The use of different formulations with specific functions for designing of multi-functional nanocarrier systems
Recent developments in molecular biology and cell biology have led to the discovery of novel genes and proteins having therapeutic potentials for various diseases including cancers. Based on these findings, novel categories of therapeutic biomacromolecules including genes, small interfering RNA (siRNAs), antisense oligonucleic acids, bioactive proteins and peptides have been developed. These macromolecules can be more advantageous than small-molecular-weight therapeutic agents in terms of their specificity and high potency to the target molecules [Nakase et al., 2010]. Gene therapy is the newest therapeutic strategy for treating human diseases. The basic idea of gene therapy is a gene or gene product that can be selectively delivered to a specific cell/tissue with minimal toxicity. This product can inhibit the expression of a specific defective gene or express a normal gene. Efficient and safe delivery is one of the key issues for the clinical application of nucleic acids as therapeutic agents [Du et al., 2010]. The goal of the Pharmaceutical Industry is to have a gene therapy medical product that can be delivered systemically.
Delivery is one of the most difficult challenges facing the gene therapy field. It is needed to use an efficient transfer system that can stabilize, transduce and express a transgene in the target tissue. Recently, non-viral technologies have been widely used as a significant alternative for gene delivery. The non-viral delivery systems have reduced adverse immune responses, are easier to manufacture and can be produced for the pharmaceutical industry in large quantities. The current progresses of gene therapy are further focused on synthesized nano-particle technologies. Some of these new chemical compositions are cationic molecules such as polymers, lipids and peptides [Scanlon, 2004; Gao et al., 2007].
However, for successful clinical trial, ideal non-viral vectors should be degradable into low molecular weight components, in response to biological stimuli, for lowered cytotoxicity and effective systemic clearance. They should also be efficient in overcoming extracellular and intracellular barriers, tissue/cell-targeted for specific accumulations and multi-functional for synergistic therapeutic and diagnostic outcomes. Recently, a broad range of different stimuli-responsive strategies (virus-mimicking gene delivery systems) has been employed to develop non-viral nucleic acid carriers that efficiently enhance multiple extracellular and intracellular gene delivery pathways by altering their physico-chemical properties in response to a variety of extra- and intra-cellular stimuli (e.g., pH, redox potential, and enzyme), as well as external triggers (e.g., light) [Zhu and Torchilin, 2012].
Therefore, numerous challenges remain to be overcome before gene therapy becomes available as a safe and effective treatment option. For instance, the cationic molecules (e.g., polymers, lipids and peptides) have the potential to be systemically delivered but selectivity for the target tissue needs to be validated. The best method for delivering genes will depend on the type of tissue targeted [Scanlon, 2004]. Recently, the development of virus-mimicking, multi-functional gene delivery systems is considered to be a potent strategy in the future, in particular for intravenous administration. This chapter summarizes the challenges for cancer gene therapy as well as addressing the advances in the multi-functional nano-carriers as a potent non-viral delivery system.
2. Gene therapy
Medicine has a long history of treating patients with cell therapies (i.e., blood transfusions) and protein therapies (i.e., growth factors and cytokines). Gene therapies are the newest therapeutic strategy for treating human diseases especially cancer [Scanlon, 2004]. This technology has been used to develop new strategies for killing cells selectively or inhibiting their growth. The field of cancer gene therapy comprises a range of technologies from direct attack on tumor cells to inducing the immune response to tumor antigens [McCormick, 2001]. However, there are serious doubts about gene therapy; for example, short-lived nature of gene therapy, immune response to a foreign object, problems with viral vectors and insertional mutagenesis inducing a tumor [Korthof, 1999].
In 1972, Friedmann and Roblin published a paper in Science entitled as "Gene therapy for human genetic disease" [Friedmann and Roblin, 1972; Rogers, 1970]. The first approved gene therapy case in the United States took place in 1990, at the National Institute of Health. It was performed on a four year old girl with a genetic defect associated to an immune system deficiency. The effects were only temporary, but successful [Blaese et al., 1995]. In addition, sickle cell disease was successfully treated in mice [Fisher, 1995]. At the same time, the researchers were able to create tiny liposomes 25 nanometers (nm) that can carry therapeutic DNA through pores in the nuclear membrane [www. newscientist.com, 2002]. In 1992, Claudio Bordignon performed the first procedure of gene therapy using hematopoietic stem cells as vectors to deliver genes aimed to correct hereditary diseases [Abbott, 1992]. In 2002, this work led to the publication of the first successful gene therapy treatment for adenosine deaminase-deficiency (SCID) [Cavazzana-Calvo et al., 2004]. In 2003, a Los Angeles research team inserted genes into the brain using liposomes coated in a polymer called polyethylene glycol (PEG). The transfer of genes into the brain is a significant achievement because viral vectors are too big to get across the blood–brain barrier. This method had potential for treating Parkinson's disease. At the same time, RNA interference or gene silencing was considered as a new way to treat Huntington's disease [www. newscientist.com, 2003]. In 2006, scientists at the National Institutes of Health have successfully treated metastatic melanoma in two patients using killer T cells genetically retargeted to attack the cancer cells. For the first time, this study demonstrated that gene therapy can be effective in treating cancer. In November 2006, Preston Nix reported on VRX496, a gene-based immunotherapy for the treatment of human immunodeficiency virus (HIV) that uses a lentiviral vector for delivery of an antisense gene against the HIV envelope [Levine et al., 2006; www.eurekalert.org, 2009].
Leber's congenital amaurosis is an inherited blinding disease caused by mutations in the RPE65 gene. In 2007-2008, the world's first gene therapy trial was announced for inherited retinal disease. They determined the safety of the sub-retinal delivery of recombinant adeno-associated virus (AAV) carrying RPE65 gene and found the positive results in vision and without the apparent side-effects [www. news.bbc.co.uk, 2007]. In 2009, the researchers were succeeded at arresting a fatal brain disease, adrenoleukodystrophy, using a vector derived from HIV to deliver the gene for the missing enzyme [Kaiser, 2009]. A paper published in 2010, deals with gene therapy for a form of achromatopsia in dogs. Achromatopsia, or complete color blindness, is presented as an ideal model to develop gene therapy directed to cone photoreceptors [Komáromy et al., 2010]. In 2011, a study carried out using genetically modified T cells to fight the chronic lymphocytic leukemia (CLL) disease. Moreover, Human HGF plasmid DNA therapy of cardiomyocytes was examined as a potential treatment for coronary artery disease as well as myocardial infarction [www. nature.com, 2011; Yang et al., 2008; Hahn et al., 2011]. However, most of the approved European and United States gene therapy protocols are for cancer (~ 66%), in contrast to monogenetic diseases (~ 11%) and cardiovascular diseases (~ 8%). The focus of cancer gene therapy has been on melanoma, prostate and ovarian cancer and leukemia [Scanlon, 2004].
2.2. The challenges of gene therapy
Recently, the use of gene therapy in medicine using plasmid DNA (pDNA), oligodeoxynucleotide (ODN) or small interfering RNA (siRNA) represents a promising new approach for treating a variety of genetic and acquired diseases (e.g., cancer). To date, more than 1000 different gene-therapy clinical trials for the treatment of many different diseases are in progress worldwide, but, the success with gene therapy has been limited. This lack of success can be assigned to difficulties related to the effective delivery of nucleic acids into target cells [Mastrobattista et al., 2006]. Naked DNA is unable to efficiently cross cellular barriers by passive diffusion because of its large size, strong negative charge, hydrophilicity and susceptibility to nuclease attack. Hence, the major challenge for
3. Delivery systems in gene therapy
3.1. Viral delivery
Delivery is one of the most difficult challenges facing the gene therapy field. An efficient transfer system has not yet been found to stabilize, transduce and express a transgene in the target tissue. Limitations of the present vector technologies have slowed the progress of clinical gene therapy [Scanlon, 2004]. Various viruses, such as
However, there are several problems to overcome before this therapy can be successful in clinical settings such as a) Enhanced lytic properties of these viruses; b) Improved yields with better manufacturing procedures of production for clinical studies; c) Systemic delivery; d) limited DNA-carrying capacity; e) lack of target-cell specificity; f) immunogenicity and h) for some viral vectors, insertional mutagenesis [Scanlon, 2004; Mastrobattista et al., 2006].
3.2. Non-viral delivery
There are some non-viral technologies that offer several advantages over the viral methodologies. Non-viral delivery systems have reduced adverse immune responses (relatively safe), are easier to manufacture, can be produced for the pharmaceutical industry in large quantities and modified by the incorporation of ligands for targeting to specific cell types [Scanlon, 2004; Mastrobattista et al., 2006]. Chemically synthesized nanoparticles constitute a new technology and offer several new strategies for successful systemic gene therapy delivery. Synthetic gene-delivery systems consist of a self-assembling complex of DNA with positively charged molecules (for example, polymers, peptides, lipids or their combinations). These complexes are small in size (40-150 nm) and usually have a net positive surface charge, which enables adsorption-mediated cell binding and internalization [Mastrobattista et al., 2006]. Some of these new chemical compositions are polymers containing either DNA/stearyl polylysine-coated lipids or peptoids (DNA coated with glycine oligomers) or cationic molecules (DNA/combined with positively charged B-cyclodextrin/adamantane and PEG). These molecules have been shown to be effective in cancer-related angiogenesis. These new agents have the potential to be systemically delivered, but their selectivity for the target tissue needs to be validated [Scanlon, 2004]. However, the levels of gene expression and the transfection efficiency mediated by synthetic vectors are low compared to viral vectors [Mastrobattista et al., 2006].
3.3. Cell delivery
One of the opportunities for gene therapy is to combine therapeutic genes with a cell to overcome the delivery to target tissues. The advantages of cell delivery of therapeutic products include minimal immune response; tissue directed therapy; selectivity and improved potency of the product. However, there are several problems to be solved: a) Determining optimal transduction of cells; b) Gene-transformed cells will require a selective growth advantage over defective cells to re-populate the host; c) DNA repair genes action (minimized mutations in the gene-transformed cells); d) Genomic stability (for optimal gene expression); e) Determining cell type for therapy (e.g., embryonic stem cells or activated, differentiated cells); f) Incorporation of a safety mechanism (
4. The challenges of nucleic acid delivery
There are many biological barriers for efficient gene delivery that need to be overcome [Mastrobattista et al., 2006]. This inefficiency of gene delivery is primarily a result of the inability of these vectors to overcome the numerous barriers encountered between the site of administration and localization in the cell nucleus. This series of barriers to efficient non-viral gene delivery is thought to include: a) the physical and chemical stability of DNA and its delivery vehicle in the extracellular space, b) cellular uptake by endocytosis, c) escape from the endosomal compartments prior to trafficking to lysosomes, d) cytosolic transport and e) nuclear localization of the plasmid for transcription. In addition to these physical and chemical obstacles, biological barriers, such as immunogenic responses to the vector itself as well as immune stimulation by certain DNA sequences containing a central un-methylated CpG motif, are present. The studies have shown that it is possible to minimize biological barriers by optimizing the plasmid sequence, thus physical and chemical barriers appear to be the negative factors to successful non-viral gene delivery [Wiethoff and Middaugh, 2003]. These barriers are briefly described as following:
4.1. Stability in extracellular compartments
The stability of non-viral delivery systems in the extracellular milieu, such as intercellular or intravascular spaces, is related to the chemical stability of the DNA as well as the physical stability of the delivery system [Wiethoff and Middaugh, 2003]. In extracellular environment, the carrier is exposed to blood components such as nucleases. This can result in premature destabilization with simultaneous release and degradation of the plasmid DNA. The half-life of naked plasmid DNA in blood is on the order of minutes. Therefore, carrier-mediated protection during transport through the blood circulation is a prerequisite to make the DNA inaccessible to degradative enzymes [Mastrobattista et al., 2006]. Condensing the DNA with a variety of polycations or by complexing with polymers that bind to DNA protects it from degradation [Wiethoff and Middaugh, 2003].
4.2. Cellular association of DNA
Irrespective of the injection route, the gene carriers should be able to bind to cells for allowing cellular uptake [Mastrobattista et al., 2006]. Association of DNA with the cell surface is typically very low in the absence of any delivery agent as an immediate consequence of the relatively high negative charge density of both the DNA and the cell surface. Polycations have been shown to substantially increase the cellular association of DNA by neutralization of the DNA negative charge, with the charge ratio of the complex modulating the extent of this contact. The degree of cellular association has also been shown to be related to the colloidal stability of these delivery systems, with those that aggregate often manifesting a greater degree of cellular association
In general, binding of nano-carrier to the cell surfaces is not, a problem if the gene carriers have a net positive surface charge, which readily induces adsorption onto negatively charged cell membranes. However, this form of binding is random and does not allow restricted delivery to target cells (e.g., tumor cells) [Mastrobattista et al., 2006]. Several additional strategies have been attempted to increase the specificity of DNA binding to cells and induce targeting to particular cell types [Wiethoff and Middaugh, 2003]. Active targeting can be achieved by the functionalization of NPs with ligands such as antibodies, peptides, nucleic acids (aptamers), carbohydrates and small molecules [Gu et al., 2007].
