Magnetic nanoparticle mediated delivery of neurotherapeutic genes to multipotent neural stem cell transplant populations

Lead Research Organisation: Keele University
Department Name: Inst for Science and Tech in Medicine


The adult brain and spinal cord (together called the central nervous system or CNS) repair poorly after injury and disease. This leads to limited recovery for patients with severe brain/spinal cord injury and disease with profound consequences for their quality of life. Stem cells found in the nervous system called neural stem cells or NSCs have enormous potential for increasing repair at injury/disease sites in the CNS as they can give rise to new stem cells, replace lost cell types of the CNS and can also suppress injury responses. These cells can migrate long distances in the CNS and are particularly attracted into areas of damage in the nervous system. Therefore, an important use for these engineered stem cells is to function as as transport 'vehicles' to deliver therapeutic molecules to injury/disease areas in the CNS.

In experimental neurology research, the most common way of delivering genes to stem cells is to use modified viruses as vehicles. Infection of cells with these viruses enables the transfer of genes of interest into the infected cells. However, experimental methods using viruses can be be technically difficult, time consuming and expensive and can alter the basic cell properties of neural stem cells such as their division capability and their genesis of new cell types. This method also has other drawbacks including major safety issues (such as reactions from the immune system and cancer causing effects) and difficulties in achieving the large scale production that is required for human and veterinary clinical therapies.

State-of-the-art delivery systems using small particles with an iron core called magnetic nanoparticles (MNPs) have many benefits in this regard and can be used effectively for delivery of genes. Preliminary clinical trials have shown that injecting MNPs into the body appears to be safe. MNPs can be linked with genes and taken up into cells to mediate gene delivery. Although MNP based systems are thought to be less effective than viruses for delivering genes, we have shown that applying static or oscillating magnetic fields (a method called 'magnetofection') can dramatically increase gene delivery by MNPs. DMC's group proved recently that MNPs can be used to safely deliver genes to several major cell types (of both rodent and canine origin) that are used in neural cell transplantation therapies. The methods used were not associated with safety issues and did not alter basic stem cell properties like their division or migration capability or the types/numbers of daughter cells they give rise to. The methods we have developed for MNP based gene transfer are very safe, technically simple, quick and relatively inexpensive, therefore their use will provide an effective and convenient method for delivering genes and can result in significant cost savings to laboratories and funding agencies.

This study will build on recent findings to develop methods to use MNPs to deliver genes coding for therapeutic molecules to stem cells (that will then be used for transplantation). We will assess different strategies to optimise uptake of MNPs coated with genes into stem cells and establish if this procedure has adverse effects on the survival and development of the cells. A further goal is to develop a new, technically easy and cheap method to examine the interactions of stem cells and MNPs at high magnification using a special 'electron' microscope. We will also investigate the underlying mechanisms by which oscillating magnetic fields increase gene delivery by MNPs. The repair enhancing potential of the engineered stem cells will be tested in a 'living' 3D slice model of the spinal cord, that can function well as injured 'host' tissue for transplant cells. This method provides a robust alternative to the use of surgical transplantation to study stem cell transplantation therapies in living animals, thereby significantly reducing animal usage and suffering in experimental research.

Technical Summary

Transplanted neural stem cells (NSCs) offer key dual advantages for neural regeneration and delivery of therapeutic biomolecules to injury foci. Their features include self-renewal and multipotentiality, ease of derivation from embryonic stem cells, migratory capacity (notably towards pathology sites and across the blood brain barrier), amenability to genetic engineering, ability to differentiate and integrate in injury foci and suppression of injury mechanisms (eg. astrocytosis and inflammation). From a technical perspective, most studies in transplantation neurobiology use viral vectors for gene delivery. While efficient, such vectors have several drawbacks including safety issues and large scale production limitations highlighting a major need for development/optimisation of alternative, nonviral approaches for basic research and translational applications. We recently provided 'proof-of-concept' that MNPs safely mediate single/combinatorial gene delivery (including that of plasmids encoding therapeutic growth factors) to NSCs without adverse effects on stem cell viability, migration, proliferation and differentiation. Gene transfer was dramatically enhanced using 'multifection' (repeat transfections) and 'magnetofection' (application of static/oscillating magnetic fields to assist transfection, the latter method using a patented oscillating field system manufactured by Keele spin-off company nanoTherics Ltd). Transfection levels were found to be similar or superior to many viral/nonviral methods currently used in neurobiology with the critical advantage of cell safety. Transfected cells could survive, migrate and differentiate normally post-transplantation. We will optimise MNP based delivery of neurotherapeutic genes to NSCs derived for transplantation and investigate the cellular mechanisms for oscillating field enhanced transfection. The repair potential of 'engineered' NSCs will be tested in a novel 3D injury model in organotypic spinal cord slice cultures.

