Since the inception of gene therapy in 1972, much progress has been made to underpin its present-day potential as a treatment for diseases of genetic origin (1). The capacity for foreign DNA to induce functional changes to the host’s intracellular machinery is well established. However, DNA can only exert its therapeutic potential if delivered to the nucleus of the target cells. The major stumbling block in the development of gene therapies has been the dearth of efficacious delivery systems (2, 3).
Viruses are naturally adept at hijacking the host cells’ machinery to enable their own proliferative agenda. Tailoring of known human viruses as potential gene delivery vectors therefore became the initial focus of research within the field (4). However, despite attempts to remove the pathogenic components of the viral apparatus, progress of these vectors has been thwarted due to safety concerns stemming from their use, and indeed patient perception (5). Non-viral based strategies manage to overcome some of the safety concerns associated with the use of viral vectors but have, as of yet, failed to match the efficacy of their viral counterparts (6). The use of naturally occurring and/or synthetic peptides whose designs are based upon viral sequences, therefore present an attractive alternative for nucleic acid delivery. Multi-functional peptide-based nanoparticles comprising distinct motifs with specific functionalities designed to overcome the extra- and intra-cellular barriers could, in theory, be used for safe and efficient gene delivery (7). Here we discuss the various hurdles that a gene delivery vector must overcome, functional peptides that are capable of facilitating their circumvention, and strategies to combine these peptides to develop effective bio-inspired gene delivery vectors.
Barriers to gene therapy
The ideal gene delivery system should be non-toxic, biodegradable, targeted, non-immunogenic and easily manufactured. In order to design such a peptide delivery system, numerous biological barriers must be understood. Following systemic injection, prospective peptides must firstly protect the therapeutic gene from the action of mononuclear phagocytes, complement and reticulo-endothelial systems, all of which results in rapid clearance from the body (8). Additionally the peptide must be able to extravasate from the circulation, pass through the fibrous extracellular matrix and reach the target tissue whilst ensuring any off-target effects are limited (9). Once the therapeutic reaches the target site, the peptide has to penetrate the cell membrane in order to deliver the genetic cargo. The mechanism of internalization has a consequential impact on its intercellular fate thereafter.
Peptides can be internalized by two main pathways: A) Endocytic or energy dependent pathways (clathrin-mediated, caveolae/lipid raft-mediated, clathrin and caveolae-independent endocytosis and macropinocytosis) and 2) direct penetration or energy independent pathways (e.g., inverted micelle model, pore formation, carpet model) (Figure 1) (10). If internalization is by endocytosis, as is predominantly the case, the objective is to escape the endosome, otherwise the genetic material will be degraded and expelled via a lysosome (11). If endosomal escape is successful then the DNA must be delivered to the nucleus where it can finally exact a sustained therapeutic effect (12). Each of these barriers must be overcome otherwise failure of the therapy is inevitable. Consequently, an in depth knowledge of these barriers is fundamental to effective peptide design.
Nucleic acid condensation
The capacity to effectively bind to and condense DNA into stable nanoparticles is essential in order to protect the cargo from enzymatic degradation in the systemic circulation and in the cytoplasm (13, 14). The use of condensing motifs can significantly enhance stability in vivo, protect DNA from the action of lytic enzymes, and ensure an appropriate nanoparticle size (<200 nm) to facilitate cellular uptake (15, 16).
Gratton et al. (17) employed a technique known as particle replication in non-wetting templates (PRINT) to define the significance of particle size, shape and surface charge on non-specific cellular uptake. In the study a clear correlation is evidenced between particle size and the extent of cellular uptake. Particles with a size >1 micrometer exhibited significantly reduced internalization kinetics compared to those that occur within the nanometer scale. However, size, although significant, was not the only defining characteristic with regard to cellular uptake profiles of PRINT particles. Both surface charge and shape were also shown to be important determinants of internalization, with rod-shaped, high aspect-ratio PRINT particles carrying a positive zeta potential the most readily internalized. These, therefore, represent the fundamentals towards which scientists involved in the field of particle design should strive.