4.3. Intracellular trafficking of non-viral gene delivery systems
Since many chemotherapeutic drugs and particularly gene therapeutics, would benefit from intracellular targeting, nano-carrier systems can be designed for receptor-mediated cell uptake, intracellular drug protection and intracellular target delivery [van Vlerken et al., 2007]. Once inside the cell, several intracellular barriers need to be crossed before the foreign DNA can be transcribed and translated [Mastrobattista et al., 2006]. These barriers contain endosomal escape, cytosolic transport of DNA and nuclear localization of plasmid DNA that described briefly in below:
4.3.1. Endosomal escape
The endocytic pathway is one of the uptake mechanisms of cells. In general, non-viral nano vectors have been developed to mimic the receptor-mediated cell entry mechanism of viruses and the main mechanism of internalization was confirmed to be endocytosis. This pathway is composed of vesicles known as endosomes with an internal pH around 5 that mature in a uni-directional manner from early endosomes to late endosomes before fusing with intracellular organelles called lysosomes which contain certain digestive enzymes. Thus, particles entering the cells via the endocytic pathway become entrapped in endosomes and eventually end up in the lysosome, where active enzymatic degradation processes take place [Varkouhi et al., 2011]. The entrapment of internalized DNA carriers in endocytic compartments prevents further intracellular transport towards the nucleus and will often result in degradation of the carrier and its associated DNA in the endosomal/lysosomal compartments [Mastrobattista et al., 2006]. While many viruses have evolved quite efficient systems for endosomal release, the situation is different for non-viral vectors, where in many cases the lack of endosomal escape is a major obstacle for efficient biological delivery, implying that more efficient methods for endosomal release would lead to improvements in designing synthetic transfection systems. In contrast to synthetic vectors, viral vectors are known to be efficient both for
4.3.2. Cytosolic transport of DNA
After endosomal escape, DNA must traverse the cytosol to access the nucleus. Those DNA carriers that manage to escape the endosomal compartments are then challenged by the complex environment of the cytosol, which contains many filamentous structures that hinder the free diffusion of large particles such as DNA carriers. Dissociation of the carrier at this stage might be required to allow further transport of the plasmid DNA molecules. However, several studies have found evidence that plasmid DNA is largely immobile in the cytosol and is rapidly degraded by cytosolic nucleases [Mastrobattista et al., 2006]. Diffusion of DNA in the cytoplasm has been found to be substantially less than that observed in dilute solution. For DNA >2000 base pairs in length, the diffusion co-efficient in the cytosol is <1% of that in water, suggesting a substantial diffusional barrier. This decreased mobility has been ascribed to molecular crowding of the plasmid, but may also reflect an increased viscosity of the cytoplasm. As expected, the diffusion coefficient of DNA in the cytoplasm is inversely related to the size of the plasmid, suggesting smaller plasmids may be more desirable. No evidence for active transport of DNA in the cytoplasm has been reported. In addition to the considerable diffusional barrier for DNA in the cytosol, the presence of calcium-sensitive cytosolic nucleases pose a significant metabolic barrier. Micro-injection of DNA into the cytoplasm results in significant degradation of the DNA with a half-life of 50-90 min [Wiethoff and Middaugh, 2003].
4.3.3. Nuclear localization of plasmid DNA
Ultimately, delivery of DNA to the nucleus must occur for transcription of the transgene to take place. The mechanism of DNA nuclear translocation and whether the DNA is still associated with the delivery system are not fully understood but appear to depend on the type of delivery vehicle employed. At least three possible routes exist for DNA transport to the nucleus. The DNA can pass into the nucleus through nuclear pores, it can become physically associated with chromatin during mitosis when the nuclear envelope breaks down or it could traverse the nuclear envelope. Of these three possibilities, the latter seems impossible and without experimental support. Nuclear pores are embedded in the nuclear envelope at fairly high surface densities (3000-4000/nucleus) and exist in at least two conformational states. The closed state permits the passive diffusion of molecules of < 9 nm in diameter, whereas the open state facilitates transport of particles < 26 nm. This latter state could certainly assist the ‘‘threading’’ of supercoiled plasmid through the nuclear pore but not passage of typical non-viral gene delivery complexes [Wiethoff and Middaugh, 2003]. Small molecules (< 40 kDa) can diffuse freely through the pores of the nuclear pore complexes (NPC), whereas larger molecules and particles (up to 40 nm in size) can only be imported through the NPC by an active transport mechanism [Mastrobattista et al., 2006]. In a few cases, collapsed particles of < 30 nm have been produced, which could presumably enter the nucleus by this mechanism. The second and perhaps quite widespread mechanism by which DNA is thought to gain access to the nucleus is by association with nuclear material on breakdown of the nuclear envelope during mitosis [Wiethoff and Middaugh, 2003]. In this case, the nuclear barrier breaks down, which explains the increased levels of transfection in dividing cells as compared to their growth-arrested counterparts. However, as most cells do not divide or divide only slowly, an active transport mechanism is needed to carry the DNA from the cytosol into the nucleus [Mastrobattista et al., 2006].
4.4. Toxicity of non-viral delivery system
An important obstacle to effective non-viral gene delivery is the cytotoxicity of the delivery vectors [Wiethoff and Middaugh, 2003]. Inside the nucleus, the transgene encoded on the plasmid vector should be expressed to establish therapeutic levels of recombinant proteins within the affected cell. This requires gene transcription regulatory elements such as promoters and enhancers, to drive the expression of the transgene in mammalian cells. Viral promoters are often used because of their strong transcriptional activation. However, their constitutive nature does not allow control over the level of transgene expression. For the expression of proteins with a narrow therapeutic window, tight control over the level of transgene expression is essential. In addition, the introduction of foreign DNA into mammalian cells can induce a profound immune response, presumably triggered by differences in the degree of methylation of the foreign DNA compared with the mammalian genome [Mastrobattista et al., 2006]. Induction of innate immune responses by un-methylated CpG sequences in plasmid DNA is perhaps a more serious concern because these species have been demonstrated to greatly reduce the efficiency of non-viral gene delivery. This immunotoxicity is thought to be related to the efficient transfection of immune cells because polycation/DNA complexes evoke a considerably greater immune response than DNA or cationic vector alone. Initial observations suggest that delivery systems that are phagocytosed, due to their large size, may promote a greater immune response. Removal of CpG motifs from DNA containing the transgene has proven a successful means of improving gene expression. The methods involved in producing vectors without these motifs are not insignificant, and currently present a major limitation to their widespread usage. It has therefore been proposed that avoidance of transfection of immune cells either by specific targeting to particular cell types or by manipulation of the physicochemical properties of the delivery systems is necessary for significant improvements in non-viral gene delivery [Wiethoff and Middaugh, 2003].
5. Multi-functional nanocarriers
Currently used pharmaceutical nanocarriers, such as liposomes, micelles, nanoemulsions, polymeric nano-particles and many others demonstrate a broad variety of useful properties including: a) longevity in the blood allowing for their accumulation in pathological areas; b) specific targeting to certain disease sites due to various targeting ligands attached to the surface of the nanocarriers; c) enhanced intracellular penetration with the help of surface-attached cell-penetrating molecules; d) contrast properties due to the carrier loading with various contrast materials allowing for direct carrier visualization
The use of cationic lipids and cationic polymers as transfection vectors for efficient intracellular delivery of DNA was suggested in 1987. Complexes between cationic lipids (such as Lipofectin®, an equimolar mixture of N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride–DOTMA and dioleoyl phosphatidylethanolamine –DOPE) and DNA (lipoplexes) and complexes between cationic polymers, such as PEI and DNA (polyplexes) are formed because of strong electrostatic interactions between the positively charged carrier and negatively charged DNA. A slight net positive charge of formed lipoplexes and polyplexes is believed to facilitate their interaction with negatively charged cells and improve transfection efficiency. Endocytosis (including the receptor mediated endocytosis) was repeatedly confirmed as the main mechanism of lipoplex/polyplex internalization by cells [Torchilin, 2006]. Of special importance is the fact that despite of endocytosis-mediated uptake lipolexes and polyplexes, DNA does not end in lysosomes but releases in the cytoplasm due to the destabilization of the endosomal membrane provoked by lipid or polymeric component of the complexes. In particular, lipoplexes fuse with the endosomal membrane when they contain a fusogenic lipid, dioleylphosphatidylethanolamine (DOPE), which easily undergoes the transition from bilayer to hexagonal phase facilitating the fusion. In case of polyplexes, which cannot directly destabilize the endosomal membrane, the mechanism of DNA escape from endosomes, is associated with the ability of polymers, strongly protonate under the acidic pH inside endosome and create a charge gradient ultimately provoking a water influx and endosomal swelling and disintegration. In both cases, DNA-containing complexes when released into the cytosol, dissociate allowing for nuclear entry of free DNA. Nuclear translocation of the plasmid DNA is relatively inefficient because of the barrier function of the nuclear membrane and small size of nuclear pores (25 nm), as well as DNA degrades fast under the action of cytoplasmic nucleases. It was estimated that only 0.1% of plasmids undergo nuclear translocation from the cytosol. The attachment of nuclear localization sequences to plasmid DNA may significantly enhance its nuclear translocation and transfection efficiency. New approaches in using multifunctional carriers for DNA delivery include the application of bi-metallic nano-rods that can simultaneously bind compacted DNA plasmid and targeting ligands in a spatially defined manner [Torchilin, 2006].
DNA–lipid amphiphiles self-assemble into novel “DNAsomes”-liposome-like core-shell structures with subunits composed of branched DNA-lipid hybrid molecules. These DNAsomes can be precisely modified over a wide range in terms of both size and surface charge. More importantly, DNAsome is a natural carrier of small interfering RNA (siRNA) due to DNA-RNA base-pairing, enabling efficient co-delivery of drugs and siRNA. The DNAsome represents a universal multi-functional drug vector for simultaneous delivery of drugs, tracer dyes, or antibodies, along with genes, siRNA or antisense nucleic acids [Roh et al., 2011].
6. Different types of nanocarrier systems
Over the last eight years, a laboratory at Northeastern University has developed an array of multi-functional nanocarriers for the delivery of genes, drugs and imaging modalities. These flexible platforms consist of polymeric and lipid systems that combine different modalities and stimuli-responsive release properties [Jabr-Milane et al., 2008].
The diagnostic and/or therapeutic objectives of a multi-functional nanocarrier system determine the design of the formulation. A review of the literature shows that there are many different types of nanocarrier formulations for the diagnosis, imaging and treatment of a wide spectrum of diseases. These multi-functional carriers share three main design components: platform (core) material, encapsulated payload/biologically active agents and targeting/surface properties. Nanocarrier platforms can be categorized as organic-based, inorganic-based or a hybrid combination. Organic nano-platforms include polymeric nanocarriers, lipid-based nanocarriers (e.g., liposomes and nanoemulsions), dendrimers and carbon-based nanocarriers (e.g., fullerenes and carbon nanotubes) [Jabr-Milane et al., 2008]. Inorganic nano-platforms include metallic nanostructures, silica nanoparticles and quantum dots. An example of a hybrid platform is colloidal gold encapsulated in liposomes or superparamagnetic iron oxide particles encapsulated in polymeric nanoparticles. Selection of the core material is highly dependent on the properties of the biologically active agents. Inherent and dynamic properties of the agents such as therapeutic index, lipophilicity, charge and size should be considered. When combining therapeutic agents with each other, with an imaging/diagnostic modality, or with energy delivery, the interaction of system components (i.e., synergy, quenching, enhanced toxicity) and release kinetics should also be considered. Surface properties are the third design component of multi-functional nanocarriers. A common surface modification technique that decreases reticuloendothelial system (RES) clearance is the physical or covalent attachment of PEG chains to the nanocarrier platform. Since tumor microvasculature is known to be highly fenestrated, colloidal particles can accumulate by the enhanced permeability and retention (EPR) effect. PEG surface modification increases circulatory residence time, which increases the probability of accumulation at the target. Block co-polymers of poly (ethylene oxide) (PEO) and poly (propylene oxide) (PPO) (e.g., Pluronics®) have also been used as surface conjugates to enhance circulation and achieve passive targeted delivery [Jabr-Milane et al., 2008].