Planned Impact

(1) We will develop methodologies for MNP mediated gene transfer of therapeutic biomolecules to neural stem cell transplant populations. Based on this work, we will report protocols that can be adopted by other workers in the field of neuroscience/neural tissue engineering. We have provided extensive proof of the feasibility of MNP gene transfer to neural cells, so this methodological 'step-change' could be rapidly realised in 2-5 years. This will benefit researchers as a simple and novel methodological tool for gene transfer to neural cells, providing a robust alternative to the viral methods currently used widely (that are technically challenging, time consuming, require special experimental permissions and can have adverse effects on stem cell survival and development). Nonviral vectors can mediate stable gene delivery in neural cells, therefore this work could be expanded easily to transplantation therapies (for short and long term gene transfer) to promote repair in models of various neurodegenerative conditions (eg. Parkinson's Disease, Huntington's Disease), injury (transection, crush, hypoxia), genetic diseases of the CNS (leukodystrophies), various mutant models and veterinary clinical transplantation approaches. In addition, this method can be adapted for use in both the neonatal/adult CNS in a range of experimental model organisms and be expanded to the study of regenerative mechanisms in peripheral nerve injury and disease.
(2) Development of protocols to optimise MNP gene transfer will reduce costs associated with experimental research as this methodology is relatively inexpensive compared with viral methods. Additionally, our ongoing work to develop and refine the use of organotypic slice culture injury models as 'host' tissue for screening purposes in cell transplantation therapies will reduce animal usage and suffering in experimental research, in keeping with the 3R's principles of Reduction, Refinement and Replacement of animal experimentation.
(3) Our work will highlight areas for materials chemistry and nanotechnology laboratories for the development of magnetofection compatible, transfection grade MNPs for neural cell applications. The OTOTO-SEM method we will develop, can offer a simple tool to study MNP-stem cell interactions, an area of high biomedical interest. Despite the major advantages of MNP based vector systems, few companies are involved with the commercial manufacture and sale of such particles in the UK, highlighting a need for development of such commercial ventures. Data from my laboratory has been used for international marketing by the company nanoTherics Ltd who have identified a global market for the equipment particularly for neural applications.
(4) In the long term, this work will benefit human and veterinary patients with CNS injury as first step in the development of an important methodological tool to augment the clinical use of neural cell transplantation therapies for neurological repair. If successful, this approach could significantly impact on their quality of life. Any effective clinical transplantation therapy to enhance CNS repair will reduce future costs to the NHS for patient care and rehabilitation. Various neural cell transplantation therapies have reached the stage of clinical trials and iron oxide nanoparticles are widely used currently for biomedical applications, therefore we can predict that the translation of MNP applications to the clinical neural transplantation setting (in human and veterinary medicine) could be achieved in the next decade.
(5) Staff employed under the grant will develop generic experimental techniques in gene delivery methods, electron microscopy, stem cell and organotypic slice culture methods and molecular biology that can be used in several experimental areas. Additionally, staff will develop oral presentation and scientific writing skills along with data acquisition and analysis, of benefit across many professional sectors.


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Fernandes AR (2014) A multicellular, neuro-mimetic model to study nanoparticle uptake in cells of the central nervous system. in Integrative biology : quantitative biosciences from nano to macro

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Jenkins SI (2016) 'Stealth' nanoparticles evade neural immune cells but also evade major brain cell populations: Implications for PEG-based neurotherapeutics. in Journal of controlled release : official journal of the Controlled Release Society

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Jenkins SI (2015) Development of a nanomaterial bio-screening platform for neurological applications. in Nanomedicine : nanotechnology, biology, and medicine

Description The aim of this project was to develop a method by which magnetic nanoparticles can be used to deliver DNA that can code for therapeutic molecules to stem cells. These 'engineered' stem cells can then act as a factory to produce molecules that can increase repair in areas of brain and spinal cord injury.
Magnetic nanoparticles have many advantages for engineering transplant stem cells, but we found in 2011, that they cannot deliver larger pieces of DNA, such as those coding for therapeutic molecules are not delivered as effectively as smaller DNA pieces. We have found a solution to the problem by using new DNA molecules called 'DNA Minicircles' and proved that these can be used very effectively to engineer stem cells. We recently proved that such engineered cells can also be grown in neurosurgical grade biomaterials to act as stem cell delivery devices functioning as advanced hybrid biomaterials.
Exploitation Route We consider that our findings have wide application in field of tissue engineering and neural transplantation as a novel and safe way to genetically engineer stem cell transplant populations. Along with surgical grade materials, we can use engineered cells as advanced implants for neurological injury.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description EPSRC Landscape Fellowship
Amount £200,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 01/2015 
End 12/2017
Title Novel multicellular cell systems 
Description Developed twomulticellular model to propagate major support cell types of CNS 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact We have described 2 in vitro systems by which the interactions of nanomaterials with the major cell types of the central nervous system can be studies 
Title Novel spinal cord injury model 
Description Developed a novel model of spinal cord injury based on organotypic slices that can be used to screen regenerative materials and cells. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2014 
Provided To Others? Yes  
Impact Can be used as a high throughput screening method to test novel therapeutic cells and materials within an in vitro injury paradigm 
Description genetically engineered cell delivery in hydrogels 
Organisation Biogelx
Country United Kingdom 
Sector Private 
PI Contribution fellowship proposal to genetically engineer neural transplant cells in hydrogels using data from BBSRC funded awards
Collaborator Contribution provide new hydrogel material for 3-D cell growth
Impact none yet, collaboration between neuroscientists and industry
Start Year 2014
Description genetically engineered cell delivery in hydrogels 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution fellowship application from my lab to genetically engineer transplant cells in hydrogels, using data from bbsrc funded grants
Collaborator Contribution supply novel tuneable hydrogel for the studies for 3-D cell growth
Impact none, yes multidisciplinary collaboration between medicinal chemists and neuroscientists
Start Year 2014