A Protocol for Preparing Mucoadhesive Emulsion Microgels and Assessing Their Mucoadhesion Properties In Vitro.

Intravesical instillation is an efficient therapeutic technique based on targeted administration of a drug directly into the lesion for the treatment of bladder diseases. This is an alternative to traditional systemic administration of drugs. However, this technique requires repeated procedures, which can lead to even greater inflammation and infection of the urethra. To date, novel systems that allow prolonged drug retention in the bladder cavity are actively being developed. We recently reported a targeted drug delivery system based on the mucoadhesive emulsion microgels consisting of the natural component whey protein isolate. Such micron-sized carriers possess high loading capacity, a prolonged drug release profile, and efficient mucoadhesive properties to the bladder urothelium. As a continuation of this work, we present a protocol for the synthesis of mucoadhesive emulsion microgels. Detailed procedures for preparing precursor solutions as well as studying the physico-chemical parameters of microgels (including loading capacity and drug release rate) and the mucoadhesive properties using the model of porcine bladder urothelium are discussed. Precautionary measures and nuances that are worth paying attention to during each experimental stage are given as well. Key features • The protocol for the synthesis of mucoadhesive emulsion microgels based on whey protein isolate is presented. The experimental conditions of emulsion microgels synthesis are discussed. • Methods for studying the physico-chemical properties of mucoadhesive emulsion microgels (size of emulsion microgels particles, loading capacity, release kinetics) are described. • The method for assessing mucoadhesive properties of emulsion microgels is demonstrated using the porcine bladder tissue model ex vivo.

Effect of photoconversion conditions on the spectral and cytotoxic properties of photoconvertible fluorescent polymer markers.

Fluorescence labeling of cells is a versatile tool used to study cell behavior, which is of significant importance in biomedical sciences. Fluorescent photoconvertible markers based on polymer microcapsules have been recently considered as efficient and perspective ones for long-term tracking of individual cells. However, the dependence of photoconversion conditions on the polymeric capsule structure is still not sufficiently clear. Here, we have studied the structural and spectral properties of fluorescent photoconvertible polymeric microcapsules doped with Rhodamine B and irradiated using a pulsed laser in various regimes, and shown the dependence between the photoconversion degree and laser irradiation intensity. The effect of microcapsule composition on the photoconversion process was studied by monitoring structural changes in the initial and photoconverted microcapsules using X-ray diffraction analysis with synchrotron radiation source, and Fourier transform infrared, Raman and fluorescence spectroscopy. We demonstrated good biocompatibility of free-administered initial and photoconverted microcapsules through long-term monitoring of the RAW 264.7 monocyte/macrophage cells with unchanged viability. These data open new perspectives for using the developed markers as safe and precise cell labels with switchable fluorescent properties.

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.

Combination of Machine Learning and Raman Spectroscopy for Determination of the Complex of Whey Protein Isolate with Hyaluronic Acid.

Macromolecules and their complexes remain interesting topics in various fields, such as targeted drug delivery and tissue regeneration. The complex chemical structure of such substances can be studied with a combination of Raman spectroscopy and machine learning. The complex of whey protein isolate (WPI) and hyaluronic acid (HA) is beneficial in terms of drug delivery. It provides HA properties with the stability obtained from WPI. However, differences between WPI-HA and WPI solutions can be difficult to detect by Raman spectroscopy. Especially when the low HA (0.1, 0.25, 0.5% w/v) and the constant WPI (5% w/v) concentrations are used. Before applying the machine learning techniques, all the collected data were divided into training and test sets in a ratio of 3:1. The performances of two ensemble methods, random forest (RF) and gradient boosting (GB), were evaluated on the Raman data, depending on the type of problem (regression or classification). The impact of noise reduction using principal component analysis (PCA) on the performance of the two machine learning methods was assessed. This procedure allowed us to reduce the number of features while retaining 95% of the explained variance in the data. Another application of these machine learning methods was to identify the WPI Raman bands that changed the most with the addition of HA. Both the RF and GB could provide feature importance data that could be plotted in conjunction with the actual Raman spectra of the samples. The results show that the addition of HA to WPI led to changes mainly around 1003 cm-1 (correspond to ring breath of phenylalanine) and 1400 cm-1, as demonstrated by the regression and classification models. For selected Raman bands, where the feature importance was greater than 1%, a direct evaluation of the effect of the amount of HA on the Raman intensities was performed but was found not to be informative. Thus, applying the RF or GB estimators to the Raman data with feature importance evaluation could detect and highlight small differences in the spectra of substances that arose from changes in the chemical structure; using PCA to filter out noise in the Raman data could improve the performance of both the RF and GB. The demonstrated results will make it possible to analyze changes in chemical bonds during various processes, for example, conjugation, to study complex mixtures of substances, even with small additions of the components of interest.

