Magnetic nanoparticles: Applications in gene delivery and gene therapy

Abstract

Gene therapy is defined as the direct transfer of genetic material to tissues or cells for the treatment of inherited disorders and acquired diseases. For gene delivery, magnetic nanoparticles (MNPs) are typically combined with a delivery platform to encapsulate the gene, and promote cell uptake. Delivery technologies that have been used with MNPs contain polymeric, viral, as well as non-viral platforms. In this review, we focus on targeted gene delivery using MNPs.

Keywords::

• cell uptake
• gene therapy
• magnetic nanoparticles
• non-viral platforms

Introduction

Nanotechnology defines the formation and application of materials, devices, and systems through the control of nanometer-sized materials, and their application in physics, chemistry, biology, engineering, applied science and other activities. In particular, rigorous efforts are in progress to develop nanomaterials for medical use as means that can be targeted to specific cells, tissues, and organs (Chouly et al. 1996, Schlorf et al. 2010, Kami et al. 2011).

Many different types of nanoparticles, magnetic nanoparticles (MNPs) being just a class among them, present exciting opportunities for technologies at the interfaces between biology, physics, and chemistry. A number of MNPs have already been used in clinical practice as contrast-enhancing agents for magnetic resonance imaging (MRI) (Prijic and Sersa 2011). Furthermore, several methods have been used to create these complexes, comprising hydrophobic interactions (Namiki et al. 2009) and electrostatic interactions (Zheng et al. 2009, Li et al. 2012).

The base of this therapeutic technique is to introduce a gene encoding a practical protein, altering the expression of an endogenous gene or possessing the capacity to cure or prevent the development of a disease (Rapti et al. 2011, Pouton and Seymour 2001, Robbins and Ghivizzani 1998, Dizaj et al. 2014).

The procedure, based on the association of MNPs with gene vectors, is called magnetofection, which is used in order to enhance gene transfer in the presence of a magnetic field. It was developed by Christian Plank and coworkers for gene transfer in cell cultures and in vivo, using MNP-naked DNA complexes or MNP-viral vector complexes (Scherer et al. 2002). In this situation, the approach of magnetofection in cells was assumed to be simple: the MNP-DNA complex is added to a culture of adherent cells. The magnetic complexes are attracted to the bottom by a magnet, placed close below the bottom of the flask or plate, where they come in close contact with the cells and are physically internalized, without any particular influence of the magnetic force on the endocytic uptake mechanism (Figure 1) (Huth et al. 2004, Schwerdt et al. 2012).

Figure 1. Diagrammatic representation of the magnetofection principle in cells. MNPs are complexed to RAds and the complex is attracted to cells by a magnetic field. (Kindly provided by OZ Biosciences, Marseille, France, www.ozbiosciences.com) (Schwerdt et al. 2012).

In vivo, magnetic fields focused over the target site have the potential to not only increase transfection but also target the therapeutic gene to a specific organ or location within the body (Figure 2). Commonly, particles carrying the therapeutic gene are injected intravenously, and high-gradient external magnets are used to capture the particles as they flow through the bloodstream. Once captured by the field, the particles are detained at the target, where they are taken up by the tissue (Dobson 2006).

Figure 2. Schematic representation (side view section) of magnetic nanoparticle-based gene targeting in vivo. Dashed gray rings indicate the lines of magnetic flux due to the ex vivo permanent disk magnet. F mag is the magnetic force vector exerted on the particles as they flow through the bloodstream (Dobson 2006).

The delivery carriers are required to be small enough to be internalized into the cells and enter the nucleus, crossing through the cytoplasm and escaping the endosome/lysosome process following endocytosis (Figure 3). The use of nanoparticles in gene delivery can benefit both the targeted and maintained gene delivery by protecting the gene against nuclease degradation and improving its stability (Dizaj et al. 2014, Dobson 2006, Zhou et al. 2011, Sinha et al. 2006, Morachis et al. 2012).

Figure 3. Internalization of non-viral vectors into cell and passage to nucleus through the cytoplasm, following endocytosis (Dizaj et al. 2014).

