Biodegradable calcium carbonate carriers for the topical delivery of clobetasol propionate.

Inflammatory dermatoses represent a global problem with increasing prevalence and recurrence among the world population. Topical glucocorticoids (GCs) are the most commonly used anti-inflammatory drugs in dermatology due to a wide range of their therapeutic actions, which, however, have numerous local and systemic side effects. Hence, there is a growing need to create new delivery systems for GCs, ensuring the drug localization in the pathological site, thus increasing the effectiveness of therapy and lowering the risk of side effects. Here, we propose a novel topical particulate formulation for the GC clobetasol propionate (CP), based on the use of porous calcium carbonate (CaCO3) carriers in the vaterite crystalline form. The designed carriers contain a substantially higher CP amount than conventional dosage forms used in clinics (4.5% w/w vs. 0.05% w/w) and displayed a good biocompatibility and effective cellular uptake when studied in fibroblasts in vitro. Hair follicles represent an important reservoir for the GC accumulation in skin and house the targets for its action. In this study, we demonstrated successful delivery of the CP-loaded carriers (CP-CaCO3) into the hair follicles of rats in vivo using optical coherent tomography (OCT). Importantly, the OCT monitoring revealed the gradual intrafollicular degradation of the carriers within 168 h with the most abundant follicle filling occurring within the first 48 h. Biodegradability makes the proposed system especially promising when searching for new CP formulations with improved safety and release profile. Our findings evidenced the great potential of the CaCO3 carriers in improving the dermal bioavailability of this poorly water-soluble GC.

In vivo skin optical clearing efficacy quantification of clinically compatible agents using line-field confocal optical coherence tomography.

Significance: The clinical use of optical methods for in vivo skin imaging is limited by skin strong scattering properties, which reduce image contrast and probing depth. The efficiency of optical methods can be improved by optical clearing (OC). However, for the use of OC agents (OCAs) in a clinical setting, compliance with acceptable non-toxic concentrations is required. Aim: OC of in vivo human skin, combined with physical and chemical methods to enhance skin permeability to OCAs, was performed to determine the clearing-effectiveness of biocompatible OCAs using line-field confocal optical coherence tomography (LC-OCT) imaging. Approach: Nine types of OCAs mixtures were used in association with dermabrasion and sonophoresis for OC protocol on three volunteers hand skin. From 3D images obtained every 5 min for 40 min, the intensity and contrast parameters were extracted to assess their changes during the clearing process and evaluate each OCAs mixture's clearing efficacy. Results: The LC-OCT images average intensity and contrast increased over the entire skin depth with all OCAs. The best image contrast and intensity improvement was observed using the polyethylene glycol, oleic acid, and propylene glycol mixture. Conclusions: Complex OCAs featuring reduced component concentrations that meet drug regulation-established biocompatibility requirements were developed and proved to induce significant skin tissues clearing. By allowing deeper observations and higher contrast, such OCAs in combination with physical and chemical permeation enhancers may improve LC-OCT diagnostic efficacy.

Impact of optical clearing on ex vivo human skin optical properties characterized by spatially resolved multimodal spectroscopy.

A spatially resolved multimodal spectroscopic device was used on a two-layered "hybrid" model made of ex vivo skin and fluorescent gel to investigate the effect of skin optical clearing on the depth sensitivity of optical spectroscopy. Time kinetics of fluorescence and diffuse reflectance spectra were acquired in four experimental conditions: with optical clearing agent (OCA) 1 made of polyethylene glycol 400 (PEG-400), propylene glycol and sucrose; with OCA 2 made of PEG-400 and dimethyl sulfoxide (DMSO); with saline solution as control and a "dry" condition. An increase in the gel fluorescence back reflected intensity was measured after optical clearing. Effect of OCA 2 turned out to be stronger than that of OCA 1, possibly due to DMSO impact on the stratum corneum keratin conformation. Complementary experimental results showed increased light transmittance through the skin and confirmed that the improvement in the depth sensitivity of the multimodal spectroscopic approach is related not only to the dehydration and refractive indices matching due to optical clearing, but also to the mechanical compression of tissues caused by the application of the spectroscopic probe.

