Multiple dyes applications for fluorescent convertible polymer capsules as macrophages tracking labels.

Tracing individual cell pathways among the whole population is crucial for understanding their behavior, cell communication, migration dynamics, and fate. Optical labeling is one approach for tracing individual cells, but it typically requires genetic modification to induce the generation of photoconvertible proteins. Nevertheless, this approach has limitations and is not applicable to certain cell types. For instance, genetic modification often leads to the death of macrophages. This study aims to develop an alternative method for labeling macrophages by utilizing photoconvertible micron-sized capsules capable of easy internalization and prolonged retention within cells. Thermal treatment in a polyvinyl alcohol gel medium is employed for the scalable synthesis of capsules with a wide range of fluorescent dyes, including rhodamine 6G, pyronin B, fluorescein, acridine yellow, acridine orange, thiazine red, and previously reported rhodamine B. The fluorescence brightness, photostability, and photoconversion ability of the capsules are evaluated using confocal laser scanning microscopy. Viability, uptake, mobility, and photoconversion studies are conducted on RAW 264.7 and bone marrow-derived macrophages, serving as model cell lines. The production yield of the capsules is increased due to the use of polyvinyl alcohol gel, eliminating the need for conventional filtration steps. Capsules entrapping rhodamine B and rhodamine 6G meet all requirements for intracellular use in individual cell tracking. Mass spectrometry analysis reveals a sequence of deethylation steps that result in blue shifts in the dye spectra upon irradiation. Cellular studies on macrophages demonstrate robust uptake of the capsules. The capsules exhibit minimal cytotoxicity and have a negligible impact on cell motility. The successful photoconversion of RhB-containing capsules within cells highlights their potential as alternatives to photoconvertible proteins for individual cell labeling, with promising applications in personalized medicine.

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.

Time-Delayed Anticancer Effect of an Extremely Low Frequency Alternating Magnetic Field and Multimodal Protein-Tannin-Mitoxantrone Carriers with Brillouin Microspectroscopy Visualization In Vitro.

The effect of an extremely low frequency alternating magnetic field (ELF AMF) at frequencies of 17, 48, and 95 Hz at 100 mT on free and internalized 4T1 breast cancer cell submicron magnetic mineral carriers with an anticancer drug, mitoxantrone, was shown. The alternating magnetic field (100 mT; 17, 48, 95 Hz; time of treatment-10.5 min with a 30 s delay) does not lead to the significant destruction of carrier shells and release of mitoxantrone or bovine serum albumin from them according to the data of spectrophotometry, or the heating of carriers in the process of exposure to magnetic fields. The most optimal set of factors that would lead to the suppression of proliferation and survival of cells with anticancer drug carriers on the third day (in comparison with the control and first day) is exposure to an alternating magnetic field of 100 mT in a pulsed mode with a frequency of 95 Hz. The presence of magnetic nanocarriers in cell lines was carried out by a direct label-free method, space-resolved Brillouin light scattering (BLS) spectrometry, which was realized for the first time. The analysis of the series of integrated BLS spectra showed an increase in the magnetic phase in cells with a growth in the number of particles per cell (from 10 to 100) after their internalization. The safety of magnetic carriers in the release of their constituent ions has been evaluated using atomic absorption spectrometry.

Labeling and Tracking of Individual Human Mesenchymal Stromal Cells Using Photoconvertible Fluorescent Microcapsules.

The behavior and migration of human mesenchymal stromal cells (hMSCs) are focal points of research in the biomedical field. One of the major aspects is potential therapy using hMCS, but at present, the safety of their use is still controversial owing to limited data on changes that occur with hMSCs in the long term. Fluorescent photoconvertible proteins are intensively used today as "gold standard" to mark the individual cells and study single-cell interactions, migration processes, and the formation of pure lines. A crucial disadvantage of this method is the need for genetic modification of the primary culture, which casts doubt on the possibility of exploring the resulting clones in personalized medicine. Here we present a new approach for labeling and tracking hMSCs without genetic modification based on the application of cell-internalizable photoconvertible polyelectrolyte microcapsules (size: 2.6 ± 0.5 µm). These capsules were loaded with rhodamine B, and after thermal treatment, exhibited fluorescent photoconversion properties. Photoconvertible capsules demonstrated low cytotoxicity, did not affect the immunophenotype of the hMSCs, and maintained a high level of fluorescent signal for at least seven days. The developed approach was tested for cell tracking for four days and made it possible to trace the destiny of daughter cells without the need for additional labeling.

Upconversion Nanoparticles Intercalated in Large Polymer Micelles for Tumor Imaging and Chemo/Photothermal Therapy.

