Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine.

The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief and for monitoring of disease progression. One route of realizing this approach is through carefully constructed nanoparticles. Due to the small size inherent to the nanoparticles a proper characterization is not trivial. This review highlights the application of time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM) for the analysis of nanoparticles, covering aspects ranging from molecular properties to particle detection in tissue samples. The latter technique is particularly important as FLIM allows for distinguishing of target molecules from the autofluorescent background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle or its interactions with other biomolecules. Thus, these techniques offer highly suitable tools in the fields of particle development, such as organic chemistry, and in the fields of particle application, such as in experimental dermatology or pharmaceutical research.

Interactions of hyaluronic Acid with the skin and implications for the dermal delivery of biomacromolecules.

Hyaluronic acid (HA) hydrogels are interesting delivery systems for topical applications. Besides moisturizing the skin and improving wound healing, HA facilitates topical drug absorption and is highly compatible with labile biomacromolecules. Hence, in this study we investigated the influence of HA hydrogels with different molecular weights (5 kDa, 100 kDa, 1 MDa) on the skin absorption of the model protein bovine serum albumin (BSA) using fluorescence lifetime imaging microscopy (FLIM). To elucidate the interactions of HA with the stratum corneum and the skin absorption of HA itself, we combined FLIM and Fourier-transform infrared (FTIR) spectroscopy. Our results revealed distinct formulation and skin-dependent effects. In barrier deficient (tape-stripped) skin, BSA alone penetrated into dermal layers. When BSA and HA were applied together, however, penetration was restricted to the epidermis. In normal skin, penetration enhancement of BSA into the epidermis was observed when applying low molecular weight HA (5 kDa). Fluorescence resonance energy transfer analysis indicated close interactions between HA and BSA under these conditions. FTIR spectroscopic analysis of HA interactions with stratum corneum constituents showed an α-helix to ß-sheet interconversion of keratin in the stratum corneum, increased skin hydration, and intense interactions between 100 kDa HA and the skin lipids resulting in a more disordered arrangement of the latter. In conclusion, HA hydrogels restricted the delivery of biomacromolecules to the stratum corneum and viable epidermis in barrier deficient skin, and therefore seem to be potential topical drug vehicles. In contrast, HA acted as an enhancer for delivery in normal skin, probably mediated by a combination of cotransport, increased skin hydration, and modifications of the stratum corneum properties.

Application of single molecule fluorescence microscopy to characterize the penetration of a large amphiphilic molecule in the stratum corneum of human skin.

We report here on the application of laser-based single molecule total internal reflection fluorescence microscopy (TIRFM) to study the penetration of molecules through the skin. Penetration of topically applied drug molecules is often observed to be limited by the size of the respective drug. However, the molecular mechanisms which govern the penetration of molecules through the outermost layer of the skin are still largely unknown. As a model compound we have chosen a larger amphiphilic molecule (fluorescent dye ATTO-Oxa12) with a molecular weight >700 Da that was applied to excised human skin. ATTO-Oxa12 penetrated through the stratum corneum (SC) into the viable epidermis as revealed by TIRFM of cryosections. Single particle tracking of ATTO-Oxa12 within SC sheets obtained by tape stripping allowed us to gain information on the localization as well as the lateral diffusion dynamics of these molecules. ATTO-Oxa12 appeared to be highly confined in the SC lipid region between (intercellular space) or close to the envelope of the corneocytes. Three main distinct confinement sizes of 52 ± 6, 118 ± 4, and 205 ± 5 nm were determined. We conclude that for this amphiphilic model compound several pathways through the skin exist.

Detecting and Quantifying Biomolecular Interactions of a Dendritic Polyglycerol Sulfate Nanoparticle Using Fluorescence Lifetime Measurements.

