Photo-triggerable liposomes based on lipid-porphyrin conjugate and cholesterol combination: Formulation and mechanistic study on monolayers and bilayers.

Lipid-porphyrin conjugates are considered nowadays as promising building blocks for the conception of drug delivery systems with multifunctional properties such as photothermal therapy (PTT), photodynamic therapy (PDT), phototriggerable release, photoacoustic and fluorescence imaging. For this aim, we have recently synthesized a new lipid-porphyrin conjugate named PhLSM. This was obtained by coupling pheophorbide-a (Pheo-a), a photosensitizer derived from chlorophyll-a, to egg lyso-sphingomyelin. The pure PhLSMs were able to self-assemble into vesicle-like structures that were however not stable and formed aggregates with undefined structures due to the mismatch between the length of the alkyl chain in sn-1 position and the adjacent porphyrin. Herein, stable PhLSMs lipid bilayers were achieved by mixing PhLSMs with cholesterol which exhibits a complementary packing parameter. The interfacial behavior as well as the fine structures of their equimolar mixture was studied at the air/buffer interface by the mean of Langmuir balance and x-ray reflectomerty (XRR) respectively. Our XRR analysis unraveled the monolayer thickening and the increase in the lateral ordering of PhLSM molecules. Interestingly, we could prepare stable vesicles with this mixture that encapsulate hydrophilic fluorescent probe. The light-triggered release kinetics and the photothermal conversion were studied. Moreover, the obtained vesicles were photo-triggerable and allowed the release of an encapsulated cargo in an ON-OFF fashion.

Changes in Phospholipid/Ceramide Profiles and Eicosanoid Levels in the Plasma of Rats Irradiated with UV Rays and Treated Topically with Cannabidiol.

Chronic UV radiation causes oxidative stress and inflammation of skin and blood cells. Therefore, in this study, we assessed the effects of cannabidiol (CBD), a natural phytocannabinoid with antioxidant and anti-inflammatory properties, on the phospholipid (PL) and ceramide (CER) profiles in the plasma of nude rats irradiated with UVA/UVB and treated topically with CBD. The results obtained showed that UVA/UVB radiation increased the levels of phosphatidylcholines, lysophospholipids, and eicosanoids (PGE2, TxB2), while downregulation of sphingomyelins led to an increase in CER[NS] and CER[NDS]. Topical application of CBD to the skin of control rats significantly upregulated plasma ether-linked phosphatidylethanolamines (PEo) and ceramides. However, CBD administered to rats irradiated with UVA/UVB promoted further upregulation of CER and PEo and led to significant downregulation of lysophospholipids. This was accompanied by the anti-inflammatory effect of CBD, manifested by a reduction in the levels of proinflammatory PGE2 and TxB2 and a dramatic increase in the level of anti-inflammatory LPXA4. It can therefore be suggested that topical application of CBD to the skin of rats exposed to UVA/UVB radiation prevents changes in plasma phospholipid profile resulting in a reduction of inflammation by reducing the level of LPE and LPC species and increasing antioxidant capacity due to upregulation of PEo species.

Effect of a Radiotherapeutic Megavoltage Beam on Ultrasound Contrast Agents.

Collateral damage to healthy surrounding tissue during conventional radiotherapy increases when deviations from the treatment plan occur. Ultrasound contrast agents (UCAs) are a possible candidate for radiation dose monitoring. This study investigated the size distribution and acoustic response of two commercial formulations, SonoVue/Lumason and Definity/Luminity, as a function of dose on clinical megavoltage photon beam exposure (24 Gy). SonoVue samples exhibited a decrease in concentration of bubbles smaller than 7 µm, together with an increase in acoustic attenuation and a decrease in acoustic scattering. Definity samples did not exhibit a significant response to radiation, suggesting that the effect of megavoltage photons depends on the UCA formulation. For SonoVue, the influence of the megavoltage photon beam was especially apparent at the second harmonic frequency, and can be captured using pulse inversion and amplitude modulation (3.5-dB decrease for the maximum dose), which could eventually be used for dosimetry in a well-controlled environment.

Mimicking Photosystem I with a Transmembrane Light Harvester and Energy Transfer-Induced Photoreduction in Phospholipid Bilayers.