Frontiers in theranostics are driving the demand for multifunctional nanoagents. Upconversion nanoparticle (UCNP)-based systems activated by near-infrared (NIR) light deeply penetrating biotissue are a powerful tool for the simultaneous diagnosis and therapy of cancer. The intercalation into large polymer micelles of poly(maleic anhydride-alt-1-octadecene) provided the creation of biocompatible UCNPs. The intrinsic properties of UCNPs (core@shell structure NaYF4:Yb3+/Tm3+@NaYF4) embedded in micelles delivered NIR-to-NIR visualization, photothermal therapy, and high drug capacity. Further surface modification of micelles with a thermosensitive polymer (poly-N-vinylcaprolactam) exhibiting a conformation transition provided gradual drug (doxorubicin) release. In addition, the decoration of UCNP micelles with Ag nanoparticles (Ag NPs) synthesized in situ by silver ion reduction enhanced the cytotoxicity of micelles at cell growth temperature. Cell viability assessment on Sk-Br-3, MDA-MB-231, and WI-26 cell lines confirmed this effect. The efficiency of the prepared UCNP complex was evaluated in vivo by Sk-Br-3 xenograft regression in mice for 25 days after peritumoral injection and photoactivation of the lesions with NIR light. The designed polymer micelles hold promise as a photoactivated theranostic agent with quattro-functionalities (NIR absorption, photothermal effect, Ag NP cytotoxicity, and Dox loading) that provides imaging along with chemo- and photothermal therapy enhanced with Ag NPs.

Proteolytic Resistance Determines Albumin Nanoparticle Drug Delivery Properties and Increases Cathepsin B, D, and G Expression.

Proteolytic activity is pivotal in maintaining cell homeostasis and function. In pathological conditions such as cancer, it covers a key role in tumor cell viability, spreading to distant organs, and response to the treatment. Endosomes represent one of the major sites of cellular proteolytic activity and very often represent the final destination of internalized nanoformulations. However, little information about nanoparticle impact on the biology of these organelles is available even though they represent the major location of drug release. In this work, we generated albumin nanoparticles with a different resistance to proteolysis by finely tuning the amount of cross-linker used to stabilize the carriers. After careful characterization of the particles and measurement of their degradation in proteolytic conditions, we determined a relationship between their sensitivity to proteases and their drug delivery properties. These phenomena were characterized by an overall increase in the expression of cathepsin proteases regardless of the different sensitivity of the particles to proteolytic degradation.

Hybrid (Bovine Serum Albumin)/Poly(N-vinyl-2-pyrrolidone-co-acrylic acid)-Shelled Microbubbles as Advanced Ultrasound Contrast Agents.

Microbubbles are routinely used ultrasound contrast agents in the clinic. While a soft protein shell is commercially preferable for imaging purposes, a rigid polymer shell demonstrates prolonged agent stability. Hence, combining polymers and proteins in one shell composition can advance microbubble properties. We formulated the hybrid "protein-copolymer" microbubble shell with a complex of bovine serum albumin and an amphiphilic copolymer of N-vinyl-2-pyrrolidone and acrylic acid. The resulting microbubbles demonstrated advanced physicochemical and acoustic properties, preserving in vitro biocompatibility. Adjusting the mass ratio between protein and copolymer allowed fine tuning of the microbubble properties of concentration (by two orders, up to 1010 MBs/mL), mean size (from 0.8 to 5 µm), and shell thickness (from 28 to 50 nm). In addition, the minimum air-liquid surface tension for the "protein-copolymer" solution enabled the highest bubble concentration. At the same time, a higher copolymer amount in the bubble shell increased the bubble size and tuned duration and intensity of the contrast during an ultrasound procedure. Demonstrated results exemplify the potential of the hybrid "protein-polymer" microbubble shell, allowing tailoring of microbubble properties for image-guided applications, combining advances of each material involved in the formulation.

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.

Nanocurcumin-Loaded UCNPs for Cancer Theranostics: Physicochemical Properties, In Vitro Toxicity, and In Vivo Imaging Studies.