Interactions of nanoparticles with biomaterials determine the biological activity that is key for the physiological response. Dendritic polyglycerol sulfates (dPGS) were found recently to act as an inhibitor of inflammation by blocking selectins. Systemic application of dPGS would present this nanoparticle to various biological molecules that rapidly adsorb to the nanoparticle surface or lead to adsorption of the nanoparticle to cellular structures such as lipid membranes. In the past, fluorescence lifetime measurements of fluorescently tagged nanoparticles at a molecular and cellular/tissue level have been proven to reveal valuable information on the local nanoparticle environment via characteristic fluorescent lifetime signatures of the nanoparticle bound dye. Here, we established fluorescence lifetime measurements as a tool to determine the binding affinity to fluorescently tagged dPGS (dPGS-ICC; ICC: indocarbocyanine). The binding to a cell adhesion molecule (L-selectin) and a human complement protein (C1q) to dPGS-ICC was evaluated by the concentration dependent change in the unique fluorescence lifetime signature of dPGS-ICC. The apparent binding affinity was found to be in the nanomolar range for both proteins (L-selectin: 87 ± 4 nM and C1q: 42 ± 12 nM). Furthermore, the effect of human serum on the unique fluorescence lifetime signature of dPGS-ICC was measured and found to be different from the interactions with the two proteins and lipid membranes. A comparison between the unique lifetime signatures of dPGS-ICC in different biological environments shows that fluorescence lifetime measurements of unique dPGS-ICC fluorescence lifetime signatures are a versatile tool to probe the microenvironment of dPGS in cells and tissue.

Nanodynamics of dendritic core-multishell nanocarriers.

The molecular dynamics of polymeric nanocarriers is an important parameter for controlling the interaction of nanocarrier branches with cargo. Understanding the interplay of dendritic polymer dynamics, temperature, and cargo molecule interactions should provide valuable new insight for tailoring the dendritic architecture to specific needs in nanomedicine, drug, dye, and gene delivery. Here, we have investigated polyglycerol-based core-multishell (CMS) nanotransporters with incorporated Nile Red as a fluorescent drug mimetic and CMS nanotransporters with a covalently bound fluorophore (Indocarbocyanine) using fluorescence spectroscopy methods. From time-resolved fluorescence depolarization we have obtained the rotational diffusion dynamics of the incorporated dye, the nanocarrier, and its branches as a function of temperature. UV/vis and fluorescence lifetime measurements provided additional information on the local dye environment. Our results show a distribution of the cargo Nile Red within the nanotransporter shells that depends on solvent and temperature. In particular, we show that the flexibility of the polymer branches in the unimolecular state of the nanotransporter undergoes a temperature-dependent transition which correlates with a larger space for the mobility of the incorporated hydrophobic drug mimetic Nile Red and a higher probability of cargo-solvent interactions at temperatures above 31 °C. The measurements have further revealed that a loss of the cargo molecule Nile Red occurred neither upon dilution of the CMS nanotransporters nor upon heating. Thus, the unimolecular preloaded CMS nanotransporters retain their cargo and are capable to transport and respond to temperature, thereby fulfilling important requirements for biomedical applications.

Skin barrier disruptions in tape stripped and allergic dermatitis models have no effect on dermal penetration and systemic distribution of AHAPS-functionalized silica nanoparticles.

The skin is a potential site of entry for nanoparticles (NP) but the role of disease-associated barrier disturbances on the path and extent of skin penetration of NP remains to be characterized. Silica nanoparticles (SiO2-NP) possess promising potential for various medical applications. Here, effects of different skin barrier disruptions on the penetration of N-(6-aminohexyl)-aminopropyltrimethoxysilane (AHAPS) functionalized SiO2-NP were studied. AHAPS-SiO2-NP (55±6 nm diameter) were topically applied on intact, tape stripped or on inflamed skin of SKH1 mice with induced allergic contact dermatitis for one or five consecutive days, respectively. Penetration of AHAPS-SiO2-NP through the skin was not observed regardless of the kind of barrier disruption. However, only after subcutaneous injection, AHAPS-SiO2-NP were incorporated by macrophages and transported to the regional lymph node only. Adverse effects on cells or tissues were not observed. In conclusion, AHAPS-SiO2-NP seem to not cross the normal or perturbed mouse skin. From the clinical editor: Skin is a potential site of entry for nanoparticles; however, it is poorly understood how skin diseases may alter this process. In tape-stripped skin and allergic contact dermatitis models the delivery properties of AHAPS-SiO2 nanoparticles remained unchanged, and in neither case were these NP-s able to penetrate the skin. No adverse effects were noted on the skin in these models and control mice.

Iopromide for Contrast-Enhanced Mammography: A Systemic Review and Meta-Analysis of Pertinent Literature.

