Photoacoustic imaging of elevated glutathione in models of lung cancer for companion diagnostic applications.

Companion diagnostics (CDx) are powerful tests that can provide physicians with crucial biomarker information that can improve treatment outcomes by matching therapies to patients. Here, we report a photoacoustic imaging-based CDx (PACDx) for the selective detection of elevated glutathione (GSH) in a lung cancer model. GSH is abundant in most cells, so we adopted a physical organic chemistry approach to precisely tune the reactivity to distinguish between normal and pathological states. To evaluate the efficacy of PACDx in vivo, we designed a blind study where photoacoustic imaging was used to identify mice bearing lung xenografts. We also employed PACDx in orthotopic lung cancer and liver metastasis models to image GSH. In addition, we designed a matching prodrug, PARx, that uses the same SNAr chemistry to release a chemotherapeutic with an integrated PA readout. Studies demonstrate that PARx can inhibit tumour growth without off-target toxicity in a lung cancer xenograft model.

Reactivity and DNA Damage by Independently Generated 2'-Deoxycytidin-N4-yl Radical.

Oxidative stress produces a variety of radicals in DNA, including pyrimidine nucleobase radicals. The nitrogen-centered DNA radical 2'-deoxycytidin-N4-yl radical (dC·) plays a role in DNA damage mediated by one electron oxidants, such as HOCl and ionizing radiation. However, the reactivity of dC· is not well understood. To reduce this knowledge gap, we photochemically generated dC· from a nitrophenyl oxime nucleoside and within chemically synthesized oligonucleotides from the same precursor. dC· formation is confirmed by transient UV-absorption spectroscopy in laser flash photolysis (LFP) experiments. LFP and duplex DNA cleavage experiments indicate that dC· oxidizes dG. Transient formation of the dG radical cation (dG+•) is observed in LFP experiments. Oxidation of the opposing dG in DNA results in hole transfer when the opposing dG is part of a dGGG sequence. The sequence dependence is attributed to a competition between rapid proton transfer from dG+• to the opposing dC anion formed and hole transfer. Enhanced hole transfer when less acidic O6-methyl-2'-deoxyguanosine is opposite dC· supports this proposal. dC· produces tandem lesions in sequences containing thymidine at the 5'-position by abstracting a hydrogen atom from the thymine methyl group. The corresponding thymidine peroxyl radical completes tandem lesion formation by reacting with the 5'-adjacent nucleotide. As dC· is reduced to dC, its role in the process is traceless and is only detectable because of the ability to independently generate it from a stable precursor. These experiments reveal that dC· oxidizes neighboring nucleotides, resulting in deleterious tandem lesions and hole transfer in appropriate sequences.

Photo- and thermo-responsive multicompartment hydrogels for synergistic delivery of gemcitabine and doxorubicin.

Hydrogels have found promising applications in drug delivery due to their biocompatibility, high drug loading capability, and tunable release profiles. However, hydrogel-based carriers are primarily employed for delivering hydrophilic payloads while hydrophobic drugs cannot be efficiently delivered due to the lack of hydrophobic domains within conventional hydrogel matrices. Herein, we report that thermo- and photo-responsive hydrogels could be constructed from amphiphilic triblock copolymers, poly(N-isopropylacrylamide)-b-poly(4-acryloylmorpholine)-b-poly(2-((((2-nitrobenzyl)oxy)carbonyl) amino)ethyl methacrylate) (PNIPAM-b-PNAM-b-PNBOC), and the resulting hydrogels could be further engineered a new carrier for both hydrophilic gemcitabine (GCT) and hydrophobic doxorubicin (DOX). PNIPAM-b-PNAM-b-PNBOC triblock copolymers were first self-assembled into micelles with hydrophobic photosensitive PNBOC cores, hydrophilic PNAM inner shells, and thermoresponsive PNIPAM coronas below the lower critical solution temperature (LCST), while hydrogels of physically cross-linked micellar nanoparticles were achieved at elevated polymer concentrations and high temperatures above the critical gelation temperature (CGT). Rheological experiments revealed that the CGT was highly dependent on polymer compositions and concentrations, that is, a longer hydrophobic PNBOC block or a higher polymer concentration led to a decreased CGT. However, the CGT prior to UV irradiation (CGT0) could be drastically elevated after UV irradiation (CGTUV) as a result of UV irradiation-induced concurrently cross-linking and hydrophobic-to-hydrophilic transition within PNBOC cores. As such, gel-to-sol transition could be accomplished by either temperature decrease or exposure to UV irradiation at a fixed temperature lower than the CGTUV. Note that both GCT and DOX could be simultaneously encapsulated into the hydrogels due to the coexistence of extramicellar aqueous phase and hydrophobic micellar cores. Intriguingly, the subsequent co-release of GCT and DOX could be regulated by taking advantage of either temperature or UV irradiation-mediated gel-to-sol transitions.

