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.

Resurrecting essential amino acid biosynthesis in mammalian cells.

Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly, raising the possibility that these pathways were lost from organisms with access to abundant EAAs. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in mammalian cells. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese hamster ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation. 13C-tracing verified de novo biosynthesis of valine and further revealed build-up of pathway intermediate 2,3-dihydroxy-3-isovalerate. Increasing the dosage of downstream ilvD boosted pathway performance and allowed for long-term propagation of second-generation cells in valine-free medium at 3.2 days per doubling. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage.

Synthetic regulatory reconstitution reveals principles of mammalian Hox cluster regulation.

Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce "synthetic regulatory reconstitution," a conceptual framework for studying gene regulation, and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130 to 170 kilobases) to an ectopic location in the mouse genome. We found that a minimal HoxA cluster recapitulated correct patterns of chromatin remodeling and transcription in response to patterning signals, whereas the addition of distal enhancers was needed for full transcriptional output. Synthetic regulatory reconstitution could provide a generalizable strategy for deciphering the regulatory logic of gene expression in complex genomes.

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.

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.

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.

Advanced Technique for In Situ Raman Spectroscopy Monitoring of the Freezing-Induced Loading Process.

The freezing-induced loading (FIL) method is a promising technique for encapsulation of bioactive substances as well as for preparation of nanocomposite materials. A critically important aspect for this method is the remote control of the freezing process. The knowledge of the moment of freezing process ending can allow us to increase the quality of loading and reduce the process duration, thus making this approach more controllable. Herein, we present a photonic technique based on Raman spectroscopy as one of the optimal solutions for remote control of FIL. As a result of our study, the setup for obtaining Raman spectra during the process of liquid vehicle crystallization in suspensions has been developed, which allowed us to analyze the sorption of nanoparticles onto micro- and submicron particles by the FIL method in situ. The main focus of the present work is the in situ Raman spectroscopy monitoring of the crystallization process, including technologically important parameters such as the ice/water interface velocity in water colloids/suspensions and the moment of the final adsorption of the nanoparticles on the microparticles. In contrast to other approaches, Raman spectroscopy allows to directly observe the hydrogen bond formation during crystallization. Additionally, a schematic and a detailed description of the setup are presented here. Thus, the developed technique has a good perspective for scaling up the FIL approach and increasing the area of application of this technology.

Multispectral sensing of biological liquids with hollow-core microstructured optical fibres.

The state of the art in optical biosensing is focused on reaching high sensitivity at a single wavelength by using any type of optical resonance. This common strategy, however, disregards the promising possibility of simultaneous measurements of a bioanalyte's refractive index over a broadband spectral domain. Here, we address this issue by introducing the approach of in-fibre multispectral optical sensing (IMOS). The operating principle relies on detecting changes in the transmission of a hollow-core microstructured optical fibre when a bioanalyte is streamed through it via liquid cells. IMOS offers a unique opportunity to measure the refractive index at 42 wavelengths, with a sensitivity up to ~3000 nm per refractive index unit (RIU) and a figure of merit reaching 99 RIU-1 in the visible and near-infra-red spectral ranges. We apply this technique to determine the concentration and refractive index dispersion for bovine serum albumin and show that the accuracy meets clinical needs.

Microstructured Optical Waveguide-Based Endoscopic Probe Coated with Silica Submicron Particles.

Microstructured optical waveguides (MOW) are of great interest for chemical and biological sensing. Due to the high overlap between a guiding light mode and an analyte filling of one or several fiber capillaries, such systems are able to provide strong sensitivity with respect to variations in the refractive index and the thickness of filling materials. Here, we introduce a novel type of functionalized MOWs whose capillaries are coated by a layer-by-layer (LBL) approach, enabling the alternate deposition of silica particles (SiO2) at different diameters-300 nm, 420 nm, and 900 nm-and layers of poly(diallyldimethylammonium chloride) (PDDA). We demonstrate up to three covering bilayers consisting of 300-nm silica particles. Modifications in the MOW transmission spectrum induced by coating are measured and analyzed. The proposed technique of MOW functionalization allows one to reach novel sensing capabilities, including an increase in the effective sensing area and the provision of a convenient scaffold for the attachment of long molecules such as proteins.