Photosystem I (PS I) is a transmembrane protein that assembles perpendicular to the membrane, and performs light harvesting, energy transfer, and electron transfer to a final, water-soluble electron acceptor. We present here a supramolecular model of it formed by a bicationic oligofluorene 12+ bound to the bisanionic photoredox catalyst eosin Y (EY2- ) in phospholipid bilayers. According to confocal microscopy, molecular modeling, and time dependent density functional theory calculations, 12+ prefers to align perpendicularly to the lipid bilayer. In presence of EY2- , a strong complex is formed (Ka =2.1±0.1×106 m-1 ), which upon excitation of 12+ leads to efficient energy transfer to EY2- . Follow-up electron transfer from the excited state of EY2- to the water-soluble electron donor EDTA was shown via UV-Vis absorption spectroscopy. Overall, controlled self-assembly and photochemistry within the membrane provides an unprecedented yet simple synthetic functional mimic of PS I.

Ultraviolet A irradiation induces ultraweak photon emission with characteristic spectral patterns from biomolecules present in human skin.

Oxidative stress is associated with photoaging of the skin as well as with skin cancer, and is therefore, critical to monitor. Ultraweak photon emission (UPE) is extremely weak light generated during the oxidative process in the living body and has been used as a non-invasive and label-free marker for the evaluation of oxidative stress. However, the mechanism of UPE generation is not clear. Therefore, we aimed to elucidate the molecular mechanism underlying UPE generation by analyzing the spectra of UPE generated from biomolecules in the skin during ultraviolet A (UVA) exposure. The spectra of UVA-induced UPE generated from linoleic acid, linolenic acid, elastin, phospholipids, and 5,6-dihydroxyindole-2-carboxylic acid were measured, and the spectrum of human skin tissue was also obtained. The spectral patterns varied for the different biomolecules and the peaks were distinct from those of the skin tissue. These results suggested that the UPE generated from skin tissue is a collection of light emitted by biomolecules. Moreover, we proposed that UPE is generated through a photosensitization reaction and energy transfer. The identified characteristic spectral patterns of UPE can be useful to elucidate UVA-induced oxidative stress in the skin, with implications for prevention and treatment of photoaging and skin diseases.

Risk estimation of the low-dose fast neutrons on the molecular structure of the lipids of peripheral blood mononuclear cells.

Our immune system can be exposed to neutral ionizing radiation. This work proposed to investigate the impacts of low-dose fast neutrons on the molecular structure of the lipids of peripheral blood mononuclear cells (PBMCs) as the primary immune cells. The effects have been followed at the time of 0, 2, 4, and 8 days after neutron exposure. Sixty-four female Wistar rats were used in this work. Thirty-two of them were irradiated using a low-dose fast neutron (241Am-Be, 0.2 mGy/h). The other thirty-two were utilized as a control group. The changes in their lipids molecular structure were investigated using Fourier transform infrared (FTIR) spectroscopy. Besides, lipids were extracted, and the total phospholipids contents were estimated. At the time of 2 and 4 days after irradiation, the results showed significant changes in the molecular structure of lipids in those irradiated samples compare with their control samples, and multivariate analysis succeeded in differentiating between control and irradiated rats. In contrast, no alterations between control and irradiated were detected at the time of 0 and 8 days after. The low-dose fast neutron could induce free radicals, which indirectly affect the molecular structure of the lipids and could damage the phospholipids molecules, and the damage could be repaired.

An Ultrasound Enhancing Agent with Nonlinear Acoustic Activity that Depends on the Presence of an Electric Field.

The nonlinear acoustic properties of microbubble ultrasound enhancing agents have allowed for the development of subharmonic, second harmonic, and contrast-pulse sequence ultrasound imaging modes, which enhance the quality, reduce the noise, and improve the diagnostic capabilities of clinical ultrasound. This study details acoustic scattering responses of perfluorobutane (PFB) microbubbles, an un-nested perfluoropentane (PFP) nanoemulsion, and two nested PFP nanoemulsions-one comprising a negatively charged phospholipid bilayer and another comprising a zwitterionic phospholipid bilayer-when excited at 1 or 2.25 MHz over a peak negative pressure range of 200 kPa to 4 MPa in the absence and presence of a 1-Hz, 1-V/cm electric field. The only sample that exhibited an increase in nonlinear activity in the presence of an electric field at both excitation frequencies was the negatively charged nested PFP nanoemulsion; the most pronounced effect was observed at an excitation of 2.25 MHz. Interestingly, the application of an electric field not only increased the nonlinear acoustic activity of the negatively charged nested PFP nanoemulsion but increased it beyond that seen when the nanoemulsion is un-nested and on the same scale as PFB microbubbles.

