Cherenkov Radiation induced photodynamic therapy - repurposing older photosensitizers, and radionuclides.

CONTEXT: Old-generation photosensitizers are minimally used in current photodynamic therapy (PDT) because they absorb in the UV/blue/green region of the spectrum where biological tissues are generally highly absorbing. The UV/blue light of Cherenkov Radiation (CR) from nuclear disintegration of beta-emitter radionuclides shows promise as an internal light source to activate these photosensitizers within tissue. Outline of the study: 1) radionuclide choice and Cherenkov Radiation, 2) Photosensitizer choice, synthesis and radiolabeling, 3) CR-induced fluorescence, 4) Verification of ROS formation, 5) CR-induced PDT with either free eosine and free CR emitter, or with radiolabelled eosin. RESULTS: Cherenkov Radiation Energy Transfer (CRET) from therapeutic radionuclides (90Y) and PET imaging radionuclides (18F, 68Ga) to eosin was shown by spectrofluorimetry and in vitro, and was shown to result in a PDT process. The feasibility of CR-induced PDT (CR-PDT) was demonstrated in vitro on B16F10 murine melanoma cells mixing free eosin (λabs = 524 nm, ΦΔ 0.67) with free CR-emitter [18F]-FDG under their respective intrinsic toxicity levels (0.5 mM/8 MBq) and by trapping singlet oxygen with diphenylisobenzofuran (DPBF). An eosin-DOTAGA-chelate conjugate 1 was synthesized and radiometallated with CR-emitter [68Ga] allowed to reach 25 % cell toxicity at 0.125 mM/2 MBq, i.e. below the toxicity threshold of each component measured on controls. Incubation time was carefully examined, especially for CR emitters, in light of its toxicity, and its CR-emitting yield expected to be 3 times as much for 68Ga than 18F (considering their ß particle energy) per radionuclide decay, while its half-life is about twice as small. PERSPECTIVE: This study showed that in complete darkness, as it is at depth in tissues, PDT could proceed relying on CR emission from radionuclides only. Interestingly, this study also repurposed PET imaging radionuclides, such as 68Ga, to trigger a therapeutic event (PDT), albeit in a modest extent. Moreover, although it remains modest, such a PDT approach may be used to achieve additional tumoricidal effect to RIT treatment, where radionuclides, such as 90Y, are strong CR emitters, i.e. very potent light source for photosensitizer activation.

Enhanced stability and photothermal efficiency of Indocyanine Green J-aggregates by nanoformulation with Calix[4]arene for photothermal therapy of cancers.

Photothermal therapy (PTT) is a method of growing attention, owing to its controllable process, high efficiency and minimal side effect. Indocyanine Green (ICG) is as Food and Drug Administration (FDA) approved agent that stands on the frontline of further developments of PTT toward clinics. However, the applicability of ICG-mediated PTT is limited by the rapid in vivo clearance and photo-degradation of ICG. To improve those parameters, nanosized ICG-loaded nanoparticles (ICG-J/CX) were fabricated in this study by co-assembly of anionic ICG J-aggregates (ICG-J) with cationic tetraguanidinium calix[4]arene (CX). This very simple approach produces ICG-J/CX with a well-defined nanometer range size and a close to neutral charge. The nanoparticles demonstrate high photothermal conversion efficiency (PCE) and dramatically improved photostability, as compared with ICG. The in vitro cellular uptake and cytotoxicity studies further demonstrated that the ICG-J/CX nanoparticles enhance uptake and photothermal efficiency in comparison with ICG or non-formulated ICG-J, overall demonstrating that ICG-J/CX mediated photothermal therapy have significant potential for attaining cancer treatment.

Non-activated Esters as Reactive Handles in Direct Post-Polymerization Modification.

Non-activated esters are prominently featured functional groups in polymer science, as ester functional monomers display great structural diversity and excellent compatibility with a wide range of polymerization mechanisms. Yet, their direct use as a reactive handle in post-polymerization modification has been typically avoided due to their low reactivity, which impairs the quantitative conversion typically desired in post-polymerization modification reactions. While activated ester approaches are a well-established alternative, the modification of non-activated esters remains a synthetic and economically valuable opportunity. In this review, we discuss past and recent efforts in the utilization of non-activated ester groups as a reactive handle to facilitate transesterification and aminolysis/amidation reactions, and the potential of the developed methodologies in the context of macromolecular engineering.

Poly(N-allyl acrylamide) as a Reactive Platform toward Functional Hydrogels.

