Highly aminated iron oxide nanoworms for simultaneous manufacturing and labeling of chimeric antigen receptor T cells.

Cell based therapies including chimeric antigen receptor (CAR) T cells are promising for treating leukemias and solid cancers. At the same time, there is interest in enhancing the functionality of these cells via surface decoration with nanoparticles (backpacking). Magnetic nanoparticle cell labeling is of particular interest due to opportunities for magnetic separation, in vivo manipulation, drug delivery and magnetic resonance imaging (MRI). While modification of T cells with magnetic nanoparticles (MNPs) was explored before, we questioned whether MNPs are compatible with CAR-T cells when introduced during the manufacturing process. We chose highly aminated 120 nm crosslinked iron oxide nanoworms (CLIO NWs, ~36,000 amines per NW) that could efficiently label different adherent cell lines and we used CD123 CAR-T cells as the labeling model. The CD123 CAR-T cells were produced in the presence of CLIO NWs, CLIO NWs plus protamine sulfate (PS), or PS only. The transduction efficiency of lentiviral CD123 CAR with only NWs was ~23% lower than NW+PS and PS groups (~33% and 35%, respectively). The cell viability from these three transduction conditions was not reduced within CAR-T cell groups, though lower compared to non-transduced T cells (mock T). Use of CLIO NWs instead of, or together with cationic protamine sulfate for enhancement of lentiviral transduction resulted in comparable levels of CAR expression and viability but decreased the proportion of CD8+ cells and increased the proportion of CD4+ cells. CD123 CAR-T transduced in the presence of CLIO NWs, CLIO NWs plus PS, or PS only, showed similar level of cytotoxicity against leukemic cell lines. Furthermore, fluorescence microscopy imaging demonstrated that CD123 CAR-T cells labeled with CLIO NW formed rosettes with CD123+ leukemic cells as the non-labeled CAR-T cells, indicating that the CAR-T targeting to tumor cells has maintained after CLIO NW labeling. The in vivo trafficking of the NW labeled CAR-T cells showed the accumulation of CAR-T labeled with NWs primarily in the bone marrow and spleen. CAR-T cells can be magnetically labeled during their production while maintaining functionality using the positively charged iron oxide NWs, which enable the in vivo biodistribution and tracking of CAR-T cells.

[Factors Affecting the Labeling of NIH 3T3 Cells with Magnetic Nanoparticles].

The factors that affect the labeling of NIH 3T3 murine fibroblasts with Fe3O4-based magnetic nanoparticles (MNPs) were studied using MNPs produced by the gas condensation and solution precipitation methods and MNPs surface-modified with 3-aminopropylsilane or L-lysine. The production method, surface modifications, the particle concentration and size, the state of the cell population, and the method of MNP introduction were found to substantially affect the efficiency of MNP binding by cells. In particular, large MNP clusters may occur in MNP suspensions in DMSO, and their disruption by sonication increased the percent yield of magnetically labeled cells. Static incubation of a cell suspension led to a more efficient labeling as compared with continuous agitation. Cells attached to a plastic support could be labeled to a higher degree than cells in suspension, but required substantially longer incubations with MNPs. MNP centrifugation on cell layers (magnetic spinoculation) significantly increased the rate and efficiency of labeling. The stability of magnetic labeling was shown to depend on the MNP dose during labeling. Electron microscopy studies demonstrated that MNPs were associated with the cell surface after 20-min incubation with cells and were mostly in the cell interior after 4-h incubation. The results of the study may be useful for preparation and application of magnetized cell samples.

Biosensing System for Concentration Quantification of Magnetically Labeled E. coli in Water Samples.

Bacterial contamination of water sources (e.g., lakes, rivers and springs) from waterborne bacteria is a crucial water safety issue and its prevention is of the utmost significance since it threatens the health and well-being of wildlife, livestock, and human populations and can lead to serious illness and even death. Rapid and multiplexed measurement of such waterborne pathogens is vital and the challenge is to instantly detect in these liquid samples different types of pathogens with high sensitivity and specificity. In this work, we propose a biosensing system in which the bacteria are labelled with streptavidin coated magnetic markers (MPs-magnetic particles) forming compounds (MLBs-magnetically labelled bacteria). Video microscopy in combination with a particle tracking software are used for their detection and quantification. When the liquid containing the MLBs is introduced into the developed, microfluidic platform, the MLBs are accelerated towards the outlet by means of a magnetic field gradient generated by integrated microconductors, which are sequentially switched ON and OFF by a microcontroller. The velocities of the MLBs and that of reference MPs, suspended in the same liquid in a parallel reference microfluidic channel, are calculated and compared in real time by a digital camera mounted on a conventional optical microscope in combination with a particle trajectory tracking software. The MLBs will be slower than the reference MPs due to the enhanced Stokes' drag force exerted on them, resulting from their greater volume and altered hydrodynamic shape. The results of the investigation showed that the parameters obtained from this method emerged as reliable predictors for E. coli concentrations.