Emerging strategies to target cancer metabolism and improve radiation therapy outcomes.

Cancer-specific metabolic changes support the anabolic needs of the rapidly growing tumor, maintain a favorable redox balance, and help cells adapt to microenvironmental stresses like hypoxia and nutrient deprivation. Radiation is extensively applied in a large number of cancer treatment protocols but despite its curative potential, radiation resistance and treatment failures pose a serious problem. Metabolic control of DNA integrity and genomic stability can occur through multiple processes, encompassing cell cycle regulation, nucleotide synthesis, epigenetic regulation of gene activity, and antioxidant defenses. Given the important role of metabolic pathways in oxidative damage responses, it is necessary to assess the potential for tumor-specific radiosensitization by novel metabolism-targeted therapies. Additionally, there are opportunities to identify molecular and functional biomarkers of vulnerabilities to combination treatments, which could then inform clinical decisions. Here, we present a curated list of metabolic pathways in the context of ionizing radiation responses. Glutamine metabolism influences DNA damage responses by mechanisms such as synthesis of nucleotides for DNA repair or of glutathione for ROS detoxification. Repurposed oxygen consumption inhibitors have shown promising radiosensitizing activity against murine model tumors and are now in clinical trials. Production of 2-hydroxy glutarate by isocitrate dehydrogenase1/2 neomorphic oncogenic mutants interferes with the function of α-ketoglutarate-dependent enzymes and modulates Ataxia Telangiectasia Mutated (ATM) signaling and glutathione pools. Radiation-induced oxidative damage to membrane phospholipids promotes ferroptotic cell loss and cooperates with immunotherapies to improve tumor control. In summary, there are opportunities to enhance the efficacy of radiotherapy by exploiting cell-inherent vulnerabilities and dynamic microenvironmental components of the tumor.

Active S2168 and inactive S21IRS pinholin interact differently with the lipid bilayer: A 31P and 2H solid state NMR study.

Pinholins are a family of lytic membrane proteins responsible for the lysis of the cytosolic membrane in host cells of double stranded DNA bacteriophages. Protein-lipid interactions have been shown to influence membrane protein topology as well as its function. This work investigated the interactions of pinholin with the phospholipid bilayer while in active and inactive confirmations to elucidate the different interactions the two forms have with the bilayer. Pinholin incorporated into deuterated DMPC-d54 lipid bilayers, along with 31P and 2H solid state NMR (SS-NMR) spectroscopy were used to probe the protein-lipid interactions with the phosphorus head group at the surface of the bilayer while interactions with the 2H nuclei were used to study the hydrophobic core. A comparison of the 31P chemical shift anisotropy (CSA) values of the active S2168 pinholin and inactive S21IRS pinholin indicated stronger head group interactions for the pinholin in its active form when compared to that of the inactive form supporting the model of a partially externalized peripheral transmembrane domain (TMD) of the active S2168 instead of complete externalized TMD1 as suggested by Ahammad et al. JPC B 2019. The 2H quadrupolar splitting analysis showed a decrease in spectral width for both forms of the pinholin when compared to the empty bilayers at all temperatures. In this case the decrease in the spectral width of the inactive S21IRS form of the pinholin showed stronger interactions with the acyl chains of the bilayer. The presence of the inactive form's additional TMD within the membrane was supported by the loss of peak resolution observed in the 2H NMR spectra.

Analysis of acoustic nonlinearity parameter B/A in liquids containing ultrasound contrast agents.

The acoustic nonlinearity parameter B/A plays a significant role in the characterization of acoustic properties of various biomaterials and biological tissues. It has the potential to be a favorable imaging modality in contrast ultrasound imaging with coated microbubbles. However, the development of effective means for evaluating the nonlinearity parameter of suspensions of ultrasound contrast agents (UCAs, also known as bubbly liquids) remains open. The present paper formulates a new equation based on the thermodynamic method that correlates both attenuation and phase velocity of linear ultrasound. The simplicity of the present method makes the B/A estimation possible with a relatively rigorous mathematical derivation. The calculated nonlinearity parameter contains the contribution of dynamic effects of bubbles, and its low-frequency limit agrees with B/A estimated by the method of mixture law when the volume fraction is below 10-4. Furthermore, the maximum B/A in bubbly liquids can reach up to105, while the minimum can be as low as -105. The negative nonlinearity parameter indicates significantly different thermodynamic properties of bubbly liquids.

Novel hyaluronic acid-modified temperature-sensitive nanoparticles for synergistic chemo-photothermal therapy.