Enabling magnetic resonance imaging of hollow-core microstructured optical fibers via nanocomposite coating.

Optical fibers are widely used in bioimaging systems as flexible endoscopes that are capable of low-invasive penetration inside hollow tissue cavities. Here, we report on the technique that allows magnetic resonance imaging (MRI) of hollow-core microstructured fibers (HC-MFs), which paves the way for combing MRI and optical bioimaging. Our approach is based on layer-by-layer assembly of oppositely charged polyelectrolytes and magnetite nanoparticles on the inner core surface of HC-MFs. Incorporation of magnetite nanoparticles into polyelectrolyte layers renders HC-MFs visible for MRI and induces the red-shift in their transmission spectra. Specifically, the transmission shifts up to 60 nm have been revealed for the several-layers composite coating, along with the high-quality contrast of HC-MFs in MRI scans. Our results shed light on marrying fiber-based endoscopy with MRI to open novel possibilities for minimally invasive clinical diagnostics and surgical procedures in vivo.

Big DNA as a tool to dissect an age-related macular degeneration-associated haplotype.

Age-related Macular Degeneration (AMD) is a leading cause of blindness in the developed world, especially in aging populations, and is therefore an important target for new therapeutic development. Recently, there have been several studies demonstrating strong associations between AMD and sites of heritable genetic variation at multiple loci, including a highly significant association at 10q26. The 10q26 risk region contains two genes, HTRA1 and ARMS2, both of which have been separately implicated as causative for the disease, as well as dozens of sites of non-coding variation. To date, no studies have successfully pinpointed which of these variant sites are functional in AMD, nor definitively identified which genes in the region are targets of such regulatory variation. In order to efficiently decipher which sites are functional in AMD phenotypes, we describe a general framework for combinatorial assembly of large 'synthetic haplotypes' along with delivery to relevant disease cell types for downstream functional analysis. We demonstrate the successful and highly efficient assembly of a first-draft 119kb wild-type 'assemblon' covering the HTRA1/ARMS2 risk region. We further propose the parallelized assembly of a library of combinatorial variant synthetic haplotypes covering the region, delivery and analysis of which will identify functional sites and their effects, leading to an improved understanding of AMD development. We anticipate that the methodology proposed here is highly generalizable towards the difficult problem of identifying truly functional variants from those discovered via GWAS or other genetic association studies.

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.

Systemic Administration of Polyelectrolyte Microcapsules: Where Do They Accumulate and When? In Vivo and Ex Vivo Study.

Multilayer capsules of 4 microns in size made of biodegradable polymers and iron oxide magnetite nanoparticles have been injected intravenously into rats. The time-dependent microcapsule distribution in organs was investigated in vivo by magnetic resonance imaging (MRI) and ex vivo by histological examination (HE), atomic absorption spectroscopy (AAS) and electron spin resonance (ESR), as these methods provide information at different stages of microcapsule degradation. The following organs were collected: Kidney, liver, lung, and spleen through 15 min, 1 h, 4 h, 24 h, 14 days, and 30 days after intravenous injections (IVIs) of microcapsules in a saline buffer at a dosage of 2.5 × 108 capsule per kg. The IVI of microcapsules resulted in reversible morphological changes in most of the examined inner organs (kidney, heart, liver, and spleen). The capsules lost their integrity due to degradation over 24 h, and some traces of iron oxide nanoparticles were seen at 7 days in spleen and liver structure. The morphological structure of the tissues was completely restored one month after IVI of microcapsules. Comprehensive analysis of the biodistribution and degradation of entire capsules and magnetite nanoparticles as their components gave us grounds to recommend these composite microcapsules as useful and safe tools for drug delivery applications.