DNA Base Modifications Mediated by Femtosecond Laser-Induced Cold Low-Density Plasma in Aqueous Solutions.

Applications based on near-infrared femtosecond laser-induced plasma in biological materials involve numerous ionization events that inevitably mediate physicochemical effects. Here, the physical chemistry underlying the action of such plasma is characterized in a system of biological interest. We have implemented wavefront shaping techniques to control the generation of laser-induced low electron density plasma channels in DNA aqueous solutions, which minimize the unwanted thermo-mechanical effects associated with plasma of higher density. The number of DNA base modifications per unit of absolute energy deposited by such cold plasma is compared to those induced by either ultraviolet or standard ionizing radiation (γ-rays). Analyses of various photoinduced, oxidative, and reductive DNA base products show that the effects of laser-induced cold plasma are mainly mediated by reactive radical species produced upon the ionization of water, rather than by the direct interaction of the strong laser field with DNA. In the plasma environment, reactions among densely produced primary radicals result in a dramatic decrease in the yields of DNA damages relative to sparse ionizing radiation. This intense radical production also drives the local depletion of oxygen.

Tuning the size of gold nanoparticles produced by multiple filamentation of femtosecond laser pulses in aqueous solutions.

In the present study, we consider the self-regulated generation of spatially homogeneous low density plasma (LDP) micro-channels as a high intensity ionization source arising from the multi-filamentation of powerful femtosecond (fs) laser pulses in aqueous solutions. We investigate the modulation of the femtosecond laser multiple filamentation for tuning the size of gold nanoparticles (AuNPs) synthesized in an irradiated gold chloride solution. Previous studies on the radiation-induced synthesis of colloidal gold by more conventional ionization sources, such as high energy γ-rays and electron beams, highlighted the dependence of the size distribution of AuNPs on the density of energy deposited per unit of time, i.e. the dose rate. The present method of laser-induced production of AuNPs rests on a similar radiation-assisted process, i.e. the reduction of the solvated trivalent gold ions by the hydrated electrons produced upon ionization of water. We find that trivial optical manipulation varies the rate of deposited energy by laser irradiation, which can be considered equivalent to a variation of the dose rate. We investigate the influence of varying the density of energy deposited on the laser-induced gold cluster size distribution and made a comparison with the high energy radiation-induced synthesis of AuNPs. Here, our results highlight that the present method of laser irradiation, in the regime of LDP generation, mimics the radiolysis of water at an adjustable high dose rate. More generally, these spatially and temporally resolved plasmas could be developed as a tool for the unprecedented control of chemistry under ionizing radiation.

Dense ionization and subsequent non-homogeneous radical-mediated chemistry of femtosecond laser-induced low density plasma in aqueous solutions: synthesis of colloidal gold.

The "cold" low density plasma channels generated by the filamentation of powerful femtosecond laser pulses in aqueous solutions constitute a source of dense ionization. Here, we probed the radiation-assisted chemistry of water triggered by laser ionization via the radical-mediated synthesis of nanoparticles in gold chloride aqueous solutions. We showed that the formation of colloidal gold originates from the reduction of trivalent ionic gold initially present in solution by the reactive radicals (e.g. hydrated electrons) produced upon the photolysis of water. We analyzed both the reaction kinetics of the laser-induced hydrated electrons and the growth kinetics of the gold nanoparticles. Introduction of radical scavengers into the solutions and different initial concentrations of gold chloride provided information about the radical-mediated chemistry. The dense ionization results in the second order cross-recombination of the photolysis primary byproducts. Competition with recombination imposes the non-homogeneous interaction of reactive radicals with solute present in irradiated aqueous solutions. Such a laser-induced non-homogeneous chemistry suggests similarities with the radiation chemistry of water exposed to conventional densely ionizing radiation (high dose rate, high linear energy transfer).

Cancer radiotherapy based on femtosecond IR laser-beam filamentation yielding ultra-high dose rates and zero entrance dose.

Since the invention of cancer radiotherapy, its primary goal has been to maximize lethal radiation doses to the tumor volume while keeping the dose to surrounding healthy tissues at zero. Sadly, conventional radiation sources (γ or X rays, electrons) used for decades, including multiple or modulated beams, inevitably deposit the majority of their dose in front or behind the tumor, thus damaging healthy tissue and causing secondary cancers years after treatment. Even the most recent pioneering advances in costly proton or carbon ion therapies can not completely avoid dose buildup in front of the tumor volume. Here we show that this ultimate goal of radiotherapy is yet within our reach: Using intense ultra-short infrared laser pulses we can now deposit a very large energy dose at unprecedented microscopic dose rates (up to 10(11) Gy/s) deep inside an adjustable, well-controlled macroscopic volume, without any dose deposit in front or behind the target volume. Our infrared laser pulses produce high density avalanches of low energy electrons via laser filamentation, a phenomenon that results in a spatial energy density and temporal dose rate that both exceed by orders of magnitude any values previously reported even for the most intense clinical radiotherapy systems. Moreover, we show that (i) the type of final damage and its mechanisms in aqueous media, at the molecular and biomolecular level, is comparable to that of conventional ionizing radiation, and (ii) at the tumor tissue level in an animal cancer model, the laser irradiation method shows clear therapeutic benefits.

