Generation of magnetic biohybrid microrobots based on MSC.sTRAIL for targeted stem cell delivery and treatment of cancer

Abstract

Background: Combining the power of magnetic guidance and the biological activities of stem cells transformed into biohybrid microrobots holds great promise for the treatment of several diseases including cancer.

Results: We found that human MSCs can be readily loaded with magnetic particles and that the resulting biohybrid microrobots could be guided by a rotating magnetic field. Rotating magnetic fields have the potential to be applied in the human setting and steer therapeutic stem cells to the desired sites of action in the body. We could demonstrate that the required loading of magnetic particles into stem cells is compatible with their biological activities. We examined this issue with a particular focus on the expression and functionality of therapeutic genes inside of human MSC-based biohybrid microrobots. The loading with magnetic particles did not cause a loss of viability or apoptosis in the human MSCs nor did it impact on the therapeutic gene expression from the cells. Furthermore, the therapeutic effect of the gene products was not affected, and the cells also did not lose their migration potential.

Conclusion: These results demonstrate that the fabrication of guidable MSC-based biohybrid microrobots is compatible with their biological and therapeutic functions. Thus, MSC-based biohybrid microrobots represent a novel way of delivering gene therapies to tumours as well as in the context of other diseases.

Keywords: Biohybrid microrobots; Cancer; Mesenchymal stem cells; Mesenchymal stromal cells; Rotating magnetic field; sPD1HAC; sTRAIL.

Fig. 1. Generation of BHM-MSCs.

A Surface marker stain of MSCs. FACS analyses of human MSCs for surface expression of CD44, CD90, CD105, and CD45. Their specific signals are shown in red, with respective isotype controls in black. B Schematic overview of the experimental procedure of BHM-MSC generation. C Prussian blue staining of human MSCs with increasing concentrations of MPs (0 ng/ml, 0.6 ng/ml, 1.5 ng/ml, 2.5 ng/ml, 5 ng/ml, and 10 ng/ml). Blue colouring indicates ferric iron particles after 1 day. Scale bars = 125 μm. D Prussian blue staining of human MSCs with increasing concentrations of MPs (0 ng/ml, 0.6 ng/ml, 1.5 ng/ml, 2.5 ng/ml, 5 ng/ml, and 10 ng/ml). Blue colouring indicates ferric iron particles after 6 days. Scale bars = 125 μm

Fig. 2. Guidance of BHM-MSCs in rotating magnetic fields.

A Schematic overview of the generation of BHM-MSCs for magnetic guidance. B Pictures of MPs diluted in PBS supplemented with 2% FBS or DMEM with 0%, 2% or 10% FBS, respectively, and left for 24 h under cell culturing conditions. Of these samples 2 ml of the supernatants (SN) were transferred to a fresh tube. The remaining 2 ml were labelled as sediment (SM). C Brightfield images of MSCs (left) or BHM-MSCs (right) loaded with 10 ng/ml MPs. D The MFG100 magnetic micro-robotics system is employed to control the movement of the BHM-MSCs in rotating magnetic fields. The system is equipped with an optical camera (Basler, puA1280-54um) for real-time monitoring. E Dispersion of BHM-MSCs under three different magnetic fields, and the results demonstrate that higher magnetic fields lead to greater dispersions. The diagram displays the average outcomes of n = 3 videos. F Dispersion under four different MP concentrations (1.25 ng/ml, 2.5 ng/ml, 5 ng/ml and 10 ng/ml). It is noteworthy that, under comparable magnetic field conditions (frequency and field intensity), higher concentrations result in greater dispersions (n = 3 experiments)

Fig. 3. Fluorescent microscopic analysis of the effects of MPs on MSC viability over time.

A Live/dead stain of MSCs incubated with increasing concentrations of MPs (0 ng/ml, 0.6 ng/ml, 1.5 ng/ml, 2.5 ng/ml, 5 ng/ml, and 10 ng/ml) for 1 day. Dead cells are indicated by a white arrow. Ethanol (EtOH) treated cells served as control. Scale bars = 125 μm. B Live/dead stain of MSCs incubated with increasing concentrations of MPs (0 ng/ml, 0.6 ng/ml, 1.5 ng/ml, 2.5 ng/ml, 5 ng/ml, and 10 ng/ml) for 6 days. Dead cells are pointed out by a white arrow. Ethanol (EtOH) treated cells served as control. Scale bars = 125 μm. C Quantification of percentage of dead cells (red) and live cells (green) compared between day 1 and day 6 across all concentrations of MPs in MSCs. Data are plotted as mean ± SEM. For the quantification a total of 15 random fields from each sample were counted

Fig. 4. Transforming genetically modified MSCs into BHM-MSCs does not affect their transgene expression.