Photochemical stability of 4'-azido-2'-deoxy-2'-methylcytidine hydrochloride: structural elucidation of major degradation products by LC-MS and NMR analysis.

The photochemical stability of (1'R,2'S,3'S,4'R)-4'-azido-2'-deoxy-2'-methylcytidine hydrochloride, a new anti-HCV agent, was investigated. Aqueous solutions and bulk drug powder of the drug candidate were exposed to UV-visible light, complying with ICH requirements. The nucleoside analog decomposed via loss of nitrogen to yield products derived from a highly reactive azide intermediate. Major photolysis products were identified by LC-MS and NMR analysis, revealing three main photodegradation pathways. The first one led to the formation of a ring-expanded imidate ester. The other degradation pathways involved exocyclic or endocyclic bond cleavage with imine or imino lactone formation. The latter were prone to rapid hydrolysis, eventually resulting in the release of cytosine, 2-methyl malonaldehyde and (E)-cytosyl-2-methylpropenal.

pH effect on the one-electron photooxidation of 5-methylcytosine with naphthoquinone sensitizer.

The pH effect on the one-electron photooxidation of 5-methyl-2'-deoxycytidine (d(m)C) by sensitization with 1,4-naphthoquinone (NQ) was investigated. Upon photoirradiation of d(m)C in the presence of NQ under slightly acidic conditions such as pH 5.0, 5-formyl-2'- deoxycytidine (d(f)C) was formed efficiently, whereas similar NQ-photosensitized oxidation of d(m)C proceeded to lesser extent under neutral or basic conditions. Under slightly acidic conditions, dmC radical cation favors to undergo irreversible deprotonation at the C(5)-methyl group to form a methyl-centered radical, leading to a higher yield of the alkali-labile oxidation products including d(f)C. In contrast, the d(m)C radical cation competitively undergoes deprotonation at the exocyclic N(4)-amino group under neutral or basic conditions, resulting in a decreased yield of NQ-photosensitized oxidation products.

The photochemistry of thymidylyl-(3'-5')-5-methyl-2'-deoxycytidine in aqueous solution.

The photochemistry of the dinucleoside monophosphate thymidylyl-(3'-5')-5-methyl-2'-deoxycytidine (Tpm5dC) has been studied in aqueous solution using both 254 nm and UV-B radiation. A variety of dinucleotide photoproducts containing 5-methylcytosine (m5C) have been isolated and characterized. These include two cyclobutane dimers (CBD) (the cis-syn [c,s]and trans-syn forms), a (6-4) adduct and its related Dewar isomer, and two isomers of a product in which the m5C moiety was converted into an acrylamidine. Small amounts of thymidylyl-(3'-5')-thymidine (TpT) were also formed, presumably as a secondary photoreaction product. In addition, a photoproduct was characterized in which the m5C moiety was lost, thus generating 3'-thymidylic acid esterified with 2'-deoxyribose at the 5-hydroxyl on the sugar moiety. The c,s CBD of Tpm5dC readily undergoes deamination to form the corresponding CBD of TpT. The kinetics of this deamination process has been studied; the corresponding enthalpy and entropy of activation for the reaction have been evaluated at pH 7.4 as being, respectively, 73.4 kJ/mol and -103.5 J/K mol. Deamination was not observed for the other characterized photoproducts of Tpm5dC.

The reactions of cytidine and 2'-deoxycytidine with SO4.- revisited. Pulse radiolysis and product studies.