Formulation of promising anticancer herbal drug curcumin as a nanoscale-sized curcumin (nanocurcumin) improved its delivery to cells and organisms both in vitro and in vivo. We report on coupling nanocurcumin with upconversion nanoparticles (UCNPs) using Poly (lactic-co-glycolic Acid) (PLGA) to endow visualisation in the near-infrared transparency window. Nanocurcumin was prepared by solvent-antisolvent method. NaYF4:Yb,Er (UCNP1) and NaYF4:Yb,Tm (UCNP2) nanoparticles were synthesised by reverse microemulsion method and then functionalized it with PLGA to form UCNP-PLGA nanocarrier followed up by loading with the solvent-antisolvent process synthesized herbal nanocurcumin. The UCNP samples were extensively characterised with XRD, Raman, FTIR, DSC, TGA, UV-VIS-NIR spectrophotometer, Upconversion spectrofluorometer, HRSEM, EDAX and Zeta Potential analyses. UCNP1-PLGA-nanocurcumin exhibited emission at 520, 540, 660 nm and UCNP2-PLGA-nanocurmin showed emission at 480 and 800 nm spectral bands. UCNP-PLGA-nanocurcumin incubated with rat glioblastoma cells demonstrated moderate cytotoxicity, 60-80% cell viability at 0.12-0.02 mg/mL marginally suitable for therapeutic applications. The cytotoxicity of UCNPs evaluated in tumour spheroids models confirmed UCNP-PLGA-nanocurcumin therapeutic potential. As-synthesised curcumin-loaded nanocomplexes were administered in tumour-bearing laboratory animals (Lewis lung cancer model) and showed adequate contrast to enable in vivo and ex vivo study of UCNP-PLGA-nanocurcumin bio distribution in organs, with dominant distribution in the liver and lungs. Our studies demonstrate promise of nanocurcumin-loaded upconversion nanoparticles for theranostics applications.

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.

SERS Platform Based on Hollow-Core Microstructured Optical Fiber: Technology of UV-Mediated Gold Nanoparticle Growth.

Surface-enhanced Raman spectroscopy (SERS) is a powerful technique for biosensing. However, SERS analysis has several concerns: the signal is limited by a number of molecules and the area of the plasmonic substrate in the laser hotspot, and quantitative analysis in a low-volume droplet is confusing due to the change of concentration during quick drying. The usage of hollow-core microstructured optical fibers (HC-MOFs) is thought to be an effective way to improve SERS sensitivity and limit of detection through the effective irradiation of a small sample volume filling the fiber capillaries. In this paper, we used layer-by-layer assembly as a simple method for the functionalization of fiber capillaries by gold nanoparticles (seeds) with a mean diameter of 8 nm followed by UV-induced chloroauric acid reduction. We also demonstrated a simple and quick technique used for the analysis of the SERS platform formation at every stage through the detection of spectral shifts in the optical transmission of HC-MOFs. The enhancement of the Raman signal of a model analyte Rhodamine 6G was obtained using such type of SERS platform. Thus, a combination of nanostructured gold coating as a SERS-active surface and a hollow-core fiber as a microfluidic channel and a waveguide is perspective for point-of-care medical diagnosis based on liquid biopsy and exhaled air analysis.

A versatile platform for bioimaging based on colominic acid-decorated upconversion nanoparticles.

Lanthanide-doped upconversion nanoparticles (UCNPs) are promising bioimaging agents that emit light under near infra-red excitation, capable of penetrating deep in biotissues with a high signal-to-noise ratio. Their successful implementation is principally associated with surface functionalization. Here, we report on UCNP surface modification with highly hydrophilic, endogenous, non-toxic, non-immunogenic colominic acid, conferring "stealth" properties. We proposed surface functionalization of UCNPs based on a two-step strategy, which consists of hydrophilization with polyethyleneimine and attachment of colominic acid by electrostatic or covalent bond formation. Analysis revealed that regardless of the nature of the bond, colominic acid acted as a non-cytotoxic UCNP surface coating with low nonspecific blood protein adsorption. UCNP-colominic acid nanocomplexes exhibited low uptake by macrophages in vitro, which plays an active role in inflammatory reactions. We demonstrated the superiority of colominic acid compared to polyethylene glycol coating in terms of the prolonged circulation time in the bloodstream of small animals when injected intravenously. The colominic acid coating made it possible to prolong the UCNP circulation time up to 3 h. This led to the efficient UCNP accumulation in the inflammation site due to microvascular remodeling, accompanied by an enhanced uptake and retention effect. UCNP-assisted imaging of inflammation in the whole-body mode as well as local visualization of blood vessels were acquired in vivo. These collective findings validate the functional significance of UCNP decoration with colominic acid for their application in bioimaging.

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.

Freezing-Induced Loading of TiO2 into Porous Vaterite Microparticles: Preparation of CaCO3/TiO2 Composites as Templates To Assemble UV-Responsive Microcapsules for Wastewater Treatment.