The common feature shared by all DNA condensing peptides is their cationic nature, and size-suitability in the condensed form (Table 1). One of the first polypeptides, Poly-L-Lysine (PLL) consisted of biodegradable repeated lysine residues that effectively condensed DNA; endosomal entrapment limited PLL’s ability to successfully deliver genetic material (18). Subsequently numerous studies have examined the merits of using either lysine- or arginine-based peptides for gene delivery, with the more compelling evidence firmly supporting arginine. Arginine binds to the DNA in milliseconds (19), has a stronger affinity for the phospholipid phosphatidylserine (Ptd-Ser) on the inner leaflet of membranes (20), and is a superior internalizer to oligolysines (21). Furthermore, recent studies by Mann et al. (22) demonstrated that block distribution of arginine R5H7R4 results in stronger condensation of DNA but that the addition of histidine residues either by R9H7 or H4R9H3 were less effective at condensation but much better at releasing the genetic cargo giving a higher transfection. These studies support those of Hatefi’s group, who suggested that vector architecture of the amino acid sequence is critical, and that clusters of lysine and histidine (KKKHHHHKKK) were superior to interspersed (KHKHKHKHKK) sequences (23). As a cationic residue, histidine not only condenses nucleic acids to an extent but perhaps more importantly also facilitates the release of the genetic cargo from the endosome via the proton sponge effect (24).
Other arginine-rich sequences exist in nature such as protamine, which plays a key role during spermatogenesis by replacing histones, thus ensuring tight condensation of the DNA (25). The Mu peptide (MRRAHHRRRRASHRRMRGG) is a 19 mer, arginine-rich peptide first identified and isolated from the adenovirus core complex in 1976 (26). The Mu peptide has been shown to consistently bind DNA into small, stable nanoparticles, which has led to its application in a number of peptide-based delivery systems (27, 28). Nevertheless peptides such as protamine and Mu remain uni-functional and so frequently they are imported into multi-functional systems to take advantage of their DNA binding characteristics. For example protamine has been utilized as the core DNA binding component of a multifunctional envelope type nano device (MEND) incorporating the fusogenic peptide GALA and the nuclear localization signal (NLS) maltotriose which boosted transfection efficiency 15.8-fold higher than that of the commercially available in vivo-jet-PEI™-Gal (29).
Karjoo et al. (30) reported transfection efficiencies (>95%) in ovarian cancer SKOV3 cells with a viral mimetic nanoparticle system designated THG/Mu-PEG5K. The THG biopolymer, which consists of a targeting peptide (T), four repeating units of histone (H) and the fusogenic peptide GALA (G), was mixed at a ratio of 8:8 with the covalently bonded Mu-PEG5K to form stable nanoparticles with pEGFP-N1 (30).
When selecting a peptide sequence it must be noted that a fine balance is required between protecting the DNA from extracellular degradation, achieving favorable pharmacokinetics and ensuring effective intracellular release. Indeed the very characteristics that make cationic peptides powerful condensers of nucleic acids may also detrimentally affect nanoparticle biodistribution in vivo and cytoplasmic release of DNA. Whilst condensation with cationic peptides can dramatically reduce interaction of DNA with enzymatic elements in vivo, peptides too, particularly highly cationic, arginine-rich peptides, are susceptible to rapid clearance from the body and degradation by proteolytic plasma enzymes (31). Such problems may be overcome by functionalization with polyethylene glycol (PEG) (32). However, modification in this way has been shown to adversely affect intracellular kinetics (33). This problem may, in turn, be overcome by the use of sheddable PEG coatings. Zhu et al. (34) reported improved tumor accumulation and tumor-specific cleavage of a self-assembly block copolymer (PEG-pp-PEI-PE) due to the use of an MMP2 labile linker for PEG.
Therefore, with regard to the method of nucleic acid condensation, there is much to consider. And this is only the first step in a complex process.
