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.

Effect of pulsed laser parameters on photoacoustic flow cytometry efficiency in vitro and in vivo.

Photoacoustic flow cytometry is one of the most effective approaches to detect "alien" objects in the bloodstream, including circulating tumor cells, blood clots, parasites, and emboli. However, the possibility of detecting high-amplitude signals from these objects against the background of blood depends on the parameters of the laser pulse. So, the dependencies of photoacoustic signals amplitude and number on laser pulse energy (5-150 µJ), pulse length (1, 2, 5 ns), and pulse repetition rate (2, 5, 10 kHz) for the melanoma cells were investigated. First, the PA responses of a melanoma cell suspension in vitro were measured to directly assess the efficiency of converting laser light into an acoustic signal. After it, the same dependence with the developed murine model based on constant rate melanoma cell injection into the animal blood flow was tested. Both in vivo and in vitro experiments show that signal generation efficiency increases with laser pulse energy above 15 µJ. Shorter pulses, especially 1 ns, provide more efficient signal generation as well as higher pulse rates. A higher pulse rate also provides more efficient signal generation, but also leads to overheating of the skin. The results show the limits where the photoacoustic flow cytometry system can be effectively used for the detection of circulating tumor cells in undiluted blood both for in vitro experiments and for in vivo murine models.

Current Principles, Challenges, and New Metrics in pH-Responsive Drug Delivery Systems for Systemic Cancer Therapy.

The paradigm of drug delivery via particulate formulations is one of the leading ideas that enable overcoming limitations of traditional chemotherapeutic agents. The trend toward more complex multifunctional drug carriers is well-traced in the literature. Nowadays, the prospectiveness of stimuli-responsive systems capable of controlled cargo release in the lesion nidus is widely accepted. Both endogenous and exogenous stimuli are employed for this purpose; however, endogenous pH is the most common trigger. Unfortunately, scientists encounter multiple challenges on the way to the implementation of this idea related to the vehicles' accumulation in off-target tissues, their immunogenicity, the complexity of drug delivery to intracellular targets, and finally, the difficulties in the fabrication of carriers matching all imposed requirements. Here, we discuss fundamental strategies for pH-responsive drug delivery, as well as limitations related to such carriers' application, and reveal the main problems, weaknesses, and reasons for poor clinical results. Moreover, we attempted to formulate the profiles of an "ideal" drug carrier in the frame of different strategies drawing on the example of metal-comprising materials and considered recently published studies through the lens of these profiles. We believe that this approach will facilitate the formulation of the main challenges facing researchers and the identification of the most promising trends in technology development.

Cytotoxicity, Dermal Toxicity, and In Vivo Antifungal Effect of Griseofulvin-Loaded Vaterite Carriers Administered via Sonophoresis.

The search for novel therapeutic strategies to treat fungal diseases is of special importance nowadays given the emerging threat of drug resistance. Various particulate delivery systems are extensively being developing to enhance bioavailability, site-specific penetration, and therapeutic efficacy of antimycotics. Recently, we have designed a novel topical formulation for griseofulvin (Gf) drug, which is currently commercially available in oral dosage forms due to its limited skin permeation. The proposed formulation is based on vaterite carriers that enabled effective incorporation and ultrasonically assisted delivery of Gf to hair follicles improving its dermal bioavailability. Here, we evaluated the effect of ultrasound on the viability of murine fibroblasts co-incubated with either Gf-loaded carriers or a free form of Gf and investigated the influence of both forms on different subpopulations of murine blood cells. The study revealed no sufficient cyto- and hemotoxicity of the carriers, even at the highest investigated concentrations. We also conducted a series of in vivo experiments to assess their multi-dose dermal toxicity and antifungal efficiency. Visual and histological examinations of the skin in healthy rabbits showed no obvious adverse effects after US-assisted application of the Gf-loaded carriers. At the same time, investigation of therapeutic efficiency for the designed formulation in comparison with free Gf and isoconazole drugs in a guinea pig model of trichophytosis revealed that the vaterite-based form of Gf provided the most rapid and effective cure of infected animals together with the reduction in therapeutic procedure number. These findings pave the way to improving antifungal therapy of superficial mycoses and justifying further preclinical studies.

Magnetic Platelets as a Platform for Drug Delivery and Cell Trapping.