The photocatalytic degradation of organic molecules is one of the effective ways for water purification. At this point, photocatalytic microreactor systems seem to be promising to enhance the versatility of the photoassisted degradation approach. Herein, we propose photoresponsive microcapsules prepared via layer-by-layer assembly of polyelectrolytes on the novel CaCO3/TiO2 composite template cores. The preparation of CaCO3/TiO2 composite particles is challenging because of the poor compatibility of TiO2 and CaCO3 in an aqueous medium. To prepare stable CaCO3/TiO2 composites, TiO2 nanoparticles were loaded into mesoporous CaCO3 microparticles with a freezing-induced loading technique. The inclusion of TiO2 nanoparticles into CaCO3 templates was evaluated with scanning electron microscopy and elemental analysis with respect to their type, concentration, and number of loading iterations. Upon polyelectrolyte shell assembly, the CaCO3 matrix was dissolved, resulting in microreactor capsules loaded with TiO2 nanoparticles. The photoresponsive properties of the resulted capsules were tested by photoinduced degradation of the low-molecule dye rhodamine B in aqueous solution and fluorescently labeled polymer molecules absorbed on the capsule surface under UV light. The exposure of the capsules to UV light resulted in a pronounced degradation of rhodamine B in capsule microvolume and fluorescent molecules on the capsule surface. Finally, the versatility of preparation of multifunctional photocatalytic and magnetically responsive capsules was demonstrated by iterative freezing-induced loading of TiO2 and magnetite Fe3O4 nanoparticles into CaCO3 templates.

Focused ultrasound-mediated fluorescence of composite microcapsules loaded with magnetite nanoparticles: In vitro and in vivo study.

High intensity focused ultrasound (HIFU) is widely used in medical practice, including cancer therapy. Also this approach is promising for remote release of encapsulated drugs in various other biomedical applications where local treatment is needed. Our approach underpins the minimization of HIFU impact on possible degradation of biological tissues and expand the use of HIFU in the controlled release of encapsulated drugs. We demonstrated the efficient ultrasound-induced release of labeled protein (Cy7-BSA) from elaborated nanocomposite microcapsules in vitro an in vivo. The capsule fabrication was done using combination of recently developed freezing-induced loading (FIL) technique and Layer-by-Layer assembly (LbL) used for the preparation of complex multilayer BSA/tannic acid nanocomposite capsules sensitive to HIFU. These capsules contain NIR fluorescent Cy7-labeled BSA in the shell for tracking in vivo and the high concentration of labels inside the capsules resulted in self-quenching provides the real-time detection of the protein once it is released from the capsule. Ultrasound-induced release in vivo of Cy7-labeled BSA initially quenched by magnetite nanoparticles was confirmed by fluorescent tomography. The significant decrease of Cy7 fluorescence under HIFU treatment in vitro was found to be due to a generation of reactive oxygen species and fast dye oxidation. Our results demonstrate that adapted HIFU setup can be used for the directed release of encapsulated substances in vivo under tissue compatible NIR monitoring by fluorescent tomography.

High-efficiency freezing-induced loading of inorganic nanoparticles and proteins into micron- and submicron-sized porous particles.

We demonstrate a novel approach to the controlled loading of inorganic nanoparticles and proteins into submicron- and micron-sized porous particles. The approach is based on freezing/thawing cycles, which lead to high loading densities. The process was tested for the inclusion of Au, magnetite nanoparticles, and bovine serum albumin in biocompatible vaterite carriers of micron and submicron sizes. The amounts of loaded nanoparticles or substances were adjusted by the number of freezing/thawing cycles. Our method afforded at least a three times higher loading of magnetite nanoparticles and a four times higher loading of protein for micron vaterite particles, in comparison with conventional methods such as adsorption and coprecipitation. The capsules loaded with magnetite nanoparticles by the freezing-induced loading method moved faster in a magnetic field gradient than did the capsules loaded by adsorption or coprecipitation. Our approach allows the preparation of multicomponent nanocomposite materials with designed properties such as remote control (e.g. via the application of an electromagnetic or acoustic field) and cargo unloading. Such materials could be used as multimodal contrast agents, drug delivery systems, and sensors.

Fabrication and photoluminescent properties of Tb3+ doped carbon nanodots.

Carbon nanodots (CNDs) doped with Tb ions were synthesized using different synthetic routes: hydrothermal treatment of a solution containing carbon source (sodium dextran sulfate) and TbCl3; mixing of CNDs and TbCl3 solutions; freezing-induced loading of Tb and carbon-containing source into pores of CaCO3 microparticles followed by hydrothermal treatment. Binding of Tb ions to CNDs (Tb-CND coupling) was confirmed using size-exclusion chromatography and manifested itself through a decrease of the Tb photoluminescence lifetime signal. The shortest Tb photoluminescence lifetime was observed for samples obtained by hydrothermal synthesis of CaCO3 microparticles where Tb and carbon source were loaded into pores via the freezing-induced process. The same system displays an increase of Tb photoluminescence via energy transfer with excitation at 320-340 nm. Based on the obtained results, freezing-induced loading of cations into CNDs using porous CaCO3 microparticles as reactors is proposed to be a versatile route for the introduction of active components into CNDs. The obtained CNDs with long-lived emission may be used for time-resolved imaging and visualization in living biological samples where time-resolved and long-lived luminescence microscopy is required.