Functionalization and magnetonavigation of T-lymphocytes functionalized via nanocomposite capsules targeting with electromagnetic tweezers.

Modification of T-lymphocytes, which are capable of paracellular transmigration is a promising trend in modern personalized medicine. However, the delivery of required concentrations of functionalized T-cells to the target tissues remains a problem. We describe a novel method to functionalize T-cells with magnetic nanocapsules and target them with electromagnetic tweezers. T-cells were modified with the following magnetic capsules: Parg/DEX (150 nm), BSA/TA (300 nm), and BSA/TA (500 nm). T-cells were magnetonavigated in a phantom blood vessel capillary in cultural medium and in whole blood. The permeability of tumor tissues to captured T-cells was analyzed by magnetic delivery of modified T-cells to spheroids formed from 4T1 breast cancer cells. The dynamics of T-cell motion under a magnetic field gradient in model environments were analyzed by particle image velocimetry. The magnetic properties of the nanocomposite capsules and magnetic T-cells were measured. The obtained results are promising for biomedical applications in cancer immunotherapy.

Plug-and-Play Lymph Node-on-Chip: Secondary Tumor Modeling by the Combination of Cell Spheroid, Collagen Sponge and T-Cells.

Towards the improvement of the efficient study of drugs and contrast agents, the 3D microfluidic platforms are currently being actively developed for testing these substances and particles in vitro. Here, we have elaborated a microfluidic lymph node-on-chip (LNOC) as a tissue engineered model of a secondary tumor in lymph node (LN) formed due to the metastasis process. The developed chip has a collagen sponge with a 3D spheroid of 4T1 cells located inside, simulating secondary tumor in the lymphoid tissue. This collagen sponge has a morphology and porosity comparable to that of a native human LN. To demonstrate the suitability of the obtained chip for pharmacological applications, we used it to evaluate the effect of contrast agent/drug carrier size, on the penetration and accumulation of particles in 3D spheroids modeling secondary tumor. For this, the 0.3, 0.5 and 4 µm bovine serum albumin (BSA)/tannic acid (TA) capsules were mixed with lymphocytes and pumped through the developed chip. The capsule penetration was examined by scanning with fluorescence microscopy followed by quantitative image analysis. The results show that capsules with a size of 0.3 µm passed more easily to the tumor spheroid and penetrated inside. We hope that the device will represent a reliable alternative to in vivo early secondary tumor models and decrease the amount of in vivo experiments in the frame of preclinical study.

Ultrasmooth, biocompatible, and removable nanocoating for hollow-core microstructured optical fibers.

Functional nanocoatings of hollow-core microstructured optical fibers (HC-MOFs) have extended the domain of their applications to biosensing and photochemistry. However, novel modalities typically come with increased optical losses since a significant surface roughness of functional layers gives rise to additional light scattering, restricting the performance of functionalization. Here, the technique that enables a biocompatible and removable nanocoating of HC-MOFs with low surface roughness is presented. The initial functional film is formed by a layer-by-layer assembly of bovine serum albumin (BSA) and tannic acid (TA). The alkaline etching at pH 9 results in the reduction of surface roughness from 26 nm to 3 nm and decreases fiber optical losses by three times. The nanocoating can be fully removed within 7 min of the treatment. Natural biocompatibility of BSA alongside antibacterial and antifouling properties of TA makes the presented nanocoating promising for biophotonic applications.

In vitro and in vivo MRI visualization of nanocomposite biodegradable microcapsules with tunable contrast.

Microcapsules, made of biodegradable polymers, containing magnetite nanoparticles with tunable contrast in both the T1 and T2 MRI modes, were successfully prepared using a layer-by-layer approach. The MRI contrast of the microcapsules was shown to depend on the distance between magnetite nanoparticles in the polymeric layers, which is controlled by their concentration in the microcapsule shell. A fivefold increase in the average distance between the nanoparticles in the microcapsule shell led to a change in the intensity of the MR signal of 100% for both the T1 and T2 modes. Enzyme treatment of biodegradable shells resulted in a change of the microcapsules' MRI contrast. In vivo degradation of nanocomposite microcapsules concentrated in the liver after intravenous injection was demonstrated by MRI. This method can be used for the creation of a new generation of drug delivery systems, including drug depot, with combined navigation, visualization and remote activated release of bioactive substances in vivo.

Engineered multicompartment vesicosomes for selective uptake by living cells.