Recently, nanoparticulate systems have been also developed for therapeutic gene delivery. Some researchers have developed glutathione-responsive nanoparticles for the delivery of plasmid DNA. The nanocarrier platform consisted of thiolated gelatin which was synthesized using 2-iminothiolane to covalently modify type B gelatin (pI ~ 4.5). The release profiles of cross-linked and non-cross-linked thiolated gelatin nanoparticles and gelatin nanoparticles (control) loaded with fluorescein isothiocyanate-conjugated dextran (FITC-dextran) were assessed in the presence of different glutathione concentrations (from 0.1 mM to 5 mM). A higher percentage of FITC–dextran was released from non-crosslinked nanoparticles compared to the cross-linked nanoparticles. The rate of FITC–dextran release from thiolated gelatin nanoparticles enhanced with increasing concentrations of glutathione. Glutathione in the media enhanced the release of FITC-dextran from thiolated gelatin nanoparticles by about 40%, while only a 20% enhancement was seen with gelatin nanoparticles [Jabr-Milane et al., 2008]. After establishing the rapid, stimuli-responsive release profile of these particles, the thiolated gelatin nanoparticles were loaded with plasmid DNA expressing green fluorescent protein (GFP). Upon incubation of the formulations with murine fibroblast cells, fluorescence imaging revealed transfection and protein expression after 6 h continuing as long as 96 h. Flow cytometry indicated that the cross-linked thiolated gelatin nanoparticles had the highest transfection efficiency. These nanocarriers are capable of rapid DNA delivery in response to intracellular glutathione. This nanocarrier platform was further modified to develop an anti-angiogenic gene therapy for the treatment of cancer. This platform consisted of PEG-modified thiolated gelatin nanoparticles loaded with plasmid DNA encoding the soluble form of the extracellular domain of vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1). VEGF receptor over-expression in cancer is associated with neo-vascularization; sFlt-1 was selected as it blocks the VEGF receptor and the associated signal cascade [Jabr-Milane et al., 2008]. The PEG modified thiolated gelatin nanoparticles showed superior
Five days after administration, the rats were sacrificed and the GI tract was harvested for analysis. The results were compared to unloaded NiMOS formulations, naked plasmid and loaded gelatin nanoparticles. Transgene expression was evident in the small and large intestines with both reporter plasmids although GFP expression was more prominent with the loaded NiMOS formulation relative to the controls. Similar results were obtained in studies with Balb/c mice. Clearly, NiMOS is a promising system for targeted delivery of therapeutic DNA to the GI tract [Jabr-Milane et al., 2008].
7. Design of multi-functional nanocarrier system
An artificial gene-delivery vector, with a multi-component architecture is able to overcoming all the barriers, in which each component performs a different task in a planned fashion. The design of multi-functional nano-carrier is a multidisciplinary task that requires a profound understanding of the physico-chemical mechanisms that drive the assembly of such nano-particles. One among many prerequisites for a successful carrier system for nucleic acids is high stability in the extracellular environment. In addition, the biological processes that elicit the cellular uptake and intracellular processing accompanied by an efficient release of the cargo in the intracellular compartment of these gene delivery systems have to be well understood. A promising strategy to create such an interactive delivery system is to exploit the various biological stimuli. With greater understanding of physiological differences between normal and disease tissues and advances in material design, there is an opportunity to develop nano-carrier systems for target-specific drug and gene delivery that will respond to the local stimuli [Qiao et al., 2010; Mastrobattista et al., 2006]. At present, many examples of versatile, self-assembling nano-particles for the delivery of DNA can be found in literature and this number is continuously growing. In this section, emphasis is placed on the functional components that are needed for effective gene delivery and also the biological stimuli such as pH and redox potential for the synthesis of multi-functional intelligent delivery systems. Briefly, Table 1 indicates these functional carriers.
7.1. Bio-compatible, bio-degradable nano-carriers
The artificial virus should preferably be constructed from materials that are biocompatible and biodegradable to prevent carrier-induced toxicities and the accumulation of carrier components in the body. In general, lipids are well tolerated. Synthetic polymers, on the other hand, have shown to induce some cytotoxicity
|Bio-compatible, biodegradable nanocarrier||Synthetic polymers:|
Poly (D, L-lactide-coglycolide), Poly (L-lactic acid),
Gelatin, Chitosan, Hyaluronic acid
|van Vlerken et al., 2007; Mastrobattista et al., 2006|
|Packaging nucleic acids into compact nanoparticles||Polyethyleimine (PEI),|
|Wagner, 2011; Walker et al., 2005|
|Long-circulation of nanocarrier||Polysaccharides, Polyacrylamide,|
PEG [PEGylated polyplexes]
|van Vlerken et al., 2007; Mastrobattista et al., 2006|
|Targeting molecules for the development of targeted NPs||Monoclonal antibody, Affibody, Aptamer, Oligopeptides, Growth factor, Vitamin||Wang et al., 2011; Wagner, 2011; Mastrobattista et al., 2006; Gu et al., 2007; van Vlerken et al., 2007; Peer et al., 2007; Alexis et al., 2008; Farokhzad et al., 2006; Zhang et al., 2001; Askoxylakis et al., 2005; Soudy et al., 2011; Kim et al., 2006; Ogris et al., 2003|
|Intracellular delivery||Escape from the endosomal compartment:|
Influenza HA2 peptide,
T-domain of the diphtheria toxin, GALA peptide,
pH-sensitive polymers: PEI
Cytosolic un-packaging: Reduction-sensitive polyplexes
SV40 from the large tumor antigen Simian virus 40,
M9 from nuclear ribonucleoprotein
|Eliyahu et al., 2005; Shim and Kwon, 2012; Vercauteren et al., 2012; Du et al., 2010; Schaffer et al., 2000; Bauhuber et al., 2009|
Polymeric nano-particles offer significant advantages over other nano-carrier platforms primarily since a remarkable flexibility in polymer matrices allows for tailoring of the nano-particle properties to meet the specific planed need. Other advantages of polymeric nano-particles include ease of production, ease of surface modification, encapsulation efficiency of the payload, payload protection, large area-to-volume, slow or fast polymer degradation and stimuli-responsive polymer erosion for temporal control over the release of drugs and feasibility of scale-up and manufacturing. Some examples of the most commonly used polymers for nano-carriers include the synthetic polymers such as poly (D, L-lactide-coglycolide) (PLGA), poly (L-lactic acid) (PLL), poly (epsiloncaprolactone) (PCL), poly (alkylcyanoacrylates) and natural polymers such as gelatin, chitosan and hyaluronic acid [van Vlerken et al., 2007; Mastrobattista et al., 2006].
7.2. Packaging nucleic acids into compact nano-particles
A prerequisite for every systemic nucleic-acid delivery system is stability in the blood stream prior to reaching its target cell. Therefore, the carrier must prevent the premature release of its load. Among the different delivery systems, nano-sized drug carriers are receiving considerable attention. Through precise selection of candidate therapeutics and appropriate functionalization of the nano-carrier systems, it is possible to develop fairly sophisticated multi-functional systems that can provide optimized anticancer therapy, a function that imparts particular use in intracellular delivery and sub-cellular localization of drugs. Reversible nucleic acid condensation by cationic proteins is a common natural process, e.g., in packaging of whole mammalian genomes into chromatin, or RNA into organelles. Compaction is also a key function of viral cores for protection against the degradative environment during infection. Nucleic acids as macromolecules are subjected to a variety of environmental factors such as pH or enzymes (e.g., nucleases) that can degrade or destroy them. Complexation of nucleic acids by cationic polymers or lipids is a widely used method to reduce their sizes and prevent their destruction by nucleases.
Reversibility is important; the delivered nucleic acid has to be accessible for subsequent transcription [Wagner, 2011]. Polyplexes are non-viral vectors consisting of DNA and polycations have shown potential in systemic targeted gene delivery in various animal models, but suffer from far lower gene transfer efficiency than viral vectors. To improve efficiency, researchers are trying to engineer synthetic vectors with virus-like qualities. For successful condensation of DNA into virus size particles, an excess of polycations is necessary, which results in a positive surface charge. The positive surface charge on particles has been shown to be generally advantageous for cell uptake [Walker et al., 2005]. Polyionic interactions, hydrogen bonding and hydrophobic interactions control the condensation of nucleic acids. In electrostatic complexes of plasmid DNA (pDNA) with polycations such as polylysine (pLys) or polyethylenimine (PEI), neutralization of approximately 10,000 negative phosphate charges of one pDNA molecule by approximately 100 polycation molecules results in compaction into “polyplexes” with sizes of 20 to > 100 nm (depending on aggregation events). The polymer/ pDNA core may be regarded as the engine of the delivery vehicle; for efficient and specific delivery, like in natural viruses, additional domains for cell entry and endosomal escape are required [Fig.1A & B; Wagner, 2011].
7.3. Long-circulation of nanocarrier
Both naked DNA and lipoplexes have showed rapid hepatic clearance during systemic administration. The liver elimination of lipoplexes was due to phagocytosis by Kupffer cells. Absence of any hydrophilic surface group on the particles, may lead to their interaction with plasma proteins, opsonization and removal from the circulation. The mononuclear phagocytic system (MPS) plays a key role in systemic removal of hydrophobic particles. In addition to biodegradability and biocompatibility, the non-viral carrier should be ‘invisible’ to the innate and acquired immune system of the patient in order to prevent unwanted immune reactions against the carrier and, consequently, rapid clearance of carriers from the blood circulation after intravenous administration. This can be achieved by adding a hydrophilic coat around the carrier. The coat can consist of a lipid bilayer or hydrophilic polymers grafted onto the surface of the carriers [Mastrobattista et al., 2006]. Among several strategies to impart particles with stealth-shielding, including surface modification with polysaccharides, polyacrylamide, and polyvinyl alcohol, surface modification with PEG and PEG co-polymers proved to be most effective, fueling its wide-spread use. PEG has a general structure of HO–(CH2CH2O)n–CH2CH2–OH, including a polyether backbone that is chemically inert, with terminal hydroxyl groups that can be activated for conjugation to different types of polymers and drugs. PEG offers the advantage that it is non-toxic and non-immunogenic, leading to approval by the United States Food and Drug Administration (FDA) for internal use in humans and inclusion in the list of inactive ingredients for oral and parenteral applications [Fig. 1 A & B; van Vlerken et al., 2007]. The protective (stealth) action of PEG is mainly due to the formation of a dense, hydrophilic cloud of long flexible chains on the surface of the colloidal particle that reduces the hydrophobic interactions with the RES. The chemically anchored PEG chains can undergo spatial conformations, thus preventing the opsonization of particles by the macrophages of the RES, which leads to preferential accumulation in the liver and spleen. PEG surface modification, therefore, enhances the circulation time of molecules and colloidal particles in the blood. The mechanism of steric hindrance by the PEG modified surface has been thoroughly examined. The water molecules form a structured shell through hydrogen bonding to the ether oxygen molecules of PEG. The tightly bound water forms a hydrated film around the particle and prevents the protein interactions. In addition, PEG surface modification may also increase the hydrodynamic size of the particle decreasing its clearance, a process that is dependent on the molecular size as well as particle volume. Ultimately, this helps in greatly increasing circulation half-life of the particles [van Vlerken et al., 2007]. In cancer therapy, PEGylated polyplexes with elongated plasma circulation may take advantage of the “enhanced permeability and retention” (EPR) effect. Long-term circulating nano-particles can extravasate and passively accumulate at tumor sites due to the leakiness of tumor vessels and ineffective lymphatic efflux (“passive tumor targeting”) [Wagner, 2011].
7.4. Targeting molecules for the development of targeted NPs
Appropriate packaging of nucleic acids and the use of PEG as a shield can help the complex survive within the circulation without being degraded or taken up by the mononuclear phagocyte system MPS. However, the next challenge for a PEGylated gene complex is to specifically target to cells or tissue of interest. By taking advantage of increased expression levels of receptors or antigens in diseased conditions, such as cancer, gene complexes can be targeted using specific ligands, such as antibodies, peptides, proteins, small molecules and RNA aptamer that recognize and bind to the cells of interest, resulting in high transfection efficiency [Wang et al., 2011].
Viruses are optimized for surviving in the relevant body fluids. Their surface is decorated with ligands for attachment to their target cell surface receptors. Often they use more than one receptor type for intracellular uptake into host cells. In design of synthetic nano-particles, such multivalent recognition and cell uptake mechanisms for nucleic acid delivery can be utilized [Wagner, 2011]. A simultaneous effect of the surface modification of gene carriers are that the positive surface charges are shielded, which significantly reduces the non-specific adsorption onto cell membranes. This enables targeting of the gene carriers towards specific cell types by conjugating ligands to the hydrophilic coat around the gene carrier that specifically bind internalizing cell-surface receptors. In this way, delivery of the transgene and subsequent expression can be restricted to target cells. Several different types of targeting ligands have been used for this purpose, including peptides, antibodies and vitamins. If targeting ligands are directed towards internalizing receptors, receptor binding will lead to receptor-mediated endocytosis of the targeted gene carriers, as they are small enough (< 200-300 nm). This route of uptake is to be preferred as it ensures intracellular accumulation of gene carriers in a receptor-specific way [Mastrobattista et al., 2006]. While it has been demonstrated that PEG surface modification of nano-carriers causes a greater accumulation of drug at the tumor-site by passive targeting, active targeting of the carrier can help in selection of the target cell-type within the tumor site and internalization of the nano-particles to a greater extent inside the target cells. Active targeting can be achieved by the functionalization of NPs with ligands such as proteins (mainly antibodies and their fragments), nucleic acids (aptamers), or other receptor ligands (peptides, carbohydrates and vitamins) [Gu et al., 2007]. Regardless of the targeting moiety, the principle outcome is essentially the same, mainly improved tumor cell recognition, improved tumor cell uptake, and reduced recognition at non-specific sites. PEG surface modification provides an advantage whereby the terminal groups of PEG can be functionalized to reactive groups for covalent coupling. Most commonly, PEG is functionalized to reactive carboxylic acids, amine, or sulfhydryl groups, allowing for efficient covalent attachment of the wide variety of targeting ligands by amide bonding or disulfide bridge formation [van Vlerken et al., 2007]. In this section, we describe some classes of targeting molecules.