Mucoadhesive Emulsion Microgels for Intravesical Drug Delivery: Preparation, Retention at Urothelium, and Biodistribution Study.

The intravesical instillation procedure is a proven method in modern urology for the treatment of bladder diseases. However, the low therapeutic efficiency and painfulness of the instillation procedure are significant limitations of this method. In the present study, we propose an approach to solving this problem by using microsized mucoadhesive macromolecular carriers based on whey protein isolate with the possibility of prolonged release of drugs as a drug delivery system. The optimal water-to-oil ratio (1:3) and whey protein isolate concentration (5%) were determined to obtain emulsion microgels with sufficient loading efficiency and mucoadhesive properties. The droplet diameter of emulsion microgels varies from 2.2 to 3.8 µm. The drug release kinetics from the emulsion microgels was evaluated. The release of the model dye in saline and artificial urine in vitro was observed for 96 h and reached up to 70% of loaded cargo for samples. The effect of emulsion microgels on the morphology and viability of two cell lines was observed: L929 mouse fibroblasts (normal adherent cells) and THP-1 human monocytes (cancer suspension cells). Developed emulsion microgels (5%, 1:3 and 1:5) showed sufficient mucoadhesion to a porcine bladder urothelium ex vivo. The biodistribution of emulsion microgels (5%, 1:3 and 1:5) in mice (n = 3) after intravesical (instillation) and systemic (intravenous) administration was assessed in vivo and ex vivo using near-infrared fluorescence live imaging for real time. It was demonstrated that intravesical instillation allows approximately 10 times more efficient accumulation of emulsion microgels in the mice urinary bladder in vivo 1 h after injection compared to systemic injection. The retention of the emulsion of mucoadhesive microgels in bladders after the intravesical instillation was observed for 24 h.

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.

Non-invasive transcutaneous influenza immunization using vaccine-loaded vaterite particles.

Transcutaneous immunization receives much attention due to the recognition of a complex network of immunoregulatory cells in various layers of the skin. The elaboration of non-invasive needle-free approaches towards antigen delivery holds especially great potential here while searching for a hygienically optimal vaccination strategy. Here, we report on a novel protocol for transfollicular immunization aiming at delivery of an inactivated influenza vaccine to perifollicular antigen presenting cells without disrupting the stratum corneum integrity. Porous calcium carbonate (vaterite) submicron carriers and sonophoresis were utilized for this purpose. Transportation of the vaccine-loaded particles into hair follicles of mice was assessed in vivo via optical coherence tomography monitoring. The effectiveness of the designed immunization protocol was further demonstrated in an animal model by means of micro-neutralization and enzyme-linked immunosorbent assays. The titers of secreted virus-specific IgGs were compared to those obtained in response to intramuscular immunization using conventional influenza vaccine formulation demonstrating no statistically significant differences in antibody levels between the groups. The findings of our pilot study render the intra-follicular delivery of the inactivated influenza vaccine by means of vaterite carriers a promising alternative to invasive immunization.

Degradation of Hybrid Drug Delivery Carriers with a Mineral Core and a Protein-Tannin Shell under Proteolytic Hydrolases.

Hybrid carriers with the mineral CaCO3/Fe3O4 core and the protein-tannin shell are attractive for drug delivery applications due to reliable coupling of anticancer drugs with protein-tannin complex and the possibility of remote control over drug localization and delivery by the external magnetic field. This study aims to elucidate the mechanisms of drug release via enzymatic degradation of a protein-tannin carrier shell triggered by proteolytic hydrolases trypsin and pepsin under physiological conditions. To do this, the carriers were incubated with the enzyme solutions in special buffers to maintain the enzyme activity. The time-lapse spectrophotometric and electron microscopy measurements were carried out to evaluate the degradation of the carriers. It was established that the protein-tannin complex demonstrates the different degradation behavior depending on the enzyme type and buffer medium. The incubation in trypsin solution mostly resulted in the protein shell degradation. The incubation in pepsin solution did not affect the protein component; however, the citric buffer stimulates the degradation of the mineral core. The presented results allow for predicting the degradation pathways of the carriers including the release profile of the loaded cargo under physiological conditions. The viability of 4T1 breast cancer cells with mineral magnetic carriers with protein-tannin shells was investigated, and their movement in the fields of action of the permanent magnet was shown.

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.

Osteogenic Capability of Vaterite-Coated Nonwoven Polycaprolactone Scaffolds for In Vivo Bone Tissue Regeneration.