Small extracellular vesicles (sEVs) have attracted tremendous interest in recent years due to their exceptional properties for therapeutic and diagnostic applications. Although much research was focused on the quantity and content of sEVs, less efforts have been put into discovering the interaction between sEVs and cells. Here we engineered multicompartment particles, termed vesicosomes, by deposition of sEVs derived from MCF7, CHO cells and human plasma onto the surface of polyelectrolyte (PE)-coated silica (SiO2) microparticles. Uptake of the PE-coated SiO2 microparticles by parent cells was significantly enhanced by coating them with sEVs, compared to PE-coated SiO2 microparticles independent of the terminated polyelectrolyte layer. This study highlights the emerging role of sEVs membrane receptors in the sEV-cells interaction and demonstrates the potential application of sEV-like multicompartment particles as therapeutic carriers.

Sentinel lymph node detection by combining nonradioactive techniques with contrast agents: State of the art and prospects.

The status of sentinel lymph nodes (SLNs) has a substantial prognostic value because these nodes are the first place where cancer cells accumulate along their spreading route. Routine SLN biopsy ("gold standard") involves peritumoral injections of radiopharmaceuticals, such as technetium-99m, which has obvious disadvantages. This review examines the methods used as "gold standard" analogs to diagnose SLNs. Nonradioactive preoperative and intraoperative methods of SLN detection are analyzed. Promising photonic tools for SLNs detection are reviewed, including NIR-I/NIR-II fluorescence imaging, photoswitching dyes for SLN detection, in vivo photoacoustic detection, imaging and biopsy of SLNs. Also are discussed methods of SLN detection by magnetic resonance imaging, ultrasonic imaging systems including as combined with photoacoustic imaging, and methods based on the magnetometer-aided detection of superparamagnetic nanoparticles. The advantages and disadvantages of nonradioactive SLN-detection methods are shown. The review concludes with prospects for the use of conservative diagnostic methods in combination with photonic tools.

Effect of Surface Modification of Multifunctional Nanocomposite Drug Delivery Carriers with DARPin on Their Biodistribution In Vitro and In Vivo.

We present a targeted drug delivery system for therapy and diagnostics that is based on a combination of contrasting, cytotoxic, and cancer-cell-targeting properties of multifunctional carriers. The system uses multilayered polymer microcapsules loaded with magnetite and doxorubicin. Loading of magnetite nanoparticles into the polymer shell by freezing-induced loading (FIL) allowed the loading efficiency to be increased 5-fold, compared with the widely used layer-by-layer (LBL) assembly. FIL also improved the photoacoustic signal and particle mobility in a magnetic field gradient, a result unachievable by the LBL alone. For targeted delivery of the carriers to cancer cells, the carrier surface was modified with a designed ankyrin repeat protein (DARPin) directed toward the epithelial cell adhesion molecule (EpCAM). Flow cytometry measurements showed that the DARPin-coated capsules specifically interacted with the surface of EpCAM-overexpressing human cancer cells such as MCF7. In vivo and ex vivo biodistribution studies in FvB mice showed that the carrier surface modification with DARPin changed the biodistribution of the capsules toward epithelial cells. In particular, the capsules accumulated substantially in the lungs─a result that can be effectively used in targeted lung cancer therapy. The results of this work may aid in the further development of the "magic bullet" concept and may bring the quality of personalized medicine to another level.

Indocyanine green dye based bimodal contrast agent tested by photoacoustic/fluorescence tomography setup.

Multimodal imaging systems are in high demand for preclinical research, experimental medicine, and clinical practice. Combinations of photoacoustic technology with other modalities including fluorescence, ultrasound, MRI, OCT have been already applied in feasibility studies. Nevertheless, only the combination of photoacoustics with ultrasound in a single setup is commercially available now. A combination of photoacoustics and fluorescence is another compelling approach because those two modalities naturally complement each other. Here, we presented a bimodal contrast agent based on the indocyanine green dye (ICG) as a single signalling compound embedded in the biocompatible and biodegradable polymer shell. We demonstrate its remarkable characteristics by imaging using a commercial photoacoustic/fluorescence tomography system (TriTom, PhotoSound Technologies). It was shown that photoacoustic signal of the particles depends on the amount of dye loaded into the shell, while fluorescence signal depends on the total amount of dye per particle. For the first time to our knowledge, a commercial bimodal photoacoustic/fluorescence setup was used for characterization of ICG doped polymer particles. Additionally, we conducted cell toxicity studies for these particles as well as studied biodistribution over time in vivo and ex vivo using fluorescent imaging. The obtained results suggest a potential for the application of biocompatible and biodegradable bimodal contrast agents as well as the integrated photoacoustic/fluorescence imaging system for preclinical and clinical studies.