Genetic medicine can prospectively benefit many diseases ranging from cancer (Rapti et al. 2011, Pouton and Seymour 2001) to hemophilia (Robbins and Ghivizzani 1998). This therapy is not only used in genetic deficits, but also in other complicated illnesses, such as autoimmunity (rheumatoid arthritis), viral infection (human immunodeficiency virus), artery disease, diabetes, and coronary diseases (Hendriks et al. 2004). With the development of this method, gene therapy will become an effective therapeutic technique for neurodegenerative conditions, AIDS, asthma, and the myriad of other genetic and developed diseases that affect humanity (Pouton and Seymour 2001, Dizaj et al. 2014). A key hurdle to its clinical application is the deficiency of safe and operative delivery systems (Park et al. 2006, Thomas et al. 2003). There are many barriers to gene delivery, comprising intracellular barriers such as intracellular uptake, DNA release, nuclear uptake, and endosomal escape, and extracellular barriers such as targeting to specific tissues and/or cells of interest, avoidance of particle clearance mechanisms, and protection of DNA from degradation (Putnam 2006, Pack et al. 2005, Harris et al. 2010).

In this study, we focus on targeted gene delivery via MNPs (called as magnetofection) and the application of these particles as therapeutic agents for some diseases. Moreover, gene delivery to cells and organs has been investigated (Fekri Aval et al. 2014, Zohre et al. 2014, Valizadeh et al. 2014, Mellatyar et al. 2014, Dadashzadeh et al. 2014, Rahimzadeh et al. 2014, Ebrahimi et al. 2014, Badrzadeh et al. 2014).

Non-viral and viral vectors

In gene delivery, it is fairly common to follow biomimetic methods. Biological systems contain modified viruses and non-pathogenic bacteria. In the investigations of magnetic carriers for gene therapy, a viral vector which carries the therapeutic gene is coated onto the magnetic carrier's surface. By holding the carrier at the target location using external magnetic fields, the virus is kept in contact with the tissue for a longer period of time, increasing the efficiency of gene transfection and expression (Mah et al. 2000, Mah et al. 2002). New magnetic carriers are being developed specially for these applications (Hughes et al. 2001), and this is an area which shows great promise (Pankhurst et al. 2003). Viral vectors are more effective than non-viral vectors for DNA delivery, but may display a significant threat to patients, although non-viral carriers are inherently safer than viral carriers (Higashi et al. 2009, Kostarelos and Miller 2005, Mastrobattista et al. 2006). In contrast to the viral gene delivery structures, the non-viral carriers are expected to be less immunogenic, with simple preparation and a possible adaptable surface modification (Philippi et al. 2010).

The non-viral vectors are usually made of lipids or polymers with or without using other inorganic substances, where they can also be prepared from a lipid-polymer or lipid-polymer-inorganic hybrid (Lu 2009). Naked DNA, generally in plasmid form, is the most basic form of non-viral transfer of a gene into a target cell (Conwell and Huang 2005, Niidome and Huang 2002, Bigger et al. 2001, Mayrhofer et al. 2009). Non-viral delivery vectors can be classified as organic systems such as lipid complexes and conjugated polymers, and inorganic systems such as MNPs and gold nanoparticles (Lee et al. 2013).

In considering the viral gene delivery vector, with its safety concerns regarding the risk of extreme immune response (adenovirus) and supplement mutagenesis, the usage of non-viral vectors can overcome the safety problems mentioned (Bharali et al. 2005). Owing to the low transferring efficiency of a naked plasmid, several chemical (liposomes) and physical (electroporation) approaches have been exploited, to increase their transferring efficiency (Dizaj et al. 2014, Deelman and Sharma 2009).

Targeted gene delivery in vivo

Gene delivery methods efficiently present a gene of interest in order to express its encoded protein in an appropriate host or host cell (Kami et al. 2011). In the case of magnetofection, the gene is attached directly to the carrier. These carriers commonly consist of a magnetic iron-oxide either dispersed within a polymer matrix – such as silica, polyvinyl alcohol (PVA), or dextran – or encapsulated within a polymer or metallic shell (Dobson 2006, Harris et al. 2003, Neuberger et al. 2005).

To obtain a large-sized nucleic acid molecule, the cytoplasm, or even the nucleus, an appropriate carrier system, such as virosomes, cationic liposomes, and nanoparticles, are required to deliver genes to cells, which improve cell internalization and protect the DNA molecule from nuclease enzymatic degradation. To achieve a suitable carrier structure, nanoparticles can be considered as good candidates for therapeutic applications because of several reasons, as follows (Akbarzadeh et al. 2012b): They exist in the same size range as proteins (Wu et al. 2008), they have large surface areas and ability to attach to a large number of surface functional groups (Indira and Lakshmi 2010), and they have controllable absorption and release properties, as also surface characteristics and particle size (Dizaj et al. 2014, Nitta and Numata 2013).