The synthesis of poly(N-allyl acrylamide) (PNAllAm) as a platform for the preparation of functional hydrogels is described. The PNAllAm was synthesized via organocatalyzed amidation of poly(methyl acrylate) (PMA) with allylamine and characterized by 1H NMR spectroscopy, size exclusion chromatography (SEC), and turbidimetry, which allowed an estimation of the lower critical solution temperature of ∼26 °C in water. The PNAllAm was then used to make functional hydrogels via photoinitiated thiol-ene chemistry, where dithiothreitol (DTT) was used to cross-link the polymer chains. In addition, mercaptoethanol (ME) was added as a functional thiol to modulate the hydrogel properties. A decrease of the volume-phase transition temperature of the resulting hydrogels was observed with increasing ME content. Altogether this work introduces a straightforward way for the preparation of PNAllAm from PMA and demonstrates its value as a reactive polymer platform for the generation of functional hydrogels.

Perturbation of Lipid Bilayers by Biomimetic Photoswitches Based on Cyclocurcumin.

The use of photoswitches which may be activated by suitable electromagnetic radiation is an attractive alternative to conventional photodynamic therapy. Here, we report all-atom molecular dynamics simulation of a biomimetic photoswitch derived from cyclocurcumin and experiencing E/Z photoisomerization. In particular, we show that the two isomers interact persistently with a lipid bilayer modeling a cellular membrane. Furthermore, the interaction with the membrane is strongly dependent on the concentration, and a transition between ordered and disordered arrangements of the photoswitches is observed. We also confirm that the structural parameters of the bilayer are differently affected by the two isomers and hence can be modulated through photoswitching, offering interesting perspectives for future applications.

Biomimetic Photo-Switches Softening Model Lipid Membranes.

We report the synthesis and characterization of a novel photo-switch based on biomimetic cyclocurcumin analogous and interacting with the lipid bilayer, which can be used in the framework of oxygen-independent light-induced therapy. More specifically, by using molecular dynamics simulations and free energy techniques, we show that the inclusion of hydrophobic substituents is needed to allow insertion in the lipid membrane. After having confirmed experimentally that the substituents do not preclude the efficient photoisomerization, we show through UV-vis and dynamic light scattering measurements together with compression isotherms that the chromophore is internalized in both lipid vesicles and monomolecular film, respectively, inducing their fluidification. The irradiation of the chromophore-loaded lipid aggregates modifies their properties due to the different organization of the two diastereoisomers, E and Z. In particular, a competition between a fast structural reorganization and a slower expulsion of the chromophore after isomerization can be observed in the kinetic profiles recorded during E to Z photoisomerization. This report paves the way for future investigations in the optimization of biomimetic photoswitches potentially useful in modern light-induced therapeutic strategies.

Accelerated Post-Polymerization Amidation of Polymers with Side-Chain Ester Groups by Intramolecular Activation.

The catalytic conversion of esters to amides represents new opportunities in the synthetic diversification and upcycling of polymers, as esters are commonly featured in various polymer structures. Yet, direct amidation is typically hampered by poor reaction kinetics and the effects of polymer structure on the reactivity remain poorly understood. We report the accelerated amidation for amines with additional hydrogen bond donating or accepting groups. These amines facilitate the expeditious (co)amidation of polymers with pendant ester groups, displaying at least a 400-fold higher reactivity relative to polyesters with esters in the main chain. Furthermore, a positive correlation between the reactivity and degree of polymerization for poly(methyl acrylate) suggests a hydrogen-bond mediated intramolecular activation of the esters, which was confirmed by FT-IR spectroscopy and basic molecular mechanics modeling. The reported method paves the way to synthesize diverse (co)polymers with amide side chains from readily available polymeric precursors.

Poly(2-allylamidopropyl-2-oxazoline)-Based Hydrogels: From Accelerated Gelation Kinetics to In Vivo Compatibility in a Murine Subdermal Implant Model.

A rapid photo-curing system based on poly(2-ethyl-2-oxazoline-co-2-allylamidopropyl-2-oxazoline) and its in vivo compatibility are presented. The base polymer was synthesized from the copolymerization of 2-ethyl-2-oxazoline (EtOx) and the methyl ester containing 2-methoxycarboxypropyl-2-oxazoline (C3MestOx) followed by amidation with allylamine to yield a highly water-soluble macromer. We showed that spherical hydrogels can be obtained by a simple water-in-oil gelation method using thiol-ene coupling and investigated the in vivo biocompatibility of these hydrogel spheres in a 28-day murine subdermal model. For comparison, hydrogel spheres prepared from poly(ethylene glycol) were also implanted. Both materials displayed mild, yet typical foreign body responses with little signs of fibrosis. This is the first report on the foreign body response of a poly(2-oxazoline) hydrogel, which paves the way for future investigations into how this highly tailorable class of materials can be used for implantable hydrogel devices.