Traversing the cellular membrane is the next critical step for successful gene delivery. Cell-penetrating peptides (CPPs) are a class of peptides that can facilitate the permeabilzation of biological membranes. Endocytosis is the predominant mechanism of membrane translocation by CPPs with subsequent entrapment in the endosome, acidification and degradation of the genetic cargo unless escape to the cytosol can occur (35). Alternatively if CPPs enter the cell via direct membrane translocation, the endosome is by-passed and the need for an escape mechanism in the design of the peptide is circumvented. Elucidation of the mechanisms by which specific peptides are internalized is therefore crucial to effective vector design.
In the literature, CPPs have been categorized either according to their origin as protein-derived, chimeric or synthetic; or, according to their amphipathic profile as primary amphipathic, secondary amphipathic or non-amphipathic (Table 2). Their designation as such depends largely on the specific amino acid engineering and physiochemical properties of the peptide in question (10). Unsurprisingly arginine abundance is a common feature of CPPs, characterized by the presence of the guanidine head group, thus facilitating formation of strong bidendate hydrogen linkages with anionic components of the cell membrane (36–38). There is to date no consensus on the precise mechanism of cellular internalization of arginine-rich CPPs. However the degree and manner of initial interaction with the cell surface membrane ultimately dictates the eventual pathway of internalization and is generally recognized as being the first step of the CPP internalization process.
In a landmark study, cell surface activity and internalization was examined using Penetratin (RQIKI WFQNR RMKWK K-amide), a 16 amino acid peptide derived from the third helix of the Antennapedia homeodomain, PenArg (RQIR IWFQ NRRM RWRR-amide) and PenLys (KQIK IWFQ NKKM KWKK-amide) (39). Studies revealed that the levels of internalization with PenArg were 10 times higher than those seen with PenLys, indicating that arginine interacted more strongly with phospholipid membranes than lysine. Åmand et al. (39) then went on to quantify the relationship between strength of CPP interaction with cell membrane and degree of cellular internalization, demonstrating conclusively an almost linear correlation between the two factors and verifying the previously unsubstantiated supposition that strength of interaction with the cell membrane is the crucial first consideration in deciphering CPP mechanism of internalization. Self-stimulated macropinocytosis was also shown to be the primary mechanism of membrane translocation for the PenArg CPP. Yet the cellular uptake of a chimera hybrid consisting only of D- and L-arginine isomers has been shown to be via direct membrane translocation following inhibition of endocytic pathways by both physical and pharmacological means (40). Direct translocation was further confirmed when the transmembrane potential was eliminated resulting in a drastic reduction in chimeric oligoarginine in the cytosol. Transmembrane potential is therefore another critical factor to consider in the translocation of guanidium-rich peptides (41).
The differences in transmembrane activity exhibited by arginine-rich CPPs can also be related to the amphipathic profile of the peptide (42, 43). For example the presence of two tryptophan (W) residues in the Penetratin backbone has led to its designation as a secondary amphipathic peptide, described as such due to the distribution of hydrophobic and hydrophilic charges on its secondary structure following interaction with phospholipid membranes (44). The presence, size and hydrophobic character of W has been shown to functionally enhance the internalization activity of CPPs primarily through improved anchoring of peptides to cell membranes (45). To what degree this amphipathic quality dictates the route of internalization is not clear. However, complete loss of function of Penetratin was observed following substitution of tryptophan (W6) for phenylalanine (46). It was then postulated that Penetratin followed a two-step model for internalization that involved initial electrostatic interaction followed by tryptophan-dependent membrane destabilization. The evidence is mounting that tryptophan therefore has a key role to play in the design of secondary amphipathic peptides. Indeed studies by Jafari et al. (47) demonstrated that replacing three leucine residues in the 18 mer C6 peptide with tryptophan to give C6M1 not only increased peptide solubility and secondary helical structure but also reduced cytotoxicity and increased intracellular uptake. Incorporation of tryptophan and leucine residues into modified Tat 48–60 markedly enhanced leakage from plasma membrane vesicles compared to those lacking a hydrophobic component (48).