Inorganic nanoparticles, polymer-based nanoparticles, lipid-based nanoparticles and hybrid nanoparticles are four major groups exploited in gene delivery. MNPs are inorganic nanoparticles which are normally utilized as gene delivery carriers. The previous study reports have demonstrated that they are not subject to microbial attack and show also good storage stability (Dizaj et al. 2014, Jin et al. 2014).

Magnetism-based targeted delivery was first defined in 1978 (Widder et al. 1978). However, techniques similar to those used for drug delivery have important potential to be used for gene therapy. For these applications, the approach must be adapted to account for the size and charge of nucleic acids (Li et al. 2012).

Currently, there are three primary gene delivery methods that use viral vectors, nucleic acid electroporation, and nucleic acid transfection. These systems vary in efficiency (Table I). It has been demonstrated that gene delivery by viral vectors can be highly effective, but may supplement viral vector nucleic acid sequences into the host genome, potentially causing undesirable effects, such as unsuitable expression of deleterious genes (Kami et al. 2011).

Table I. Gene delivery systems (Kami et al. 2011).

	Expression type	Efficiency (%)	Cell Viability (%)	Safety
Virus *	Stable, or Transient	80–90%	80–90%	Low
Electroporation	Transient	50–70%	40–50%	High
TF reagent **	Transient	20–30%	80–90%	High

The use of MNPs to increase the effectiveness of the cell-fusion vector hemagglutinating virus Japan envelope (HVJ-E) was represented by Morishita and others. They found that by associating protamine sulfate (PS)-coated MNPs to HVJ-E, transfection was improved in vitro in BHK21 cells, even with a reduction in the amount of HVJ-E and no proof of toxicity (Dobson 2006, Morishita et al. 2005). However, in order for MNPs to act as efficient carriers for DNA or pharmaceutical drugs, the external surface of the particles must first be modified to allow attachment of the target molecules. Molecules can be attached to the surface of the particles in some ways, such as employing cleavable linkers or utilizing electrostatic interactions between the particle surface and the therapeutic agent (McBain et al. 2008).

MNPs can be coated with compounds such as natural polymers (proteins and carbohydrates) (Akbarzadeh et al. 2012c, Valizadeh et al. 2012, Akbarzadeh et al. 2013, Akbarzadeh et al. 2012a, Akbarzadeh et al. 2012d, Akbarzadeh et al. 2012e), synthetic organic polymers (polyethylene glycol, PVA, poly-L-lactic acid) (Akbarzadeh et al. 2013, Mollazade et al. 2013, Nejati-Koshki et al. 2013, Rezaei-Sadabady et al. 2013), silica (Fallahzadeh et al. 2010), and gold (Kami et al. 2011, Ebrahimnezhad et al. 2013, Pourhassan-Moghaddam et al. 2013). In the case of in vitro magnetofection, the particles are generally coated with polyethylenimine (PEI), which attaches DNA to the particle's surface via charge interactions (Dobson 2006). In the first study to show targeted delivery of DNA using MNPs, Cathryn Mah, Barry Byrne, and coworkers coated the adeno-associated virus (AAV) encoding Green Fluorescent Protein (GFP) to the surface of MNPs using a cleavable heparin sulfate linker (Mah et al. 2000, McBain et al. 2008).

Although the use of target-specific linkers undoubtedly supplies an elegant approach to the attachment of target molecules, it is not always possible. An optional approach for binding DNA to the surface of particles is to employ the electrostatic interactions between the negatively charged phosphate backbone of DNA and the positively charged molecules connected to the particle surface. A current choice for this approach is the cationic polymer PEI (Ahmadi et al. 2014). It is now understood that particle DNA complexes normally enter the cell by endocytosis through clathrin-dependent pits (Davaran et al. 2013), it is possible that this property of PEI may remain favorable for PEI-coated particles (McBain et al. 2008).

Another innovative and interesting approach to nanoparticle-mediated gene delivery is the use of nanotubes, which has been reported by (Ghasemali et al. 2013). This approach is based upon using nickel-embedded carbon nanotubes covered in DNA. When the nanotubes are hosted to cells in the presence of a specially oriented magnetic field, the nanotubes align with the magnetic flux lines as they are pulled towards the cells. This allows the nanotubes to spear the cells, pass through the membrane, and deliver the target DNA, and this technique has been successfully used to transfect a number of various cell types, while maintaining a high rate of cell viability after transduction (McBain et al. 2008).