In current orthopedic practice, bone implants used to-date often exhibit poor osteointegration, impaired osteogenesis, and, eventually, implant failure. Actively pursued strategies for tissue engineering could overcome these shortcomings by developing new hybrid materials with bioinspired structure and enhanced regenerative potential. In this study, the osteogenic and therapeutic potential of bioactive vaterite is investigated as a functional component of a fibrous polymeric scaffold for bone regeneration. Hybrid two-layered polycaprolactone scaffolds coated with vaterite (PCL/CaCO3 ) are studied during their 28-days implantation period in a rat femur defect. After this period, the study of tissue formation in the defected area is performed by the histological study of femur cross-sections. Immobilization of alkaline phosphatase (ALP) into PCL/CaCO3 scaffolds accelerates new bone tissue formation and defect repair. PCL/CaCO3 and PCL/CaCO3 /ALP scaffolds reveal 37.3% and 62.9% areas, respectively, filled with newly formed bone tissue in cross-sections compared to unmineralized PCL scaffold (17.5%). Bone turnover markers are monitored on the 7th and 28th days after implantation and reveal an increase of osteocalcin level for both PCL/CaCO3 and PCL/CaCO3 /ALP compared with PCL indicating the activation of osteogenesis. These findings indicate that vaterite, as an osteoconductive component of polymeric scaffolds, promotes osteogenesis, supports angiogenesis, and facilitates bone defect repair.

Effect of liraglutide on microcirculation in rat model with absolute insulin deficiency.

INTRODUCTION: The investigations of angiotropic effects of liraglutide are an issue of significant scientific and practical interest. The successful application of liraglutide has been shown in glycemic control in patients with the type 2 diabetes mellitus (DM), but the effect of liraglutide in patients with type 1 DM has not been completely studied yet in clinical practice. Therefore, the present study is aimed to investigate the effect of liraglutide which is agonist of glucagon-like peptide-1 receptors, on microcirculation in white outbred rats with the alloxan-induced diabetes. MATERIALS AND METHODS: The study was performed with 70 white outbred rats, divided into 4 groups: 1) control group (intact animals (Control)); 2) comparison group (diabetes mellitus (DM)) - animals with the alloxan-induced diabetes; 3) experimental group no. 1 (liraglutide low dose (LLD)) - animals with the alloxan-induced diabetes, which were injected by liraglutide at dosage of 0.2 mg/kg of animal weight per a day; 4) experimental group no. 2 (liraglutide high dose (LHD)) - animals with the alloxan-induced diabetes, which were injected by liraglutide at dosage of 0.4 mg/kg of animal weight per a day. The carbohydrate metabolism disorders, the microcirculation of posterior paw skin, as well as the concentration of catecholamines and markers of endothelial alteration in blood were estimated at the 42nd day of the experiment in the comparison and experimental groups. RESULTS: It was found that the correction of carbohydrate metabolism by liraglutide is succeeded by the normalization of skin perfusion of posterior paw skin of the experimental animals. Recovery of microcirculation is associated with a decrease in vascular tone and stimulation of endothelium-dependent vasodilation, caused by simultaneous decrease of catecholamines, endothelin-1 and asymmetric dimethylarginine (ADMA) concentrations in blood serum. At the same time, the administration of liraglutide on the background of insulin-deficiency results in decrease of endothelial cell alteration markers concentration in blood, such as sE-selectin, syndecan-1, and vascular endothelial growth factor (VEGF). CONCLUSION: Administration of liraglutide leads to the normalization of the carbohydrate metabolism simultaneously with the correction of microcirculation in rats with the absolute insulin deficiency. The demonstrated recovery of microcirculation by liraglutide, which represents an analogue of glucagon-like peptide-1, provides new prospects for its approval as a potential drug for pathogenetic correction of microcirculatory disorders in patients with the type 1 DM.

Influence of the new formulation based on silver alginate microcapsules loaded with tannic acid on the microcirculation of the experimental periodontitis in rats.

The microvascular changes caused by disorders of host immune response to oral microorganisms resulting in long-lasting inflammation of gums play a critical role in the periodontal lesion in the pathogenesis of chronic periodontitis. Current strategies of non-surgical periodontal therapy are aimed at the attainment of anti-inflammatory effects. We hypothesized that the usage of the microencapsulated form of anti-inflammatory substances with vasoactive effects could enhance the efficiency of the therapy by the prolonged release of active components. The prepared suspension of silver-alginate microcapsules loaded with tannic acid in the hydrogel was applied in vivo to the experimental model of periodontitis in rats induced by a ligature. The effect of this formulation was assessed by monitoring changes in local microcirculation performed by the Laser Doppler Flowmetry (1 and 24 h after application of hydrogel on intact gums and 21-days after the start of periodontitis' modeling). Application of the hydrogel containing multicomponent microcapsules to the affected area of gums allows correction of inflammatory microcirculatory disorders in model periodontitis. Immobilization of tannic acid into microcapsules allows increasing the correction of the following parameters: perfusion disorders, neurogenic tone of arterioles, myogenic tone of precapillary sphincters, as well as a venous outflow in the microvasculature of the gums. The hydrogel containing multicomponent microcapsules reduces the vascular inflammatory response in the model of periodontitis. Loading of silver-alginate microcapsules with tannic acid enhances the efficiency of microvascular disorders' correction in the model of periodontitis that suggests the prospects for application of this drug delivery system for non-surgical treatment of periodontitis.

