Magnetically navigated microbubbles coated with albumin/polyarginine and superparamagnetic iron oxide nanoparticles.

While microbubbles (MB) are routinely used for ultrasound (US) imaging, magnetic MB are increasingly explored as they can be guided to specific sites of interest by applied magnetic field gradient. This requires the MB shell composition tuning to prolong MB stability and provide functionalization capabilities with magnetic nanoparticles. Hence, we developed air-filled MB stabilized by a protein-polymer complex of bovine serum albumin (BSA) and poly-L-arginine (pArg) of different molecular weights, showing that pArg of moderate molecular weight distribution (15-70 kDa) enabled MB with greater stability and acoustic response while preserving MB narrow diameters and the relative viability of THP-1 cells after 48 h of incubation. After MB functionalization with superparamagnetic iron oxide nanoparticles (SPION), magnetic moment values provided by single MB confirmed the sufficient SPION deposition onto BSA + pArg MB shells. During MB magnetic navigation in a blood vessel mimicking phantom with magnetic tweezers and in a Petri dish with adherent mouse renal carcinoma cell line, we demonstrated the effectiveness of magnetic MB localization in the desired area by magnetic field gradient. Magnetic MB co-localization with cells was further exploited for effective doxorubicin delivery with drug-loaded MB. Taken together, these findings open new avenues in control over albumin MB properties and magnetic navigation of SPION-loaded MB, which can envisage their applications in diagnostic and therapeutic needs.

Two-in-one sensor of refractive index and Raman scattering using hollow-core microstructured optical waveguides for colloid characterization.

Hollow-core microstructured optical waveguides (HC-MOW) have recently emerged in sensing technologies, including the gas and liquid detection for industrial as well as clinical applications. Antiresonant HC-MOW provide capabilities for applications in refractive index (RI) sensing, while the long optical path for analyte-light interaction in HC-MOW leads to increased sensitivity of sensor based on Raman scattering signal measurements. In this study, we developed a two-in-one sensor device using HC-MOW for RI and Raman scattering detection. The performance of the sensor was evaluated by characterizing protein-copolymer multicomponent colloids, specifically, bovine serum albumin (BSA) and poly(N - vinyl-2 -pyrrolidone-co-acrylic acid) P(VP-AA) nano-sized complexes and microbubbles of the corresponding shell. Monocomponent solutions showed linear dependencies of RI and characteristic Raman peak intensities on mass concentration. Multicomponent Raman sensing of BSA@P(VP-AA) complexes and microbubbles revealed that changes in P(VP-AA) characteristic peak intensities can describe interactions between components needed to produce colloid systems. RI sensing of multicomponent colloids demonstrated linear dependence on total mass concentrations for BSA@P(VP-AA) complexes, while corresponding BSA@P(VP-AA) microbubbles can be detected with concentrations as high as 4.0 × 108 MB/mL. Therefore, the developed two-in-one sensor of RI and Raman scattering can be used the robust characterization of albumin-based colloids designed for therapeutic and diagnostic needs.

Albumin microbubbles conjugated with zinc and aluminum phthalocyanine dyes for enhanced photodynamic activity.

Gas-liquid interfaces are reaching a particular interest in biomedicine. Microbubbles, ultrasound contrast agents of clinical routine, gained increasing attention as theranostic platforms due to the preserved acoustic response, drug conjugation capabilities, and applicability in biological barrier opening. A combination of microbubbles and photodynamic therapy agents can enhance the photodynamic effect, yet the evaluation of agent conjugation on microbubble stabilization and photodynamic effect is needed. Hence, two commercially available phthalocyanine photosensitizers - Holosens® (ZnPc) and Photosens® (AlPc) - were coupled with bovine serum albumin before microbubble synthesis. We demonstrated an albumin: phthalocyanine ratio of 1:1 and covalent attachment for ZnPc, a ratio of 1:3 with electrostatic binding for AlPc. Submicron-sized microbubbles (air- and SF6- filled) had a diameter of 0.8 µm. Albumin-phthalocyanine conjugates increased the microbubble concentration and shelf-life stability compared to plain ones. We hypothesized that phthalocyanine fluorescence lifetime values decreased after conjugation with microbubbles due to narrow distance between conjugates in the shell. Agents based on AlPc demonstrated higher photodynamic activity than agents based on ZnPc, and microbubbles preserved acoustic stability in human blood plasma. The biodistribution of AlPc-conjugated microbubbles was evaluated. We conclude that our microbubble platforms demonstrate greater photodynamic activity and prolonged stability for further applications in photodynamic therapy.