Improved detection sensitivity of D-mannitol crystalline phase content using differential spectral phase shift terahertz spectroscopy measurements.

We report quantitative measurement of the relative proportion of δ- and ß-D-mannitol crystalline phases inserted into polyethylene powder pellets, obtained by time-domain terahertz spectroscopy. Nine absorption bands have been identified from 0.2 THz to 2.2 THz. The best quantification of the δ-phase proportion is made using the 1.01 THz absorption band. Coherent detection allows using the spectral phase shift of the transmitted THz waveform to improve the detection sensitivity of the relative δ-phase proportion. We argue that differential phase shift measurements are less sensitive to samples' defects. Using a linear phase shift compensation for pellets of slightly different thicknesses, we were able to distinguish a 0.5% variation in δ-phase proportion.

Two-photon absorption cross section of excited phthalocyanines by a femtosecond Ti-sapphire laser.

In the past few years, photodynamic therapy (PDT) has become a major treatment for neovascular age-related macular degeneration (AMD) in which there is abnormal growth of choroidal neovasculature (CNV) that eventually obscures central vision, leading to blindness. However, one of the main limitations of current PDT is the relatively low specificity of the photosensitizer (PS) and light for pathological tissue which may induce damage to adjacent healthy tissue. An alternative approach to circumvent the specificity limitation is to improve the irradiation process. In particular two photon (2-gamma) excitation promises a more precise illumination of the target tissue. PS are activated by the simultaneous absorption of 2-gamma delivered by ultra-fast pulses of near infrared light. In order to evaluate the efficiency of phthalocyanine (Pc) dyes for 2-gamma absorption we measured 2-gamma absorption cross sections (sigma(2)) of a number of metalated Pc (MPc) dyes at lambda(ex) = 800 nm using a femtosecond laser. The studied Pc molecules vary by the type of the central metal ion (Al or Zn) and the number of peripheral sulfo substituents (MPcS). Each MPc dye of our series shows an improved 2-gamma absorption sigma(2) as compared to that obtained for Photofrin (3.1 +/- 0.1 GM, with 1 GM = 10(-50) cm(4) s photon(-1) mol(-1)), the PS currently approved for 1-gamma PDT. Our data show an 2.5-fold enhancement for AlPcCl, AlPcS(2adj) and ZnPcS(3)C(9), up to 10-fold (28.6 +/- 0.72 GM) for the ZnPcS(4) dye relative to Photofrin. These findings confirm the efficiency of Pc for 2-gamma absorption processes and represent the first detailed comparison study of 2-gamma absorption sigma(2) between Photofrin and Pc dyes.

Oxygen dependence of two-photon activation of zinc and copper phthalocyanine tetrasulfonate in Jurkat cells.

Photodynamic therapy (PDT), the use of light-activated drugs, is a promising treatment of cancer as well as several nonmalignant conditions. However, the efficacy of one-photon (1-gamma) PDT is limited by hypoxia, which can prevent the production of the cytotoxic singlet oxygen ((1)O(2)) species, leading to tumor resistance to PDT. To solve this problem, we propose an irradiation protocol based on a simultaneous, two-photon (2-gamma) excitation of the photosensitizer (Ps). Excitation of the Ps triplet state leads to an upper excited triplet state T(n) with distinct photochemical properties, which could inflict biologic damage independent of the presence of molecular oxygen. To determine the potential of a 2-gamma excitation process, Jurkat cells were incubated with zinc or copper phthalocyanine tetrasulfonate (ZnPcS(4) or CuPcS(4)). ZnPcS(4) is a potent (1)O(2) generator in 1-gamma PDT, while CuPcS(4) is inactive under these conditions. Jurkat cells incubated with either ZnPcS(4) or CuPcS(4) were exposed to a 670 nm continuous laser (1-gamma PDT), 532 nm pulsed-laser light (2-gamma PDT), or a combination of 532 and 670 nm (2-gamma PDT). The efficacy of ZnPcS(4) to photoinactivate the Jurkat cells decreased as the concentration of oxygen decreased for both the 1-gamma and 2-gamma protocols. In the case of CuPcS(4), cell phototoxicity was measured only following 2-gamma irradiation, and its efficacy also decreased at a lower oxygen concentration. Our results suggest that for CuPcS(4) the T(n) excited state can be populated after 2-gamma irradiation at 532 nm or the combination of 532 and 670 nm light. Dependency of phototoxicity upon aerobic conditions for both 1-gamma and 2-gamma PDT suggests that reactive oxygen species play an important role in 1-gamma and 2-gamma PDT.