Background: Contrast-enhanced mammography (CEM) is an emerging breast imaging modality. Clinical data is scarce. Objectives: To summarize clinical evidence on the use of iopromide in CEM for the detection or by systematically analyzing the available literature on efficacy and safety. Design: Systematic review and meta-analysis. Data sources and methods: Iopromide-specific publications reporting its use in CEM were identified by a systematic search within Bayer's Product Literature Information (PLI) database and by levering a recent review publication. The literature search in PLI was performed up to January 2023. The confirmatory-supporting review publication was based on a MEDLINE/EMBASE + full text search for publications issued between September 2003 and January 2019. Relevant literature was selected based on pre-defined criteria by 2 reviewers. The comparison of CEM vs traditional mammography (XRM) was performed on published results of sensitivity and specificity. Differences in diagnostic parameters were assessed within a meta-analysis. Results: Literature search: A total of 31 studies were identified reporting data on 5194 patients. Thereof, 19 studies on efficacy and 3 studies on safety. Efficacy: in 11 studies comparing iopromide CEM vs XRM, sensitivity was up to 43% higher (range 1%-43%) for CEM. Differences in specificity were found to be in a range of -4% to 46% for CEM compared with XRM. The overall gain in sensitivity for CEM vs XRM was 7% (95% CI [4%, 11%]) with no statistically significant loss in specificity in any study assessed. In most studies, accuracy, positive predictive value, and negative predictive value were found to be in favor of CEM. In 2 studies comparing CEM with breast magnetic resonance imaging (bMRI), both imaging modalities performed either equally well or CEM tended to show better results with respect to sensitivity and specificity. Safety: eight cases of iopromide-related adverse drug reactions were reported in 1022 patients (0.8%). Conclusions: Pertinent literature provides evidence for clinical utility of iopromide in CEM for the detection or confirmation of breast cancer. The overall gain in sensitivity for iopromide CEM vs XRM was 7% with no statistically significant loss in specificity.

Time-resolved fluorescence microscopy (FLIM) as an analytical tool in skin nanomedicine.

The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief, and for monitoring of disease progression. Topical application of drug-loaded nanoparticles for the treatment of skin disorders is a promising strategy to overcome the stratum corneum, the upper layer of the skin, which represents an effective physical and biochemical barrier. The understanding of drug penetration into skin and enhanced penetration into skin facilitated by nanocarriers requires analytical tools that ideally allow to visualize the skin, its morphology, the drug carriers, drugs, their transport across the skin and possible interactions, as well as effects of the nanocarriers within the different skin layers. Here, we review some recent developments in the field of fluorescence microscopy, namely Fluorescence Lifetime Imaging Microscopy (FLIM)), for improved characterization of nanocarriers, their interactions and penetration into skin. In particular, FLIM allows for the discrimination of target molecules, e.g. fluorescently tagged nanocarriers, against the autofluorescent tissue background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle and its interactions with other biomolecules. Thus, FLIM shows the potential to overcome several limits of intensity based microscopy.

Determination of nanostructures and drug distribution in lipid nanoparticles by single molecule microscopy.

Drug loading capacity in nanostructured lipid carriers (NLC) depends on the formation of nanostructures within the lipid matrix. However, investigation of these nanostructures with sizes below the diffraction limit of visible light is quite challenging. Thus, until now the determination of structures and drug distribution within NLCs was not possible. Therefore, we aimed at developing a method to visualize the nanostructures within the lipid carriers. Model NLCs loaded with a lipophilic fluorescent drug mimetic, ATTO-Oxa12, were produced and investigated by single-molecule tracking and localization-based superresolution microscopy. Results revealed spherical ATTO-Oxa12-filled nanostructures with diameters of ∼70nm and 120-130nm, both smaller than the NLC size (∼160nm). The ATTO-Oxa12 diffusion constant was calculated from the single-molecule traces (D⩾1µm2/s) and indicated the distribution of the model drug in the oily component. Together these data suggest the existence of drug-loaded oily nanocompartments, which could fill up to ∼50% of the model NLCs' volume. In conclusion, a novel tool based on single-molecule microscopy is now available that allows for the precise determination of drug distribution and the characterization of lipid nanostructures, information that is paramount for optimizing lipid nanoparticle formulations.

Increased permeability of reconstructed human epidermis from UVB-irradiated keratinocytes.

Extrinsic (photo) aging accelerates chronologically aging in the skin due to cumulative UV irradiation. Despite recent insights into the molecular mechanisms of fibroblast aging, age-related changes of the skin barrier function have been understudied. In contrast, the constantly increasing subpopulation of aged patients causes a clinical need for effective and safe (dermatological) treatment. Herein, we reconstructed human epidermis from UVB-irradiated keratinocytes (UVB-RHE). UVB-irradiated keratinocytes show higher activity of senescence associated ß-galactosidase, less cell proliferation, and reduced viability. Higher amounts of ß-galactosidase are also detectable in UVB-RHE. Moreover, UVB-RHE release more interleukin-1α and -8 into the culture medium and present altered differentiation with a thinner stratum corneum compared to normal RHE. For the first time, the permeation of testosterone and caffeine through UVB-irradiated RHE indicate a clear influence of the UVB stress on the skin barrier function. Impaired barrier function was confirmed by the increased permeation of testosterone and caffeine as well as by the increased penetration of dendritic core-multishell nanocarriers into the constructs. Taken together, UVB-RHE emulate hallmarks of skin aging and might contribute to an improved non-clinical development of medicinal or cosmetic products.