Multifunctional nanostructured drug delivery carriers for cancer therapy: Multimodal imaging and ultrasound-induced drug release.

Development of multimodal systems for therapy and diagnosis of neoplastic diseases is an unmet need in oncology. The possibility of simultaneous diagnostics, monitoring, and therapy of various diseases allows expanding the applicability of modern systems for drug delivery. We have developed hybrid particles based on biocompatible polymers containing magnetic nanoparticles (MNPs), photoacoustic (MNPs), fluorescent (Cy5 or Cy7 dyes), and therapeutic components (doxorubicin). To achieve high loading efficiency of MNP and Dox to nanostructured carriers, we utilized a novel freezing-induced loading technique. To reduce the systemic toxicity of antitumor drugs and increase their therapeutic efficacy, we can use targeted delivery followed by the remote control of drug release using high intensity-focused ultrasound (HIFU). Loading of MNPs allowed performing magnetic targeting of the carriers and enhanced optoacoustic signal after controlled destruction of the shell and release of therapeutics as well as MRI imaging. The raster scanning optoacoustic mesoscopy (PA, RSOM), MRI, and fluorescent tomography (FT) confirmed the ultrasound-induced release of doxorubicin from capsules: in vitro (in tubes and pieces of meat) and in vivo (after delivery to the liver). Disruption of capsules results in a significant increase of doxorubicin and Cy7 fluorescence initially quenched by magnetite nanoparticles that can be used for real-time monitoring of drug release in vivo. In addition, we explicitly studied cytotoxicity, intracellular localization, and biodistribution of these particles. Elaborated drug delivery carriers have a good perspective for simultaneous imaging and focal therapy of different cancer types, including liver cancer.

Golden Vaterite as a Mesoscopic Metamaterial for Biophotonic Applications.

Mesoscopic photonic systems with tailored optical responses have great potential to open new frontiers in implantable biomedical devices. However, biocompatibility is typically a problem, as engineering of optical properties often calls for using toxic compounds and chemicals, unsuitable for in vivo applications. Here, a unique approach to biofriendly delivery of optical resonances is demonstrated. It is shown that the controllable infusion of gold nanoseeds into polycrystalline sub-micrometer vaterite spherulites gives rise to a variety of electric and magnetic Mie resonances, producing a tuneable mesoscopic optical metamaterial. The 3D reconstruction of the spherulites demonstrates the capability of controllable gold loading with volumetric filling factors exceeding 28%. Owing to the biocompatibility of the constitutive elements, "golden vaterite" paves the way to introduce designer-made Mie resonances to cutting-edge biophotonic applications. This concept is exemplified by showing efficient laser heating of gold-filled vaterite spherulites at red and near-infrared wavelengths, highly desirable in photothermal therapy, and photoacoustic tomography.

Barnase encapsulation into submicron porous CaCO3 particles: studies of loading and enzyme activity.

The present study focuses on the immobilization of the bacterial ribonuclease barnase (Bn) into submicron porous calcium carbonate (CaCO3) particles. For encapsulation, we apply adsorption, freezing-induced loading and co-precipitation methods and study the effects of adsorption time, enzyme concentration and anionic polyelectrolytes on the encapsulation efficiency of Bn. We show that the use of negatively charged dextran sulfate (DS) and ribonucleic acid from yeast (RNA) increases the loading capacity (LC) of the enzyme on CaCO3 particles by about 3-fold as compared to the particles with Bn itself. The ribonuclease (RNase) activity of encapsulated enzyme depends on the LC of the particles and transformation of metastable vaterite to stable calcite, as studied by the assessment of enzyme activities in particles.

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.

Optical clearing for photoacoustic lympho- and angiography beyond conventional depth limit in vivo.