7.4.1. Monoclonal antibodies
Monoclonal antibodies (mAb) are the first and are still the preferred class of targeting molecules. The mAb Rituximab was approved by the FDA for treating B-cell lymphoma in 1997. Another successful therapeutic mAb is Trastuzumab (Herceptin), an anti-HER2 mAb which binds to ErbB2 receptors and was approved by the FDA for treating breast cancer in 1998. Cetuximab, which binds to epidermal growth factor receptors (EGFR), was approved for treating colorectal cancer in 2004 and head/neck cancer in 2006. Bevacizumab, a tumor angiogenesis inhibitor that binds to vascular endothelial growth factor (VEGF), was approved for treating colorectal cancer in 2004. Trastuzumab and rituximab have been conjugated to poly (lactic acid) (PLA) NPs resulting in conjugates that exhibit a six-fold increase in the rate of particle uptake compared with similar particles lacking the mAb targeting. Today, over 200 delivery systems based on antibodies or their fragments are in preclinical and clinical trials. Antibodies may be used in their native state or as fragments for targeting [Imai and Takaoka, 2006; Peer et al., 2007].
7.4.2. Affibodies as targeting ligands
In recent times, a novel class of small molecules called “affibodies,” which can be considered antibody mimics, have been examined for targeting. Affibodies are a class of polypeptide ligands that are potential candidates for tissue-specific targeting of drug-encapsulated controlled release polymeric nanoparticles. Affibody molecules are relatively small proteins (6-8 kDa) that offer the advantage of being extremely stable, highly soluble, and readily expressed in bacterial systems or produced by peptide synthesis. The binding affinities of affibody molecules are considerably higher compared with the corresponding antibodies. The binding pocket of an affibody is composed of 13 amino acids, which can be randomized to bind a variety of targets. In contrast to monoclonal antibody, affibody has following advantages as a targeting ligand. First, the small size of affibody (MW: 6 kDa) guarantees its tissue/cell penetration ability. Second, its functional end groups for chemical conjugation are distanced from its binding site. Moreover, affibody has a robust structure, and can be easily synthesized in a large-scale manner. All of these advantages make the affibody a valuable ligand for targeted drug delivery [Alexis et al., 2008; Manjappa et al., 2011]. Recently, anti-HER2 affibody was also employed as a targeting ligand for nano-scaled drug delivery systems. Alexis et al. conjugated the anti-HER2 affibody to poly-(D, L-lactic acid)-poly (ethylene glycol)-maleimide (PLA-PEG-Mal) copolymer for targeted delivery to cells that over-express the HER-2 antigen [Alexis et al., 2008].
7.4.3. Aptamer targeting molecules
A novel class of molecules, referred to as nucleic acid ligands (aptamers), has been developed to generate targeting agents. Aptamers are short single-stranded DNA or RNA oligonucleotides or modified DNA or RNA oligonucleotides that fold by intramolecular interaction into unique conformations with ligand-binding characteristics. Like antibodies, aptamers can be prepared to bind target antigens with high specificity and affinity. The use of aptamers as targeting molecules has several potential advantages over antibodies. Aptamers with high affinity for a target can be prepared through
7.4.4. Oligopeptide-based targeting molecules
Recently, a number of tumor homing peptides have been reported that specifically target cancer cells and show promising results for tumor targeted drug delivery. Peptides, being smaller than other targeting ligands, have excellent tissue penetration properties and can be easily conjugated to drugs and oligonucleotides by chemical synthesis. Peptides are nearly invisible to the immune system and are not taken up in the reticuloendothelial system like antibodies and so are expected to cause minimal or no side effects to bone marrow, liver, and spleen [Gu et al., 2007]. For example, Cilengitide® is a cyclic arginine-glycine-aspartic acid (RGD) peptide that binds to the cell adhesion integrin αvβ3 on endothelial cells results in increased intracellular drug delivery in different murine tumor models and is currently in phase II clinical trials for the treatment of non-small cell lung cancer and pancreatic cancer. Despite the success mentioned, RGD-targeted therapy still encounters many challenges. First challenge is the limitation associated with the non-specific adhesive nature of the RGD-integrin targeting system. Integrins are extracellular receptors that are not only expressed on cancer cells but also on nearly all epithelial cells and are therefore not cancer specific. Recent development of phage display screening methods has successfully isolated peptide ligands with high specificity and affinity to cell-surface hormone receptors (LHRH receptors, somatostatin receptors) and tumor vasculature antigens [Gu et al., 2007]. One of those is a dodecapeptide identified through phage display by Zhang
7.4.5. Growth factor or vitamin-based targeting molecules
Growth factor or vitamin interactions with cancer cells represent a commonly used targeting strategy, as cancer cells over-express the receptors for nutrition to maintain their fast-growing metabolism. Epidermal growth factor (EGF) has been shown to block and reduce tumor expression of the EGF receptor, which is over-expressed in a variety of tumor cells such as breast and tongue cancer [Peer et al., 2007].
One of the most extensively studied small molecule targeting moieties for drug delivery is folic acid (folate). The high-affinity vitamin is a commonly used ligand for cancer targeting because folate receptors (FRs) are frequently over-expressed in a range of tumor cells. It has been used as a targeting moiety combined with a wide array of drug delivery vehicles including liposomes, protein toxins, polymeric NPs, linear polymers, and dendrimers to deliver drugs selectively into cancer cells using FR-mediated endocytosis [Gu et al., 2007]. The folic acid-PEGylated PEI polyplex was evaluated as a gene carrier, and successfully delivered siRNA and pDNA to tumour cells [Kim et al., 2006].
Transferrin (Tf), an iron-binding glycoprotein interacts with Tf receptors (TfRs), which are overexpressed on a variety of tumor cells (including pancreatic, colon, lung, and bladder cancer) owing to increased metabolic rates. Direct coupling of these targeting agents to nano-carriers has improved intracellular delivery and therapeutic outcome in animal models. Tf-linked PEG-PEI was developed for tumor-selective gene delivery. The surface charge of the complexes was shielded by either PEG or a higher density of linked Tf to block undesired non-specific interactions with blood components, followed by selective targeting to tumor cells. This approach resulted in a 100-fold higher gene expression in tumor cells compared with other tissues [Ogris et al., 2003].
One challenge with targeting receptors whose expression correlates with metabolic rate, such as folate and Tf, is that these receptors are also expressed in fast growing healthy cells such as fibroblasts, epithelial and endothelial cells. Therefore, NP delivery system needs to be further refined and tuned to increase tumor selectivity [Gu et al., 2007; Peer et al., 2007].
7.5. Intracellular delivery
Non-viral vectors have to overcome multiple intracellular barriers subsequently after cellular uptake usually via endocytosis. After cellular internalization, a critical intracellular obstacle to non-viral gene delivery is degradation in the endosome/lysosome where nucleic acids are easily degraded by a mildly acidic pH and acid activated enzymes. The ability of non-viral vectors to release the nucleic acids in their intracellular targets, while surviving through low pH and digestive processes and avoiding unwanted premature decomplexation, is a key efficiency-determining requirement. The most widely believed theory in designing non-viral vectors for efficient endosomal escape is employing the “proton sponge effect” [Eliyahu et al., 2005]. Importantly, intracellular targets, where the nucleic acids are released from the vector, determine the overall gene delivery efficiency because the action sites are different for nucleic acid types. For example, plasmid DNA must be localized in the nucleus, where gene expression is initiated by transcription. However, nuclear entry is one of the major obstacles to non-viral gene delivery although nuclear translocation of plasmid DNA is crucial to achieve desired transfection efficiency. Moreover, the nuclear translocation mechanism of plasmid DNA in the cytoplasm has not yet been fully elucidated. Passive diffusion of macromolecules (e.g., plasmid DNA) in the cytoplasm is restricted by a complex network of microtubules, proteins, and various sub-cellular organelles. One explanation is the nuclear localization of plasmid DNA during cellular mitosis when the nuclear envelope disassembles. For non-dividing cells, the transport of polyplexes into the nucleus occurs only via an active transport mechanism through a nuclear pore complex that prevents molecules larger than 40 kDa from passively diffusing into the nucleus. In contrast to plasmid DNA delivery, it is obvious that transnuclear localization of siRNA should be prevented to achieve efficient RNA interference (RNAi) since siRNA acts in the cytosol, where it targets mRNA with matching sequences [Shim and Kwon, 2012]. As nano-medicines often require delivery of their therapeutic payload to specific sub-cellular locations, knowledge about intracellular trafficking might prove useful for the control of the intracellular processing of nano-particles and lead to optimization of their design [Vercauteren et al., 2012].
7.5.1. Escape from the endosomal compartment
Following internalization of peptide-DNA condensates by endocytosis, the polyplex must be able to escape the endosome so that the DNA can be delivered to the nucleus for gene expression. After endocytosis, entrapment of the vector within the acidified vesicles of the endosomal/lysosomal system is a critical barrier to non-viral gene delivery systems. Unfortunately, the environment of the lysosomal interior is harmful for nucleic acid (NA) integrity, unless the carrier offers sufficient protection against the degradation by the acid hydrolases [Vercauteren et al., 2012]. Viruses and some pathogenic bacteria have pH-sensitive surface proteins that change conformation in mildly acidic environments such as in endosomes, and exhibit membrane-disruptive (fusogenic or endosomolytic) properties. Synthetic fusogenic peptides that mimic the sequences of these natural proteins have been confirmed to increase cytoplasmic gene delivery [Du et al., 2010]. Since escape from the endosomes is essential for efficient NA therapy, much attention has been paid to this issue, resulting in a variety of endosomolytic carriers. For example, cationic and pH-responsive lipids can be added to phospholipid carriers to assist in releasing the NAs into the cytoplasm by destabilizing the endosomal lipid bilayer in the acidic environment of the endolysosomes. Another approach is based on the coupling of fusogenic peptides to the carriers, which undergo conformational changes after their exposure to the decreasing endosomal pH values, exposing hydrophobic faces of the fusion peptide which destabilizes the endosomal membrane. Examples of these endosome-disruptive peptides are the influenza HA2 peptide, melittin, the T-domain of the diphtheria toxin or the GALA peptide [Vercauteren et al., 2012]. In an effort to translate the proton sponge activity to gene delivery peptides, histidine has been added to peptide sequences. The imidazole group of histidine has a pKa of ~ 6.0, therefore allowing it to become protonated in the acidic environment of the endosome. At physiological pH the histidines will remain neutrally charged, thereby imparting selective membrane disruption in the acidic endosome. In the past two decades, synthetic pH-sensitive polymers as endosomolytic agents have attracted great interest due to their low or non immunogenicity, which is a concern of using fusogenic proteins or peptides. Polymers such as PEI contain several secondary amines that are easily protonated in the acidic environment of the endosome. As protons are pumped in, PEI absorbs the protons leading to endosomal swelling and membrane disruption. In general, these smart polymers have both hydrophobic parts and weakly acidic groups, which afford them pH-dependent endosomolytic properties. At physiological pH, the polymers have little endosomolytic activity but undergo a conformational change at endosomal pH and show membrane-disruptive properties. This provides the polymers with reduced toxicity to the other biomembranes at neutral pH, but with the ability to facilitate endosomal escape [Fig. 1A; Du et al., 2010].
7.5.2. Cytosolic un-packaging
The final step of the gene delivery process, un-packaging of the polyplex, can limit the efficiency of gene delivery and expression. For
7.5.3. Nuclear import
The nuclear envelope that separates the cell’s genetic material from the surrounding cytoplasm represents a physical barrier for nuclear import of macromolecules such as pDNA. The nuclear envelope contains openings in the form of nuclear pore complexes, which allow free diffusion of molecules up to 50 kDa, corresponding to a hydrodynamic diameter of approximately 10 nm. There are several lines of evidence showing that nuclear import is a rate-limiting step for transfection of pDNA. Nuclear import of pDNA may be more challenging for transfection of non-dividing cells. Indeed, non-dividing cells showed a 90% lower expression level as compared to actively dividing cells. Strategies for nuclear import of genes have been developed following further elucidation of the endogenous nuclear import machinery [Wang et al., 2011]. In nature, large molecules with sizes up to 30 nm in diameter that require trans-nuclear transport contain nuclear localization signals (NLS) that are recognized by nuclear transport receptors like importins or transportins, and the whole complex is thereafter actively transported through NPCs [Vercauteren et al., 2012]. The nuclear localization sequence is a major player that shuttles protein–plasmid complexes through the nuclear pore. NLS-mediated active nuclear translocation involves a process starting from its interaction with cytoplasmic importins to binding of the NLS to the nuclear pore complex and the passage through the pore. Identification of the NLSs, such as SV40 from the larger tumor antigen Simian virus 40 and M9 from nuclear ribonucleoprotein, enabled design of non-viral gene vectors with nuclear targeting properties [Wang et al., 2011].