Stoichiometric Control over Partial Transesterification of Polyacrylate Homopolymers as Platform for Functional Copolyacrylates.

Only recently, post-polymerization modification reactions of unactivated polyacrylates have been emerging as an attractive alternative to utilizing reactive monomers, enabling the synthetic upcycling of these widely applied polymers. Within this contribution, the triazabicyclodecene-catalyzed transesterification of polyacrylates is reported, including the reaction kinetics and the broad scope for macromolecular design of functional copolyacrylates. More specifically, the transesterification is performed under equilibrium conditions with a set of primary alcohols whereby the reaction kinetics and the obtained conversion as a function of stoichiometric excess of alcohol are evaluated. The results show that the obtained conversion is dependent on the polarity of the solvent and of the alcohol. Through this approach, the transesterification degree can be accurately controlled by stoichiometry, enabling the precise modulation of the macromolecular structure. Finally, the utility of this approach is demonstrated to incorporate functional side chains that are incompatible with radical polymerization, to facilitate Diels-Alder and thiol-ene reactions, enabling access to a broad range of functional materials from simple polyacrylate homopolymer precursors.

Poly(2-ethyl-2-oxazoline) Conjugates with Salicylic Acid via Degradable Modular Ester Linkages.

Conjugation of drugs to polymers is a widely used approach to gain control over the release of therapeutics. In this contribution, salicylic acid, a multipurpose model drug, is conjugated to the biocompatible poly(2-ethyl-2-oxazoline) (PEtOx). The drug is attached to the side chains of a polymer carrier through a hydrolytically cleavable ester linker, via a sequential postpolymerization modification. The chemical modulation of this ester, i.e., by primary or secondary alcohols, is demonstrated to greatly influence the ester hydrolysis rate. This crucial parameter allows us to tune the in vitro kinetics of the sustained drug release for periods exceeding a month in phosphate-buffered saline (PBS). The synthetic accessibility of the cleavable linker, together with the modularity of the drug release rate offered by this approach, highlights the utility of this class of polymers in the field of long-lasting drug delivery systems for persistent and chronic disease treatment.

Reductive Amination/Cyclization of Methyl Levulinate with Aspartic Acid: Towards Renewable Polyesters with a Pendant Lactam Unit.

Environmental regulation and depletion of fossil resources are boosting the search for new polymeric materials produced from biomass. Here, the synthesis of a new diester bearing a pendant lactam unit from methyl levulinate and aspartic acid is reported. The palladium-catalyzed reductive amination/cyclization sequence was carefully optimized to afford the diacid with high yield (>95 %). In a second step, the compound was esterified to give the corresponding diester. The latter monomer was copolymerized with α-ω linear diols, yielding polyesters with molecular weights up to 20.5 kg mol-1 .

Cellular imaging using BODIPY-, pyrene- and phthalocyanine-based conjugates.

Fluorescent Probes aimed at absorbing in the blue/green region of the spectrum and emitting in the green/red have been synthesized (as the form of dyads-pentads), studied by spectrofluorimetry, and used for cellular imaging. The synthesis of phthalocyanine-pyrene 1 was achieved by cyclotetramerization of pyrenyldicyanobenzene, whereas phthalocyanine-BODIPY 2c was synthesized by Sonogashira coupling between tetraiodophthalocyanine and meso-alkynylBODIPY. The standard four-steps BODIPY synthesis was applied to the BODIPY-pyrene dyad 3 starting from pyrenecarbaldehyde and dimethylpyrrole. 1H, 13C, 19F, 11BNMR, ICP, MS, and UV/Vis spectroscopic analyses demonstrated that 2c is a mixture of BODIPY-Pc conjugates corresponding to an average ratio of 2.5 BODIPY per Pc unit, where its bis, tris, tetrakis components could not be separated. Fluorescence emission studies (µM concentration in THF) showed that the design of the probes allowed excitation of their antenna (pyrene, BODIPY) in the blue/green region of the spectrum, and subsequent transfer to the acceptor platform (BODIPY, phthalocyanine) followed by its emission in the green/red (with up to 140-350 nm overall Stokes shifts). The fluorescent probes were used for cellular imaging of B16F10 melanoma cells upon solubilization in 1% DMSO containing RPMI or upon encapsulation in liposomes (injection method). Probes were used at 1-10 µM concentrations, cells were fixed with methanol and imaged by biphoton and/or confocal microscopy, showing that probes could achieve the staining of cells membranes and not the nucleus.

Nickel-Catalyzed α-Allylation of Aldehydes and Tandem Aldol Condensation/Allylation Reaction with Allylic Alcohols.

