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Engineering multifunctional superparamagnetic nanoparticles for long-term stem cell tracking

Final Report Summary - STEMTRACK (Engineering multifunctional superparamagnetic nanoparticles for long-term stem cell tracking)

The main goal of this project was to develop a method to monitor stem cells following in vivo administration using a non-invasive method called magnetic resonance imaging (MRI) that will not cause harm to the patient. The technology proposed is based on superparamagnetic iron oxide nanoparticles, or ‘SPIONs’ and iron-based genetic reporter genes The main advantage of SPIONs is that, because of their nanoscale dimensions, they can be easily introduced into cells without detrimental side effects. Furthermore, because iron is a natural substance in the human body, being an essential component of the oxygen-carryng molecule, haemoglobin, it is unlikely to cause any harm to patients.
Work performed since the start of the project
We have assessed the current practices for using SPIONs as means for labelling and imaging stem cells and identified that this is the most promising imaging technique for clinical use given its safety and unrestricted penetration depth, with some small clinical trials confirming its potential for cell tracking. Caveats, however, also exist for this modality, which are related to the possibility of the label (SPIONs) being released from stem cells and taken up by host cells, resulting in false positives. Another limitation is the dissolution of the contrast agents upon cell division resulting in lower contrast and potential loss of signal in the long term. Iron-based reporter genes can help in overcoming these problems, although at a lower detection sensitivity.
A range of commercially available SPIONs-based contrast agents were evaluated as a means to label and image mesenchymal stem cells with a focus on the effect of particle size on cell labelling and imaging properties. We have established appropriate protocols for cell labelling leading to the internalisation of nano- and micro-sized contrast agents and demonstrated for the first time that photothermal microscopy can be used as a very sensitive method for imaging the dynamics of SPION uptake in cells. By combining this technique with immunohistochemistry, we have shown their accumulation in the cell’s lysosomes. Most importantly, we have seen no adverse effects on mesenchymal stem cell self-renewal or differentiation potential when labelled with such contrast agents. Under regular culture conditions, nano- and micro-sized particles stay within the lysosomes and are not excreted although they tend to be evenly distributed between daughter cells upon cell division, effectively diluting the concentration of contrast agent in the cell.
Concomitantly, together with a team of chemists, we have successfully developed a a new class of SPIONs bearing a biomimetic shell composed of a biomimetic polymer (poly[2-(methacryloyloxy)ethylphosphorylcholine], pMPC). Such polymers have been proven extremely useful in biomedical applications given their biocompatibility and water solubility and have been shown to be very efficacious in providing a non-biofouling surface. We were one of the first research teams to graft pMPC to SPIONs and show that they are not only effective as an MRI contrast agent, but can be used to label stem cells. A detailed investigation of the effect of chain length on its anti-biofouling properties suggest that longer chains result in a stealthier surface that prevents spontaneous uptake of SPIONs by stem cells. That has allowed us to produce SPIONs that are stealth when bearing a pMPC chain but that can be further functionalised for specific targeting of e.g. cells exhibiting a specific marker.
On the reporter gene line of study, we have assessed the efficacy of overexpressing three iron regulatory genes as a means to produce biogenic iron-based nanoparticles. The genes of choice where ferritin, transferrin receptor and magA, a bacterial gene, extracted from a magnetotactic bacterium, that is thought to be involved in iron biomineralisation. We have found that magA appears to be toxic for most adult stem cells, which might pose a limitation on what concerns its use for regenerative therapies. Ferritin and transferrin receptor, endogenous genes of mammalian cells, did not pose any direct toxic effect. We have been able to increase the intracellular amount of iron of different stem cells by purely culturing them in iron-supplemented culture media. When overexpressing ferritin, transferrin receptor or a combination of those, a slight increase in intracellular iron content was observed in respect to naïve cells which could lead to stronger contrast in MRI.
By using two different animal models, we have shown that the SPION labelled cells maintain their viability once implanted in the host animal, and that MR imaging can be used to identify the location of those cells with a strong increase in relaxation rate (and hence contrast generation) at the point of administration. Of particular importance was the identification that cells which are administrated intravenously in mouse models get entrapped in the lung, precluding their migration to the organs of interest. This outcome generated a strong impact in our current kidney regenerative therapy studies and has led to the development of new stem cell administration routes which might aid in bypassing the lung and thus, the entrapment of therapeutic cells in this organ. We anticipate that several regenerative medicine therapies under study in animals and humans will benefit from imaging techniques based on SPIONs and reporter genes allowing the identification of stem cell localisation and the development of optimal modes of administration for reaching the target organ.

Main results:

1. We have evaluated the potential of range of novel SPIONs and micro-sized magnetic particles using a suite of in vitro techniques and identified particles that have potential for in vivo use.
2. We have established a novel method based on photothermal microscopy for monitoring the uptake of magnetic particles in living cells in vitro.
3. We have established a chick model for evaluating the performance of the magnetic particles in vivo.
4. We have assessed the suitability of the magnetic particles in the chick model as well as in a mouse model and identified particles that could be suitable for longer term tracking.
5. We have generated a range of magnetic reporter cell lines and identified reporters that could be suitable for use in stem cells.
6. We have evaluated the potential of magnetic reporters for enabling MR-based cell tracking and found that the reporters increase intracellular iron concentration to a limited extent, suggesting that they will have limited utility for cell tracking in vivo.

Final results and impact:
We have identified magnetic particles that are useful for MR-based tracking and have accurately evaluated the usefulness of genetic reporters. This information will have greatest impact on researchers in the stem cell and regenerative medicine fields who are intending to use MR-based cell tracking techniques, as they will know which particles and reporter systems are likely to be most useful.