8. Designing switchable nanosystems for medical application
To enhance therapeutic efficacy while minimizing side effects, a large number of nanomaterial based platforms have been developed that allow simple delivery of genes. Based on important earlier work in the field of liposomal gene delivery and inorganic nanomaterials, the last decade has brought a broad array of new and improved nanoscale carrier platforms such as biodegradable and non-degradable polymers, dendrimers, carbon nanotubes, metallic and organic nanoparticles, quantum dots, nanogels or peptidic nanoparticles. Ideally, gene vectors should be capable of self-assembly with nucleic acids and accommodate with any type of nucleic acid or their combination. They should also target cells of interest, escape from endosomes and/or transport into nuclei. A viable gene vector for systemic delivery needs to minimize toxicity and phagocytosis and avoid non-specific interactions and self-aggregation. For potential gene delivery applications, ideal gene carriers need to combine both the targeting property and the stimulus responsiveness to enhance the bioavailability of the gene as well as to reduce the side effects. Therefore, designing stimulus-responsive nanoparticles for programmed gene delivery, which release the gene on arrival at the targeted site, is highly desired. Stimulus-responsive nanoparticles produce physical or chemical changes when subjected to external signals, including variations of macromolecular structures, solubility, surface properties, swelling and dissociation [Lehner et al., 2012]. Stimulus-responsive nanoparticles can be classified based on the type of stimulus as internally and externally controllable materials. Internal stimuli (e.g. activation by pH, redox potential, enzymes) might be controlled by a molecular mechanism highly specific for a disease and therefore improve on targeting properties. However, absolutely disease-specific internal molecular triggers are difficult to find for certain diseases. External stimuli like light, ultrasound, electromagnetic fields or ionizing radiation have the advantage of being focusable on certain body areas. This may be a significant advantage where a target cell is strongly involved in pathogenesis at one location (e.g., cancer stem cells in a cancer tissue), but of vital importance in other locations (e.g., stem cells in the bone marrow). A key challenge in externally controlled nanomaterials is tissue penetration and avoidance of undesired tissue damage in the radiation path from radiation source to target tissue. The ease of temporal control in external stimuli may represent a particular advantage for certain applications [Lehner et al., 2012]. Herein, we focus on the use of internal and biological stimuli that can be used to incorporate switch functionality into such nanocarriers and describe the clinical experience with various nanosize carrier systems as a basis for the design of new, improved, functional and “intelligent” nanosystems for gene delivery.
8.1. pH differences for stimuli-responsive delivery
The main property that is the basis of utilization of pH responsive polymers in gene delivery is the significant change in pH value within the cellular compartments. There are numerous pH gradients in physiological and pathological processes [Du et al., 2010]. The pH profile of pathological tissues, such as inflammation, infection and cancer, is significantly different from that of the normal tissue. The pH at systemic sites of infections, primary tumors, and metastasized tumors is lower than the pH of normal tissue. The pH, surface charge and density of low density lipoprotein receptors are the factors that show notable differences among the normal and tumor tissues. All these properties are known to influence the drugs’ physicochemical properties and are exploited for enhanced delivery to the target site. The extracellular pH values of solid tumor are in general slightly lower than in blood or other normal tissues. Since tumors proliferate very rapidly, the vasculature of tumor is often insufficient to supply enough nutritional and oxygen needs for the expanding population of tumor cells. This results in difference in metabolic environment between the various solid tumors and the surrounding normal tissue. The insufficient oxygen in tumor leads to hypoxia and causes production of lactic acid and hydrolysis of ATP in an energy-deficient environment contributes to an acidic microenvironment, which has been found in many tumors. Most of the solid tumors have lower extracellular pH (6.5) than the surrounding tissues (pH= 7.5). The pH is compartmentalized in tumor tissue into an intracellular component (pHi), which is similar in tumor and normal tissues and an extracellular component (pHe), which is relatively acidic in tumors [Ganta et al., 2008]. This behavior can be utilized for the preparation of stimuli responsive drug or gene-delivery systems, which can exploit the biochemical properties at the contaminated site for targeted delivery. Cellular components such as the cytoplasm, endosomes, lysosomes, endoplasmic reticulum, golgi bodies, mitochondria and nuclei are known to maintain their own characteristic pH values. It is well known that the lower pH values are found in endosomes (5.5-6.0) and lysosomes (4.5-5.5). On the basis of these discoveries, various pH/acid-sensitive polymers have been developed as carriers for pDNA, ODN or siRNA delivery [Du et al., 2010; Ganta et al., 2008].
8.1.1. Low pH-sensitive reversible shielding/masking
This part focuses on the design and synthesis of polymeric carriers that can condense a large dose of therapeutic nucleic acids into particles that can sense the differences in environmental pH. The pH-modulated or self-regulated polymers are designed to use these pH differences by incorporating appropriate structural or functional features into the basic scaffold of the polymers to improve the efficacy of gene delivery. In general, polycations (PCs), such as PEI or dendrimers (polyplexes) or with cationic lipids (lipoplexes) with positively charged surfaces are preferred for gene delivery in cell culture [Du et al., 2010]. Although, these positively charged particles are favorable for gene transfer efficiency
Walker and colleagues reported reversible shielding of polyplexes with pH-triggered deshielding properties, enabled by conjugating PEG to the polycations poly-L-lysine (PLL) and PEI via a pH-sensitive hydrazone bond (PC-HZN-PEG) of acyl hydrazides or 2-pyridyl hydrazines. The reversibility of the hydrazone bond within conjugates was determined at physiological and endosomal acidic pH, identifying suitable linker systems for endosomal deshielding. The polyplexes with the acid-sensitive linkages showed much higher plasmid gene delivery efficiencies (1-2 orders of magnitude) than those with stable linkages, both
In 2007, Knorr
8.1.2. pH-dependent endosomolytic polymers
Following internalization of lipoplexes or polyplexes via the endocytic pathway, endosomal entrapment and subsequent lysosomal degradation are a major blockage that limits the efficiency of gene delivery. After cellular uptake of the gene carriers, the carrier should escape from the endosomal compartment in order to reach the cytosol and then, the nucleus. This requires dissociation of the internalized carrier from the receptors that triggered the internalization process. In addition, the membranes of the endosomes should be destabilized to allow translocation of the carriers into the cytosol [Mastrobattista et al., 2006]. Identification of endosomolytic or fusogenic components and their integration into non-viral gene delivery systems are major strategies being exploited to facilitate endosomal escape.
In the case of polyplexes, PEI and polyamidoamine (PAMAM) are two representative cationic polymers with a high efficiency of gene transfer due in part to their capability to facilitate endosomal escape. A “proton sponge effect” provides a sound explanation for the intrinsic endosomolytic activity. Upon PEI-based or PAMAM-based polyplex entry into acidic endosomes, the polymer behaves as a sponge that absorbs protons as a result of protonation of the polymer-containing amine groups (primary, secondary and tertiary). Accumulation of protons subsequently drives an influx of counter chloride ion into endosomes, leading to increased osmotic pressure and subsequent flow of water into the endosomal interior and eventually swells and ruptures endosomal membrane [Eliyahu et al., 2005].
PEIs are available in a wide range of molecular weights (MW) from 423 Da to 800 kDa and with different branching degree (from linear to branched). Generally, high MW, branched PEIs have high transfection efficiency but also high toxicity due to high cationic charge. By contrast, low MW PEIs are less toxic but less efficient as gene delivery agents. Many efforts have been directed towards creating PEI derivatives combining higher transfection efficacy and good biocompatibility. One approach to reduce the cytotoxicity, biodegradable polyethylenimine with imine linkages as acid-labile moieties were synthesized and investigated for pDNA delivery. The half-life of the acid-labile PEI was 1.1 h at pH= 4.5 and 118 h at pH= 7.4, suggesting that the acid-labile PEI may be rapidly degraded into non-toxic low molecular weight PEI in acidic endosome. Acid-labile PEIs showed close transfection efficiency to PEI 25KDa, but much less toxicity due to the degradation of acid-labile linkage. Therefore, the acid-labile PEIs may be useful for the development of a non-toxic polymeric gene carrier [Kim et al., 2005].
The cationic lipids have been shown to destabilize the endosomal membrane. Addition of dioleoyl phosphatidylethanolamine (DOPE) or cholesteryl hemisuccinate (CHEMS) to non-viral vectors affected the pH-sensitivity of the formulations. The mechanism behind the phenomenon, is that electrical interaction between cationic lipids and anion endosomal membranes results in the formation of ion-pairs that promote the formation of the inverted hexagonal (HII) phase and disrupt endosomal membrane. Many preparations of lipoplexes contain DOPE as a helper lipid for fusogenic functionality. The ability of DOPE to destabilize endosomal membranes is based on its propensity to acquire an inverted hexagonal phase (HII). DOPE has a small cross-section head group and a large hydrocarbon area that favors a non-bilayer structure with a cone shape that facilitates the destabilization of endosomal membranes and gene transfection. The pH-responsive component, CHEMS, being negatively charged at neutral pH stabilizes the bilayer structure, however, at acidic pH it becomes protonated and loses its stabilization property [Ma et al., 2007; Wu and Zhao, 2007].
Another approach to reduce the cytotoxicity of PEI is modification with hydrophobic moieties, such as lipids. Since lipids are the main component of cell membrane, modification with hydrophobic moieties may result in additional hydrophobic interaction between polyplexes and cell membranes, which in turn would facilitate the delivery of a payload into cells. Various hydrophobic modifications have been tried, including modification with cholesterol, myristate, dodecyl iodide, hexadecyl iodide, palmitic acid, oleic acid, stearic acid, and phosphatidylcholine. In a recent study, Sawant
In order to take the advantages of both polycationic polymers and liposomes, Nie
The endosome-escape potential of poly-histidine increases their use for delivery of nucleic acids. The imidazole ring within histidine is a major component. Under the action of an acidic endosomal interior, the weak basic nature of the imidazole ring with pKa around 6 allows its protonation and acquires cationic charges which trigger the destabilization of endosomal membranes. Accumulation of histidine residues within endosomes could elicit a proton sponge effect and destroy endosomes as a result of their increased osmolarity. Both chemistry conjugation and genetic engineering have produced a series of histidine-rich polymers and peptides as well as lipids with imidazole, imidazolinium or imidazolium polar heads. These histidylated carriers have been used to deliver nucleic acids including pDNA, mRNA or siRNA duplex
The histidine-rich peptide H5WYG is a derivative of the N-terminal sequence of the HA-2 subunit of the
In an attempt to facilitate endosome escape, many strategies have been developed to mimic the viral mechanism for endosome destabilization. Viruses have acquired efficient solutions for escaping from the maturating acidifying endosomes. For example, glycoproteins of enveloped viruses contain hidden fusion peptides which are exposed after endocytosis, to trigger fusion of the viral with the endosomal membrane [Wagner, 2011]. It is well-established that the influenza virus utilizes the pH-sensitive membrane-destabilizing Hemagglutinin protein (HA2) displayed on the viral coat to disrupt the endosomal membrane and enter the cytoplasm. Hemagglutinin and other fusion proteins are characterized by their unique ability to switch from an ionized and hydrophilic conformation at physiologic pH to a hydrophobic and membrane-active one in response to acidic endosomal pH gradients, which destabilizes the endosomal membrane leading to leakage of endosomal contents into the cytoplasm [Lin et al., 2010]. At neutral pH, the HA2 subunit adapts a non-helical conformation due to charge repulsion arising from ionization of glutamic and aspartic acid residues. Within the interior of the acidic endosomal compartment, however, the HA2 subunit transitions into a stable helical secondary structure due to the protonation of glutamic and aspartic acids. The hydrophobic and hydrophilic faces of the helical conformation favor endosomal membrane destabilization.
Endocytosed non-enveloped viruses such as
Synthetic peptides mimicking a virus’s fusogenic peptides have also been designed for delivery of nucleic acids. The amphipathic peptide, GALA, was synthesized with 30 amino acid residues with a repeated amino acid sequence (e.g., glutamic acid-alanine-leucine-alanine) that demonstrated pH sensitive fusogenic properties. A GALA peptide with a formulation based on DNA/cationic liposome/ Transferrin (Tf) complexes induced enhanced gene transfection. It is assumed that Tf-triggered internalization of the complexes by receptor-mediated endocytosis and the GALA peptide promoted endosomal destabilization and release of the genetic material into the cytoplasm [Park et al., 2010].