Magnetically modified microalgae and their applications.

The majority of algal cells can interact with a wide range of nano- and microparticles. Upon interaction the modified cells usually maintain their viability and the presence of foreign material on their surfaces or in protoplasm can provide additional functionalities. Magnetic modification and labeling of microalgal biomass ensures a wide spectrum of biotechnological, bioanalytical and environmental applications. Different aspects of microalgal cell magnetic modification are covered in the review, followed by successful applications of magnetic algae. Modified cells can be employed during their harvesting and removal, applied in toxicity microscreening devices and also as efficient adsorbents of different types of xenobiotics.

On-Chip Sonoporation-Based Flow Cytometric Magnetic Labeling.

Tracing magnetically labeled cells with magnetic resonance imaging (MRI) is an emerging and promising approach to uncover in vivo behaviors of cells in cell therapy. Today, existing methods for the magnetic labeling of cells are cumbersome and time-consuming, which has greatly limited the progress of such studies on cell therapy. Thus, in this study, using the flow cytometric loading technology, we develop a sonoporation-based microfluidic chip (i.e., a microfluidic chip integrated with ultrasound; MCU), to achieve the safe, instant, convenient, and continuous magnetic labeling of cells. For the MCU we designed, a suitable group of operating conditions for safely and efficiently loading superparamagnetic iron oxide (SPIO) nanoparticles into DC2.4 cells was identified experimentally. Under the identified operating conditions, the DC2.4 cells could be labeled in approximately 2 min with high viability (94%) and a high labeling quantity of SPIO nanoparticles (19 pg of iron per cell). In addition, the proliferative functions of the cells were also well maintained after labeling. Furthermore, the in vivo imaging ability of the DC2.4 cells labeled using the MCU was verified by injecting the labeled cells into the leg muscle of the C57BL/6 mice. The results show that the excellent imaging outcome can be continuously achieved for 7 days at a density of 106 cells/mL. This work can provide insight for the design of magnetic cell labeling devices and promote the MRI-based study of cell therapies.

L-Lysine-modified Fe3O4 nanoparticles for magnetic cell labeling.

The efficiency of magnetic labeling with L-Lys-modified Fe3O4 magnetic nanoparticles (MNPs) and the stability of magnetization of rat adipose-derived mesenchymal stem cells, lineage-negative (Lin(-)) hematopoietic progenitor cells from mouse bone marrow and human leukemia K562 cells were studied. For this purpose, covalent modification of MNPs with 3-aminopropylsilane and N-di-Fmoc-L-lysine followed by removal of N-protecting groups was carried out. Since the degree of hydroxylation of the surface of the starting nanoparticles plays a crucial role in the silanization reaction and the possibility of obtaining stable colloidal solutions. In present work we for the first time performed a comparative qualitative and quantitative evaluation of the number of adsorbed water molecules and hydroxyl groups on the surface of chemically and physically obtained Fe3O4 MNPs using comprehensive FTIR spectroscopy and thermogravimetric analysis. The results obtained can be further used for magnetic labeling of cells in experiments in vitro and in vivo.

A Safe and Effective Magnetic Labeling Protocol for MRI-Based Tracking of Human Adult Neural Stem Cells.