This study has developed a versatile nano-system with the combined advantages of photothermal effect, active tumor-targeting, temperature-sensitive drug release, and photoacoustic imaging. The nano-system consists of the core of the phase change material (PCM), the outer polypyrrole (PPY) shell and the hyaluronic acid (HA) modified in the PPY shell. The obtained composite nanoparticles (denoted as DTX/PPN@PPY@HA) were spherical with a mean diameter of about 232.7 nm. In vivo and in vitro photoacoustic imaging experiments show that DTX/PPN@PPY@HA is an effective photoacoustic contrast agent, which can be used for accurate localization of tumor region and real-time guidance of photothermal chemotherapy. DTX/PPN@PPY@HA shows good photothermal effects and temperature-sensitive drug release. In addition, cellular experiments showed that DTX/PPN@PPY@HA could be efficiently internalized into tumor cells and produce significant cytotoxicity with the help of near-infrared (NIR) laser. Furthermore, the remarkable inhibition of DTX/PPN@PPY@HA against tumor growth was achieved in 4T1 tumor-bearing mice model.

Conformational photoswitching of a synthetic peptide foldamer bound within a phospholipid bilayer.

The dynamic properties of foldamers, synthetic molecules that mimic folded biomolecules, have mainly been explored in free solution. We report on the design, synthesis, and conformational behavior of photoresponsive foldamers bound in a phospholipid bilayer akin to a biological membrane phase. These molecules contain a chromophore, which can be switched between two configurations by different wavelengths of light, attached to a helical synthetic peptide that both promotes membrane insertion and communicates conformational change along its length. Light-induced structural changes in the chromophore are translated into global conformational changes, which are detected by monitoring the solid-state (19)F nuclear magnetic resonance signals of a remote fluorine-containing residue located 1 to 2 nanometers away. The behavior of the foldamers in the membrane phase is similar to that of analogous compounds in organic solvents.

Interfacial Rheological Properties of Contrast Microbubble Targestar P as a Function of Ambient Pressure.

In this Technical Note, we determine the interfacial rheological parameters of the encapsulation of the contrast agent Targestar P using ultrasound attenuation. The characteristic parameters are obtained according to two interfacial rheological models. The properties-surface dilatational elasticity (0.09 ± 0.01 N/m) and surface dilatational viscosity (8 ± 0.1E-9 N·s/m)-are found to be of similar magnitude for both models. Contrast microbubbles experience different ambient pressure in different organs. We also measure these parameters as functions of ambient pressure using attenuation measured at different overpressures (0, 100 and 200 mm Hg). For each value of ambient hydrostatic pressure, we determine the rheological properties, accounting for changes in the size distribution caused by the pressure change. We discuss different models of size distribution change under overpressure: pure adiabatic compression or gas exchange with surrounding medium. The dilatational surface elasticity and viscosity are found to increase with increasing ambient pressure.

Targeted gene delivery to the synovial pannus in antigen-induced arthritis by ultrasound-targeted microbubble destruction in vivo.

The purpose of this study was to optimize an ultrasound-targeted microbubble destruction (UTMD) technique to improve the in vivo transfection efficiency of the gene encoding enhanced green fluorescent protein (EGFP) in the synovial pannus in an antigen-induced arthritis rabbit model. A mixture of microbubbles and plasmids was locally injected into the knee joints of an antigen-induced arthritis (AIA) rabbits. The plasmid concentrations and ultrasound conditions were varied in the experiments. We also tested local articular and intravenous injections. The rabbits were divided into five groups: (1) ultrasound+microbubbles+plasmid; (2) ultrasound+plasmid; (3) microbubble+plasmid; (4) plasmid only; (5) untreated controls. EGFP expression was observed by fluorescent microscope and immunohistochemical staining in the synovial pannus of each group. The optimal plasmid dosage and ultrasound parameter were determined based on the results of EGFP expression and the present and absent of tissue damage under light microscopy. The irradiation procedure was performed to observe the duration of the EGFP expression in the synovial pannus and other tissues and organs, as well as the damage to the normal cells. The optimal condition was determined to be a 1-MHz ultrasound pulse applied for 5 min with a power output of 2 W/cm(2) and a 20% duty cycle along with 300 µg of plasmid. Under these conditions, the synovial pannus showed significant EGFP expression without significant damage to the surrounding normal tissue. The EGFP expression induced by the local intra-articular injection was significantly more increased than that induced by the intravenous injection. The EGFP expression in the synovial pannus of the ultrasound+microbubbles+plasmid group was significantly higher than that of the other four groups (P<0.05). The expression peaked on day 5, remained detectable on day 40 and disappeared on day 60. No EGFP expression was detected in the other tissues and organs. The UTMD technique can significantly enhance the in vivo gene transfection efficiency without significant tissue damage in the synovial pannus of an AIA model. Thus, this could become a safe and effective non-viral gene transfection procedure for arthritis therapy.