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.

Microbubbles Stabilized by Protein Shell: From Pioneering Ultrasound Contrast Agents to Advanced Theranostic Systems.

Ultrasound is a widely-used imaging modality in clinics as a low-cost, non-invasive, non-radiative procedure allowing therapists faster decision-making. Microbubbles have been used as ultrasound contrast agents for decades, while recent attention has been attracted to consider them as stimuli-responsive drug delivery systems. Pioneering microbubbles were Albunex with a protein shell composed of human serum albumin, which entered clinical practice in 1993. However, current research expanded the set of proteins for a microbubble shell beyond albumin and applications of protein microbubbles beyond ultrasound imaging. Hence, this review summarizes all-known protein microbubbles over decades with a critical evaluation of formulations and applications to optimize the safety (low toxicity and high biocompatibility) as well as imaging efficiency. We provide a comprehensive overview of (1) proteins involved in microbubble formulation, (2) peculiarities of preparation of protein stabilized microbubbles with consideration of large-scale production, (3) key chemical factors of stabilization and functionalization of protein-shelled microbubbles, and (4) biomedical applications beyond ultrasound imaging (multimodal imaging, drug/gene delivery with attention to anticancer treatment, antibacterial activity, biosensing). Presented critical evaluation of the current state-of-the-art for protein microbubbles should focus the field on relevant strategies in microbubble formulation and application for short-term clinical translation. Thus, a protein bubble-based platform is very perspective for theranostic application in clinics.

AuNP Aptasensor for Hodgkin Lymphoma Monitoring.

A liquid biopsy based on circulating small extracellular vesicles (SEVs) has not yet been used in routine clinical practice due to the lack of reliable analytic technologies. Recent studies have demonstrated the great diagnostic potential of nanozyme-based systems for the detection of SEV markers. Here, we hypothesize that CD30-positive Hodgkin and Reed-Sternberg (HRS) cells secrete CD30 + SEVs; therefore, the relative amount of circulating CD30 + SEVs might reflect classical forms of Hodgkin lymphoma (cHL) activity and can be measured by using a nanozyme-based technique. A AuNP aptasensor analytics system was created using aurum nanoparticles (AuNPs) with peroxidase activity. Sensing was mediated by competing properties of DNA aptamers to attach onto surface of AuNPs inhibiting their enzymatic activity and to bind specific markers on SEVs surface. An enzymatic activity of AuNPs was evaluated through the color reaction. The study included characterization of the components of the analytic system and its functionality using transmission and scanning electron microscopy, nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and spectrophotometry. AuNP aptasensor analytics were optimized to quantify plasma CD30 + SEVs. The developed method allowed us to differentiate healthy donors and cHL patients. The results of the CD30 + SEV quantification in the plasma of cHL patients were compared with the results of disease activity assessment by positron emission tomography/computed tomography (PET-CT) scanning, revealing a strong positive correlation. Moreover, two cycles of chemotherapy resulted in a statistically significant decrease in CD30 + SEVs in the plasma of cHL patients. The proposed AuNP aptasensor system presents a promising new approach for monitoring cHL patients and can be modified for the diagnostic testing of other diseases.

Air-Filled Microbubbles Based on Albumin Functionalized with Gold Nanocages and Zinc Phthalocyanine for Multimodal Imaging.

Microbubbles are intravascular contrast agents clinically used in diagnostic sonography, echocardiography, and radiology imaging applications. However, up to date, the idea of creating microbubbles with multiple functionalities (e.g., multimodal imaging, photodynamic therapy) remained a challenge. One possible solution is the modification of bubble shells by introducing specific compounds responsible for such functions. In the present work, air-core microbubbles with the shell consisting of bovine serum albumin, albumin-coated gold nanocages, and zinc phthalocyanine were prepared using the sonication method. Various physicochemical parameters such as stability over time, size, and concentration were investigated to prove the potential use of these microbubbles as contrast agents. This work shows that hybrid microbubbles have all the necessary properties for multimodal imaging (ultrasound, raster-scanning microscopy, and fluorescence tomography), which demonstrate superior characteristics for potential theranostic and related biomedical applications.

Oxygen Generation Using Catalytic Nano/Micromotors.