Rydberg et al. (49) took the studies with CPPs one step further by examining the effect of arginine and tryptophan positioning within the peptide. Results indicated that positioning 1–4 tryptophans at the N-terminus significantly impaired efficacy; while cellular uptake was highest for the RWmix (RWRRWRRWRRWR), attributable to greater secondary amphipathicity afforded by equal spacing of the residues, cytotoxicity was lower in a RWR (RRRRWWWWRRRR) sequence that achieved greater accumulation in the cytoplasm and nucleus, aided by the non-endocytic uptake route (49). The positioning of the amino acid residues then becomes a critical factor with only the RWmix entering via endocytosis. Taken together it becomes apparent that when designing a peptide for nucleic acid therapeutics, the distribution of the selected residues can have a profound influence on the mechanism of uptake.
The effects of cargo and cell membrane composition on internalization are also key considerations that cannot be overlooked. The size and type of cargo, as well as the manner of binding to the CPP, can influence CPP translocation characteristics (50–52). Much information to this regard has been gleaned from the implementation of unilamellar vesicles as model membranes to analyze the interaction of CPPs with lipid membranes. A recent publication by Vasconcelos et al. (53) used large unilamellar vesicles (LUVs) to delineate the relationship between peptide hydrophobicity and membrane perturbation characteristics of sterylated analogues of Transportan 10. They demonstrate that the interaction between the peptide and its given cargo can have an important influence on CPP secondary structure and therefore internalization profiles (53). However, the use of liposomal models as a tool to elucidate CPP mechanism of action is discouraged by some, who claim they do not adequately represent the environmental complexity of live cells (48).
Cell membrane glycosiaminoglycan (GAG) content has also been cited as a crucial mediator of internalization (54, 55). Naik et al. (56) examined the effect of surface-bound and free GAGs on the permeabilization characteristics of R16 and K16 homo-peptides in live cells. Results found that DNA complexed with the R16 peptide enter cells via non-endocytic and endocytic pathways, but that both are GAG independent. Complexes of DNA and the K16 peptide enter primarily via an endocytotic pathway, and is dependent on GAG presence (56, 57). Subrizi et al. (48) further challenged the role played by GAGs in cellular uptake, producing evidence that they actually inhibit movement of the Tat peptide across biological membranes in live cells. Indeed many of the methods used to analyze CPP behavior exhibit a high degree of analytical variability that only fuels the debate surrounding the mechanisms of cellular uptake (36).
Therefore until standardized methods are agreed amongst the field for evaluating the CPP phenomenon, correlations between peptide composition and cellular uptake will be difficult to elucidate. What is commonly accepted is that CPP permeabilzation occurs via two or more pathways and the propensity of any given CPP toward a particular pathway is highly variable and depends on a number of factors. These factors include CPP size, distribution of charge, hydrophobicity and peptide conformation, as well as considerations of cell membrane composition and cargo. These variables need to be accurately accounted for in experimental design to ensure reproducibility and consistency of results. Once this is achieved peptides can be tailored to ensure maximal accumulation within the desired intracellular location.
Endocytosis is the primary mechanism for movement of extracellular material across biological membranes (11). Once endocytosed, the transported material is engulfed within an endosome. Lysosomes fuse with endosomes resulting in the acid degradation of the endosomal contents. This presents a significant barrier to gene delivery, one that must be overcome in order for DNA to reach its site of action, namely the nucleus. Fusogenic peptides are a class of amphipathic peptides derived from the N-terminal segment of the HA-2 subunit of the influenza virus hemagglutinin (58). The HA2 peptide (GLFGAIAGFIENGWEGMIDG) forms an α-helix under acidic conditions and fuses with the endosomal membrane, enabling cargo delivery into the cytosol (59). At physiological pH, the lytic activity of the HA2 peptide is negligible, which confers a level of targeting for endosome of the target cell. This then renders HA2 peptide and subsequent derivatives suitable for systemic administration.
