Applications of nanoparticle systems in gene delivery and gene therapy

Abstract

For successful gene therapy, expansion of appropriate gene delivery systems could be one of the factors of major significance. Gene therapy provides large opportunities for treating diseases, including genetic disorders, infections, and cancer. Polymeric carriers have relatively low cytotoxicity and immunogenicity. Polymeric gene carriers are a potential substitute to using viral vectors. Overall, polymeric carriers can contain large-sized DNA, be conjugated with suitable functionalities, and be administered frequently. However, polymeric gene carriers have some restrictions, such as low gene transfection efficiencies and a moderately short period of gene expression.

This study explores the current status of development of polymeric gene carriers, and presents guidelines for the prospective use of the polymer-based gene delivery systems in gene therapy.

Keywords::

• gene delivery
• gene therapy
• gene carriers
• immunogenicity

Introduction

Gene therapy has gathered a lot of attention in the fields of medicine, pharmacology, and biotechnology, due to its potential to treat chronic diseases and genetic disorders (Park et al. 2006, Ledley 1996), and its ability to facilitate localized delivery to target tissues, strongly improving the efficacy of various therapies. In recent years, significant effort has been made in the area of nanotechnology for drug delivery, since it offers a suitable means of delivering small molecular drugs, such as proteins, peptides or genes to the organ or tissue of interest, by either localized or targeted delivery (Bengali et al. 2005, Yang et al. 2001). Nanocarriers have provided a novel stage for target-specific delivery of therapeutic agents. Several delivery tools have been designed based on different nanoparticles, such as polymers (Lai et al. 2009), dendrimers (Ensign et al. 2012), liposomes (Knowles et al. 1995), and gold nanoparticles (Ghosh et al. 2008).

Gene delivery systems should be organized to protect the genetic materials from premature degradation in the systemic blood stream, and to effectively transfer the therapeutic genes to target cells (Park et al. 2006, Han et al. 2000).

Nanoparticle systems can be engineered to have a number of attractive qualities for therapy, including: (i) sustained and controlled release of drugs locally (Krebs et al. 2010, Kievit et al. 2009), (ii) deep tissue penetration due to their nanoscale size (Park et al. 2006, Pissuwan et al. 2011), (iii) cellular uptake and sub-cellular trafficking (Malafaya et al. 2007, Jeong et al. 2007), and (iv) protection of cargo therapeutics at both the extracellular and intracellular levels (Park et al. 2006, Jeong et al. 2007, Panyam and Labhasetwar 2003).

The size of nanoparticles (NPs) ranges from 10 to 200 nm, which is the same as the size of protein molecules. The small size offers the potential of targeted gene delivery, allowing the deep penetration of tissues at the proper site, like solid tumors, with a high level of specificity (Jin et al. 2009).

With the small size, NPs can easily interact with biomolecules on the cell surface or inside cells, and deliver genetic materials such as DNA, RNA, or small interfering RNA (siRNA) into target cells or tissues for gene expression (Kaul and Amiji 2005, Kaul 2005, Kneuer et al. 2000a, 2000b).

This review will discuss the existing status of use of natural polymers, dendrimers, liposomes, and gold nanoparticles (AuNPs) for gene delivery. Gene therapy involves intracellular transfer of nucleic acid drugs to adjust cellular functions and responses by expressing exogenous proteins, by inhibiting instruction of a specific gene, or by controlling undesirable genomic mutations.

Polymers in gene delivery

Biomaterial sciences have already explained that polymers can be considered to have a very biocompatible, nontoxic form, with no immunogenicity, which in theory enables repeated administration and continuing therapeutic effects (Schaffert and Wagner 2008).

Natural polymers have been used for a wide range of gene therapies, from NPs to three-dimensional scaffolds. These particles have been used successfully for oral and intramuscular delivery as non-viral gene therapy systems (Dang and Leong 2006, Caplan 2005). Polymeric NPs are used in adenosine deaminase enzyme deficiency (ADA) and acute lymphoblastic leukemia (Anajwala et al. 2010).