Modification of multifunctional envelope-type nano-devices (MENDs) with GALA peptide facilitating endosomal escape, leads to the enhanced transfection efficiency of pDNA and siRNA duplex
A cationic counterpart of GALA is the peptide KALA which is formed by substitution of the alanine of GALA with lysine and a decrease in content of glutamic acid. KALA was the first designed peptide that could bind DNA, destabilize membranes, and mediate significant gene delivery. KALA undergoes a pH-dependent amphipathic α-helix to random coil conformational change, when the environmental pH decreased from 7.5 to 5.0. KALA can deliver both ODN and pDNA into cells [Park et al., 2010].
Several groups have focused their efforts on the development of synthetic, polymeric carriers that mimic the endosomolytic properties of fusogenic proteins and enhance the cytoplasmic delivery of therapeutic macromolecules. Recently, the ability of natural phospholipids to self-assemble into organized membrane-enclosed structures has been mimicked by amphiphilic co-polymers. The combination of a hydrophobic monomer and an ionizable co-monomer that has a more hydrophilic nature is one of the interesting strategies that have been adopted frequently for pH-responsive gene delivery. A change in pH and subsequent adjustment in the net charge causes the phase transformation depending on the hydrophobic and hydrophilic balance of the copolymer. These synthetic amphiphiles form assemblies that are remarkably similar to biological analogues, such as vesicles. Polymeric amphiphiles have much higher molecular weights than phospholipids and can self-assemble into more entangled membranes, imparting improved mechanical properties to the final structure [Park et al., 2010]. Polymer vesicles or polymersomes can combine several different polymeric compositions provided that they have the correct hydrophile/hydrophobe ratio. Particularly interesting for biomedical applications, are those copolymers that combine hydrophobic blocks with the non-antigenic properties of either PEG or biomimetic poly (2-(methacryloyloxy) ethyl phosphorylcholine) (PMPC). Indeed, the macromolecular nature of such polymersomes offers several advantages as compared to low-molecular-mass amphiphilic systems such as liposomes. PEG or PMPC polymersomes can be decorated by denser and higher molecular mass hydrophilic polymeric corona, with consequent longer circulation times than more traditional delivery systems such as stealth liposomes and other nanoparticles. Typical examples are the copolymers of methyl methacrylate (MMA) with methacrylic acid (MAc) or dimethylaminoethyl methacrylate (DMAEMA). The MMA is the hydrophobic section while MAc is the hydrophilic part of the chains. MAc is more hydrophilic at high pH when the carboxylic groups (COOH) are deprotonated, but becomes more hydrophobic when the carboxylic groups are protonated. The phase change occurs around the pKa value of the carboxylic groups, which is around 4.5-5.5. The copolymers of MMA with DMAEMA, which are hydrophilic at low pH, when the amino groups are protonated, but more hydrophobic when the amino groups are deprotonated. The key feature of these polymers is their ability to directly enhance the intracellular delivery of DNA, by destabilizing biological membranes in response to pH changes within the vesicular compartment [Park et al., 2010].
These copolymers are characterized by their unique ability to “sense” the changes in environment pH where they undergo a change from a hydrophilic, stealth-like conformation at physiologic pH to a hydrophobic and membrane-destabilizing one in response to acidic endosomal pH gradients [Lin et al., 2010]. Poly(ethylacrylic acid) (PEAA) is the first reported polymer to display a pH-dependent disruption of synthetic lipid vesicles at acidic pH= 6.3 or lower. The family of poly (alkyl acrylic acid) like poly (methyl acrylic acid) (PMAA), poly (ethyl acrylic acid) (PEAA), poly (propyl acrylic acid) (PPAA), and poly (butyl acrylic acid) (PBAA) were provided with pH-dependent, membrane destabilizing activities. These polymers are hydrophilic and stealth-like at physiological pH, but become membrane-destabilizing after uptake into the endosomal compartment where they enhance the release of therapeutic cargo into the cytoplasm [Lin et al., 2010].
Diblock copolymers PMPC-PDPA as biomimetic polymersomes were used for gene delivery made of pH-sensitive poly (2-(methacryloyloxy) ethyl-phosphorylcholine)-co-poly (2-(diisopropylamino) ethyl-methacrylate) (PMPC-PDPA) diblock copolymers. The high biocompatibility features are ascribed to PMPC residue whereas the PDPA block imparts pH responsiveness. These diblock copolymers exist as stable vesicles at physiological pH but these vesicles rapidly dissociate at around pH= 5-6 to form unimers. The phase transition of vesicles to unimers takes place because of protonation of the tertiary amine groups of PDPA chains, which transforms hydrophobic PDPA chains into a hydrophilic entity from physiological pH to mild acidic conditions. pH-responsive PMPC-PDPA vesicles release their contents upon exposure to the low pH (5.5) media in endosomes or lysosomes [Lomas et al., 2007].
Comb-like diblock copolymers with a robust membrane-destabilizing activity in response to a mildly acidic pH in the endosome were also developed. Acid-cleavable hydrazone linker has also been frequently utilized to achieve the facilitated release of nucleic acids from polyplexes. The first pH-sensitive block was the copolymer of ethyl acrylic acid (EAA) and hydrophobic methacrylate, and the second block was the copolymer of hexyl methacrylate (HMA) and trimethyl aminoethyl methacrylate (TMAEMA) grafted with N-acryloxy succinimide or β-benzyl L-aspartate N-carboxy-anhydride via acid-cleavable hydrazone linkages. These comb-like polymers exhibited a high concentration-dependent hemolytic activity in acidic solutions and degraded into smaller fragments via acid-hydrolysis of hydrazone linkages, resulting in minimized toxicity and facilitated elimination by renal excretion
8.1.3. Low pH-sensitive siRNA/ODN–polymer conjugates
Small interfering RNA (siRNA)-based therapies have great potential for the treatment of diseases such as cancer, but an effective delivery strategy for siRNA is unclear. Benoit
Acid-degradable and targetable polyion complex (PIC) micelles increased the gene silencing in hepatoma cells. This multi-functional carrier was synthesized by assembling lactosylated-PEG-siRNA conjugates via acid-labile β-thiopropionate linkages into PIC micelles through the mixing with poly (L-lysine). The lactosylated-PEG-siRNA/PLL polyplexes were successfully transported into hepatoma cells in a receptor-mediated manner, releasing hundreds of active siRNA molecules into the cellular interior responding to the pH decrease in the endosomal compartment. This carrier exhibited almost 100 times enhancement in gene silencing activity and facilitating the practical utility of siRNA therapeutics [Oishi et al., 2005].
8.2. Redox-responsive nanocarriers
One of the several micro-environmental features, which have been widely exploited for improving the efficiency of nucleic acid delivery, is the redox potential gradient existing between extracellular environment and various subcellular organelles in normal as well as pathological states. The existence of a high redox potential gradient between oxidizing extracellular space and the reducing environment of subcellular organelles has been exploited mainly by incorporating a disulfide bond(s) into the structure of the delivery vectors to provide them with a capability to release the therapeutic nucleotides selectively in the subcellular reducing space. The original interest in gene delivery systems controlled by redox potential gradients was guided by the need to transiently enhance stability of the vectors during the delivery [Cheng et al., 2011; Meng et al., 2009].
The design of reduction-sensitive polymers and conjugates usually involves incorporation of disulfide linkage(s) in the main chain, at the side chain or the cross-linker in the structure of the polymers of either linear or branched structure. There are only a few examples of polyplexes where the disulfide bonds are associated with the nucleic acids [Soundara Manickam and Oupický, 2006]. Reduction-sensitive polymers and conjugates are characterized by an excellent stability in the circulation and in extracellular fluids, whereas they are prone to rapid degradation under a reductive environment present in intracellular compartments such as the cytoplasm and the cell nucleus at a time scale from minutes to hours, through thiol-disulfide exchange reactions. This quick-response chemical degradation behavior is distinct from common hydrolytically degradable polymers such as aliphatic polyesters and polycarbonates in which the ester and carbonate bonds usually exhibit gradual degradation kinetics inside body with degradation times ranging from days to weeks/or to months. This remarkable feature renders them extremely for the controlled cytoplasmic delivery of a variety of bioactive molecules including DNA, siRNA, antisense oligonucleotide (ODN), proteins, drugs, etc [Du et al., 2010].
Disulfide bonds present in the structure of polyplexes are readily reduced in the reducing intracellular environment, while largely preserved in the predominantly oxidizing extracellular space. The intracellular reduction of disulfide bonds is most likely mediated by small redox molecules like glutathione (GSH) and thioredoxin, either alone or with the help of redox enzymes. Glutathione tripeptide (g-glutamyl-cysteinyl-glycine; GSH) is the most abundant intracellular sulfhydryl present in millimolar concentrations inside the cell but only in micromolar concentrations in the blood plasma and GSH/glutathione disulfide (GSSG) is the major redox couple in animal cells [Meng et al., 2009]. Glutathione has multiple direct and indirect functions in many critical cellular processes like synthesis of proteins and DNA, amino acid transport, enzyme activity, metabolism and protection of cells. Glutathione also serves as a reductant by functioning to destroy the free radicals, hydrogen peroxide and other peroxides. It also functions as a storage form of cysteine. The intracellular glutathione concentration is an additive function of both the oxidized (GSSG) and the reduced forms (GSH) of glutathione. The glutathione redox ratio is maintained and determined by the activity of glutathione reductase, NADPH concentrations and transaldolase activity. The redox state of the GSH/GSSG couple is often used as an indicator of the overall redox environment of the cell. The large difference in reducing potential between the intracellular and extracellular milieu may be exploited for triggered intracellular delivery of a variety of bioactive molecules including DNA, siRNA, antisense oligonucleotide (ODN), proteins and low molecular weight drugs. Furthermore, of particular interest is that tumor tissues are highly reducing and hypoxic compared with normal tissues, with at least 4-fold higher concentrations of GSH in the tumor tissues over normal tissues, rendering the reducible bioconjugates valuable for tumor-specific drug and gene delivery [Meng et al., 2009]. Some examples are mentioned for the use of redox responsive nanocarrier in below:
8.2.1. The use of responsive sensitive nanocarrier for the stabilization of the carrier and decrease the cytotoxicity
A prerequisite for every systemic nucleic-acid delivery system is stability in the blood stream prior to reaching its target cell. Therefore, the carrier must prevent the premature release of its load. In order to enhance the stability of the delivery system, either the surface of the carriers was crosslinked, or the low-molecular-weight materials were sulfhydryl polymerized. Peptides containing many lysines and histidines were used for the complexation of nucleic acids, while cysteines were introduced to obtain stable and reductively degradable carriers. A series of different vehicles was built from sequences of many lysines, tryptophan, and a variable number of cysteines. All peptides obtained were able to condense DNA into particles, and exhibited increased stability after disulfide formation. The disulfide crosslinked carriers conveyed 5- to 60-fold higher gene expression as compared to their non-crosslinked analogs. The degree of gene expression was dependent on the number of incorporated cysteines, with a maximum for terminally inserted cysteines. The modification of the e-amino groups of pLL with 3-(2-aminoethyldithio) propionyl residues or their crosslinking with Dimethyl 3,3-dithiobispropionimidate (DTBP) comprises another approach utilizing reductively degradable gene carriers. These molecules were able to complex nucleotides into particles that were stable under physiological conditions but disintegrated upon treatment with GSH [Meng et al., 2009].
Low molecular weight DNA condensing polypeptides were developed by substituting one to four lysine residues of Cys-Trp-Lys18 (CWK18) with cysteine groups. These polypeptides could spontaneously oxidize to form interpeptide disulfide crosslinks after binding to plasmid DNA, resulting in small stabilized DNA condensates. These reversibly cross-linked polypeptide DNA condensates were 5-60-fold more potent at mediating gene expression in HepG2 and COS-7 cells as compared to the un-crosslinked alkylated CWK18 (AlkCWK18) DNA condensates [Fig. 2 A; McKenzie et al., 2000]. In another study, in order to improve the endosomolytic properties, Histidine containing reducible polycations based on CH6K3H6C monomers (His6 RPCs) was developed, which are highly effective DNA transfection agents, provided sufficient buffering capacity and enhanced
High molecular weight polypeptides containing disulfide bonds in the backbone were synthesized by an oxidative copolymerization of a histidine-rich peptide (HRP) and a nuclear localization sequence (NLS) peptide derived from the importin α-binding SV40 T antigen sequence. The synthetic approach allowed an easy synthesis of reducible copolypeptides (rCPP) with different relative contents of the HRP and NLS sequences. The rCPPs were synthesized by DMSO-mediated oxidative polycondensation. To combine two functional peptides into a single polymeric carrier, multiblock reducible copolypeptides were synthesized by randomly connecting HRP and NLS peptides via disulfide bonds into a linear polypeptide chain. Mild oxidation of the terminal Cys residues with DMSO was used. The copolypeptides show minimal cytotoxicity and transfection activity comparable to or better than control PEI polyplexes [Manickam and Oupický, 2006].