Magnetic resonance imaging (MRI) provides a unique tool for in vivo visualization and tracking of stem cells in the brain. This is of particular importance when assessing safety of experimental cell treatments in the preclinical or clinical setup. Yet, specific imaging requires an efficient and non-perturbing cellular magnetic labeling which precludes adverse effects of the tag, e.g., the impact of iron-oxide-nanoparticles on the critical differentiation and integration processes of the respective stem cell population investigated. In this study we investigated the effects of very small superparamagnetic iron oxide particle (VSOP) labeling on viability, stemness, and neuronal differentiation potential of primary human adult neural stem cells (haNSCs). Cytoplasmic VSOP incorporation massively reduced the transverse relaxation time T2, an important parameter determining MR contrast. Cells retained cytoplasmic label for at least a month, indicating stable incorporation, a necessity for long-term imaging. Using a clinical 3T MRI, 1 × 103 haNSCs were visualized upon injection in a gel phantom, but detection limit was much lower (5 × 104 cells) in layer phantoms and using an imaging protocol feasible in a clinical scenario. Transcriptional analysis and fluorescence immunocytochemistry did not reveal a detrimental impact of VSOP labeling on important parameters of cellular physiology with cellular viability, stemness and neuronal differentiation potential remaining unaffected. This represents a pivotal prerequisite with respect to clinical application of this method.

Magnetic Nanoparticle Labeling of Human Platelets from Platelet Concentrates for Recovery and Survival Studies.

Platelets are the smallest blood cells and important for hemostasis. Platelet concentrates (PC) are medicinal products transfused to prevent or treat bleeding. Typically, platelets in PCs are assessed by in vitro tests for their function. However, in vivo testing of these platelets is highly desirable. To distinguish transfused platelets from patients or probands own cells after PC transfusions within the scope of clinical studies, platelets need to be efficiently labeled with minimal preactivation prior to transfusion. Here we report on a method for improved cell uptake of ferucarbotran magnetic nanoparticles contained in Resovist, an FDA-approved MRI contrast agent, by modifying the nanoparticle shell with human serum albumin (HSA). Both HSA-ferucarbotran nanoparticles and magnetically labeled platelets were produced according to EU-GMP guidelines. Platelet function after labeling was evaluated by light transmission aggregometry and by determination of expression of CD62P as platelet activation marker. Magnetic labeling does not impair platelet function and platelets showed reasonable activation response to agonists. Platelet survival studies in NOD/SCID-mice resulted in comparable survival behavior of magnetically labeled and nonlabeled platelets. Additionally, labeled platelets can be recovered from whole blood by magnetic separation.

Highly Efficient Labeling of Human Lung Cancer Cells Using Cationic Poly-l-lysine-Assisted Magnetic Iron Oxide Nanoparticles.

Cell labeling with magnetic iron oxide nanoparticles (IONPs) is increasingly a routine approach in the cell-based cancer treatment. However, cell labeling with magnetic IONPs and their leading effects on the biological properties of human lung carcinoma cells remain scarcely reported. Therefore, in the present study the magnetic γ-Fe2O3 nanoparticles (MNPs) were firstly synthesized and surface-modified with cationic poly-l-lysine (PLL) to construct the PLL-MNPs, which were then used to magnetically label human A549 lung cancer cells. Cell viability and proliferation were evaluated with propidium iodide/fluorescein diacetate double staining and standard 3-(4,5-dimethylthiazol-2-diphenyl-tetrazolium) bromide assay, and the cytoskeleton was immunocytochemically stained. The cell cycle of the PLL-MNP-labeled A549 lung cancer cells was analyzed using flow cytometry. Apoptotic cells were fluorescently analyzed with nuclear-specific staining after the PLL-MNP labeling. The results showed that the constructed PLL-MNPs efficiently magnetically labeled A549 lung cancer cells and that, at low concentrations, labeling did not affect cellular viability, proliferation capability, cell cycle, and apoptosis. Furthermore, the cytoskeleton in the treated cells was detected intact in comparison with the untreated counterparts. However, the results also showed that at high concentration (400 µg mL-1), the PLL-MNPs would slightly impair cell viability, proliferation, cell cycle, and apoptosis and disrupt the cytoskeleton in the treated A549 lung cancer cells. Therefore, the present results indicated that the PLL-MNPs at adequate concentrations can be efficiently used for labeling A549 lung cancer cells and could be considered as a feasible approach for magnetic targeted anti-cancer drug/gene delivery, targeted diagnosis, and therapy in lung cancer treatment.

Magnetic ligation method for quantitative detection of microRNAs.

A magnetic ligation method is utilized for the detection of microRNAs among a complex biological background without polymerase chain reaction or nucleotide modification. The sandwich probes assay can be adapted to analyze a panel of microRNAs associated with cardiovascular diseases in heart tissue samples.