Gaseous oxygen plays a vital role in driving the metabolism of living organisms and has multiple agricultural, medical, and technological applications. Different methods have been discovered to produce oxygen, including plants, oxygen concentrators and catalytic reactions. However, many such approaches are relatively expensive, involve challenges, complexities in post-production processes or generate undesired reaction products. Catalytic oxygen generation using hydrogen peroxide is one of the simplest and cleanest methods to produce oxygen in the required quantities. Chemically powered micro/nanomotors, capable of self-propulsion in liquid media, offer convenient and economic platforms for on-the-fly generation of gaseous oxygen on demand. Micromotors have opened up opportunities for controlled oxygen generation and transport under complex conditions, critical medical diagnostics and therapy. Mobile oxygen micro-carriers help better understand the energy transduction efficiencies of micro/nanoscopic active matter by careful selection of catalytic materials, fuel compositions and concentrations, catalyst surface curvatures and catalytic particle size, which opens avenues for controllable oxygen release on the level of a single catalytic microreactor. This review discusses various micro/nanomotor systems capable of functioning as mobile oxygen generators while highlighting their features, efficiencies and application potentials in different fields.

Optoacoustic/Fluorescent/Acoustic Imaging Probe Based on Air-Filled Bubbles Functionalized with Gold Nanorods and Fluorescein Isothiocyanate.

Liquid/surfactant/gas interfaces are promising objects for nanoengineered multimodal contrasts, which can be used for biomedical imaging in preclinical and clinical applications. Microbubbles with the gaseous core and shell made of lipids/proteins have already acted as ultrasound (US) contrast agents for angiography. In the present work, microbubbles with a shell composed of Span 60 and Tween 80 surfactants functionalized with fluorescein isothiocyanate and gold nanorods to achieve a multimodal combination of US, fluorescence, and optoacoustic imaging are described. Optimal conditions for microbubble generation by studying the surface tension of the initial solutions and analyzing the size, stability, and charge of the resulting bubbles were found. By controlling and modifying bubbles' surface properties, an increase in stability and storage time can be achieved. The functionalization of bubbles with gold nanoparticles and a dye by using an optimally selected sonication protocol was performed. The biomedical application's potential in imaging modalities of functionalized microbubbles using a medical US device with a frequency of 50 MHz, fluorescence tomography, and raster-scanning optoacoustic mesoscopy measurements was evaluated. The obtained results are important for optimum stabilization and functionalization of gas/liquid interfaces and the following applications in the multimodal biomedical imaging.

Air-Filled Bubbles Stabilized by Gold Nanoparticle/Photodynamic Dye Hybrid Structures for Theranostics.

Microbubbles have already reached clinical practice as ultrasound contrast agents for angiography. However, modification of the bubbles' shell is needed to produce probes for ultrasound and multimodal (fluorescence/photoacoustic) imaging methods in combination with theranostics (diagnostics and therapeutics). In the present work, hybrid structures based on microbubbles with an air core and a shell composed of bovine serum albumin, albumin-coated gold nanoparticles, and clinically available photodynamic dyes (zinc phthalocyanine, indocyanine green) were shown to achieve multimodal imaging for potential applications in photodynamic therapy. Microbubbles with an average size of 1.5 ± 0.3 µm and concentration up to 1.2 × 109 microbubbles/mL were obtained and characterized. The introduction of the dye into the system reduced the solution's surface tension, leading to an increase in the concentration and stability of bubbles. The combination of gold nanoparticles and photodynamic dyes' influence on the fluorescent signal and probes' stability is described. The potential use of the obtained probes in biomedical applications was evaluated using fluorescence tomography, raster-scanning optoacoustic microscopy and ultrasound response measurements using a medical ultrasound device at the frequency of 33 MHz. The results demonstrate the impact of microbubbles' stabilization using gold nanoparticle/photodynamic dye hybrid structures to achieve probe applications in theranostics.

Gold nanoparticle-carbon nanotube multilayers on silica microspheres: Optoacoustic-Raman enhancement and potential biomedical applications.