Other carriers, such as chitosan (deacetylated chitin) were also stabilized via disulfide bonds. For this purpose, the primary amino groups of low-molecular-weight chitosan were thiolated with 2-iminothiolane. These modified polymers, such as chitosanthiobutylamidines, were mixed with pDNA to form coacervates in the nanometer range [Bauhuber et al., 2009]. In a recent study, Ho
A dilemma in non-viral nucleic acid delivery is demonstrated by considering the polymeric transfection agent PEI, which is often referred to as the gold standard for polymer-based gene carriers due to the relatively high transfection efficacy of its polyplexes. Unfortunately, efficacy and adverse reactions seem to be strongly associated with the use of PEI. A popular strategy to reduce the toxicity of polyplexes is to use low MW polycations or cationic monomers that are less cytolytic, and crosslink them with agents that can be cleaved or activated by the intracellular environment. Numerous reports describe the synthesis of carrier systems containing disulfide bonds that allow for a compaction and protection of the nucleic acid in the extracellular environment accompanied by reduced toxicity due to intracellular polymer degradation. Peng
In another study, Son
8.2.2. Disulfides for the attachment of a shielding moiety
DOPE and N-[2-methoxypoly (ethylene glycol)-α-aminocarbonylethyl-dithiopropionate] formed a liposome that was covalently coupled to distearoylphosphatidylethanolamine (mPEG-SS-DSPE). The incorporation of the mPEG-SS-DSPE stabilized these liposomes at low pH, but the stabilizing effect was quickly reversed when the carrier was incubated with either DTT or cell-free extracts. Under these conditions, the disulfides were reduced, and thus the protecting PEG chains were cleaved, and the vehicle became degradable at low pH, releasing its load. Disulfides were used for the formation of copolymers consisting of bPEI 25 kDa and PEG of 20 or 30 kDa. The connection of PEG via DSPE led to a 125% increase in blood levels and decreased hemolysis compared to bPEI 25 kDa [Bauhuber et al., 2009].
Polyion complex (PIC) micelles are self-assembling particles with a core-shell structure formed by complexation between a pair of oppositely charged polymers having hydrophilic PEG segments. They can be utilized as gene delivery vectors with high water-solubility and colloidal stability, employing negatively charged genetic materials and cationic polymers. One approach to enhance the stability of PIC micelles is glutathione (GSH)-sensitive stabilization of PIC micelles with the core cross-linked through disulfide bonds, composed of antisense oligonucleotide and thiolated PEG-PLL block copolymer. The micelles showed sufficient colloidal stability due to the PEG shell and core cross-linking. Moreover, ODN entrapped in the micelles also displayed highly increased stability against nuclease, compared to that in the micelles without cross-linking. Release of ODN from the dissociated micelles at intracellular GSH concentration suggested the potential for intracellular ODN delivery. It is reported that PEG-peptide (PEG-SS-Cys-Trp-Lys(18): PEG-SS-CWK18) having disulfide cross-linker displayed much enhanced gene transfer efficiency compared to PEG-peptide having non-reducible cross-linker (PEG-VS-CWK18) when complexed with plasmid DNA [Kim and Kim, 2011].
8.2.3. Disulfides for the attachment of a targeting moiety
For a successful accumulation of the nucleic acid in the target cell, the delivery system requires the attachment of a specific “recognition element”. However, since many targeting moieties are rather large, they hinder effective unpacking of the gene vector. Thus, inside cells, it is very important to remove them to facilitate the disassembly of the load. Here, disulfides can be quite useful, as they can be cleaved at the cell surface during cellular entry or in the cytosol [Bauhuber et al., 2009]. The frequency of sulfhydryl occurrence in Antibodies/proteins or other molecules is usually low as compared to other groups like amines or carboxylates. The use of sulfhydryl reactive chemistries thus can restrict modification to only a limited number of sites within a target molecule. N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP) is one of the most popular heterobi-functional cross-linking agents. The NHS ester end of SPDP reacts with amine groups to form an amide linkage, while the 2-pyridyldithiol group at the other end can react with sulfhydryl residues to form a disulfide linkage. The reagent is also useful in creating sulfhydryls in proteins and other molecules. Once modified with SPDP, a protein can be treated with DTT (or other disulfide reducing agents, to release the pyridine-2-thione leaving group and form the free sulfhydryl [Manjappa et al., 2011].
Iminothiolane (Traut's reagent) can react with primary amines in a ring-opening reaction that regenerates the free sulfhydryl. An example is the thiolation of antibody using Traut's reagent in the preparation of immunoliposomes. It is an excellent thiolation reagent for use in the preparation of immunotoxins. It has also been used to modify and introduce sulfhydryls into oligosaccharides from asparagines linked glycans [Manjappa et al., 2011].
8.2.4. Disulfide bonds to enhance the intracellular release of the nucleic acid
SiRNA has generated great interest as a research tool and a therapeutic agent because of its ability to efficiently silence specific genes by the mechanism of RNA interference [Vidugirienė et al., 2007]. However, siRNA cannot penetrate the cellular membrane alone and can be easily degraded by RNase; therefore, effective siRNA carriers are needed for siRNA-based therapies. In this regard, poly ion complexes (PICs) formed from nucleic acids and oppositely charged polycations have been widely studied as a promising nucleic acids carrier because of the variety of chemical designs of polycations. PICs protect nucleic acids from enzymatic degradation and show facilitated cellular uptake. Thus, PICs have demonstrated to be useful for plasmid DNA (pDNA) delivery
The conjugation of nucleic acids including siRNA and antisense oligodeoxynucleotide to polymer such as PEG and hyaluronic acid through a disulfide bond represents a approach to construct GSH-responsive gene delivery systems [Cheng et al., 2011]. Lee
9. Stimuli-sensitive multi-functional nano-particulate nucleic acid delivery systems
As mentioned in the previous sections, there are some conflicting demands for overcoming different extra- and intracellular barriers met by nucleic acid-loaded nano-particles during delivery. The delivery systems, which response to only one stimulus and have less functionality, may not be efficient enough to achieve a satisfactory therapeutic effect
The ligands on the particle surface can be used to recognize a specific cell/tissue, and facilitate cellular uptake through receptor-mediated endocytosis. A reversibly removable hydrophilic pole, such as PEG chains, provides stealth protection of the nanoparticles during their circulation in the blood as well as travelling through the ECM, and may reduce toxicity. Moieties that are responsive to different stimulus must be combined to result in an appropriate set of various trigger mechanisms at different time points. Such moieties might be cleaved in acidic environments, show good buffering capacity, be redox active or enzymatically cleavable, the pole can be removed, which might enhance the cellular uptake and/or endosomal escape. In the inner part of the nanoparticles, nucleic acids can be temporally loaded through electrostatic interaction, covalent conjugation, or physical encapsulation, which will protect the nucleic acid against enzymatic degradation. The inner part should be stable enough until delivery to the correct site (cytoplasm and/or nucleus) where it is disassembled to release the naked nucleic acid upon some kind of stimulus. In addition, it is preferred that the inner part contains endosomolytic components that help the endosomal escape. If cell penetrating peptide (CPP) is incorporated onto the surface of the inner part, cellular uptake may be further improved [Du et al., 2010]. Many experimental results emphasize that the main obstruction of CPPs is the absence of specific cellular delivery [Vivès et al., 2008]. Ideally, the design of a smart delivery system should be built in such a way that during the first phase of delivery, a non-specific cell-penetrating function is shielded by the function of cell-recognition motif which favors the concentration of the drug at the targeted cell type. Upon accumulating in the target, protecting polymer (or specific ligand) attached to the surface of the smart nanocarrier via the stimuli-sensitive bond should detach under the action of local conditions and expose the previously hidden second function (CPP) allowing for the subsequent delivery of the carrier and its cargo inside cells [Torchilin, 2008]. Such engineering of smart nanodevices has been illustrated in the work by Sawant
Another example of a triggerable system was created by the synthesis of a liposomal carrier that was comprised of a membrane-permeable ligand and a reductively detachable PEG coating. The surface cover consisted of PEG coupled to DOPE via a thiolytically cleavable linker, while the ligand, an ariginine octamer, was immobilized onto cholesteryl hemisuccinate (CHEMS). These conjugates were mixed with dipalmitoylphosphatidylcholin (DPPC) and unmodified DOPE to form liposomes. DOPE/mPEG–DTP–DSPE liposomes were stable in plasma whereas in the presence of a reducing agent the PEG coating was effectively detached off the liposomal surface, leading to vesicle destabilization and fusion as well as complete release of the entrapped contents. Upon activation of the trigger, the arginine octamer would be exposed, causing cellular uptake of the liposome. Once in the cytosol, the remaining disulfide bonds would be cleaved, ultimately resulting in the destabilization the whole carrier [Fig. 2B; Bauhuber et al., 2009].
10. Nanoparticles mediated brain delivery systems
Nanoparticles have emerged as potential drug delivery carriers to tissues throughout the body. Yet passing the BBB is particularly difficult. The proper design of such engineered ‘nanocarriers’ becomes very important in transversing the impermeable membranes to facilitate drug delivery. At the same time, it is also required to retain the drug stability and ensure that early degradation of drugs from the nanocarriers does not take place. Therefore, for drugs to be successfully delivered to their target, many factors such as its size, biocompatibility, target specific affinity, avoidance of reticuloendothelial systems, stability in blood, or ability to facilitate controlled drug release need to be considered during manufacture of the NPs. As for nanocarriers to serve as good candidates for drug delivery across the BBB can be summarized as following [Bhaskar et al., 2010]: a) Particle diameter less than 100 nanometers; b) Non-toxic, biodegradable and biocompatible; c) Stable in blood (i.e., no opsonisation by proteins); d) BBB-targeted (i.e., use of cell surface, ligands, and receptor mediated endocytosis); e) No activation of neutrophils, non-inflammatory; f) No platelet aggregation; h) Avoidance of the reticuloendothelial systems; i) Prolonged circulation time; j) Scalable and cost effective with regard to manufacturing process; m) Agreeable to small molecules, peptides, proteins or nucleic acids; n) Controlled drug release or modulation of drug release profiles [Bhaskar et al., 2010].
One of the most important challenges in nano-based diagnostics and drug delivery is the functionalization of nanoparticles. At first, the combination of effective conjugation strategies is needed to develop, in a highly controlled way, specific biomolecules to the surface of nanoparticles. Some of the most prominent candidate biomolecules are cell penetrating peptides such as SynB vectors, penetratin and Tat that facilitate enhanced intracellular delivery, fluorescent dyes (rhodamine, alexa, Cy5.5), tumoral markers for brain and gene therapeutic agents for genetic therapy such as siRNA [Bhaskar et al., 2010].
Functionalization itself requires a profound knowledge of the target organ and its transport mechanisms. The BBB has several transport molecules that can potentially increase the efficiency and kinetics of nanocarriers towards brains such as, growth factors (e.g. epidermal growth factor, vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factors (IGF-I and -II), biotin binding proteins (avidin, streptavidin, or neutravidin), insulin, albumin, leptin, lactoferrin, iron binding protein p97 (melanotransferrin), transferrin and Angiopep-2. Some agents play a pivotal role in enhancing the permeability of nanoprobes through BBB. Moreover, by altering the surface of polymeric nanoparticles on coating them with different hydrophilic surfactants, such as polysorbate 80 (Tween® 80) or other polysorbates with 20 polyoxyethylene units, biocompatible coatings of non-viral gene delivery systems, e.g. by PEG attachment for siRNA delivery show significant advantage in brain targeting [Bhaskar et al., 2010].
11. Cell penetrating peptides as efficient delivery systems
Generally, the nucleic acid delivery techniques comprise various physical and chemical methods, viral and non-viral vector systems, and uptake of naked nucleic acids [Veldhoen et al., 2008; Bolhassani et al., 2011]. All of them have certain advantages and dis-advantages and might only be appropriate if particular requirements are performed. For instance, physical and chemical methods like microinjection, electroporation or particle bombardment as well as calcium phosphate co-precipitation are highly efficient but rather harmful for the target cells and lack the potential to be applicable
Peptides acting as shuttles for a controlled cellular delivery of nucleic acids represent a new concept to bypass the problem of poor bio-availability and clinical efficacy of such macromolecules [Veldhoen et al., 2008]. The idea of using peptides as carriers goes back some 20 years when two groups discovered by chance that the HIV-1 transactivating protein Tat is taken up by mammalian cells [Frankel and Pabo, 1998; Green and Loewenstein, 1988]. Just a few years later, the Antennapedia homeodomain of
Up to now numerous CPPs have been described. According to their origin, they can be grouped into three classes. The first group contains CPPs originating from naturally occurring proteins “protein derived CPPs”, the second consists of chimeric CPPs composed of different protein domains and the third class includes so-called “model CPPs” which were developed according to structure-fuction relationships without any homology to natural sequences. All known CPPs are basic amino acids causing a net positive charge at physiological pH [Langel, 2002].
