Origins of linewidth in ¹H magic-angle spinning NMR

A detailed study of the factors determining the linewidth (and hence resolution) in 1H solid-state magic-angle spinning NMR is described. Although it has been known from the early days of magic-angle spinning (MAS) that resolution of spectra from abundant nuclear spins, such as 1H, increases approximately linearly with increasing sample rotation rate, the difficulty of describing the dynamics of extended networks of coupled spins has made it difficult to predict a priori the resolution expected for a given sample. Using recently developed, highly efficient methods of numerical simulation, together with experimental measurements on a variety of test systems, we propose a comprehensive picture of 1H resolution under MAS. The "homogeneous" component of the linewidth is shown to depend primarily on the ratio between an effective local coupling strength and the spin rate, modified by geometrical factors which loosely correspond to the "dimensionality" of the coupling network. The remaining "inhomogeneous" component of the natural linewidth is confirmed to have the same properties as in dilute-spin NMR. Variations in the NMR frequency due to chemical shift effects are shown to have minimal impact on 1H resolution. The implications of these results for solid-state NMR experiments involving 1H and other abundant-spin nuclei are discussed

Commercial articulated collaborative in situ 3D bioprinter for skin wound healing

In situ bioprinting is one of the most clinically relevant techniques in the emerging bioprinting technology because it could be performed directly on the human body in the operating room and it does not require bioreactors for post-printing tissue maturation. However, commercial in situ bioprinters are still not available on the market. In this study, we demonstrated the benefit of the originally developed first commercial articulated collaborative in situ bioprinter for the treatment of full-thickness wounds in rat and porcine models. We used an articulated and collaborative robotic arm from company KUKA and developed original printhead and correspondence software enabling in situ bioprinting on curve and moving surfaces. The results of in vitro and in vivo experiments show that in situ bioprinting of bioink induces a strong hydrogel adhesion and enables printing on curved surfaces of wet tissues with a high level of fidelity. The in situ bioprinter was convenient to use in the operating room. Additional in vitro experiments (in vitro collagen contraction assay and in vitro 3D angiogenesis assay) and histological analyses demonstrated that in situ bioprinting improves the quality of wound healing in rat and porcine skin wounds. The absence of interference with the normal process of wound healing and even certain improvement in the dynamics of this process strongly suggests that in situ bioprinting could be used as a novel therapeutic modality in wound healing.publishersversionPeer reviewe

Interaction of mTHPC liposomal formulations with serum proteins

The use of liposomes as drug delivery systems is an accepted approach to improve the photosensitizer efficacy. Because of their characteristic small size (between 50 and 200 nm) and good solubility and stability, liposomes represent an ideal delivery system for nonpolar photodrugs. In this perspective, a clinically approved photosensitizer, meta-tetra(hydroxyphenyl)chlorin (mTHPC) has been loaded into liposomes with or without addition of PEGylated lipid. The present study addresses the distribution pattern of liposomal mTHPC (Foslip(R) and Fospeg(R)) in blood serum assessed by gel-filtration chromatography It was found that the affinity of pure and liposome-based mTHPC towards different plasma proteins is almost identical. The major part of the photosensitizer localizes in the high density lipoproteins fractions, while a minor fraction passes through the column with low-density lipoproteins. Only a small part of mTHPC molecules is found in the albumin fraction. As opposed to conventional liposomes with a very rapid disruption of the lipid vesicles and fast release of the drugs, mTHPC loaded DPPC/DPPG liposomes show a very slow release of the active component. After 30 min of Foslip(R) incubation with serum only a small percentage of mTHPC redistributes from the liposomes. Increasing incubation time to 6 h results in a significant reduction of the mTHPC fluorescence signal associated with mTHPC embedded into liposomes and concomitant increase of the signal associated with the protein- based bands. After 24 h incubation the distribution pattern was similar to the elution profile of serum containing free mTHPC. In contrast to Foslip(R), short incubation of PEGylated liposomes containing mTHPC (Fospeg(R)) with serum results in a release of approximately half of mTHPC from the lipid carriers. The kinetics of release is clearly two-phased: rapid release followed by slow redistribution. The slow phase is decelerated compared to Foslip(R)

Molecular Mechanisms of Changes in Homeostasis of the Dermal Extracellular Matrix: Both Involutional and Mediated by Ultraviolet Radiation

Skin aging is a multi-factorial process that affects nearly every aspect of skin biology and function. With age, an impairment of structures, quality characteristics, and functions of the dermal extracellular matrix (ECM) occurs in the skin, which leads to disrupted functioning of dermal fibroblasts (DFs), the main cells supporting morphofunctional organization of the skin. The DF functioning directly depends on the state of the surrounding collagen matrix (CM). The intact collagen matrix ensures proper adhesion and mechanical tension in DFs, which allows these cells to maintain collagen homeostasis while ECM correctly regulates cellular processes. When the integrity of CM is destroyed, mechanotransduction is disrupted, which is accompanied by impairment of DF functioning and destruction of collagen homeostasis, thereby contributing to the progression of aging processes in skin tissues. This article considers in detail the processes of skin aging and associated changes in the skin layers, as well as the mechanisms of these processes at the molecular level

Age-Related Changes in the Fibroblastic Differon of the Dermis: Role in Skin Aging