Gene delivery using MNPs and magnetic force

MNPs are already in use by some researchers to enhance transfection efficiencies of cultured cells. Therefore, MNP-nucleic acid complexes are added to cell culture media and then onto the cell surface by applying a magnetic force (Figure 4) (McBain et al. 2008).

Figure 4. MNP gene delivery system (Magnetofection). Plasmids are bound to MNPs, which are then moved from the media to the cell surface by applying a magnetic force (Sadat et al. 2014).

Today, research has made progress in finding a way to use MNPs which are ultra-small and biocompatible, to improve the overall uptake of genetically engineered cells like monocytes or macrophages by tumors, following their systemic administration (Davaran et al. 2014). Muthana and coworkers suggested that this new magnetic targeting method could be used to target ‘therapeutically armed’ monocytes or other forms of cellular gene delivery vehicles to tumors, and thus overcome the difficulty of poor targeting in current cell-based gene therapy protocols (Figure 5) (Davaran et al. 2014).

Figure 5. Schematic representation of the possible role of MNPs in enhancing monocyte-based gene delivery to tumors. MNP-loaded monocytes injected into the bloodstream of the patient circulate and are then drawn out of the blood vessels in the tumor under the influence of a local magnetic field (Davaran et al. 2014).

As early as 1960, Freeman (Kouhi et al. 2014) proposed that such MNPs could be transported through the vascular system and concentrated in a special part of the body using an externally-applied magnetic field. Since then, MNPs have been conjugated to several therapeutic agents like the anti-cancer drugs, and a magnetic field applied to the target tissue (Abbasi et al. 2014b, Pourhassan-Moghaddam et al. 2014). The hurdle with this approach has been that although the drug is concentrated within the target tissue by the magnet, comparatively little pierces beyond the perivascular areas, and thus the deeper regions of the tissue remain untreated (Abbasi et al. 2014c, Eatemadi et al. 2014b).

Many researchers have reported magnetofection approaches (Table II). They improved the surface of iron oxide-based MNPs to enhance transfection efficiency and decrease cytotoxicity (Kami et al. 2011).

Table II. Summary of magnetofection literature(Kami et al. 2011).

Author	Year	MNPs	Modifying Agent	Vector	Targeting Cell, or Tissue	TF Efficiency
MorishitaN (Hosseininasab et al. 2014)	2005	Iron oxide (γ-Fe2O3)	HVJ-E, heparin sulfate	Plasmid	Liver, BALB/c mice	** 3-fold
Yang SY (Davoudi et al. 2014)	2008	Iron oxide (Fe3O4)	DOTAP:DOPE	Plasmid	He99	−
Namiki Y (Anganeh et al. 2014b)	2009	Magnetite	Oleic acid, Phospholipid	Plasmid	HSC45	** 8-fold
Arsianti M (Alimirzalu et al. 2014)	2010	Iron oxide	Branch PEI (MW: 25 k)	Plasmid	BHK-21	−
Kami D (Mollazade et al. 2013)	2011	Iron oxide (γ-Fe2O3)	PEI max (MW: 25 k)	Plasmid	P19CL6	* 82%

Topical and systemic delivery

Topical delivery

Magnetofection proposes two potential advantages for regional delivery to tumors. First, it can enhance the cellular uptake and retention of payloads at the injection region. A second advantage of magnetofection for topical delivery is tumor diffusion. Current delivery techniques cannot efficiently deliver therapeutic genes to all regions of tumors, specifically the hypoxic focus, due in part to the complex nature of the vasculature inside many tumors (Li et al. 2012, Eatemadi et al. 2014b).

Systemic delivery

Efficient prolonged systemic gene therapy needs effective gene transfer, suitable expression, and long-standing survival of transduced cells. This technique might be used to prevent or treat cardiovascular diseases, such as thromboembolic disease, hypertension, hypercholesterolemia, or diabetes mellitus (DM). For the treatment of DM or hypertension, the ability to achieve physiologic regulation of expression will be essential prior to use these treatments in humans. Current gene therapy methods with retroviral and AAV vectors are restricted by their inability to achieve sufficient levels of expression for many disease, whereas adenoviral vectors are limited by short-term expression (Jalil et al. 2014).

Magnetofection in cells

Scherer et al. (Scherer et al. 2002) and Plank et al. (Alizadeh et al. 2014) reported that using MNPs to carry gene vectors to various cells showed significantly increased uptake of these vectors, followed by high target protein expression. The magnetic field applied on the gene vector-magnetic particle complex may raise the accumulation of these complexes on the surface of several cells. Specifically, cell lines that have only imperfect efficiencies regarding target gene expression, such as human endothelial cells, can be well adapted to magnetofection (Nejati-Koshki et al. 2014, Alizadeh et al. 2014, Abbasi et al. 2014a, Ebrahimi et al. 2014a).