Ultrafast 2-dimensional image monitoring and array-based passive cavitation detection for ultrasound contrast agent destruction in a variably sized region.

OBJECTIVES: A combined approach was proposed, based on programmable ultrasound equipment, to simultaneously monitor surviving microbubbles and detect cavitation activity during microbubble destruction in a variably sized region for use in ultrasound contrast agent (UCA)-enhanced therapeutic ultrasound applications. METHODS: A variably sized focal region wherein the acoustic pressure was above the UCA fragmentation threshold was synthesized at frequencies of 3, 4, 5, and 6 MHz with a linear broadband imaging probe. The UCAs' temporal and spatial distribution during the microbubbles' destruction was monitored in a 2-dimensional imaging plane at 5 MHz and a frame rate of 400 Hz, and simultaneously, broadband noise emissions during the microbubbles' fragmentation were extracted by using the backscattered signals produced by the focused release bursts (ie, destruction pulses) themselves. Afterward, the temporal evolution of broadband noise emission, the surviving microbubbles in a region of interest (ROI), and the destruction area in a static UCA suspension were computed. Then the inertial cavitation dose, destruction rate of microbubbles in the ROI, and area of the destruction region were determined. RESULTS: It was found that an increasing pulse length and a decreasing transmit aperture and excitation frequency were correlated with an increased inertial cavitation dose, microbubble destruction rate, and destruction area. Furthermore, it was obvious that the microbubble destruction rate was significantly correlated with the inertial cavitation dose (P < .05). In addition, the intensity decrease in the ROI was significantly correlated with the destruction area (P < .05). CONCLUSIONS: By the proposed strategy, microbubbles could be destroyed in a variably sized region, and destruction efficiency as well as the corresponding inertial cavitation dose could be regulated by manipulating the transmission parameters.

The delayed onset of subharmonic and ultraharmonic emissions from a phospholipid-shelled microbubble contrast agent.

Characterizing the non-linear response of microbubble contrast agents is important for their efficacious use in imaging and therapy. In this article, we report that the subharmonic and ultraharmonic response of lipid-shelled microbubble contrast agents exhibits a strong temporal dependence. We characterized non-linear emissions from Targestar-p microbubbles (Targeson Inc., San Diego, CA, USA) periodically for 60 min, at 10 MHz excitation frequency. The results revealed a considerable increase in the subharmonic and ultraharmonic response (nearly 12-15 and 5-8 dB) after 5-10 min of agent preparation. However, the fundamental and the harmonic response remained almost unchanged in this period. During the next 50 min, the subharmonic, fundamental, ultraharmonic, and harmonic responses decreased steadily by 2-5 dB. The temporal changes in the non-linear behavior of the agent appeared to be primarily mediated by gas-exchange through the microbubble shell; temperature and prior acoustic excitation based mechanisms were ruled out. Further, there was no measurable change in the agent size distribution by static diffusion. We envisage that these findings will help obtain reproducible measurements from agent characterization, non-linear imaging, and fluid-pressure sensing. These findings also suggest the possibility for improving non-linear imaging by careful design of ultrasound contrast agents.

Measuring absolute blood pressure using microbubbles.

Gas microbubbles are highly compressible, which makes them very efficient sound scatterers. As another consequence of their high compressibility, the radii of the microbubbles are affected by the pressure of the fluid around them, which changes their resonance frequency. Although the pressures present within the human body cause only minor variations in the radii of uncoated microbubbles (∼0.2% per 10 mmHg) and, therefore, very small variations in the resonance frequency (∼1 kHz per 10 mmHg), it was found in the work described here, through both simulations and in vitro measurements, that large changes in resonance frequency can occur in phospholipid-coated microbubbles for small blood pressure variations because of the exotic buckling dynamics of phospholipid monolayers (up to 240 kHz per 10 mmHg). This method should allow non-invasive measurement of the gauge blood pressure in deep blood vessels as long as the microbubble physical properties are well controlled.

In vitro acoustic characterization of three phospholipid ultrasound contrast agents from 12 to 43 MHz.