Skin aging is a multi-factorial process that affects nearly every aspect of skin biology and function. The processes developing in the skin during aging are based on fundamental molecular mechanisms associated with fibroblasts, the main cellular population of the dermis. It has been revealed that the amount of fibroblasts decreases markedly with age and their functional activity is also reduced. This inevitably leads to a decrease in the regenerative abilities of the skin and the progression of its aging. In this review we consider the mechanisms underlying these processes, mainly the changes observed with age in the stem/progenitor cells that constitute the fibroblastic differon of the dermis and form their microenvironment (niches). These changes lead to the depletion of stem cells, which, in turn, leads to a decrease in the number of differentiated (mature) dermal fibroblasts responsible for the production of the dermal extracellular matrix and its remodeling. We also describe in detail DNA damages, their cellular and systemic consequences, molecular mechanisms of DNA damage response, and also the role of fibroblast senescence in skin aging

Insights into homonuclear decoupling from efficient numerical simulation: Techniques and examples

A combination of techniques, including rational number synchronisation and pre-diagonalisation of the time-dependent periodic Hamiltonian, are described which allow the efficient simulation of NMR experiments involving both magic-angle spinning (MAS) and RF irradiation, particularly in the important special case of phase-modulated decoupling sequences. Chebyshev and conventional diagonalisation approaches to calculating propagators under MAS are also compared, with Chebyshev methods offering significant advantages in cases where the Hamiltonian is large and time-dependent but not block-diagonal (as is the case for problems involving combined MAS and RF). The ability to simulate extended coupled spin systems efficiently allows H-1 spectra under homonuclear decoupling to be calculated directly and compared to experimental results. Reasonable agreement is found for the conditions under which homonuclear decoupling is typically applied for rigid solids (although the increasing deviation of experimental results from the predictions of theory and simulation at higher RF powers is still unexplained). Numerical simulations are used to explore three features of these experiments: the interaction between the magic-angle spinning and RF decoupling, the effects of tilt pulses in acquisition windows and the effects of "phase propagation delays" on tilted axis precession. In each case, the results reveal features that are not readily anticipated by previous analytical studies and shed light on previous empirical observations. (c) 2008 Elsevier Inc. All rights reserved

Resonance light scattering spectra of meta-tetra(hydroxyphenyl)chlorin in liposomes

International audienc

Comparison of techniques of drug redistribution registration from nanosized carriers in biological systems

International audienceNanosized liposomes are popular pharmaceutical carriers for drugs with poor water solubility, since they can be solubilized in the hydrophobic lipid bilayer. The use of liposomal formulations requires the development of non-invasive methods of drug release control in biological systems. In our study, a clinically approved photosensitizer, meta-tetra(hydroxyphenyl)chlorin (mTHPC) has been loaded into dimiristoylphosphatidylcholine-based liposomes. A high local concentration of the photosensitizer in liposomes produces the fluorescence features which may be used for analyzing the rate of pigment release from lipid carriers. We compare three different techniques to analyze the redistribution of mTHPC from liposomes to biological substrates: mTHPC anisotropy measurements, photoinduced mTHPC fluorescence quenching and excitation energy transfer from the fluorescent probe diphenylhexatrien (DPH) to mTHPC. Each method was shown to possess a characteristic range of sensitivity, with photoinduced quenching providing a better dynamic response

Vascular damage photoinduced by liposomal formulations of mTHPC in xenografted CAM: influence of drug release

Tumoricidal photodynamic therapy (PDT) efficacy implicates the combination of direct cellular and indirect vascular damage supported by an immune activation. It was demonstrated that the success of mTHPC-PDT depends particularly on tumoral vascular damage (Garrier et al., 2010). Recently developed liposomal formulations of mTHPC (Foslip®, Fospeg®) aim to improve the pharmacokinetic properties of the photosensitizer. However, it was shown in vitro, in human plasma, that the mTHPC release significantly depends on the liposomal formulation (Reshetov et al., 2011). Indeed, a part of mTHPC was released much faster from PEGylated liposomes compared to conventional ones. In this context, the kinetic of mTHPC release from lipid nanovesicles is an essential point in the estimation of the photoinduced vascular damage. The chick chorioallantoic membrane (CAM) model, particularly adapted for the study of vascular events, was used for the experiments. Foslip® and Fospeg® were either intravenously administered into CAM (in ovo) or incubated in chick plasma (ex ovo). In both cases, photosensitizer release was estimated by photoinduced fluorescence quenching technique in plasma (Kachatkou et al, 2009). PDT treatment was realized on xenografted CAM with EMT6 murine mammary carcinoma cells, a model developed in our laboratory in accordance with previous studies (Mitra et al, 2005). Vascular damage was evaluated by macroscopy and histology of CAM and tumors. Foslip® exhibited no significant difference in terms of mTHPC release when it was incubated in ovo and ex ovo. The plateau of release was obtained after 2 hours of incubation. In contrast, incubation with human plasma yielded a plateau after 6 hours, indicating an important influence of the plasma composition. Concerning Fospeg®, ex ovo studies confirmed the two-phase release of photosensitizer observed previously in human plasma. Different drug light intervals (DLIs) corresponding to partial or complete mTHPC redistribution were tested for Foslip® and Fospeg®-based PDT activity