Could LASCA-imaging of GB-speckles be applied for a high discrimination and typing of pathogenic bacteria?

In this article, the method of analysis of GB-speckles (gene-based speckles) has been adapted to the problem of detecting the differences in a group of genes (usually 5-7), used in Multi Locus Sequence Typing (MLST). This method is based on s-LASCA imaging (spatial Laser Speckle Contrast Analysis) of virtual GB-speckle and on the technique of RGB coordinates for GB-speckles, processed by the s-LASCA method. A very high sensitivity and accuracy of the new method for detecting gene polymorphism as a great alternative to classical MLST has been demonstrated. The analysis of GB-speckles, obtained for the concatenated sequences of seven genes (gatA, gidA, enoA, fumC, hemN, hflX, oppA) of three different Chlamydia trachomatis strains (E/Bour, ST94; G/9301, ST95; G/11222, ST94) has been applied as the model. The high efficiency of usage of s-LASCA-imaging of GB-speckles has been shown. The data obtained represent a significant progress in digital biology as a whole and improvements in the bio-digitalization of bacterial DNA.

Discrimination of the SARS-CoV-2 strains using of coloured s-LASCA-imaging of GB-speckles, developed for the gene "S" nucleotide sequences.

Background: A recent bioinformatics technique involves changing nucleotide sequences into 2D speckles. This technique produces speckles called GB-speckles (Gene Based speckles). All classical strategies of speckle-optics, namely speckle-interferometry, subtraction of speckle-images as well as speckle-correlometry have been inferred for processing of GB-speckles. This indicates the considerable improvement in the present tools of bioinformatics.   Methods: Colour s-LASCA imaging of virtual laser GB-speckles, a new method of high discrimination and typing of pathogenic viruses, has been developed. This method has been adapted to the detecting of natural mutations in nucleotide sequences, related to the spike glycoprotein (coding the gene «S¼) of SARS-CoV-2 gene as the molecular target.    Results: The rate of the colouring images of virtual laser GB-speckles generated by s-LASCA can be described by the specific value of R. If the nucleotide sequences compared utilizing this approach the relevant images are completely identical, then the three components of the resulting colour image will be identical, and therefore the value of R will be equal to zero. However, if there are at least minimal differences in the matched nucleotide sequences, then the value of R will be positive.    Conclusion: The high effectiveness of an application of the colour images of GB-speckles that were generated by s-LASCA- has been demonstrated for discrimination between different variants of the SARS-CoV-2 spike glycoprotein gene.

Optimized skin optical clearing for optical coherence tomography monitoring of encapsulated drug delivery through the hair follicles.

Hair follicles (HF) represent a drug delivery reservoir for improved treatment of skin disorders. Although various particulate systems play an important role in HF-targeting, their optical monitoring in skin is challenging due to strong light scattering. Optical clearing is an effective approach allowing the increasing of particle detection depth in skin. The enhancement of optical probing depth (OPD) and optical detection depth (ODD) of particle localization using optical coherence tomography (OCT) was evaluated under application of various optical clearing agents (OCAs) together with skin permeability enhancers ex vivo in rats. Efficient OPD increasing was demonstrated for all investigated OCAs. However, skin dehydration under action of hyperosmotic agents led to the worsening of OCT-contrast in dermis decreasing the ODD. Lipophilic agents provided optical clearing of epidermis without its dehydration. The highest ODD was obtained at application of a PEG-400/oleic acid mixture. This OCA was tested in vivo showing beneficial ODD and OPD enhancement.

Source separation approach for the analysis of spatially resolved multiply excited autofluorescence spectra during optical clearing of ex vivo skin.

