads, in comparison to the corresponding monomers, are observed. Nonsymmetrical dyads (except bacteriochlorin-bacteriochlorin) show two distinctive Q y bands, corresponding to the absorption of each dyad component. A nearly quantitative S1-S1 energy transfer between hydroporphyrins in dyads, leading to an almost exclusive emission of hydroporphyrin with a lower S1 energy, has been determined. Several symmetrical and all nonsymmetrical dyads exhibit a significant reduction in fluorescence quantum yields in solvents of high dielectric constants; this is attributed to the photoinduced electron transfer. The complexation of one macrocycle by Cu(II) or Pd(II) enhances intersystem crossing in the adjacent, free base dyad component, which is manifested by a significant reduction in fluorescence and increase in quantum yield of 1O2 photosensitization.

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A series of energy transfer arrays, comprising a near-IR absorbing and emitting bacteriochlorin, and BODIPY derivatives with different absorption bands in the visible region (503-668 nm) have been synthesized. Absorption band of BODIPY was tuned by installation of 0, 1, or 2 styryl substituents [2-(2,4,6-trimethoxyphenyl)ethenyl], which leads to derivatives with absorption maxima at 503, 587, and 668 nm, respectively. Efficient energy transfer (>0.90) is observed for each dyad, which is manifested by nearly exclusive emission from bacteriochlorin moiety upon BODIPY excitation. Fluorescence quantum yield of each dyad in nonpolar solvent (toluene) is comparable with that observed for corresponding bacteriochlorin monomer, and is significantly reduced in solvent of high dielectric constants (DMF), most likely by photoinduced electron transfer. Given the availability of diverse BODIPY derivatives, with absorption between 500-700 nm, BODIPY-bacteriochlorin arrays should allow for construction of near-IR emitting agents with multiple and broadly tunable absorption bands. Solvent-dielectric constant dependence of Φf in dyads gives an opportunity to construct environmentally sensitive fluorophores and probes.

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BODIPY-hydroporphyrin energy transfer arrays allow for development of a family of fluorophores featuring a common excitation band at 500 nm, tunable excitation band in the deep red/near-infrared window, and tunable emission. Their biomedical applications are contingent upon retaining their optical properties in an aqueous environment. Amphiphilic arrays containing PEG-substituted BODIPY and chlorins or bacteriochlorins were prepared and their optical and fluorescence properties were determined in organic solvents and aqueous surfactants. The first series of arrays contains BODIPYs with PEG substituents attached to the boron, whereas in the second series, PEG substituents are attached to the aryl at the meso positions of BODIPY. For both series of arrays, excitation of BODIPY at 500 nm results in efficient energy transfer to and bright emission of hydroporphyrin in the deep-red (640-660 nm) or near-infrared (740-760 nm) spectral windows. In aqueous solution of nonionic surfactants (Triton X-100 and Tween 20) arrays from the second series exhibit significant quenching of fluorescence, whereas properties of arrays from the first series are comparable to those observed in polar organic solvents. Reported arrays possess large effective Stokes shift (115-260 nm), multiple excitation wavelengths, and narrow, tunable deep-red/near-IR fluorescence in aqueous surfactants, and are promising candidates for a variety of biomedical-related applications.

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Attempts to develop a credible prebiotic route to tetrapyrroles have relied on enzyme-free recapitulation of the extant biosynthesis, but this process has foundered from the inability to form the pyrrole porphobilinogen (PBG) in good yield by self-condensation of the precursor δ-aminolevulinic acid (ALA). PBG undergoes robust oligomerization in aqueous solution to give uroporphyrinogen (4 isomers) in good yield. ALA, PBG, and uroporphyrinogen III are universal precursors to all known tetrapyrrole macrocycles. The enzymic formation of PBG entails carbon-carbon bond formation between the less stable enolate/enamine of one ALA molecule (3-position) and the carbonyl/imine (4-position) of the second ALA molecule; without enzymes, the first ALA reacts at the more stable enolate/enamine (5-position) and gives the pyrrole pseudo-PBG. pseudo-PBG cannot self-condense, yet has one open α-pyrrole position and is proposed to be a terminator of oligopyrromethane chain-growth from PBG. Here, 23 analogues of ALA have been subjected to density functional theoretical (DFT) calculations, but no motif has been identified that directs reaction at the 3-position. Deuteriation experiments suggested 5-(phosphonooxy)levulinic acid would react preferentially at the 3- versus 5-position, but a hybrid condensation with ALA gave no observable uroporphyrin. The results suggest efforts toward a biomimetic, enzyme-free route to tetrapyrroles from ALA should turn away from structure-directed reactions and focus on catalysts that orient the two aminoketones to form PBG in a kinetically controlled process, thereby avoiding formation of pseudo-PBG.

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