The possibility of using magnetically labeled blood cells as carriers is a novel approach in targeted drug-delivery systems, potentially allowing for improved bloodstream delivery strategies. Blood cells already meet the requirements of biocompatibility, safety from clotting and blockage of small vessels. It would solve the important problem of the patient's immune response to embedded foreign carriers. The high efficiency of platelet loading makes them promising research objects for the development of personalized drug-delivery systems. We are developing a new approach to use platelets decorated with magnetic nanoparticles as a targeted drug-delivery system, with a focus on bloodstream delivery. Platelets are non-nuclear blood cells and are of great importance in the pathogenesis of blood-clotting disorders. In addition, platelets are able to attach to circulating tumor cells. In this article, we studied the effect of platelets labeled with BSA-modified magnetic nanoparticles on healthy and cancer cells. This opens up broad prospects for future research based on the delivery of specific active substances by this method.

Macrophage In Vitro and In Vivo Tracking via Anchored Microcapsules.

A new promising trend in personalized medicine is the use of autologous cells (macrophages or stem cells) for cell-based therapy and also as a "Trojan horse" for targeted delivery of a drug carrier. The natural ability of macrophages for chemotaxis allows them to deliver cargo to the damaged area, significantly reducing side effects on healthy organ tissues. Therefore, it is important to develop tools to track their behavior in the organism. While labeled containers can serve as anchored tags for imaging macrophages in vivo, they can affect the properties and functions of macrophages. This work demonstrates that 3 µm sized capsules based on biocompatible polyelectrolytes and fluorescently labeled with both Cy7 and RITC dyes do not affect cell functionalization in vitro, such as viability, proliferation, and movement of transformed monocyte/macrophage-like cells (RAW 264.7) and primary bone marrow derived macrophages (BMDM) at maximal loading of five capsules per cell. In addition, capsules allowed fluorescent detection of ex vivo loaded cells 24 h after the tail vein injection in vivo and visualization of microcapsule-laden macrophages ex vivo using confocal microscopy. We have delivered about 62.5% of injected BMDM containing 12.5 million capsules with 3.75 µg of high-molecular-weight cargo (0.3 pg/capsule) to the liver. Our results demonstrate that 3 µm polyelectrolyte fluorescently labeled microcapsules can be used for safe macrophage loading, allowing cell tracking and drug delivery, which will facilitate development of macrophage-based cell therapy protocols.

CaCO3-based carriers with prolonged release properties for antifungal drug delivery to hair follicles.

Superficial fungal infections are of serious concern worldwide due to their morbidity and increasing distribution across the globe in this era of growing antimicrobial resistance. The delivery of antifungals to the target regions of the skin and sustaining the effective drug concentration are essential for successful treatment of such mycoses. Topical formulations get extra benefits here if they penetrate into the hair follicles since fungal hyphae can proliferate and produce spores in such reservoirs. We designed a novel particulate system for the encapsulation and intrafollicular delivery of griseofulvin (Gf) antifungal drug, which is water-insoluble and currently commercially available in oral dosage forms. Micron-sized calcium carbonate (vaterite) carriers containing 25 ± 3% (w/w) of Gf were prepared via the wet chemical method. The successful in vivo transportation of the carriers into the hair follicles of rats was demonstrated using scanning electron and confocal laser scanning microscopy. In addition, we introduced an approach toward Gf release prolongation for the proposed system. The stabilizing coatings were formed on the surface of the obtained particles via the layer-by-layer technique. The formulations displayed sufficient biocompatibility and good cellular uptake in contact with fibroblast cells in vitro. Four different coatings were tested for their preserving ability in the course of continued carrier incubation in the model media. The best release prolonging formulation liberated 38% of the loaded Gf during 5 days, while the uncoated carriers demonstrated more than 50% drug release within the first 24 h in water. To assess the in vivo release properties, free Gf drug and Gf-loaded carriers (uncovered and covered with the stabilizing shell) were administered topically in rats and the drug excretion profiles were further studied. By comparing the daily Gf levels in urine, we verified the sustained effect (longer than a week) of the stabilizing shell formed on the carrier surface. Conversely, the application of the free drug did not provide reliable Gf detection for this period. These findings open new prospects for the efficiency enhancement of topical therapeutics. Importantly, the elaborated system could be adapted for the dermal delivery of various water-insoluble drugs beyond the scope of antifungal therapy.