Hierarchy of Hybrid Materials-The Place of Inorganics-in-Organics in it, Their Composition and Applications.

Hybrid materials, or hybrids incorporating both organic and inorganic constituents, are emerging as a very potent and promising class of materials due to the diverse, but complementary nature of the properties inherent of these different classes of materials. The complementarity leads to a perfect synergy of properties of desired material and eventually an end-product. The diversity of resultant properties and materials used in the construction of hybrids, leads to a very broad range of application areas generated by engaging very different research communities. We provide here a general classification of hybrid materials, wherein organics-in-inorganics (inorganic materials modified by organic moieties) are distinguished from inorganics-in-organics (organic materials or matrices modified by inorganic constituents). In the former area, the surface functionalization of colloids is distinguished as a stand-alone sub-area. The latter area-functionalization of organic materials by inorganic additives-is the focus of the current review. Inorganic constituents, often in the form of small particles or structures, are made of minerals, clays, semiconductors, metals, carbons, and ceramics. They are shown to be incorporated into organic matrices, which can be distinguished as two classes: chemical and biological. Chemical organic matrices include coatings, vehicles and capsules assembled into: hydrogels, layer-by-layer assembly, polymer brushes, block co-polymers and other assemblies. Biological organic matrices encompass bio-molecules (lipids, polysaccharides, proteins and enzymes, and nucleic acids) as well as higher level organisms: cells, bacteria, and microorganisms. In addition to providing details of the above classification and analysis of the composition of hybrids, we also highlight some antagonistic yin-&-yang properties of organic and inorganic materials, review applications and provide an outlook to emerging trends.

Effect of low-temperature plasma treatment of electrospun polycaprolactone fibrous scaffolds on calcium carbonate mineralisation.

This article reports on a study of the mineralisation behaviour of CaCO3 deposited on electrospun poly(ε-caprolactone) (PCL) scaffolds preliminarily treated with low-temperature plasma. This work was aimed at developing an approach that improves the wettability and permeability of PCL scaffolds in order to obtain a superior composite coated with highly porous CaCO3, which is a prerequisite for biomedical scaffolds used for drug delivery. Since PCL is a synthetic polymer that lacks functional groups, plasma processing of PCL scaffolds in O2, NH3, and Ar atmospheres enables introduction of highly reactive chemical groups, which influence the interaction between organic and inorganic phases and govern the nucleation, crystal growth, particle morphology, and phase composition of the CaCO3 coating. Our studies showed that the plasma treatment induced the formation of O- and N-containing polar functional groups on the scaffold surface, which caused an increase in the PCL surface hydrophilicity. Mineralisation of the PCL scaffolds was performed by inducing precipitation of CaCO3 particles on the surface of polymer fibres from a mixture of CaCl2- and Na2CO3-saturated solutions. The presence of highly porous vaterite and nonporous calcite crystal phases in the obtained coating was established. Our findings confirmed that preferential growth of the vaterite phase occurred in the O2-plasma-treated PCL scaffold and that the coating formed on this scaffold was smoother and more homogenous than those formed on the untreated PCL scaffold and the Ar- and NH3-plasma-treated PCL scaffolds. A more detailed three-dimensional assessment of the penetration depth of CaCO3 into the PCL scaffold was performed by high-resolution micro-computed tomography. The assessment revealed that O2-plasma treatment of the PCL scaffold caused CaCO3 to nucleate and precipitate much deeper inside the porous structure. From our findings, we conclude that O2-plasma treatment is preferable for PCL scaffold surface modification from the viewpoint of use of the PCL/CaCO3 composite as a drug delivery platform for tissue engineering.

Polymeric and Lipid Membranes-From Spheres to Flat Membranes and vice versa.

Membranes are important components in a number of systems, where separation and control of the flow of molecules is desirable. Controllable membranes represent an even more coveted and desirable entity and their development is considered to be the next step of development. Typically, membranes are considered on flat surfaces, but spherical capsules possess a perfect "infinite" or fully suspended membranes. Similarities and transitions between spherical and flat membranes are discussed, while applications of membranes are also emphasized.