Photodynamic inhibition of acetylcholinesterase after two-photon excitation of copper tetrasulfophthalocyanine.

Sequential two-photon (2-gamma) activated copper tetrasulfophthalocyanine (CuPcS(4)) was shown capable of inactivating acetylcholinesterase (ACE). ACE activity was measured photometrically by the Ellman method. Simultaneous irradiation of ACE in the presence of CuPcS(4) with 514 nm (183 mW/cm(2)) and 670 nm (86 mW/cm(2)) continuous wave (CW) light induced a 20-50% increase in enzyme inhibition as compared to one-photon (1-gamma) irradiation, using either 514- or 670-nm (CW) light at the same fluences. The enzyme activity was not affected by CuPcS(4) or light alone, decreased linearly with the irradiation time, and was shown to be oxygen-dependent. We conclude that the photoinactivation of ACE with sequential 2-gamma irradiation involves reactive oxygen species produced by the interaction of the upper excited T(n) state of CuPcS(4) with molecular oxygen. As CuPcS(4) shows little activity as a conventional 1-gamma photosensitizer, unwanted side effects such as prolonged skin sensitivity are eliminated rendering 2-gamma photodynamic therapy advantageous for the treatment of selected medical applications.

Two-photon absorption of copper tetrasulfophthalocyanine induces phototoxicity towards Jurkat cells in vitro.

The feasibility to induce oxygen-independent tumour cell kill by two-photon excitation of copper tetrasulfophthalocyanine (CuPcS4) was studied in Jurkat cells in vitro. Following incubation with CuPcS4 cells were transferred to a closed cuvette and irradiated with 532 nm pulsed-laser or 680 nm continuous-laser light to evaluate the effect of either two- or one-photon excitation, respectively. Cell survival was measured using MTT and Trypan Blue exclusion tests. Cell viability decreased 10-20% following two-photon excitation while one-photon illumination did not affect cell survival. These data confirm that two-photon excitation of CuPcS4 to the upper excited triplet state results in the formation of toxic species suggesting its potential use as a sensitizer for the photodynamic treatment of poorly oxygenated tumours.

Electron transfer in DNA duplexes containing 2-methyl-1,4-naphthoquinone.

2-methyl-1,4-naphthoquinone (menadione, MQ) was linked to synthetic oligonucleotides and exposed to near-UV light to generate base radical cations in DNA. This model system of electron transfer induced alkali-labile breaks at GG doublets, similar to anthraquinone and metallointercalators systems. In sharp contrast to other systems, the photolysis of MQ-DNA duplexes gave interstrand cross-links and alkali-labile breaks at bases on the complementary strand opposite the MQ moiety. For sequences with an internal MQ, the formation of cross-links with A and C opposite the MQ moiety was 2- to 3-fold greater than that with G and T. The yield of cross-links was more than 10-fold greater than that of breaks opposite MQ, which in turn was more than 2-fold greater than breaks at GG doublets. The yield of damage at GG doublets greatly increased for a sequence with a terminal MQ. The distribution of base damage was measured by enzymatic digestion and HPLC analysis (dAdo > dThd > dGuo > dCyd). The formation of novel products in MQ-DNA duplexes was attributed to the ability of excited MQ to generate the radical cations of all four DNA bases; thus, this photochemical reaction provides an ideal model system to study the effects of ionizing radiation and one-electron oxidants.

Ultrafast studies of the excited-state dynamics of copper and nickel phthalocyanine tetrasulfonates: potential sensitizers for the two-photon photodynamic therapy of tumors.

In order to evaluate the potential of copper and nickel phthalocyanine tetrasulfonates as sensitizers for two-photon photodynamic therapy, we conducted kinetic femtosecond measurements of transient absorption and bleaching of their excited state dynamics in aqueous solution. Samples were pumped with 620 nm and 310 nm laser light, which allowed us to study relaxation processes from both the first and second singlet (or doublet for the copper phthalocyanine) excited states. A second excitation from the first excited triplet state, approximately 685 and 105 ps after the first excitation for copper and nickel phthalocyanine tetrasulfonate respectively, was the most efficient way to bring the molecules to an upper triplet state. Presumably this highest triplet state can inflict molecular damage on adjacent biomolecules int eh absence of oxygen, resulting in the desired cytotoxic cellular response. Transient absorption spectra at different fixed delays indicate that optimum efficiency would require that the second photon has a wavelength of approximately 750 nm.