Support for the Safe Use of Zinc Oxide Nanoparticle Sunscreens: Lack of Skin Penetration or Cellular Toxicity after Repeated Application in Volunteers.

Zinc oxide is a widely used broad-spectrum sunscreen, but concerns have been raised about the safety of its nanoparticle (NP) form. We studied the safety of repeated application of agglomerated zinc oxide (ZnO) NPs applied to human volunteers over 5 days by assessing the skin penetration of intact ZnO-NPs and zinc ions and measuring local skin toxicity. Multiphoton tomography with fluorescence lifetime imaging microscopy was used to directly visualize ZnO-NP skin penetration and viable epidermal metabolic changes in human volunteers. The fate of ZnO-NPs was also characterized in excised human skin in vitro. ZnO-NPs accumulated on the skin surface and within the skin furrows but did not enter or cause cellular toxicity in the viable epidermis. Zinc ion concentrations in the viable epidermis of excised human skin were slightly elevated. In conclusion, repeated application of ZnO-NPs to the skin, as used in global sunscreen products, appears to be safe, with no evidence of ZnO-NP penetration into the viable epidermis nor toxicity in the underlying viable epidermis. It was associated with the release and penetration of zinc ions into the skin, but this did not appear to cause local toxicity.

Interrogation of metabolic and oxygen states of tumors with fiber-based luminescence lifetime spectroscopy.

The study of metabolic and oxygen states of cells in a tumor in vivo is crucial for understanding of the mechanisms responsible for tumor development and provides background for the relevant tumor's treatment. Here, we show that a specially designed implantable fiber-optic probe provides a promising tool for optical interrogation of metabolic and oxygen states of a tumor in vivo. In our experiments, the excitation light from a ps diode laser source is delivered to the sample through an exchangeable tip via a multimode fiber, and the emission light is transferred to the detector by another multimode fiber. Fluorescence lifetime of a nicotinamid adenine dinucleotide (NAD(P)H) and phosphorescence lifetime of an oxygen sensor based on an iridium (III) complex of enzothienylpyridine (BTPDM1) are explored both in model experiment in solutions and in living mice.

Non-invasive metabolic imaging of melanoma progression.

Skin cancer is associated with abnormal cellular metabolism which if identified early introduces the possibility of intervention to prevent its progress to a deadly metastatic stage. This study combines multiphoton microscopy with fluorescence lifetime imaging (FLIM) using a syngeneic melanoma mouse model, to detect changes in metabolic state of single epidermal cells as a metabolic marker to monitor the progress of tumor growth. This method utilizes imaging of the ratio of the amounts of the free and protein-bound forms of the intracellular autofluorescent metabolic co-enzyme nicotinamide adenine dinucleotide (NADH). Here, we investigate the impact of the primary tumor lesion on the epidermal layers at three different growth stages of melanoma lesion compared to normal skin as a control. We showed a significant increase in the free-to-bound NADH ratio with the growth of the solid melanoma tumor, while concurrently the short and the long lifetime components of NADH remained constant. These results demonstrate the ability of FLIM for rapid, non-invasive and sensitive assessment of melanoma progression revealing its potential as a diagnostic tool for melanoma detection and as an aid for melanoma staging.

Varying the morphology of silver nanoparticles results in differential toxicity against micro-organisms, HaCaT keratinocytes and affects skin deposition.

The use of silver nanoparticles (Ag NPs) within the healthcare sector and consumer products is rapidly increasing. There are now a range of diverse-shaped Ag NPs that are commercially available and many of the products containing nanosilver are topically applied to human skin. Currently, there is limited data on the extent to which the antimicrobial efficacy and cytotoxicity of Ag NPs is related to their shape and how the shape of the Ag NPs affects their distribution in both intact and burn wounded human skin after topical application. In this study, we related the relative Ag NP cytotoxicity to potential skin pathogens and HaCaT keratinocytes in vitro with the shape of the Ag NPs. We employed multiphoton fluorescence lifetime imaging to map the distribution of the native and unlabeled Ag NPs after topical application to both intact and burn wounded human skin using the localized surface plasmon resonance signal of the Ag NPs. Truncated plate shaped Ag NPs led to the highest cytotoxicity against both bacteria (IC50 ranges from 31.25 to 125 µg/mL depending on the bacterial species) and HaCaT keratinocytes (IC50 78.65 µg/mL [95%CI 63.88, 96.83]) thus both with similar orders of magnitude. All Ag NPs were less cytotoxic than solutions of silver nitrate (IC50 of 7.85 µg/mL [95%CI 1.49, 14.69]). Plate-shaped Ag NPs displayed the highest substantivity within the superficial layers of the stratum corneum when topically applied to intact skin and the highest deposition into the wound bed when applied to burned ex vivo human skin relative to other Ag NP shapes.

Effect of flexing and massage on in vivo human skin penetration and toxicity of zinc oxide nanoparticles.