Poly[acrylonitrile-co-(N-vinyl pyrrolidone)] nanoparticles - Composition-dependent skin penetration enhancement of a dye probe and biocompatibility.

Nanoparticles can improve topical drug delivery: size, surface properties and flexibility of polymer nanoparticles are defining its interaction with the skin. Only few studies have explored skin penetration for one series of structurally related polymer particles with systematic alteration of material composition. Here, a series of rigid poly[acrylonitrile-co-(N-vinyl pyrrolidone)] model nanoparticles stably loaded with Nile Red or Rhodamin B, respectively, was comprehensively studied for biocompatibility and functionality. Surface properties were altered by varying the molar content of hydrophilic NVP from 0 to 24.1% and particle size ranged from 35 to 244nm. Whereas irritancy and genotoxicity were not revealed, lipophilic and hydrophilic nanoparticles taken up by keratinocytes affected cell viability. Skin absorption of the particles into viable skin ex vivo was studied using Nile Red as fluorescent probe. Whilst an intact stratum corneum efficiently prevented penetration, almost complete removal of the horny layer allowed nanoparticles of smaller size and hydrophilic particles to penetrate into viable epidermis and dermis. Hence, systematic variations of nanoparticle properties allows gaining insights into critical criteria for biocompatibility and functionality of novel nanocarriers for topical drug delivery and risks associated with environmental exposure.

Overview about the localization of nanoparticles in tissue and cellular context by different imaging techniques.

The increasing interest and recent developments in nanotechnology pose previously unparalleled challenges in understanding the effects of nanoparticles on living tissues. Despite significant progress in in vitro cell and tissue culture technologies, observations on particle distribution and tissue responses in whole organisms are still indispensable. In addition to a thorough understanding of complex tissue responses which is the domain of expert pathologists, the localization of particles at their sites of interaction with living structures is essential to complete the picture. In this review we will describe and compare different imaging techniques for localizing inorganic as well as organic nanoparticles in tissues, cells and subcellular compartments. The visualization techniques include well-established methods, such as standard light, fluorescence, transmission electron and scanning electron microscopy as well as more recent developments, such as light and electron microscopic autoradiography, fluorescence lifetime imaging, spectral imaging and linear unmixing, superresolution structured illumination, Raman microspectroscopy and X-ray microscopy. Importantly, all methodologies described allow for the simultaneous visualization of nanoparticles and evaluation of cell and tissue changes that are of prime interest for toxicopathologic studies. However, the different approaches vary in terms of applicability for specific particles, sensitivity, optical resolution, technical requirements and thus availability, and effects of labeling on particle properties. Specific bottle necks of each technology are discussed in detail. Interpretation of particle localization data from any of these techniques should therefore respect their specific merits and limitations as no single approach combines all desired properties.

Penetration of normal, damaged and diseased skin--an in vitro study on dendritic core-multishell nanotransporters.

A growing intended or accidental exposure to nanoparticles asks for the elucidation of potential toxicity linked to the penetration of normal and lesional skin. We studied the skin penetration of dye-tagged dendritic core-multishell (CMS) nanotransporters and of Nile red loaded CMS nanotransporters using fluorescence microscopy. Normal and stripped human skin ex vivo as well as normal reconstructed human skin and in vitro skin disease models served as test platforms. Nile red was delivered rapidly into the viable epidermis and dermis of normal skin, whereas the highly flexible CMS nanotransporters remained solely in the stratum corneum after 6h but penetrated into deeper skin layers after 24h exposure. Fluorescence lifetime imaging microscopy proved a stable dye-tag and revealed striking nanotransporter-skin interactions. The viable layers of stripped skin were penetrated more efficiently by dye-tagged CMS nanotransporters and the cargo compared to normal skin. Normal reconstructed human skin reflected the penetration of Nile red and CMS nanotransporters in human skin and both, the non-hyperkeratotic non-melanoma skin cancer and hyperkeratotic peeling skin disease models come along with altered absorption in the skin diseases.