Frank Stracke

Nanoparticles and their interactions with the dermal barrier

The dermal application of drugs is promising due to the ease of application. In this context nano-scale carrier systems were already evaluated in several studies with respect to the skin interaction and the impact on drug penetration. At the same time the upcoming production of engineered nano-scale materials requires a thorough safety evaluation. Drug delivery as well as risk assessment depends crucially on the ability of such carriers to overcome the skin barrier and reach deeper tissue layers. Therefore, the interaction of nanoparticles with skin and especially skin models is an intriguing field. However, the data obtained do not show a clear image on the effect of nano-carriers. Especially the penetration of such particles is an open and controversially discussed topic. The literature reports different results mainly on pig or murine skin showing strong penetration (pig and mouse) or the opposite. Looking only at the sizes of the particles also no conclusive picture can be obtained. Nevertheless, size is regarded to play an important role for skin penetration. Furthermore, the state of the skin influences penetration (hydration) and the mechanical stress is of outmost importance.

Influence of Nanoencapsulation on Human Skin Transport of Flufenamic Acid

The effect of the inclusion of flufenamic acid in poly(lactide-co-glycolide) nanoparticles on the transport of flufenamic acid into excised human skin was investigated. Penetration and permeation data were acquired using two different in vitro test systems: the Saarbrücken penetration model, where the skin acts as its own receptor medium, and the Franz diffusion cell, where the receptor medium is a buffer solution. For the stratum corneum, no differences were found between nanoencapsulated and free drug. Drug accumulation in the deeper skin layers and drug transport across human epidermis were slightly delayed for the nanoencapsulated drug compared to the free drug after shorter incubation times (<12 h). In contrast, after longer incubation times (>12 h), the nanoencapsulated drug showed a statistically significantly enhanced transport and accumulation (p < 0.05). Additionally, nanoencapsulated flufenamic acid was visualized by multiphoton fluorescence microscopy. Particles were found homogeneously distributed on the skin surface and within the dermatoglyphs, but no nanoparticles were detected within or between the corneocytes.

In vivo Drug Screening in Human Skin Using Femtosecond Laser Multiphoton Tomography

The novel femtosecond laser multiphoton imaging system DermaInspect forin vivotomography of human skin was used to study the diffusion and intradermal accumulation of topically applied cosmetic and pharmaceutical components. Near-infrared 80 MHz picojoule femtosecond laser pulses were employed to excite endogenous fluorophores and fluorescent components of a variety of ointments via a two-photon excitation process. In addition, collagen was imaged by second harmonic generation. A high submicron spatial resolution and 50 ps temporal resolution was achieved using galvoscan mirrors and piezodriven focusing optics together with a time-correlated single-photon counting module with a fast microchannel plate detector. Individual intratissue cells, intracellular mitochondria, melanosomes, and the morphology of the nuclei as well as extracellular matrix elements were clearly visualized due to NAD(P)H, melanin, elastin, and collagen imaging and the calculation of fluorescence lifetime images. Nanoparticles and intratissue drugs were detected by two-photon-excited fluorescence. In addition, hydration effects and UV effects were studied by monitoring modifications of cellular morphology and autofluorescence. The system was used to observe the diffusion through the stratum corneum and the accumulation and release of functionalized nanoparticles along hair shafts and epidermal ridges. The novel noninvasive 4-D multiphoton tomography tool provides high-resolution optical biopsies with subcellular resolution, and offers for the first time the possibility to study in situthe diffusion through the skin barrier, long-term pharmacokinetics, and cellular response to cosmetic and pharmaceutical products.

Large-area, uniform, high-spatial-frequency ripples generated on silicon using a nanojoule-femtosecond laser at high repetition rate

Large-area high-spatial-frequency patterns (HSFLs) of λ/6 periodicity have been generated by a nanojoule-femtosecond laser scanning technique (80 MHz, 170 fs, 700-950 nm) at the silicon-air interface. The excellent large-area uniformity allowed reproducible and accurate measurements of the periodicity. Variation of experimental parameters as illumination geometry, and pulse energy and number showed no influence on the ripple spacing. A wavelength dependence was observed and compared to current models of HSFL formation. A particular second-harmonic model was found to match the results best but needs to take into account transient changes in the refractive index under laser exposure. A second-harmonic mechanism is further supported by direct spectroscopic observation.