An additive-free nickel-catalyzed α-allylation of aldehydes with allyl alcohol is reported. The reaction is promoted by 1 mol % of in situ formed nickel complex in methanol, and water is the sole by-product of the reaction. The experimental conditions allow the conversion of various α-branched aldehydes and α,ß-unsaturated aldehydes as nucleophiles. The same catalyst and reaction conditions enabled a tandem aldol condensation of aldehyde/α-allylation reaction.

Redshifted Cherenkov Radiation for in vivo Imaging: Coupling Cherenkov Radiation Energy Transfer to multiple Förster Resonance Energy Transfers.

Cherenkov Radiation (CR), this blue glow seen in nuclear reactors, is an optical light originating from energetic ß-emitter radionuclides. CR emitter 90Y triggers a cascade of energy transfers in the presence of a mixed population of fluorophores (which each other match their respective absorption and emission maxima): Cherenkov Radiation Energy Transfer (CRET) first, followed by multiple Förster Resonance Energy transfers (FRET): CRET ratios were calculated to give a rough estimate of the transfer efficiency. While CR is blue-weighted (300-500 nm), such cascades of Energy Transfers allowed to get a) fluorescence emission up to 710 nm, which is beyond the main CR window and within the near-infrared (NIR) window where biological tissues are most transparent, b) to amplify this emission and boost the radiance on that window: EMT6-tumor bearing mice injected with both a radionuclide and a mixture of fluorophores having a good spectral overlap, were shown to have nearly a two-fold radiance boost (measured on a NIR window centered on the emission wavelength of the last fluorophore in the Energy Transfer cascade) compared to a tumor injected with the radionuclide only. Some CR embarked light source could be converted into a near-infrared radiation, where biological tissues are most transparent.

Direct subphthalocyanine conjugation to bombesin vs. indirect conjugation to its lipidic nanocarrier.

Bombesin (BBN) was covalently bound to graftable subphthalocyanine (SubPc) or to a cholesterol derivative, a component of a liposome that encapsulates non-graftable SubPc. The latter bioconjugation approach was suitable to address the stability of SubPc and was achieved by copper-free click-chemistry on the outer-face of the liposome. Liposomes were purified (FPLC) and then analyzed in size (outer diameter about 60 nm measured by DLS). In vitro binding studies allowed to determine the IC50 13.9 nM for one component of the liposome, cholesterol, conjugated to BBN. Hence, azido- (or alkynyl-) liposomes give fluorophores with no reactive functional group available on their backbone a second chance to be (indirectly) bioconjugated (with bombesin).

Harnessing medically relevant metals onto water-soluble subphthalocyanines: towards bimodal imaging and theranostics.

Subphthalocyanine (SubPc), a putative fluorophore for optical imaging (OI), was conjugated to chelating ligands (DOTA, DTPA) affording water-soluble conjugates complexed with (non-radioactive) metals relevant to the following medical imaging techniques/therapies: MRI (Gd), PET (Cu, Ga), SPECT (In, Ga, Lu), RIT (Cu, Lu, Y), and NCT (Gd). Magneto-optical properties of ditopic gadolinium species (and optical properties of other metal containing species) were examined (brightness (ε × ΦF) and relaxivity R1) and fluorescence confocal/biphoton microscopy studies were conducted.

Subphthalocyanines: addressing water-solubility, nano-encapsulation, and activation for optical imaging of B16 melanoma cells.

Water-soluble disulfonato-subphthalocyanines (SubPcs) or hydrophobic nano-encapsulated SubPcs are efficient probes for the fluorescence imaging of cells. 20 nm large liposomes (TEM and DLS) incorporated about 13% SubPc. Moreover, some of these fluorophores were found to be pH activatable.

Inter/intramolecular Cherenkov radiation energy transfer (CRET) from a fluorophore with a built-in radionuclide.

The Cherenkov radiation (CR) from [(18)F]-FDG, [(177)Lu]-LuCl3 and [(90)Y]-YCl3 was detected and CR energy transfer (CRET) to several fluorophores was examined. Subsequent fluorescence emission was found to be a function of the position of absorption bands with respect to the CR peak, energy of emitted particles, radionuclide/fluorophore loading, and fluorophore brightness. A variant of the best fluorophore with a built-in radionuclide was synthesized to achieve inter- and intra-molecular CRET.

Magneto-optical nanomaterials: a SPIO-phthalocyanine scaffold built step-by-step towards bimodal imaging.

A SPIO-phthalocyanine nanohybrid is developed as a bimodal contrast agent for Optical and Magnetic Resonance Imaging. The organic coating was covalently attached onto SPIO in a step-by-step approach. Each coated-SPIO was thoroughly characterized. The hydrodynamic size of the SPIO-Pc is ca. 60 nm with a coverage of ca. 690 Pc/SPIO.