Spatially resolved multiply excited autofluorescence spectroscopy is a valuable optical biopsy technique to investigate skin UV-visible optical properties in vivo in clinics. However, it provides bulk fluorescence signals from which the individual endogenous fluorophore contributions need to be disentangled. Skin optical clearing allows for increasing tissue transparency, thus providing access to more accurate in-depth information. The aim of the present contribution was to study the time changes in skin spatially resolved and multiply excited autofluorescence spectra during skin optical clearing. The latter spectra were acquired on an ex vivo human skin strip lying on a fluorescent gel substrate during 37 minutes of the optical clearing process of a topically applied sucrose-based solution. A Non Negative Matrix Factorization-based blind source separation approach was proposed to unmix skin tissue intrinsic fluorophore contributions and to analyze the time evolution of this mixing throughout the optical clearing process. This spectral unmixing exploited the multidimensionality of the acquired data, i.e., spectra resolved in five excitation wavelengths, four source-to-detector separations, and eight measurement times. Best fitting results between experimental and estimated spectra were obtained for optimal numbers of 3 and 4 sources. These estimated spectral sources exhibited common identifiable shapes of fluorescence emission spectra related to the fluorescent gel substrate and to known skin intrinsic fluorophores matching namely dermis collagen/elastin and epidermis flavins. The time analysis of the fluorophore contributions allowed us to highlight how the clearing process towards the deepest skin layers impacts skin autofluorescence through time, namely with a strongest contribution to the bulk autofluorescence signal of dermis collagen (respectively epidermis flavins) fluorescence at shortest (respectively longest) excitation wavelengths and longest (respectively shortest) source-to-detector separations.

Speckle-interferometry and speckle-correlometry of GB-speckles.

A new method of coding of genetic information using laser speckles has been developed. Specific technique of transforming the nucleotide of gene into a speckle pattern (gene-based speckles or GB-speckles) is suggested. Reference speckle patterns of omp1 gene of typical wild strains of Chlamydia trachomatis of genovars D, E, F, G, and J are generated. This is the first report in which perspectives of the proposed technique in the bacterial gene identification and detection of natural genetic mutations in bacteria as a single nucleotide polymorphism (SNP) are demonstrated. The usage of GB-speckles can be viewed as the next step on the way to the era of digital biology.

Nucleophilic Ring Opening of Donor-Acceptor Cyclopropanes with the Cyanate Ion: Access to Spiro[pyrrolidone-3,3'-oxindoles].

The nucleophilic ring opening of donor-acceptor cyclopropanes with the cyanate ion is reported for the first time. Cyclopropanes, spiro-activated with oxindole fragments as acceptors, are shown to undergo transformations into biologically relevant spiro[pyrrolidone-3,3'-oxindoles] while being treated with potassium cyanate under microwave assistance.

Carbon determination in carbon-manganese steels under atmospheric conditions by Laser-Induced Breakdown Spectroscopy.

The most sensitive lines of carbon, used nowadays for its determination in steels by laser-induced-breakdown spectroscopy (LIBS), are at vacuum UV and, thereby, LIBS potential is significantly reduced. We suggested the use of the C I 833.51 nm line for carbon determination in low-alloy steels (c(C)~0.186-1.33 wt.%) in air. Double-pulse LIBS with the collinear scheme was performed for maximal enhancement of a carbon emission signal without substantial complication of experimental setup. Since this line is strongly broadened in laser plasma, it overlapped with the closest iron lines greatly. We implemented a PCR method for the construction of a multivariate calibration model under spectral interferences. The model provided a RMSECV = 0.045 wt.%. The predicted carbon content in the rail templet was in an agreement with the reference value obtained by a combustion analyzer within the relative error of 6%.

Automatic identification of emission lines in laser-induced plasma by correlation of model and experimental spectra.

We have applied an algorithm to automatically identify emission lines in laser-induced breakdown spectrometry (LIBS). A Q-switched Nd:YAG laser at 355 nm was used to ablate a high-alloy stainless steel sample. The algorithm was implemented by three parts: simulation of the set of spectra corresponding to different temperature (T) and electron density (N(e)), searching the best correlated pair of a model spectrum and an experimental one, and attributing the peaks with certain lines. In order to construct the model spectra, we used the parameters of atomic and ionic lines, levels, the mechanisms of the broadening of spectral lines, and the selected parameters of the spectrograph. The highest correlation coefficient between the model and the experimental spectrum was 0.943 for T = 0.675 eV and lg(N(e)) = 16.7 cm(-3). More than 40 emission lines were labeled automatically in the spectral region 393.34-413.04 nm.