At present, a peptide is considered a CPP, if it shows the ability to cross a biological membrane. A cargo can be bound to the CPP covalently or non-covalently. Covalent attachment can be achieved either by expression as a fusion construct or by chemical coupling [Zatsepin et al., 2005]. In some cases, cargo and carrier bind each other non-covalently through ionic interactions. Depending on the nature of both binding partners assembly of nanoparticles may occur [Veldhoen et al., 2008].
The studies have shown that the cargo-CPP complexes are taken up by directly penetrating the cell membrane or by an endocytotic pathway. However, the precise mechanism of internalization remains elusive and strongly depends on the properties of both CPP and cargo as well as the transfection conditions and the cell lines used [Maiolo et al., 2005; De Coupade et al., 2005; El-Andaloussi et al., 2007; Bolhassani, 2011]. Recent studies indicate that the uptake mechanism of CPPs can be influenced by the attachment of cargo. For example, Richard et al. [Richard et al., 2003; Richard et al., 2005] reported a co-localization of Tat 48-59 with markers of clathrin-mediated endocytosis, whereas Fittipaldi
However, further information about the exact mechanism of uptake of such delivery systems is expected in the near future. Furthermore, it has been shown that even minor changes of the physical state of a CPP (e.g., exchange of certain amino acids) can alter translocation properties significantly. This particularly holds true for the attachment of large cargo molecules [Veldhoen et al., 2008]. Thus, it might not be possible to generalize results obtained with a CPP, and it might be necessary to characterize each carrier/cargo complex individually. If CPPs are proposed to be used for therapeutic purposes in the future, it is essential to focus on the attachment of functional cargos and analyze their biological effects inside the cell. Data from our lab clearly show that uptake and biological activity of a functional cargo is everything but the same. Therefore, a quantitative comparison of cargo taken up and functionally active cargo is an essential requirement in order to improve therapeutic efficacy. Indeed, only looking for efficient internalization is not sufficient [Veldhoen et al., 2008].
The successful clinical application of nucleic acid-based therapeutic strategies has been limited by the poor delivery efficiency achieved by existing vectors. The development of alternative delivery systems for improved biological activity is, therefore, obligatory. Since two decades ago that the Tat protein, and derived peptides, can translocate across biological membranes, cell-penetrating peptides have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. Despite extensive research on the use of CPPs for this purpose, the exact mechanisms underlying their cellular uptake and that of peptide conjugates remain controversial [Trabulo et al., 2010].
There are many examples of CPP-mediated delivery of plasmid DNA into cultured cells and also
A combination of a peptide nucleic acids (PNA) with the SV40 core NLS, performed by Branden
Over the last years, a research group focused on the S413-PV cell-penetrating peptide generated from the combination of 13-amino acid cell penetrating sequence derived from the Dermaseptin S4 peptide with the SV40 large T antigen nuclear localization signal [Trabulo et al., 2010]. In these studies, complexes obtained through electrostatic association of the S413-PV cell penetrating peptide with plasmid DNA are able to very efficiently mediate transfection, particularly at high peptide/DNA charge ratios (5/1 and higher). Importantly, complexes prepared with the S413-PV or reverse NLS peptides mediate transfection at significantly higher efficiencies than those containing the scrambled version of the peptide, demonstrating the importance of the cell-penetrating sequence derived from the Dermaseptin S4 peptide (amino acids 1-13) to the transfection process. Additionally, we demonstrated that ternary complexes, resulting from association of cationic liposomes to peptide/DNA complexes, are significantly more efficient in mediating transfection than the corresponding peptide/DNA or cationic liposome/DNA complexes [Trabulo et al., 2010]. In agreement with what has been described for oligonucleotides, CPPs seem to be very efficient to mediate the uptake of plasmid DNA, as well as lipoplexes and polyplexes containing DNA, surpassing the cell membrane barrier. However, the challenge of overcoming the entrapment of complexes inside endosomes has not been solved as easily as initially predicted, even taking advantage of the capacity of direct translocation to the cytoplasm of some CPPs. Nevertheless, several of the studies described above present promising strategies to overcome this limitation, such as chemical modification of the peptide backbone or coupling of CPPs to other classes of delivery vectors. Overall, accumulated evidence suggests that CPPs used in combination with other delivery systems are more likely to be effective for gene therapy purposes than CPPs alone [Trabulo et al., 2010].
12. Bioactive peptides
As antibiotic resistance increases worldwide, there is an increasing pressure to develop novel classes of antimicrobial compounds to fight infectious disease. Peptide therapeutic represents a novel class of therapeutic agents. Some of them, such as cationic antimicrobial peptides (CAMPs) and peptidoglycan recognition proteins (PGRPs), have been identified from studies of innate immune effector mechanisms, while others are completely novel compounds generated in biological systems. Currently, only selected cationic antimicrobial peptides have been licensed, and for topical applications. However, research using new approaches to identify novel antimicrobial peptide therapeutics, and new approaches to delivery and improving stability, will result in an increased range of peptide therapeutics available in the clinic for broader applications. A potentially rich source of peptide therapeutics that is being investigated by researchers is the innate immune response, the effectors of which are produced by eukaryotes to defend themselves against microbial attack [Oyston et al., 2009].
Human cancer is one of the most important causes of death in the western countries. In advanced stages of the disease, the therapeutic opportunities are still limited due to the difficulty to target specifically only cancer cells sparing healthy ones. Cancer cells have on their surface antigens that are expressed at higher levels than their normal counterparts. Often these antigens (also called tumor-associated antigens) have receptor activity and bind to specific proteins or peptides. The latter can be used for the specific delivery of anticancer drugs to cancer cells through retargeting strategies and/or for the direct modulation of cancer cell proliferation and survival interacting with cell-surface-specific receptors. These bioactive peptides can be raised against either tumor cells themselves or to the tumor microenvironment cell components (tumor vessels, tumor-associated macrophages, and fibroblasts) [Fields et al., 2009]. However, the feasibility of pharmacological application of peptides depends on absorption and bioavailability in intact forms in target tissues.
Moreover, their correct bio-distribution is sometimes hindered by biopharmaceutical obstacles, that is, protection by circulating protease-mediated degradation or specific accumulation in tumor tissues. Chemical modification of peptide backbone can increase the stability of peptides in biological fluids. Moreover, the use of delivery systems, and in particular the use of nanotechnologies, not only protects peptides from enzymatic degradation but also improves the delivery of the bioactive peptide in the target tissue. Moreover, peptide conjugation on the surface of nano-vectors can be useful for selective delivery of conventional chemotherapeutic agents in tumor tissues. The requirements for an effective and safe dermatological therapeutic or active ingredient are included as following:
The molecule exhibits a proven specific beneficial bioactivity that would lead to a rational demonstrable effect.
The bioactivity does not have a negative consequence either theoretically or experimentally due to its mechanism of action.
The molecule does not exhibit toxicity such as cytotoxicity, inflammation, immunogenicity, or mutagenicity.
The molecule is capable of reaching its desired target intact and in its active form.
The molecule can be formulated in such a way as to be stable, compatible with other components, and be delivered effectively to the skin [Fields et al., 2009].
Collectively, these are not easily achieved criteria. For a new technology paradigm to emerge, these criteria not only have to be met but be applicable across the wide range of product-acceptable bioactivities. Peptides have significant advantages over many other technologies in addressing these criteria primarily based upon their chemistry. Peptides consist of chains of amino acids which can be modified in many ways to increase receptor binding, increase specificity, decrease toxicity, and increase skin penetration, stability, and solubility. In this way, the field of bioactive peptides for dermatological applications has changed significantly in recent years. From modest beginnings of a single peptide capable of stimulating collagen, technological advances have created newer peptides capable of targeting most aspects of dermal health. These advances include neutralizing toxins, stimulating fibroblast scaffolding, reducing inflammation and other desirable effects [Fields et al., 2009].
13. Challenges in gene delivery for DNA vaccines
The goal of DNA vaccination is transfection of an antigen presenting cells (APC) or a bystander cell to produce antigens in an immuno-stimulatory setting. The field of genetic vaccines has so far been limited by a lack of safe and effective gene delivery systems [Nguyen et al., 2008]. We attempted to mention some barriers and solutions (e.g., viral/non-viral methods) for DNA vaccines individually.
13.1. Viral and non-viral methods for gene delivery
The main recombinant viral vectors used for gene delivery are
13.2. Barriers to gene delivery for DNA vaccines
There are many potential blocks that must be overcome for successful DNA delivery, a process broadly defined as transfection. Plasmid DNA must first be packaged into particles. Requirements for gene delivery include protection of plasmid DNA from degradation, localization to the tissue and cell types of interest avoiding off-target distribution, minimal inactivation by interaction with serum proteins, low clearance from the blood or interstitial space and efficient transport through the extracellular matrix to the surface of target cells. Next, the DNA-containing particles must associate with cells and become internalized into them by cellular uptake processes [Nguyen et al., 2008]. Following uptake, DNA-containing particles must escape the endosomal/ phagosomal compartment into the cytoplasm and release their DNA cargo. DNA must finally translocate into the nucleus to be transcribed into mRNA and subsequently translated into protein antigen. Viruses have evolved to accomplish these steps and provide a framework for the design of synthetic delivery particles. Despite efficient uptake of particles of a variety of sizes,
13.3. Particle uptake by APCs and targeting gene delivery for DNA vaccines
Particle uptake by phagocytosis (particles > 500 nm), macropinocytosis, and receptor-mediated endocytosis are particularly important routes of entry into APCs. Delivery systems can be designed to exploit these ways. Cell-specific targeting can significantly enhance transfection efficiency and the desired therapeutic outcome. The direct conjugation of targeting moieties such as receptor ligands, peptides, sugars, aptamers and antibodies can increase cell and tissue specificity and transfection efficiency. Additionally, targeting can be based upon size-specific signals to avoid off-target affects. A variety of strategies exist for targeting APCs. First, APCs express Fc-receptors, which bind to the constant region of antibodies to facilitate uptake of antibody-coated foreign bodies. APCs also express complement receptors that help clear complement-opsonized particles [Nguyen et al., 2008]. Lectin-binding receptors, such as the mannose receptor and scavenger receptors that recognize apoptotic bodies, certain bacterial components and other non-self motifs are PRRs commonly found on APCs that can enhance particle uptake and may trigger innate immune activation. Second, unlike most other cell types, immature DCs constitutively sample their extracellular fluid environment non-specifically through macropinocytosis to maintain immune surveillance and vigilance for foreign particles, as well as to present endogenous proteins for maintenance of self-tolerance. This constant sampling may explain DC-targeting by particle size. Particle size may also influence the specific route of entry, as reviewed recently by Xiang
13.4. Biomaterials for DNA delivery: Non-polymeric biomaterials for gene delivery
Many materials have been developed for gene delivery. Early chemical methods of increasing the efficacy of gene delivery focused on co-precipitation of the DNA with salts such as calcium phosphate. More recently, inorganic materials have also been combined with polymers to form hybrid gene delivery nanoparticles. For example, textured surfaces and silica nano-particles have been shown to be effective for gene transfer
Gene therapy possesses great potential for combating a variety of diseases. Initial results are promising and some technologies have advanced to clinical trials. Yet challenges remain, and despite decades of study, safe and efficient gene delivery remains a major blockage in human medicine. The current progresses of gene therapy are further focused on synthesized nano-particle technologies such as polymers (PLGA, chitosan and PEI), lipids and peptides. It is necessary to understand the effects of particle size, surface characteristics and material interactions. Research on cell penetrating peptides (CPPs) as gene/ drug delivery systems has clarified their capacity to promote the efficient internalization of therapeutic biomolecules. Despite differences in size, charge and/or structure between different bioactive molecules, it is clear that CPP-based systems appear to be very versatile and efficient delivery is achievable following proper adjustment of the carrier to the transported biomolecule. Because the development of drug, oligonucleotide or gene delivery systems is aimed at a clinical application, the design of these novel delivery vectors should consider other important issues including safety, bio-distribution, ease of manufacturing, scale-up, reproducibility and analytical and physical characterization. The advance of CPP technology depends on the development of strategies that facilitate endosomal escape and that confer cell specificity to these systems. A careful investigation of the mechanisms of internalization of CPP-cargo complexes or conjugates will greatly help the improvement of this powerful technology. However, a viable non-viral gene vector for systemic delivery depends on its capacity to bypass a series of physiological barriers and its efficiency in carrying nucleic acids to a targeted site within a cell. The concept of a multifunctional delivery system helps to solve the problems associated with various barriers. The availability of delivery devices directed towards each individual barrier, provides a basis for this direction, although the complexity in the development of multifunctional non-viral vectors is much more than a merely combination of various devices into a single system. In order to achieve the optimal gene delivery, the researchers have focused largely on the evolution of “intelligent” bio-responsive materials, as well as on the advances in formulation technologies. In this process, a number of strategies have emerged including the balances between gene packing and controlled release, and optimal control between long-circulation and intracellular trafficking that promotes safer and more efficient delivery of gene/drug in a systemic context.