AIM: We assessed the effects of flexing and massage on human skin penetration and toxicity of topically applied coated and uncoated zinc oxide nanoparticles (˜75 nm) in vivo. MATERIALS & METHODS: Noninvasive multiphoton tomography with fluorescence lifetime imaging was used to evaluate the penetration of nanoparticles through the skin barrier and cellular apoptosis in the viable epidermis. RESULTS: All nanoparticles applied to skin with flexing and massage were retained in the stratum corneum or skin furrows. No significant penetration into the viable epidermis was seen and no cellular toxicity was detected. CONCLUSION: Exposure of normal in vivo human skin to these nanoparticles under common in-use conditions of flexing or massage is not associated with significant adverse events.

Time-resolved spectrometry of mitochondrial NAD(P)H fluorescence and its applications for evaluating the oxidative state in living cells.

Time-resolved fluorescence spectrometry is a highly valuable technological tool to detect and characterize mitochondrial metabolic oxidative changes by means of endogenous fluorescence (Chorvat and Chorvatova, Laser Phys Lett 6: 175-193, 2009). Here, we describe the detection and measurement of endogenous mitochondrial NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence directly in living cultured cells using fluorescence lifetime spectrometry imaging after excitation with 405 nm picosecond (ps) laser. Time-correlated single photon counting (TCSPC) method is employed.

Comparison of broadband and ultrabroadband pulses at MHz and GHz pulse-repetition rates for nonlinear femtosecond-laser scanning microscopy.

Nonlinear optical imaging of human skin and of polychromatic microspheres was carried out to compare and evaluate the imaging properties of three different excitation femtosecond lasers: a spectrally tunable 80 MHz Ti: sapphire oscillator that produced 100 fs pulses (spectral width ∼10 nm) and two ultrabroadband Ti: sapphire oscillators with repetition rates of 85 MHz and 1 GHz. The latter of these two and the 100 fs laser were combined with a laser scanning microscope (TauMap). The intensities of images of the polychromatic microsphere samples obtained with both lasers are in accordance with the usual dependence of two-photon processes on laser pulse parameters, i.e. the intensity is proportional to the square of the mean laser power and the reciprocal pulse duration. In contrast to that, skin images measured with all three different excitation sources with mean powers of each laser adjusted to the particular pulse length and repetition rate exhibited discrepancies from this relation. For characterization of the ultrabroadband GHz laser, the measurements are supplemented by spectra of second-harmonic-generation signals of urea and collagen.

Multiphoton excitation characteristics of cellular fluorophores of human skin in vivo.

In vivo multiphoton tomography with a wavelength-tunable femtosecond laser has been performed to investigate the autofluorescence intensity of major endogenous fluorophores of human skin in dependence on the excitation wavelength. In high-resolution multiphoton images of different skin layers, clear trends were found for fluorophores like keratin, NAD(P)H, melanin as well as for the elastin and collagen networks. The analysis of the measurements is supplemented by additional measurements of fluorescence lifetime imaging and signal-decay curves by time-correlated single-photon counting.

Trafficking of mature miRNA-122 into the nucleus of live liver cells.

The binding of superquencher molecular beacon (SQMB) probes to human single-stranded cellular miRNA-122 targets was detected in various single live cells with femtosecond laser microscopy. For delivery of the SQMB-probes, 3D-nanoprocessing of single cells with sub-15 femtosecond 85 MHz near-infrared laser pulses was applied. Transient nanopores were formed by focusing the laser beam for some milliseconds on the membrane of a single cell in order to import of SQMB-probes into the cells. In single cells of the human liver cell lines Huh-7D12 and IHH that expressed miRNA-122, we measured target binding in the cytoplasm by two-photon fluorescence imaging. We found increased fluorescence with time in a nonlinear manner up to the point where steady state saturation was reached. We also studied the intracellular distribution of target SQMB and provide for the first time strong experimental evidence that cytoplasmic miRNA travels into the cell nucleus. To interpret nonlinear binding, a number of individual miRNA-122 positive cells (Huh-7D12 and IHH) and negative control cells, human VA13 fibroblasts and Caco-2 cells were analyzed. Our experimental data are consistent with the cytoplasmic assembly of nuclear miRNA and provide further mechanistic insight in the regulatory function of miRNAs in cellular physiology. An open issue in the regulation of gene expression by miRNA is whether miRNA can activate gene expression in addition to the well-known inhibitory effect. A first step for such a regulatory role could be the travelling of miRNA-RISC into the nucleus.

Spectral diffusion induced by proton dynamics in pigment-protein complexes.

The fluorescence emission of individual photosystem I complexes from Synechocystis PCC 6803 in protonated and deuterated buffer shows zero-phonon lines as well as broad intensity distributions. The number and the line width of the zero phonon lines depend strongly on the solvent (H(2)O/D(2)O). The spectral diffusion rate of the whole fluorescence emission from photosystem I is significantly reduced upon deuteration of the solvent. This leads to a substantial increase of well-resolved zero-phonon lines. Since the chlorophyll a chromophores lack exchangeable protons, these observed changes in the spectral diffusion have to be assigned to exchangeable protons at the amino acids and structural water molecules in the chromophore binding pocket.