Photoacoustic (PA) imaging (PAI) is an emerging powerful tool for noninvasive real-time mapping of blood and lymphatic vessels and lymph nodes in vivo to diagnose cancer, lymphedema and other diseases. Among different PAI instruments, commercially available raster-scanning optoacoustic mesoscopy (RSOM) (iThera Medical GmbH., Germany) is useful for high-resolution imaging of different tissues with high potential of clinical translation. However, skin light scattering prevents mapping vessels and nodes deeper than 1-2 mm, that limits diagnostic values of PAI including RSOM. Here we demonstrate that glycerol-based tissue optical clearing (TOC) overcomes this challenge by reducing light scattering that improves RSOM depth penetration. In preclinical model of mouse limb in vivo, the replacement of conventional acoustic coupling agents such as water on the mixture of 70 % glycerol and 30 % ultrasound (US) gel resulted in the increase of tissue imaging depth in 1.5-2 times with 3D visualization of vessels with diameter down to 20 µm. To distinguish blood and lymphatic networks, we integrated label-free PA angiography (i.e., imaging of blood vessels), which uses hemoglobin as endogenous contrast agent, with PA lymphography based on labeling of lymphatic vessels with exogenous PA contrast agents. Similar to well-established clinical lymphography, contrast agents were injected in tissue and taken up by lymphatic vessels within a few minutes that provided quick RSOM lymphography. Furthermore, co-injection of PA contrast dye and multilayer nanocomposites as potential low-toxic drug-cargo showed selective prolonged accumulation of nanocomposites in sentinel lymph nodes. Overall, our findings open perspectives for deep and high resolution 3D PA angio- and lymphography, and for PA-guided lymphatic drug delivery using new RSOM & TOC approach.

Photoacoustic and fluorescence lifetime imaging of diatoms.

Photoacoustic and fluorescent methods are used intensely in biology and medicine. These approaches can also be used to investigate unicellular diatom algae that are extremely important for Earth's ecology. They are enveloped within silica frustules (exoskeletons), which can be used in drug delivery systems. Here, we report for the first time the successful application of photoacoustic (PA) and fluorescent visualization of diatoms. Chlorophyll a and c and fucoxanthin were found likely to be responsible for the photoacoustic effect in diatoms. The PA signal was obtained from gel drops containing diatoms and was found to increase with the diatom concentration. The fluorescence lifetime of the diatom chromophores ranged from 0.5 to 2 ns. The dynamic light scattering, absorbance, and SEM characterization techniques were also applied. The results were considered in combination to elucidate the nature of the photoacoustic signal. Possible biotechnological applications are proposed for the remote photoacoustic monitoring of algae.

Biodegradable Polymeric Multilayer Capsules for Therapy of Lung Cancer.

Formulated forms of cancer therapeutics enhance the efficacy of treatment by more precise targeting, increased bioavailability of drugs, and an aptitude of some delivery systems to overcome multiple drug resistance of tumors. Drug carriers acquire importance for anti-cancer interventions via targeting tumor-associated macrophages with active molecules capable to either eliminate them or change their polarity. Although several packaged drug forms have reached the market, there is still a high demand for novel carrier systems to hurdle limitations of existing drugs on active molecules, toxicity, bioeffect, and stability. Here, we report a facile assembly and delivery methodology for biodegradable polymeric multilayer capsules (PMC) with the purpose of further use in injectable drug formulations for lung cancer therapy via direct erosion of tumors and suppression of the tumor-promoting function of macrophages in the tumor microenvironment. We demonstrate delivery of low-molecular-weight drug molecules to lung cancer cells and macrophages and provide details on in vivo distribution, cellular uptake, and disintegration of the developed PMC. Poly-l-arginine and dextran sulfate alternately adsorb on a ∼500 nm CaCO3 sacrificial template followed by removal of the inorganic core to obtain hollow capsules for consequent loading with drug molecules, gemcitabine or clodronate. The capsules further compacted upon loading down to ∼250 nm in diameter via heat treatment. A comparative study of the capsule internalization rate in vitro and in vivo reveals the benefits of a diminished carrier size. We show that macrophages and epithelial cells of the lungs and liver internalize capsules with efficacy higher than 75%. Using an in vivo mouse model of lung cancer, we also confirm that tumor lungs better retain smaller capsules than the healthy lung tissue. The pronounced cytotoxic effect of the encapsulated gemcitabine on lung cancer cells and the ability of the encapsulated clodronate to block the tumor-promoting function of macrophages prove the efficacy of the developed capsule loading method in vitro. Our study taken as a whole demonstrates the great potential of the developed PMC for in vivo treatment of cancer via transporting active molecules, including those that are water-soluble with low molecular weight, to both cancer cells and macrophages through the bloodstream.