The reactions of SO4.- with 2'-deoxycytidine 1a and cytidine 1b lead to very different intermediates (base radicals with 1a, sugar radicals with 1b). The present study provides spectral and kinetic data for the various intermediates by pulse radiolysis as well as information on final product yields (free cytosine). Taking these and literature data into account allows us to substantiate but also modify in essential aspects the current mechanistic concept (H. Catterall, M. J. Davies and B. C. Gilbert, J. Chem. Soc., Perkin Trans. 2, 1992, 1379). SO4.- radicals have been generated radiolytically in the reaction of peroxodisulfate with the hydrated electron (and the H. atom). In the reaction of SO4.- with 1a (k = 1.6 x 10(9) dm3 mol-1 s-1), a transient (lambda max = 400 nm, shifted to 450 nm at pH 3) is observed. This absorption is due to two intermediates. The major component (lambda max approximately 385 nm) does not react with O2 and has been attributed to an N-centered radical 4a formed upon sulfate release and deprotonation at nitrogen. The minor component, rapidly wiped out by O2, must be due to C-centered OH-adduct radical(s) 6a and/or 7a suggested to be formed by a water-induced nucleophilic replacement. These radicals decay by second-order kinetics. Free cytosine is only formed in low yields (G = 0.14 x 10(-7) mol J-1 upon electron-beam irradiation). In contrast, 1b gives rise to an intermediate absorbing at lambda max = 530 nm (shifted to 600 nm in acid solution) which rapidly decays (k = 6 x 10(4) s-1). In the presence of O2, the decay is much faster (k approximately 1.3 x 10(9) dm3 mol-1 s-1) indicating that this species must be a C-centered radical. This has been attributed to the C(5)-yl radical 8 formed upon the reaction of the C(2')-OH group with the cytidine SO4(.-)-adduct radical 2b. This reaction competes very effectively with the corresponding reaction of water and the release of sulfate and a proton generating the N-centered radical. Upon the decay of 8, sugar radical 11 is formed with the release of cytosine. The latter is formed with a G value of 2.8 x 10(-7) mol J-1 (85% of primary SO4.-) at high dose rates (electron beam irradiation). At low dose rates (gamma-radiolysis) its yield is increased to 7 x 10(-7) mol J-1 due to a chain reaction involving peroxodisulfate and reducing free radicals. Phosphate buffer prevents the formation of the sugar radical at the SO4(.-)-adduct stage by enhancing the rate of sulfate release by deprotonation of 2b and also by speeding up the decay of the C(5)-yl radical into another (base) radical. Cytosine release in cytidine is mechanistically related to strand breakage in poly(C). Literature data on the effect of dioxygen on strand breakage yields in poly(C) induced by SO4.- (suppressed) and upon photoionisation (unaltered) lead us to conclude that in poly(C) and also in the present system free radical cations are not involved to a major extent. This conclusion modifies an essential aspect of the current mechanistic concept.

Chemical synthesis and biochemical properties of oligonucleotides that contain the (5'S,5S,6S)-5',6-cyclo-5-hydroxy-5,6-dihydro-2'-deoxyuridine DNA lesion.

The first chemical synthesis of (5'S,5S,6S)-5',6-cyclo-5-hydroxy-5,6-dihydro-2'-deoxyuridine [(5'S,5S,6S)-cyclo-5-OH-dHdU], a radiation-induced decomposition product of 2'-deoxycytidine in aerated solution, is reported. Subsequently, 2'-deoxycytidine was incorporated into oligodeoxyribonucleotides with defined sequences by using an optimized system of protection that takes into account the reactivity and stability of the modified building blocks. After deprotection and purification, the chemical composition of the modified DNA fragments was assessed by enzymatic digestions and mass spectrometry measurements. The MS analyses confirmed the presence and integrity of the lesion within the synthesized DNA fragments. In vitro replication and repair studies showed that (5'S,5S,6S)-cyclo-5-OH-dHdU acts as a block for DNA polymerases when inserted into DNA oligomers and is not excised by any of the tested DNA N-glycosylases. Therefore, (5'S,5S,6S)-cyclo-5-OH-dHdU may represent a potential lethal lesion within the cell if it is not removed by the nucleotide excision repair machinery.

Free radical formation in X-irradiated crystals of 2'-deoxycytidine hydrochloride. Electron magnetic resonance studies at 10 K.

Single crystals of deoxycytidine hydrochloride (CdR.HCl) have been X-irradiated at 10 K with doses up to about 150 kGy and studied using 24 GHz (K-band) EPR, ENDOR and FSE spectroscopy. In this system, the cytosine base is protonated at the N3 position. Nine different radicals were characterized and identified. Three of these are ascribed to three versions of the one-electron reduced species, probably differing in their protonation state. Radicals formed by net hydrogen addition to the cytosine C5 and C6 positions were observed at 10 K. The hydrogen-abstraction radical at the deoxyribose C1' position most probably results from initial oxidation of the base. The remaining radical species are all localized to the sugar moiety, representing products formed by net hydrogen abstraction from three of the five available carbons of the deoxyribose sugar. The lack of base-centered oxidation products as well as the structures of the one-electron reduced species is rationalized by considering the specific proton donor-acceptor properties of this crystalline lattice in comparison with similar systems.