Preparation and biological evaluation of multifunctional PLGA-nanoparticles designed for photoacoustic imaging

UNLABELLED Nanoparticulate contrast agents for molecular imaging have attracted widespread interest for diagnostic applications with high resolution in medicine. Here we introduce polymer-based multifunctional nanoparticles exhibiting a near-infrared absorption in the range of the Nd:YAG laser wavelength of 1064 nm as a novel resorbable photoacoustic (PA) contrast system and report about their biological evaluation. Submicron-sized spherical nanoparticles with a high encapsulation efficiency (>87%) were created by incorporation of near-infrared dyes (IR5/IR26) in poly[(rac-lactide)-co-glycolide] (PLGA) with 50 mol% glycolide content via a specific spray-drying process in good yield (>75%). Subsequent application of a centrifugation protocol produced two different size fractions with diameters in the ranges 445-540 nm and 253-305 nm; these were further used for investigation of PA properties and cytotoxic effects. The prepared PLGA nanoparticles exhibited PA properties using a Nd:YAG laser-based system. After exposure of particle concentrations up to 10 μg·ml(-1) for 2 days no effects on viability, mitochondrial activity and proliferation, and cell death of human hepatocarcinoma cells and monkey kidney cells were observed. The excellent PA properties in combination with the positive biological results qualify the dye-loaded PLGA particles as promising candidates for a resorbable PA contrast system. FROM THE CLINICAL EDITOR Photoacoustics (PA), a new modality, in which laser light is shined into tissue and absorbed by inherent proteins or synthetic particles is reflected back and received as ultrasound. This technique was shown to be effective with an erodible polymer particle containing near infrared dyes. In vitro, the PA properties of the PLGA particles persisted for 2 days in cell culture.

Generation of high spatial frequency ripples on silicon under ultrashort laser pulses irradiation

Periodic high spatial frequency ripples structures have been generated in silicon under femtosecond laser pulses irradiation. The period of the ripples is wavelength dependent. It increases from 110 up to 160 nm when the wavelength varies from 700 to 950 nm, respectively. We propose a refined model of the second harmonic generation ripples spacing theory Λ=λ/2nλ∗ taking into account the modified femtosecond laser excited silicon refractive index n∗ related to the Drude model. Good agreement is found between experimental results and the presented revisited model.

Intrinsic conformer jumps observed by single molecule spectroscopy in real time

Abstract Single molecule spectral dynamics originate from changes (1) within the chromophore and (2) in the chromophoric environment, hence are intrinsic and extrinsic. To interpret spectral phenomena of intrinsic origin, assignment of fluorescence behaviour to distinct molecular states is required. We realized this by means of a perylene based chromophore with an amino substituent. The fluorescence spectrum of this dye depends strongly on the amino group conformation relative to the chromophore. Thus different conformers of the chromophore can be distinguished. Here we evidence the occurrence of spectrally defined conformers and report transitions between them, revealed by single molecule spectroscopy.

Formation mechanism of femtosecond laser-induced high spatial frequency ripples on semiconductors at low fluence and high repetition rate

Periodic high spatial frequency ripples structures (HSFL) have been generated in silicon (Si) and in germanium (Ge) at very low fluence below or close to the melting fluence threshold, at different wavelengths and at high repetition rate femtosecond laser pulses (80 MHz, 700–950 nm, 170 fs). HSFL initiation, formation, and arrangement combine structural modification of the surface initiated by heat accumulation of successive pulses with second harmonic generation. HSFL are wavelength dependent and the refractive index plays a central role on their periodicities. HSFL spacing follows quite well a law of Λ=λ/2nλ*, where nλ* is the modified femtosecond laser excited refractive index as a function of the wavelength for Si and Ge.

Dynamics of single dye molecules observed by confocal imaging and spectroscopy

The fluorescence emission of single rhodamine dye molecules (rhodamine 6G and rhodamine 630) at room temperature was analyzed by using scanning confocal laser microscopy in conjunction with polarization analysis, fluorescence spectroscopy, time-resolved detection (minutes to microseconds), and excitation saturation. Results are presented and discussed 1) for samples with dye molecules at the glass-air interface and 2) covered with an additional thin protective polymer film (polyvinylbutyral). Under the polymer layer, the single-molecule fluorescence was more stable than the glass-air interface. This result may be explained by fewer spontaneous variations of the fluorescence rate, polarization changes, spectral shifts, and longer photochemical lifetimes.

Single molecule fluorescence spectroscopy of mutants of the Discosoma red fluorescent protein DsRed

We studied the emission of mutants of the red fluorescent protein DsRed by room temperature single molecule fluorescence spectroscopy. Bulk samples of the DsRed variant E8 show mixed green and red fluorescence of equivalent intensities individually spectrally similar to arrested green and mature red fluorescent forms of DsRed. Investigations at the single molecule level indicate that, like DsRed, E8 is not monomeric at single molecule concentrations. The entities visualized are composed of green and red emitting proteins without a fixed ratio of green to red fluorescing units. We find indications for only weak, if any, fluorescence resonance energy transfer (FRET) between red and green chromophores within one E8 entity.