Live Cell Poration by Au Nanostars to Probe Intracellular Molecular Composition with SERS.

A new type of flat substrate has been used to visualize structures inside living cells by surface-enhanced Raman scattering (SERS) and to study biochemical processes within cells. The SERS substrate is formed by stabilized aggregates of gold nanostars on a glass microscope slide coated with a layer of poly (4-vinyl pyridine) polymer. This type of SERS substrate provides good cell adhesion and viability. Au nanostars' long tips can penetrate the cell membrane, allowing it to receive the SERS signal from biomolecules inside a living cell. The proposed nanostructured surfaces were tested to study, label-free, the distribution of various biomolecules in cell compartments.

Fluorescent Convertible Capsule Coding Systems for Individual Cell Labeling and Tracking.

In modern biomedical science and developmental biology, there is significant interest in optical tagging to study individual cell behavior and migration in large cellular populations. However, there is currently no tagging system that can be used for labeling individual cells on demand in situ with subsequent discrimination in between and long-term tracking of individual cells. In this article, we demonstrate such a system based on photoconversion of the fluorescent dye rhodamine B co-confined with carbon nanodots in the volume of micron-sized polyelectrolyte capsules. We show that this new fluorescent convertible capsule coding system is robust and is actively uptaken by cell lines while demonstrating low toxicity. Using a variety of cellular lines, we demonstrate how this tagging system can be used for code-like marking and long-term tracking of multiple individual cells in large cellular populations.

Lightsheet-based flow cytometer for whole blood with the ability for the magnetic retrieval of objects from the blood flow.

Detection and extraction of circulating tumor cells and other rare objects in the bloodstream are of great interest for modern diagnostics, but devices that can solve this problem for the whole blood volume of laboratory animals are still rare. Here we have developed SPIM-based lightsheet flow cytometer for the detection of fluorescently-labeled objects in whole blood. The bypass channel between two blood vessels connected with the external flow cell was used to visualize, detect, and magnetically separate fluorescently-labeled objects without hydrodynamic focusing. Carriers for targeted drug delivery were used as model objects to test the device performance. They were injected into the bloodstream of the rat, detected fluorescently, and then captured from the bloodstream by a magnetic separator prior to filtration in organs. Carriers extracted from the whole blood were studied by a number of in vitro methods.

pH-Sensitive Dairy-Derived Hydrogels with a Prolonged Drug Release Profile for Cancer Treatment.

A novel versatile biocompatible hydrogel of whey protein isolate (WPI) and two types of tannic acid (TAs) was prepared by crosslinking of WPI with TAs in a one-step method at high temperature for 30 min. WPI is one common protein-based preparation which is used for hydrogel formation. The obtained WPI-TA hydrogels were in disc form and retained their integrity after sterilization by autoclaving. Two TA preparations of differing molecular weight and chemical structure were compared, namely a polygalloyl glucose-rich extract-ALSOK 02-and a polygalloyl quinic acid-rich extract-ALSOK 04. Hydrogel formation was observed for WPI solutions containing both preparations. The swelling characteristics of hydrogels were investigated at room temperature at different pH values, namely 5, 7, and 9. The swelling ability of hydrogels was independent of the chemical structure of the added TAs. A trend of decrease of mass increase (MI) in hydrogels was observed with an increase in the TA/WPI ratio compared to the control WPI hydrogel without TA. This dependence (a MI decrease-TA/WPI ratio) was observed for hydrogels with different types of TA both in neutral and acidic conditions (pH 5.7). Under alkaline conditions (pH 9), negative values of swelling were observed for all hydrogels with a high content of TAs and were accompanied by a significant release of TAs from the hydrogel network. Our studies have shown that the release of TA from hydrogels containing ALSOK04 is higher than from hydrogels containing ALSOK 02. Moreover, the addition of TAs, which display a strong anti-cancer effect, increases the cytotoxicity of WPI-TAs hydrogels against the Hep-2 human laryngeal squamous carcinoma (Hep-2 cells) cell line. Thus, WPI-TA hydrogels with prolonged drug release properties and cytotoxicity effect can be used as anti-cancer scaffolds.

Changes in Autofluorescence Level of Live and Dead Cells for Mouse Cell Lines.