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.

Photoswitchable Spasers with a Plasmonic Core and Photoswitchable Fluorescent Proteins.

Photoswitchable fluorescent proteins (PFPs) that can change fluorescence color upon excitation have revolutionized many applications of light such as tracking protein movement, super-resolution imaging, identification of circulating cells, and optical data storage. Nevertheless, the relatively weak fluorescence of PFPs limits their applications in biomedical imaging due to strong tissue autofluorecence background. Conversely, plasmonic nanolasers, also called spasers, have demonstrated potential to generate super-bright stimulated emissions even inside single cells. Nevertheless, the development of photoswitchable spasers that can shift their stimulated emission color in response to light is challenging. Here, we introduce the novel concept of spasers using a PFP layer as the active medium surrounding a plasmonic core. The proof of principle was demonstrated by synthesizing a multilayer nanostructure on the surface of a spherical gold core, with a non-absorbing thin polymer shell and the PFP Dendra2 dispersed in the matrix of a biodegradable polymer. We have demonstrated photoswitching of spontaneous and stimulated emission in these spasers below and above the spasing threshold, respectively, at different spectral ranges. The plasmonic core of the spasers serves also as a photothermal (and potentially photoacoustic) contrast agent, allowing for photothermal imaging of the spasers. These results suggest that multimodal photoswitchable spasers could extend the traditional applications of spasers and PFPs in laser spectroscopy, multicolor cytometry, and theranostics with the potential to track, identify, and kill abnormal cells in circulation.

Photoacoustic and fluorescent effects in multilayer plasmon-dye interfaces.

Progress in understanding the cell biology and diseases depends on advanced imaging and labeling techniques. Here, we address this demand by exploring novel multilayered nanocomposites (MNCs) with plasmonic nanoparticles and absorbing dyes in thin nonabsorbing shells as supercontrast multimodal photoacoustic (PA) and fluorescent agents in the near-infrared range. The proof of concept was performed with gold nanorods (GNRs) and indocyanine green (ICG) dispersed in a matrix of biodegradable polymers. We demonstrated synergetic PA effects in MNCs with the gold-ICG interface that could not be achieved with ICG and GNRs alone. We also observed ultrasharp PA and emission peaks that could be associated with nonlinear PA and spaser effects, respectively. Low-toxicity multimodal MNCs with unique plasmonic, thermal and acoustic properties have the potential to make a breakthrough in PA flow cytometry and near-infrared spasers in vivo by using the synergetic interaction of plasmonic modes with a nearby absorbing medium.

Submicron-Sized Nanocomposite Magnetic-Sensitive Carriers: Controllable Organ Distribution and Biological Effects.

Although new drug delivery systems have been intensely developed in the past decade, no significant increase in the efficiency of drug delivery by nanostructure carriers has been achieved. The reasons are the lack of information about acute toxicity, the influence of the submicron size of the carrier and difficulties with the study of biodistribution in vivo. Here we propose, for the first time in vivo, new nanocomposite submicron carriers made of bovine serum albumin (BSA) and tannic acid (TA) and containing magnetite nanoparticles with sufficient content for navigation in a magnetic field gradient on mice. We examined the efficacy of these submicron carriers as a delivery vehicle in combination with magnetite nanoparticles which were systemically administered intravenously. In addition, the systemic toxicity of this carrier for intravenous administration was explicitly studied. The results showed that (BSA/TA) carriers in the given doses were hemocompatible and didn't cause any adverse effect on the respiratory system, kidney or liver functions. A combination of gradient-magnetic-field controllable biodistribution of submicron carriers with fluorescence tomography/MRI imaging in vivo provides a new opportunity to improve drug delivery efficiency.

Label-free characterization of white blood cells using fluorescence lifetime imaging and flow-cytometry: molecular heterogeneity and erythrophagocytosis [Invited].

Blood cell analysis is one of the standard clinical tests. Despite the widespread use of exogenous markers for blood cell quantification, label-free optical methods are still of high demand due to their possibility for in vivo application and signal specific to the biochemical state of the cell provided by native fluorophores. Here we report the results of blood cell characterization using label-free fluorescence imaging techniques and flow-cytometry. Autofluorescence parameters of different cell types - white blood cells, red blood cells, erythrophagocytic cells - are assessed and analyzed in terms of molecular heterogeneity and possibilities of differentiation between different cell types in vitro and in vivo.