One of the first derivatives of the HA2 sequence was the 30 mer designer peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA), characterized by a glutamic acid-alanine-leucine-alanine repeat (60). The maximum α–helical conformation of GALA occurred at a pH 5 which gives rise to a hydrophobic face on one side of the peptide and a subsequent interaction with the endosomal membrane. This results in pore formation and endosomal escape of the cargo to the cytosol. The repeating glutamic residues in GALA render it anionic and therefore ineffective for condensing and protecting nucleic acids. Nevertheless GALA has been utilized in several multi-functional systems as a discrete endosomal-disrupting motif. For example, the GALA peptide was incorporated into a biomimetic vector that also had four repeats of histone proteins to condense DNA, a targeting motif for HER2 and a cathepsin substrate that acts as an intracellular cleavage site (61). Studies demonstrated that positioning GALA on the N-terminus of the multifunctional vector ensured fusogenic amphipathic activity. GALA has also been utilized in the R8-MEND system to significantly improve gene expression in the liver (618-fold) in nanoparticles with a pDNA/PEI negative core (62).
In a bid to increase the functionality of GALA, Wyman et al. (63) substituted the negatively charged glutamic acid residues of GALA with positively charged lysine to produce the cationic peptide KALA (WEAKLAKALAKALAKHLAKALAKALKACEA), which not only retains its fusogenic activity but can also condense negatively charged nucleic acids thanks to the positive charge conferred by the lysine. KALA has been utilized as an independent transfection agent alone and also to improve the activity of other delivery vehicles. KALA coating of PEG-g-PLL not only increased transfection efficiency but also displayed negligible toxicity compared to PEG-g-PLL alone (64). KALA has also been used to coat magnetic mesoporous silica nanoparticles capped with PEI to deliver VEGF siRNA (M-MSN-siRNA@PEI-KALA) to not only reduce cytotoxicity but also significantly delay tumor growth in A549 lung tumors in vivo (65).
Given the superiority of arginine over lysine as previously discussed, McCarthy et al. (66) went on to create another cationic peptide termed RALA (WEARLARALARALARHLARALARALRACEA) to deliver DNA. Studies demonstrated that the fusogenic activity of RALA remained pH-dependent, toxicity was reduced in vitro compared to a commercial agent and that cellular entry was via caveolin- and clathrin-mediated endocytosis. Furthermore the RALA/pDNA nanoparticles retained activity following lyophilization with trehalose giving a suitable isotonic formulation for in vivo administration. Following systemic administration, gene expression was maximally observed in the lungs and liver (66).
A recent study carried out by Nouri et al. (67) compared the fusogenic activity of GALA, KALA and a number of other synthetic HA2-derived fusogenic peptides, INF7 (GLFEAIEGFIENGWEGMIDGWYG) (68), H5WYG (GLFHAIAHFIHGGWHGLIHGWYG) (69) and RALA, each coupled with four histone repeats and a targeting motif. Although GALA outperformed the other peptides in terms of both the percentage of cells transfected and the levels of green fluorescent protein expressed, H5WYG performed best in the hemolytic assay, suggesting that of the five fusogenic peptides investigated, H5WYG is superior at disrupting endosomal membranes. This result was not unexpected as the presence of an imidazole ring in H5WYG acts as a proton sponge, thus enhancing the pH-buffering capacity of the peptide. H5WYG has a pKa value of approximately 6.0 and will be protonated at around pH 6. This property therefore facilitates early escape from weakly acidic endosomes whilst remaining in an inactive conformation at physiological pH (68). It should be noted, however, that the contribution offered by improved buffering is currently debated, with some finding that improved buffering of polymer complexes at low pH may not always enhance endosomal escape (70).
Histidines have also been employed to improve TAT as a gene delivery vehicle. Although TAT is excellent at condensing DNA and traversing the cell membrane, it cannot escape the endosome, rendering it ineffective for gene delivery. However, Lo et al. covalently added histidine residues to the C-terminus of TAT and found that the addition of 10 residues resulted in a 7000-fold increase in gene expression (71). Further modifications included the addition of two cysteine residues to improve stabilization and an equal distribution of histidine to give C-5H-Tat-5H-C which improved transfection a further 1000 fold (71).
It is important to note however, that the fusogenic activity of these peptides is a consequence of a pH-dependent shift in their conformational status, occurring in the late endosome or upon fusion with a lysosome following clathrin-mediated endocytosis. Endocytosis by non-acidic pathways such as caveolae-mediated endocytosis and macropinocytosis will nullify the membrane lytic activity of fusogenic peptides, in which case entrapment within the endosome remains a major problem. Therefore, before employing a fusogenic peptide in any delivery system, due consideration must first be given to the mechanism of cellular entry.