In condensing the anionic DNA into dense nanoparticles, the polymeric gene carrier can provide protection from DNAses and lengthen the bioavailability of the integrated DNA. In addition, polymers with available functional groups can be tailored with surface ligands to realize targeted gene delivery via receptor-mediated endocytosis (Junghans et al. 2000, Abdallah et al. 1996, Burke and Pun 2008).

A certain advantage of polymer systems is the abundance of potential chemical modifications, such as the synthesis of block and comb polymers (Mehnert and Mäder 2001, Müller et al. 2000).

The communication between the positively charged polymer backbone and negatively charged DNA leads to the spontaneous formation of nano-sized complexes (polyplexes) (Lee and Kim 2005).The structure of the polyplex core effectively prevents the entrance of nucleases into the enclosed nucleic acid drugs. They should maintain their solidity before cellular uptake by endocytosis (Jeong et al. 2007, Abdallah et al. 1996, Glover et al. 2005) (Figure 1).

Figure 1. (a) The formation of polyplexes by ionic interaction between negatively charged DNA and polycations (b) major barriers that polyplexes should overcome for efficient gene delivery Extracellular barriers include the maintenance of stability of the polyplexes during systemic circulation, and the transport from blood vessels to the tissue. Intracellular barriers for delivery include (i) cellular uptake via endocytosis, (ii) endosomal escape, (iii) transport to the perinuclear space, (iv) unpacking of the polyplexes, and (v) nuclear translocation (Molecular Design of Functional Polymers for Gene Therapy, 2007).

Polymeric non-viral carriers consist of: polyethylenimine (PEI), poly(L-lysine) (PLL), poly(2-(dimethylamino)ethyl methacrylate (pDMAEMA), dendrimers, biodegradable polymers, natural polymers, and non-ionic neutral polymers (Brem and Gabikian 2001, Choi et al. 1999).

PEI has been commonly used in paper industries, water purification, and food industries. The first time PEI was used in gene therapy was in 1995, and PEI has become one of the gold standards of non-viral gene delivery (Eliyahu et al. 2005, Wivel and Wilson 1998, Rosenberg et al. 1990). PEI is an angiogenic, endothelial cell-targeted gene delivery system (Park et al. 2006).

Not only does the molecular weight of the PEI have advantages, the degree of branching also plays a significant role in the biological properties of complexes with nucleic acids (Dang and Leong 2006, Eliyahu et al. 2005, Melo et al. 2004, Partridge and Oreffo 2004). Branched PEI molecules have primary, secondary, and tertiary amino groups, while linear PEI molecules mostly have secondary amines. On the other hand, studies with linear PEIs showed constantly higher transfection efficiency and lower cytotoxicity compared to branched PEI (Park et al. 2006, Ferrari et al. 1997, Coll et al. 1999, Wightman et al. 2001).

The PEI-grafted PEG (PEI-g-PEG) copolymer has been shown to lessen PEI's toxicity with special PEG grafting ratios, by providing a physical barrier between the cell and PEI (Kievit et al. 2009, Petersen et al. 2002, Suh et al. 2002).

The PEI-g-PEG-RGD/DNA complexes showed much lower cytotoxicity and about five times higher transfection competence in VEGF-stimulated angiogenic human dermal microvascular endothelial cells (HDMEC) than the PEI/DNA complexes.

In addition, PEI, one of the most commonly used polymeric gene carriers, can increase the transfection competence by evading DNA complexes from degradative endosome or lysosome compartments (Schaffert and Wagner 2008, Kasyanenko et al. 2012, Gyawali et al. 2012, Kim et al. 2005).

PLL is used in nonviral gene delivery, and has been utilized in physicochemical and biological experiments (Wolfert et al. 1999, Merdan et al. 2002). PLL, having a molecular weight of less than 3000, cannot shape firm complexes with DNA, suggesting that the number of primary amines in the PLL backbone is significant for the complex pattern. PLL is biodegradable, and this property exhibits modest to high toxicity (Phillips 1995, Fajac et al. 2000).