Direct effects of gamma-radiation on 2'-deoxycytidine in frozen aqueous solution.

The isolation and characterization of the main stable diamagnetic products formed upon exposure of frozen aqueous solutions of 2'-deoxycytidine at 196 K to 60 Co gamma-rays are described. The initial formation of the radical pi-anion of 2'-deoxycytidine is strongly indicated by the formation of 5,6-dihydro-2'-deoxyuridine and 5,6-dihydrouracil. The formation of radical centres within the sugar moiety is clearly implied. As reported previously, the observed formation of 5',6-cyclo-5,6-dihydro-2'-deoxyuridine indicates hydrogen atom abstraction at the C5' position. The release of cytosine, and 2-deoxy-D-ribono-1,4-lactone may result from deprotonation of a pristine radical pi-cation at C1' or from direct hydrogen abstraction at this position. In general, the structures of the final products correlate well with those of the primary radicals identified from ESR studies by other workers.

Formation of cyclobutane dimers and (6-4) photoproducts upon far-UV photolysis of 5-methylcytosine-containing dinucleotide monophosphates.

The far-UV photochemistry of 5-methylcytosine, a minor DNA base, was studied in three dinucleoside monophosphates, including m5dCpT, Tpm5dC, and m5dCpdC. The model compounds were exposed to 254-nm radiation, and the resulting photoproducts were isolated by reverse-phase HPLC and characterized as cyclobutane dimers, (6-4) adducts, and the related Dewar valence isomers by UV, mass, and 1H NMR spectroscopies. The rate of formation of the different photoproducts was compared with those obtained by photolysis of TpT and the corresponding cytosine dinucleoside monophosphates, including dCpT, TpdC, and dCpdC. The formation of deaminated m5dC-containing photoproducts was observed in each of the far-UV irradiated solution of m5dCpT, Tpm5dC, and m5dCpdC. They were shown to be generated mainly through a photochemical process since methylation of the C5 atom of the cytosine ring appeared to dramatically decrease the deamination rate of the C5-C6 saturated photoproducts.

Photodegradation of the cytosine nucleosides family by water-soluble iron(III) porphyrins.

Aqueous solutions of cytosine nucleosides such as cytidine (C),2'-deoxycytidine (dC),2',3'-dideoxycytidine (ddC), and 1-beta-D-arabinofuranosylcytosine (ara-C) were irradiated with visible light in the presence of a water-soluble iron(III) porphyrin photocatalyst (FeIIITMPyP or FeIIITSPP) in air or under an argon atmosphere. HPLC analyses revealed release of cytosine as a dominant reaction. In the case of the irradiations of isomeric C and ara-C, their interconversion was also observed as a minor reaction. Under an argon atmosphere, a stoichiometric reduction of the catalyst into FeIITMPyP or FeIITSPP was observable by UV-vis spectroscopy. Quantum yields for the cytosine release and the catalyst reduction (phi cyt and phi FeII) were very much dependent both on the presence and the stereochemistry of the 2'-OH group. For the FeIIITMPyP case, for example, (a) phi cyt and phi FeII decreased in the order C > ara-C > dC > ddC and (b) oxygen lowered the phi cyt of dC and ddC but not phi cyt of C and ara-C. These results strongly support the previously proposed mechanism, i.e., C and ara-C undergo the reaction via a C2' carbon radical (e.g., eq 4) and dC and ddC undergo the reaction via a C1' or C4' carbon radical (e.g., eq 3). The observed effects of 2-propanol and solution pH suggest that a main reactive species photogenerated from FeTSPP is a free hydroxyl radical (*OH), while that photogenerated from FeTMPyP is probably an iron-coordinated active oxygen species (crypto-OH or Fe = O).

The photochemistry of 5-methylcytosine and 5-methyl-2'-deoxycytidine in aqueous solution.