Red antenna states of photosystem I from Synechocystis PCC 6803.

Single-molecule spectroscopy at low temperatures was used to elucidate spectral properties, heterogeneities, and dynamics of the red-shifted chlorophyll a (Chl a) molecules responsible for the fluorescence from photosystem I (PSI). Emission spectra of single PSI complexes from the cyanobacterium Synechocystis PCC 6803 show zero-phonon lines (ZPLs) as well as broad intensity distributions without ZPLs. ZPLs are found most frequently on the blue side of the broad intensity distributions. The abundance of ZPLs decreases almost linearly at longer wavelengths. The distribution of ZPLs indicates the existence of at least two pools with maxima at 699 and 710 nm. The pool with the maximum at 710 nm is assigned to chlorophylls absorbing around 706 nm (C706), whereas the pool with the maximum at 699 nm (F699) can be assigned to chlorophylls absorbing at 692, 695, or 699 nm. The broad distributions dominating the red side of the spectra are made up of a low number of emitters assigned to the red-most pool C714. The properties of F699 show close relation to those of F698 in Synechococcus PCC 7002 and C708 in Thermosynechococcus elongatus. Furthermore, a high similarity is found between the C714 pool in Synechocystis PCC 6803 and C708 in Synechococcus PCC 7002 as well as C719 in T. elongatus.

Red antenna states of photosystem I from Synechococcus sp. PCC 7002.

Absorption, fluorescence and single-molecule spectroscopy at low temperatures were used to elucidate spectral properties, heterogeneities and dynamics of the red-shifted chlorophyll a (Chla) molecules responsible for the fluorescence in photosystem I (PSI) from the cyanobacterium Synechoccocus sp. PCC 7002. The 77 K absorption spectrum indicates the presence of 2-3 red-shifted Chla's absorbing at about 708 nm. The fluorescence emission spectrum is dominated by a broad band at 714 nm. The emission spectra of single PSI complexes show zero-phonon lines (ZPLs) as well as a broad intensity distribution without ZPLs. The spectral region below 710 nm often shows ZPLs, they form a spectral band with a maximum at 698 nm (F698). The region above 710 nm is dominated by broad intensity distributions and the observation of ZPLs is less frequent. The broad distributions are due to the emission of the C708 Chla's and the emission from F698 stems from a Chla species absorbing at the blue side of P700. The properties of these two emissions show a close relation to those of the C708 and C719 pools observed in T. elongatus. Therefore an assignment of F698 and C708 to Chla-species with similarities to C708 and C719 in T. elongatus is proposed.

Assignment of red antenna states in photosystem I from Thermosynechococcus elongatus by single-molecule spectroscopy.

Single-molecule spectroscopy at cryogenic temperatures was used to elucidate spectral properties, heterogeneities, and dynamics of the chlorophyll a (Chla) molecules responsible for the fluorescence in photosystem I (PSI) from the cyanobacteria Thermosynechococcus elongatus. Absorption and hole burning data suggest the presence of three pools absorbing at wavelengths greater than 700 nm with their absorption maxima at 708, 715, and 719 nm. The responsible Chla molecules are termed C708, C715, and C719. In the emission spectra of single PSI complexes, zero-phonon lines (ZPLs) were observed over the whole red emission range of PSI. The spectral region of the C708 pool is dominated by intense ZPLs; on the other hand, the broad emission of C715/C719 is unstructured and ZPLs are seen in this region much less frequently. Spectral jumps of ZPLs were observed. The dynamics as well as the spectral range covered by such jumps differ for C708 and C715/C719. This heterogeneity is likely caused by differences in the close environment of the chromophores. A tentative assignment of C708 and C715/C719 to Chla dimers and a Chla trimer is discussed, which is based on the remarkable structural differences in the environment of the most probable candidates for the red-most fluorescence.

Red pool chlorophylls of photosystem I of the cyanobacterium Thermosynechococcus elongatus: a single-molecule study.

Photosystem I reaction centers of the cyanobacterium Thermosynechococcus elongatus have been investigated using single-molecule spectroscopy. Single-molecule fluorescence emission spectra reveal a new fluorescence band located at 745 nm. Fluorescence polarization spectroscopy and fluorescence autocorrelation analysis show that only a few chlorophylls are responsible for the photoemission from the Photosystem I trimer at low temperature. Intersystem crossing parameters of the red pool chlorophylls have been determined via fluorescence autocorrelation measurements. The triplet yield of the red chlorophylls is strongly reduced in comparison to chlorophyll a in solution. Strong quenching of the triplet state indicates that the red chlorophylls are located in close contact to carotenoids.