The PLL/DNA complexes show a reasonably high cytotoxicity and are poor gene delivery vectors (Malafaya et al. 2007, Xiao et al. 2003), having an affinity to combine and precipitate, depending on the ionic potency (Lucas et al. 1999). For example, addition of chloroquine for constant reasonable transfection activity (Xiao et al. 2003).

On the other hand, the properties of the complex surface may be the cause for cytotoxicity. Moreover, the charged surface can enable the nonspecific adsorption of serum proteins, resulting in rapid clearance of the complexes from the blood stream (Ogris et al. 1999). Therefore, to solve this problem, PEG was attached, to prevent the interaction and to increase complex stability in the presence of serum protein (Lee and Kim 2005, Harada-Shiba et al. 2002).

Dendrimers

The word ‘dendrimer’ originates from two Greek words, dendra: tree, and meros: part. Dendrimers are characterized by their distinct architectural feature, in which several hyper-branched arms extend from a central core to the surface, in a controlled manner (Harada-Shiba et al. 2002).

Dendrimers of lower generations (0, 1, and 2) have extremely asymmetric shapes and have more open structures as compared to higher generation dendrimers (Klajnert and Bryszewska 2000). Dendrimers become closely packed as they pull out to the periphery, which forms a closed membrane-like structure. When a critical branched state is reached, dendrimers cannot grow because of a lack of space (Yin and Alivisatos 2004) (Figure 3).

Figure 2. Electrophoresis for DNA concentration with polymer (DNA–Polymer Complexes for Gene Therapy, 2012).

Figure 3. Representation of a fourth generation dendrimer.

Dendrimers are monodisperse macromolecules, unlike linear polymers. The solubility of dendrimers is powerfully influenced by the nature of surface groups. Dendrimers ending in hydrophilic groups are soluble in polar solvents, while dendrimers having hydrophobic end groups are soluble in nonpolar solvents (Mourey et al. 1992).

Dendrimers consist of three main parts, namely, the surface (functional peripheral group), interior (affects host-guest properties), and core (affects the 3D shape of the dendrimer) (Ma et al. 2002).

Dendrimers have been used in in vitro diagnostics. There are more than fifty families of dendrimers. They have been tested in preclinical studies, such as magnetic resonance imaging (MRI) (Fischer and Vögtle 1999).

There have been attempts to use dendrimers in the targeted delivery of drugs and other therapeutic agents (Hawker and Frechet 1992).

The biological properties of dendrimers are important because of the increasing attention in using them in biomedical applications (Frechet 1994).

Liposomes

Liposomes were discovered by Bangham et al. in the 1960s, and were introduced as drug delivery vehicles in the 1970s (Bangham and Horne 1964, Wissing et al. 2004, Eldem et al. 1991). They are spherical vesicles composed of one or more phospholipid bilayers, in most cases phosphatidylcholine. The main advantage of this carrier system is the reduction of side effects caused at the injection site (Schmidt 1997).

Particulate drug carriers investigated for many years include oil-in-water (O/W) emulsions, liposomes, microparticles and nanoparticles (Müller et al. 2000, zur Mühlen et al. 1998). The O/W emulsions have been introduced effectively in clinical practice for parenteral nutrition. Anchored in these emulsions for parenteral nutrition, drug-containing emulsion structures have been developed, e.g. containing diazepam and etomidate (Schwarz et al. 1994).

It was the anti-aging product Capture (Dior) which paved the way for liposome-based pharmaceutical products. The use of solid lipids as a matrix material for drug delivery is well-known, from the use of lipid pellets for oral drug delivery (Kakkar et al. 2011).

The nanoparticles are in the submicron size range (50–1000 nm), and they are composed of physiological lipids. At room temperature, the particles are in the solid state. Therefore, the mobility of entrapped drugs is reduced, which is a requirement for controlled drug release (Akiyoshi et al. 1998).