The nucleobase 5-methylcytosine (I) is a minor component of eukaryotic DNA thought to be important in regulation of gene expression. The photochemical reactions of this nucleobase and its 2'-deoxyribonucleoside, 5-methyl-2'-deoxycytidine (II), in water have been studied. These reactions lead, respectively, to 3-amino-2-methylacrylamidine (Ib) and 3-(2-erythro-D-pentopyranos-1-yl)amino-2-methylacrylamidine (IIb) as the main photoproducts. The structure of the photoproducts was established by spectroscopic methods (1H and 13C NMR, UV spectroscopy, electron impact and liquid secondary ion mass spectrometry); in the case of Ib, confirmatory evidence was obtained by chemical methods (photolysis of 5-methyl[2-13C]cytosine, hydrolysis of N-carbomethoxy-3-amino-2-methylacrylamidine and reaction of Ib with 1,1'-carbonyldiimidazole to give I). The quantum yield for formation of Ib was determined to be 1.8 x 10(-3) at pH 7.5 while the quantum yield for formation of IIb has a lower value of 0.2 x 10(-3) at pH 7.5. These quantum yields depend strongly on pH and reach maximum values of 2.0 x 10(-3) at pH 7.0 (Ib) and 0.6 x 10(-3) at pH 5.0 (IIb). The mechanism of formation of Ib (or IIb) is proposed to involve nucleophilic attack of water on the C-2 position of photoexcited I (or II), followed by ring opening and decarboxylation of an intermediate carbamic acid.

Direct radiation action of heavy ions on DNA as studied by ESR-spectroscopy.

The effects of heavy ions on the mechanisms of free radical formation in DNA-constituents as compared to low-LET irradiation are investigated by means of Electron Spin Resonance (ESR) spectroscopy. Dose-yield curves were measured at low (T < 100 K) and ambient temperatures in order to obtain the G-value, that is number of radicals formed per 100 eV absorbed energy. These G-values show a characteristic LET-dependence and are one to two orders of magnitude lower than for low-LET irradiation. Measurements on 2'Deoxycytidine at 300 K using combined heavy ion and X-ray irradiation methods suggested that this effect can be partially explained by a destruction of radicals during the irradiation.

Identification of the two cis-syn [2 + 2] cycloadducts resulting from the photoreaction of 3-carbethoxypsoralen with 2'-deoxycytidine and 2'-deoxyuridine.

Near-ultraviolet photolysis of 2'-deoxycytidine (dCyd) and 3-carbethoxypsoralen (3-CPs) in the dry state was found to generate two main stable photoadducts which were separated by thin-layer and high-performance liquid chromatography. Fast atom bombardment and plasma desorption mass spectrometry analyses suggested that the bound molecule to 3-CPs is dCyd. These two compounds were found to produce the corresponding 2'-deoxyuridine (dUrd) derivatives through a deamination process when left in aqueous solutions with a lifetime close to 24 h at 20 degrees C. The chemical structure of the deaminated photoadducts was confirmed by photochemical synthesis using dUrd as the substrate. UV and fluorescent measurements indicated that the furan moiety of 3-CPs is involved in the photobinding reaction. The cyclobutane type structure of the modified dUrd derivatives was established on the basis of its photoreversibility and detailed 1H NMR analysis. The cis-syn stereoconfiguration of the two photocycloadducts was inferred from coupling constant considerations and on the basis of the complete assignment of the cyclobutyl protons, requiring the synthesis of deuterated nucleosides at pyrimidine carbon C(6). Further confirmation of the diastereoisomeric relationship between the two cis-syn dUrd <54' 65'> 3-CPs was provided by circular dichroism measurements.

Heavy-ion-induced free radical formation in solid DNA-constituents: quantitative and structural aspects.

Electron spin resonance (ESR) spectroscopy was used to study free radical formation in solid, polycrystalline pellets of DNA-constituents. Dose-yield curves were obtained at low (ca 90 K) and ambient temperatures, and were analyzed for initial G-values (radicals per 100 eV absorbed energy). At both temperatures, values of one or more orders of magnitude below the corresponding x-ray values were found. Combination of x-ray and heavy-ion irradiation indicated an enhanced radical destruction as probable cause of reduced G-values. Structural aspects as analyzed from the ESR-powder spectra revealed differences in initial, low-temperature radical population and in decay reactions upon annealing between heavy-ion bombardment and low-LET irradiation.

Free radicals induced in solid DNA by heavy ion bombardment.

Free radical formation after heavy-ion bombardment was studied in solid, polycristalline pellets of DNA-constituents by analysing the dose-yield curves and the spectra obtained by ESR-spectroscopy at low (< 100 K) and ambient temperatures. The dose-yield curves were found to correlate with those found after X-irradiation but shifted to higher doses and lower saturation concentrations. The corresponding radical yields (per 100 eV) exhibit values which are one to two orders of magnitudes lower. The structural aspects as revealed from powder ESR-spectra gave a complex inter-relation between substance, LET, dose and irradiation temperature, which is discussed in terms of mechanistic models.