Endothelial and epithelial cells

Magnetofection has been described to potentiate gene delivery to cultured primary endothelial cells and to human umbilical vein endothelial cells (HUVEC). Therefore, up to a 360-fold increase in luciferase gene transfer was achieved by magnetofection, as compared to various conventional transfection procedures (Schwerdt et al. 2012, Abbasi et al. 2014d).

It has been observed in various cell lines that coupling of MNPs to gene vectors of any kind results in an effect of higher uptake of these vectors and consequently, target protein expression (McBain et al. 2008). Furthermore, it was seen that magnetofection allowed locally enhanced expression of ß-galactosidase activity in some cell lines (McBain et al. 2008).

Recently, the progress of MNPs coated with PEG and with covalently linked branched PEI (bPEI), has been reported. In HUVEC cultures, nonviral vector-hybrid MNP complexes demonstrated highly efficient magnetofection, even in serum-conditioned media (Ghalhar et al. 2014). In another study, MNPs complexed to Lipofectamine 2000 or cationic lipid 67/plasmid DNA (pDNA) liposome complexes were reported to be highly efficient for gene delivery in airway epithelial cell cultures, but less efficient than pDNA alone when applied in the murine nasal epithelium in vivo. The latter result is likely to be a consequence of the significant precipitation of the complexes achieved in vivo (Schwerdt et al. 2012, Daraee et al. 2014a).

Tumor and embryonic cells

Magnetofection of cDNA constructs and shRNA into mouse genital ridge tissue was applied as a means of gain-of-function and loss-of-function analysis, correspondingly. Ectopic expression of Sry convinced female-to-male sex-reversal, although knockdown of Sox9 expression caused male-to-female sex-reversal, consistent with the known functions of these genes. Also, the ectopic form of Tmem184a, a gene of unknown function in female genital ridges, resulted in the failure of gonocytes to arrive at meiosis. These results suggest that magnetofection may constitute a proper tool for the study of gene function in a broad range of developing tissues and organs (Schwerdt et al. 2012, Tabatabaei Mirakabad et al. 2014).

Delivery to internal organs

Several studies investigating magnetic gene targeting of internal organs have used reporter genes. Namiki et al. have represented systems that have effectively delivered reporter genes that can also be used to successfully deliver therapeutic genes once they have been improved (Daraee et al. 2014b). Gene therapy has shown promising results in treating hepatocellular carcinoma, both in vitro and in vivo. These methods consist of p53 gene replacement (Nasrabadi et al. 2014) and RNAi-mediated gene silencing (Chung et al. 2014). In both tests, gene therapy only worked when genes were directly applied to the liver (Schwerdt et al. 2012).

In the spinal cord, magnetic nanoparticle/PEI complexes have been demonstrated to be targetable following intrathecal injection (Ghalhar et al. 2014). For spinal tumors, this method offers a unique technique for targeting several regions of the spine, by increasing the effect of a therapy at the tumor site and reducing exposure at other sites (Schwerdt et al. 2012).

Conclusion

Magnetically-guided drug or gene targeting using MNPs is a favorable method for cancer gene therapy and cancer chemotherapy. The rationale behind these two treatment modalities is based on binding either chemotherapeutics or nucleic acids onto the surface of MNPs, which are then directed to the tumor by using an external magnetic field. Recently, binding of nucleic acids to MNPs has been confirmed as a successful non-viral transfection system of special cell lines in vitro. With the optimization of this technique, called magnetofection, we are confident that it will become another form of gene delivery for the treatment of cancer (Herizchi et al. 2014, Kafshdooz et al. 2014, Badrzadeh et al. 2014, Sohrabi et al. 2014, Tozihi et al.2014, Afsaneh et al. 2014, Kordi et al. 2014, Anganeh et al. 2014, Barkhordari et al. 2014, Dadashzadeh et al. 2014).

Authors’ contributions

AA conceived of the study and participated in its design and coordination. SM, FZS and MS participated in the sequence alignment and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors thank the Department of Medical Nanotechnology, Faculty of Advanced Medical Science of Tabriz University, for all support provided. This work is funded by the 2015 Drug Applied Research Center Tabriz University of Medical Sciences Grant.

Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.