Label-free characterization of cell subpopulations is a very promising biomedical approach. Nowadays, there are several label-free methods based on different physical properties such as size, density, stiffness, etc. allowing the characterization of biological objects. However, fluorescence properties are the most suitable feature for the label-free study of tissue and cells. Understanding the autofluorescence level peculiarities of normal and pathological / live and dead cells can become a helpful tool for cells' metabolic activity, viability evaluation, and diagnostics of a number of diseases. In this study, we applied a series of mouse cell lines (RAW 264.7 - macrophages, L929 - fibroblasts, C2C12 - myoblasts, and B16-F10 - melanoma) to compare cell autofluorescence of live and dead cells under 488 nm laser excitation and found the difference between their autofluorescence depending on a cell state and type.

Site-specific release of reactive oxygen species from ordered arrays of microchambers based on polylactic acid and carbon nanodots.

Non-destructive, controllable, remote light-induced release inside cells enables studying of time- and space-specific surface-mediated delivery of bioactive compounds, which is an important approach in a wide range of biomedical tasks, especially those related to the control of cell growth, regenerative medicine, and self-disinfecting structures such as catheters. In this regard, the elaboration of encapsulation and controlled release of oxidative species is in high demand due to its versatile applications. One of the obvious candidates for such species is hydrogen peroxide. However, the delivery of hydrogen peroxide to the site of interest with high temporal and spatial precision remains challenging due to the active and unstable nature of the substance. We hereby present an approach to encapsulate and store a hydrogen peroxide-containing solid compound (sodium percarbonate) in the free-standing arrays of biopolymer-based microchambers. In this regard, we use solid-state encapsulation enabling high payload ability, followed by isolated storage in order to prevent contact of the cargo with water. Monitoring of the release profiles reveals the encapsulation of sodium percarbonate with little leakage for up to 24 hours. Microchambers are fabricated with predetermined size and spatial distribution, which allows the release of extremely small amounts of cargo (10-30 pg) with high spatial accuracy. Microchambers are made of polylactic acid and functionalized by carbon nanodots, which provide biocompatibility and biodegradability of the whole system together with responsiveness towards NIR light. These chambers facilitate both ultrasound-assisted burst release and laser-driven carbon nanoparticle-assisted precise release of extremely small, controlled amounts of a few picograms of hydrogen peroxide in submerged conditions. Microchambers loaded with sodium percarbonate provided adhesion and high viability of mouse fibroblasts over 24 h of exposure. The developed system opens an exciting avenue for prospective delivery routes in a number of areas such as wound healing by time and site-specific release.

In Vitro Bioeffects of Polyelectrolyte Multilayer Microcapsules Post-Loaded with Water-Soluble Cationic Photosensitizer.

Microencapsulation and targeted delivery of cytotoxic and antibacterial agents of photodynamic therapy (PDT) improve the treatment outcomes for infectious diseases and cancer. In many cases, the loss of activity, poor encapsulation efficiency, and inadequate drug dosing hamper the success of this strategy. Therefore, the development of novel and reliable microencapsulated drug formulations granting high efficacy is of paramount importance. Here we report the in vitro delivery of a water-soluble cationic PDT drug, zinc phthalocyanine choline derivative (Cholosens), by biodegradable microcapsules assembled from dextran sulfate (DS) and poly-l-arginine (PArg). A photosensitizer was loaded in pre-formed [DS/PArg]4 hollow microcapsules with or without exposure to heat. Loading efficacy and drug release were quantitatively studied depending on the capsule concentration to emphasize the interactions between the DS/PArg multilayer network and Cholosens. The loading data were used to determine the dosage for heated and intact capsules to measure their PDT activity in vitro. The capsules were tested using human cervical adenocarcinoma (HeLa) and normal human dermal fibroblast (NHDF) cell lines, and two bacterial strains, Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Our results provide compelling evidence that encapsulated forms of Cholosens are efficient as PDT drugs for both eukaryotic cells and bacteria at specified capsule-to-cell ratios.

Detection of Rare Objects by Flow Cytometry: Imaging, Cell Sorting, and Deep Learning Approaches.