The acoustic properties of two clinical (Definity, Lantheus Medical Imaging, North Billerica, MA, USA; SonoVue, Bracco S.P.A., Milan, Italy) and one pre-clinical (MicroMarker, untargeted, Bracco, Geneva, Switzerland; VisualSonics, Toronto, ON, Canada) ultrasound contrast agent were characterized using a broadband substitution technique over the ultrasound frequency range 12-43 MHz at 20 ± 1°C. At the same number concentration, the acoustic attenuation and contrast-to-tissue ratio of the three native ultrasound contrast agents are comparable at frequencies below 30 MHz, though their size distributions and encapsulated gases and shells differ. At frequencies above 30 MHz, native MicroMarker has higher attenuation values and contrast-to-tissue ratios than native Definity and SonoVue. Decantation was found to be an effective method to alter the size distribution and concentration of native clinical microbubble populations, enabling further contrast enhancement for specific pre-clinical applications.

Ultrasound and microbubble-targeted delivery of small interfering RNA into primary endothelial cells is more effective than delivery of plasmid DNA.

Ultrasound and microbubble-targeted delivery (UMTD) is a promising non-viral technique for genetic-based therapy. We found that UMTD of small interfering RNA (siRNA) is more effective than delivery of plasmid DNA (pDNA). UMTD (1 MHz, 0.22 MPa) of fluorescently labeled siRNA resulted in 97.9 ± 1.5% transfected cells, with siRNA localized homogenously in the cytoplasm directly after ultrasound exposure. UMTD of fluorescently labeled pDNA resulted in only 43.0 ± 4.2% transfected cells, with localization mainly in vesicular structures, co-localizing with endocytosis markers clathrin and caveolin. Delivery of siRNA against GAPDH (glyceraldehyde-3-phosphate dehydrogenase) effectively decreased protein levels to 24.3 ± 7.9% of non-treated controls (p < 0.01). In contrast, 24 h after delivery of pDNA encoding GAPDH, no increase in protein levels was detected. Transfection efficiency, verified with red fluorescently labeled pDNA encoding enhanced green fluorescent protein, revealed that of the transfected cells, only 2.0 ± 0.7% expressed the transgene. In conclusion, the difference in localization between siRNA and pDNA after UMTD is an important determinant of the effectiveness of these genetic-based technologies.

Photodynamic oxidation of Staphylococcus warneri membrane phospholipids: new insights based on lipidomics.

RATIONALE: The photodynamic process involves the combined use of light and a photosensitizer, which, in the presence of oxygen, originates cytotoxic species capable of oxidizing biological molecules, such as lipids. However, the effect of the photodynamic process in the bacterial phospholipid profile by a photosensitizer has never been reported. A lipidomic approach was used to study the photodynamic oxidation of membrane phospholipids of Staphylococcus warneri by a tricationic porphyrin [5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin triiodide, Tri-Py(+)-Me-PF]. METHODS: S. warneri (10(8) colony forming units mL(-1)) was irradiated with white light (4 mW cm(-2), 21.6 J cm(-2)) in the presence of Tri-Py(+)-Me-PF (5.0 µM). Non-photosensitized bacteria were used as control (irradiated without porphyrin). After irradiation, total lipids were extracted and separated by thin-layer chromatography (TLC). Isolated fractions of lipid classes were quantified by phosphorus assay and analyzed by mass spectrometry (MS): off-line TLC/ESI-MS, hydrophilic interaction (HILIC)-LC/MS and MS/MS. RESULTS: The most representative classes of S. warneri phospholipids were identified as phosphatidylglycerols (PGs) and cardiolipins (CLs). Lysyl-phosphatidylglycerols (LPGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs) and phosphatidic acids (PAs) were also identified. After photodynamic treatment, an overall increase in the relative abundance of PGs was observed as well as the appearance of new oxidized species from CLs, including hydroxy and hydroperoxy derivatives. Formation of high amounts of lipid hydroperoxides was confirmed by FOX2 assay. Photodynamic oxidation of phospholipid standards revealed the formation of hydroperoxy and dihydroperoxy derivatives, confirming the observed CL oxidized species in S. warneri. CONCLUSIONS: Membrane phospholipids of S. warneri are molecular targets of the photoinactivation process induced by Tri-Py(+) -Me-PF. The overall modification in the relative amount of phospholipids and the formation of lipid hydroxides and hydroperoxides indicate the lethal damage caused to photosensitized bacterial cells.