A Schematic overview of the experimental procedure to generate transgene expressing BHM-MSCs. B Prussian blue stain of MSCs transduced with Ad.Luc (MSC.Luc) or Ad.sTRAIL (MSC.sTRAIL) with MP concentrations of 0 ng/ml (– MPs) and 10 ng/ml (+ MPs). Blue staining indicates presence of ferric iron particles. Scale bars = 250 μm. C Live/dead stain of untransduced MSCs (MSC), MSCs transduced with adenoviral vectors expressing luciferase (MSC.Luc) or sTRAIL (MSC.sTRAIL), with (+ MPs) or without MPs (– MPs). Live cells display green fluorescence, dead cells display red fluorescence. Examples of dead cells are indicated by a white arrow. Scale bars = 125 μm. D Quantification of percentage of dead cells (red) and live cells (green). Data are plotted as mean ± SEM. For the quantification a total of 15 random fields from each sample were counted. E Luciferase reporter assay of MSCs loaded with MPs at different concentrations and transduced with an adenoviral vector expressing luciferase (MSC.Luc). Luciferase activity is expressed as relative light units (RLU). Untransduced MSCs loaded with 5 ng/ml MPs were used as negative controls in the luciferase activity assay. Results depicted are from three independent experiments and plotted as mean ± SEM. F TRAIL expression and secretion was measured by ELISA in supernatants from MSCs transduced with adenoviral vectors expressing luciferase (MSC.Luc) or sTRAIL (MSC.sTRAIL), with or without MPs. Results depicted are from three independent experiments and plotted as mean ± SEM

Fig. 5. The sTRAIL secreted by BHM-MSCs is functional.

A Schematic overview of the experimental design to test for sTRAIL functionality. In the first approach, supernatant from transgene expressing BHM-MSCs is transferred onto cancer cells. In the second approach, transgene expressing BHM-MSCs are directly mixed with cancer cells. B Live/dead stain of MDA-MB-231 breast cancer cells treated with supernatants from MSCs transduced with an adenoviral vector expressing sTRAIL (MSC.sTRAIL) with (+ MPs) or without MPs (– MPs). Live cells display green fluorescence, dead cells display red fluorescence. Examples of dead cells are indicated by a white arrow. Scale bars = 125 μm. C Quantification of percentage of dead MDA-MB-231 breast cancer cells (red) and live MDA-MB-231cells (green). Data are plotted as mean ± SEM. For the quantification a total of 15 random fields from each sample were counted. D MDA-MB-231 breast cancer cells treated with supernatants from untransduced MSCs (MSC), MSCs transduced with adenoviral vectors expressing luciferase (MSC.Luc) or sTRAIL (MSC.sTRAIL), with (+ MPs) or without MPs (– MPs). Apoptosis was measured by DNA-hypodiploidy assay and flow cytometry. Samples were measured in triplicate. Data are plotted as mean ± SEM. E MDA-MB-231 breast cancer cells treated with different concentrations of sTRAIL secreted from BHM-MSCs. After 7 days, cell survival was assessed by crystal violet staining. The measurement values of untreated cells were set to 100. Results are from three independent experiments. Data are plotted as mean ± SEM. (n = 3). F MDA-MB-231 breast cancer cells (MDA) mixed with MSCs transduced with adenoviral vectors expressing luciferase (MSC.Luc) or sTRAIL (MSC.sTRAIL), with (+ MPs) or without MPs (– MPs). Cell death was analysed after 3 days by live/dead staining. Live cells display green fluorescence, dead cells display red fluorescence. Dead cells are indicated by a white arrow. Scale bars = 125 μm. G Quantification of percentage of dead cells (red) and live cells (green). Data are plotted as mean ± SEM. For the quantification a total of 15 random fields from each sample were counted

Fig. 6. sTRAIL expression does not affect the migratory capacity of BHM-MSCs.

A Schematic overview of the transwell migration assay. B MSCs transduced with adenoviral vectors expressing luciferase (MSC.Luc) or sTRAIL (MSC.sTRAIL), with (+ MPs) or without MPs (– MPs) were analysed for their migratory activity. The number of cells which crossed the membrane in the unloaded MSC samples were set to 100. For each group, cells from six transwells were quantified. Data are plotted as mean ± SEM