They are stabilized with non-toxic surfactants like poloxamer and lecithin (zur Mühlen et al. 1998, Gasco 1993). Rupenagunta et al., in 2011, showed that the advantages of solid lipid nanoparticles (SLNs) are the use of physiological lipids, decrease in the the danger of acute and chronic toxicity, and avoidance of organic solvents in production. Fahr and Liu, in 2007, demonstrated the improved bioavailability of poorly water soluble molecules, protection of both chemically labile agents from degradation in the gut and sensitive molecules from the outer environment, thereby achieving a high concentration of efficient compound (Fahr and Liu 2007).

The avoidance of organic solvents, a potential extensive application range (dermal, intravenous), and the high pressure homogenization as a recognized production method, allow large-scale production. Moreover, improved bioavailability, and protection of sensitive drug molecules from the outer environment (water, light), are added advantages (Jores et al. 2005).

To date, numerous studies have been conducted regarding the optimization of production parameters (Smith 1986), long term stability (Müller et al. 1996), recrystallization behaviour (Westesen and Siekmann 1997), morphological characterization (Almeida and Souto 2007), and in vivo toxicity (Wissing et al. 2004).

Solid lipid nanoparticles (SLNs) were introduced in 1991, as a novel drug delivery system for pharmaceutical drugs in a variety of application routes, and as alternative carriers (Mehnert and Mäder 2001). At the turn of the millennium, modifications of SLNs, the so-called nanostructured lipid carriers (NLC) and the lipid drug conjugate (LDC) nanoparticles, have been introduced in the literature (zur Mühlen et al. 1998). The disadvantages of SLNs include their poor drug loading capacity.

Gold nanoparticles

AuNPs are particularly interesting because biologically, they should not be cytotoxic (Daniel and Astruc 2004).

The fact that the surface of AuNPs suggests well established routes for functionality makes them particularly favored vehicles. Charged or hydrophobic motifs can be bound to the surface, and in fact can be combined with modification protocols that include targeting moieties such as antibodies and receptors. These too can be mixed with a payload such as double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA), which is then transfected into cells (Sandhu et al. 2002, Rosi et al. 2006).

AuNPs have a high affinity for biomolecules and have been used as nonviral vectors for DNA delivery (Melo et al. 2004, Thomas and Klibanov 2003). AuNPs chemically functionalized with alkylthiol-terminated oligonucleotides (Mirkin et al. 1996) are highly stable in saline solutions, and bind complementary nucleic acids in a very selective and cooperative manner (Jin et al. 2003); their equilibrium association constants can be more than two orders of magnitude greater than those observed for unmodified oligonucleotides and their complements (Rosi et al. 2006).

Many studies have shown that AuNPs have played a role as drug and vaccine carriers into target cells or specific tissues. Generally, this has been achieved by modifying the surface of the AuNPs, so that they can bind to the specific targeting drugs or other biomolecules. The delivery of drugs with nanoparticles can result in higher concentrations than possible with normal drug delivery schemes (Chen et al. 2008).

AuNPs are also attractive because of their unique properties, which we have described previously. For example, in 2001, there was an investigation of AuNPs that had been functionalized with cationic quaternary ammonium groups and then electrostatically bound to plasmid DNA. This composite particle could protect the DNA from enzymatic degradation and could regulate DNA transcription of T7 RNA polymerase (McIntosh et al. 2001, Han et al. 2006) (Figure 2).

Conclusion

Biomaterial sciences have explained that polymeric systems can be safely used in a very nontoxic, biocompatible, and non-immunogenic form, which in theory enables continual administration and continuing therapeutic effects. Nanocarriers and biomaterials have provided a narrative stage for target-specific delivery of therapeutic agents.

Authors’ contributions

AA and TK conceived of the study and participated in its design and coordination. EA 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, Tabriz University, for all support provided. This work is funded by the Grant 2011-0014246 of the National Research Foundation of Korea. This work is also funded by the 2014 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.