There has been growing interest in recent years in developing multifunctional materials for studying the structure interface in biological systems. In this regard, the multimodal systems, which possess activity in the near-infrared (NIR) region, become even more critical for the possibility of improving examined biotissue depth and, eventually, data analysis. Herein, we engineered bi-modal contrast agents by integrating carbon nanotubes (CNT) and gold nanoparticles (AuNP) around silica microspheres using the Layer-by-Layer self-assembly method. The experimental studies revealed that microspheres with CNT sandwiched between AuNP exhibit strong absorption in the visible and NIR regions and high optoacoustic contrast (OA, also called photoacoustics) and Raman scattering when illuminated with 532 nm and 785 nm lasers, respectively. The developed microspheres demonstrated amplification of the signal in the OA flow cytometry at the laser wavelength of 1064 nm. This finding was further validated with ex vivo brain tissue using a portable Raman spectrometer and imaging with the Raster-scanning OA mesoscopy technique. The obtained data suggest that the developed contrast agents can be promising in applications of localization OA tomography (LOT), OA flow cytometry, and multiplex SERS detection.

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.

Amplification of photoacoustic effect in bimodal polymer particles by self-quenching of indocyanine green.

A new type of bimodal contrast agent was made that is based on the self-quenching of indocyanine green (ICG) encapsulated in a biocompatible and biodegradable polymer shell. The increasing of a local ICG concentration that is necessary for the obtaining of self-quenching effect was achieved by freezing-induced loading and layer-by-layer assembly. As a result, an efficient photoacoustic(optoacoustic)/fluorescent contrast agent based on composite indocyanine green/polymer particles was successfully prepared and was characterized by fluorescence and photoacoustic(optoacoustic) tomography in vitro. This type of contrast agent holds good promise for clinical application owing to its high efficiency and biosafety.

Magnetic nanorods for remote disruption of lipid membranes by non-heating low frequency magnetic field.

This work presents direct evidence of disordering of liposomal membranes by magnetic nanoparticles during their exposures to non-heating alternating Extremely Low Frequency Magnetic Field (ELF MF). Changes in the lipid membrane structure were demonstrated by the Attenuated total reflection Fourier Transform Infrared and fluorescence spectroscopy. Specifically, about 50% of hydrophobic chains became highly mobile under the action of ELF MF. Magnetic field-induced increase in the membrane fluidity was accompanied by an increase in membrane permeability and release of solutes entrapped in liposomes. The effect of ELF MF on the membrane fluidity was greater in case of 70 × 12 nm magnetite nanorods adsorbed on the liposomes surface compared to liposomes with ~7 nm spherical MNPs embedded within lipid membranes. A physical model of this process explaining experimental data is suggested. The obtained results open new horizons for the development of systems for triggered drug release without dangerous heating and overheating of tissues.

Magnetic liposome design for drug release systems responsive to super-low frequency alternating current magnetic field (AC MF).

HYPOTHESIS: Magnetic liposomes are shown to release the entrapped dye once modulated by low frequency AC MF. The mechanism and effectiveness of MF application should depend on lipid composition, magnetic nanoparticles (MNPs) properties, temperature and field parameters. EXPERIMENTS: The study was performed using liposomes of various lipid composition and embedded hydrophobic MNPs. The liposomes structural changes were studied by the transmission electron microscopy (TEM) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and the leakage was monitored by the fluorescent dye release. FINDINGS: Magnetic liposomes exposure to the AC MF resulted in the clustering of the MNPs in the membranes and disruption of the lipid packaging. Addition of cholesterol diminished the dye release from the saturated lipid-based liposomes. Replacement of the saturated lipid for unsaturated one also decreased the dye release. The dye release depended on the strength, but not the frequency of the field. Thus, the oscillating motion of MNPs in AC MF ruptures the gel phase membranes of saturated lipids. As the temperature increases the disruption also increases. In the liquid crystalline membranes formed by unsaturated lipids the deformations and defects created by mechanical motion of the MNPs are more likely to heal and results in decreased release.

Enzyme-functionalized gold-coated magnetite nanoparticles as novel hybrid nanomaterials: synthesis, purification and control of enzyme function by low-frequency magnetic field.

The possibility of remotely inducing a defined effect on NPs by means of electromagnetic radiation appears attractive. From a practical point of view, this effect opens horizons for remote control of drug release systems, as well as modulation of biochemical functions in cells. Gold-coated magnetite nanoparticles are perfect candidates for such application. Herein, we have successfully synthesized core-shell NPs having magnetite cores and gold shells modified with various sulphur containing ligands and developed a new, simple and robust procedure for the purification of the resulting nanoparticles. The carboxylic groups displayed at the surface of the NPs were utilized for NP conjugation with a model enzyme (ChT). In the present study, we report the effect of the low-frequency AC magnetic field on the catalytic activity of the immobilized ChT. We show that the enzyme activity decreases upon exposure of the NPs to the field.