Flow cytometry nowadays is among the main working instruments in modern biology paving the way for clinics to provide early, quick, and reliable diagnostics of many blood-related diseases. The major problem for clinical applications is the detection of rare pathogenic objects in patient blood. These objects can be circulating tumor cells, very rare during the early stages of cancer development, various microorganisms and parasites in the blood during acute blood infections. All of these rare diagnostic objects can be detected and identified very rapidly to save a patient's life. This review outlines the main techniques of visualization of rare objects in the blood flow, methods for extraction of such objects from the blood flow for further investigations and new approaches to identify the objects automatically with the modern deep learning methods.

Effect of Systemic Polyelectrolyte Microcapsule Administration on the Blood Flow Dynamics of Vital Organs.

Polyelectrolyte microcapsules and other targeted drug delivery systems could substantially reduce the side effects of drug and overall toxicity. At the same time, the cardiovascular system is a unique transport avenue that can deliver drug carriers to any tissue and organ. However, one of the most important potential problems of drug carrier systemic administration in clinical practice is that the carriers might cause circulatory disorders, the development of pulmonary embolism, ischemia, and tissue necrosis due to the blockage of small capillaries. Thus, the presented work aims to find out the processes occurring in the bloodstream after the systemic injection of polyelectrolyte capsules that are 5 µm in size. It was shown that 1 min after injection, the number of circulating capsules decreases several times, and after 15 min less than 1% of the injected dose is registered in the blood. By this time, most capsules accumulate in the lungs, liver, and kidneys. However, magnetic field action could slightly increase the accumulation of capsules in the region-of-interest. For the first time, we have investigated the real-time blood flow changes in vital organs in vivo after intravenous injection of microcapsules using a laser speckle contrast imaging system. We have demonstrated that the organism can adapt to the emergence of drug carriers in the blood and their accumulation in the vessels of vital organs. Additionally, we have evaluated the safety of the intravenous administration of various doses of microcapsules.

New Frontiers in Diagnosis and Therapy of Circulating Tumor Markers in Cerebrospinal Fluid In Vitro and In Vivo.

One of the greatest challenges in neuro-oncology is diagnosis and therapy (theranostics) of leptomeningeal metastasis (LM), brain metastasis (BM) and brain tumors (BT), which are associated with poor prognosis in patients. Retrospective analyses suggest that cerebrospinal fluid (CSF) is one of the promising diagnostic targets because CSF passes through central nervous system, harvests tumor-related markers from brain tissue and, then, delivers them into peripheral parts of the human body where CSF can be sampled using minimally invasive and routine clinical procedure. However, limited sensitivity of the established clinical diagnostic cytology in vitro and MRI in vivo together with minimal therapeutic options do not provide patient care at early, potentially treatable, stages of LM, BM and BT. Novel technologies are in demand. This review outlines the advantages, limitations and clinical utility of emerging liquid biopsy in vitro and photoacoustic flow cytometry (PAFC) in vivo for assessment of CSF markers including circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), microRNA (miRNA), proteins, exosomes and emboli. The integration of in vitro and in vivo methods, PAFC-guided theranostics of single CTCs and targeted drug delivery are discussed as future perspectives.

Live-Cell Imaging by Confocal Raman and Fluorescence Microscopy Recognizes the Crystal Structure of Calcium Carbonate Particles in HeLa Cells.

Porous calcium carbonate (CaCO3 ) vaterite particles are very attractive templates for the encapsulation of pharmaceuticals and for the construction of hollow polyelectrolyte capsules, sensors, and enzyme-catalyzed reactors. Although CaCO3 is biocompatible and biodegradable, little is known about the intercellular behavior and properties of vaterite particles in the cytoplasm of cells. In this work, the authors combine confocal Raman and fluorescent microscopy for the imaging of porous CaCO3 vaterite particles in HeLa cells to study the uptake and status of the particles inside the cells in real time. Analysis of the fluorescence images shows that the particles penetrated the plasma membrane 3 h after being added to the cell culture and that the internalization of the particles continued up to 48 h. The crystal structure of individual vaterite particles in the cytoplasm of HeLa cells did not obviously change for 144 h. For clusters of particles, however, the authors identify Raman spectroscopic signatures of the stable calcite phase after 72 h of incubation, confirming an ion-exchange mechanism of vaterite transformation to calcite. The results indicate that these imaging approach to examining inorganic particles in living cells may have theranostic applications.