Of all the barriers to gene delivery, nuclear import is by far the most challenging to overcome. The nucleus is enveloped by a highly impermeable double lipid bilayer known as the nuclear membrane (72). Movement across this membrane is regulated by highly restrictive nuclear pore complexes, interacting protein domains that form aqueous channels between the cell cytoplasm and the nucleoplasm. At their narrowest point these channels are a diameter of around 10 nm, allowing passive diffusion of ions and small proteins (<10 nm) into the nucleus. Movement of larger molecules into the nucleus relies on nuclear localization signals (NLS), small peptide sequences that interact with components of the importin super family of proteins, which mediate macromolecular movement into the nucleus. The genetic cargo must be within close proximity to the nuclear membrane to enable binding to nuclear transport factors, a process that can be facilitated by components of the cell cytoskeleton, which coordinate movement of molecules through the “cytoplasmic sieve” (12). The challenge is therefore to identify a NLS that can: 1) Retain functionality by not binding to the genetic cargo that is to be delivered, 2) interact sufficiently with elements of the cytoskeleton to mediate accumulation of the genetic cargo around the outer membrane of the nuclear envelope and 3) bind specifically with importin adaptor proteins that arbitrate nuclear uptake.
Over the past number of decades many peptide NLSs have been identified for the purposes of active shuttling of DNA to the nucleus and their ability to enhance nuclear accumulation of cargo. However, despite numerous attempts to improve transfection with non-viral vectors through the use of NLSs, nuclear accumulation in quiescent cells remains a significant barrier to successful gene delivery (73). When selecting a NLS in peptide design, the key factors that need to be considered are 1) NLS characteristics, 2) cellular characteristics and 3) cargo characteristics.
Classical NLSs, such as the REV peptide derived from HIV (RQARRNRRNRRRRWR) and the large tumor antigen of the simian virus 40 (SV40) (PKKKRKV) are short, basic peptides that interact with importin-α adaptor proteins to mediate transport to the nucleus. Kim et al. (74) recently demonstrated enhanced transfection when a cysteine-enriched SV40 derivative (GYGPKKKRKVGGC) was complexed with pLuc DNA before cationic liposome encapsulation. Several variants of the SV40 were tested, but only the C terminal disulfide homodimer resulted in improved efficiency and DNA release (74). The human mRNA-binding protein hnRNP M9 (GNYNNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY) is an example of a non-classical NLS, which binds directly to importin-β without binding to importin-α adaptor proteins (75). The unordered M9 peptide is distinctly useful in multifunctional peptide systems because the lack of basic residues reduces interaction with the DNA cargo and therefore the NLS functionality remains intact. Canine et al. (76) utilized the M9 NLS in a multifunctional biopolymer termed FP-DCE-NLS-TM where FP is a fusogenic peptide, DCE a DNA condensing and endosomolytic sequence and the TM a targeting motif. A truncated version FP-DCE-TM evoked negligible gene expression thus proving that the M9 NLS remained functional in the biopolymer (76).
The binding affinities of nuclear transport proteins to particular NLS is cell-type dependent. Gu et al. (77) characterized the nuclear import characteristics of the HIV-1 derived Rev peptide across different cell lines, with results suggesting that HeLa, U937 and THP-1 cell lines employed transportin as the major transport receptor to rev, whereas in 293T, Jurkat or CEM cell lines, importin-β was the primary mediator of nuclear uptake. Intranuclear transport characteristics are also known to alter once cells become cancerous (78, 79). A truncated form of importin-α has been found to lack a NLS binding domain in ZR-75-1 breast cancer cells, which would restrict the efficacy of any NLS operating on that pathway, e.g., SV40 (80).
Direct conjugation of the NLS to the DNA cargo has largely failed to significantly enhance gene expression (12, 72). In its uncondensed form, DNA is susceptible to degradation by cytoplasmic enzymes and movement through the densely packed cytosol is impeded due to the unordered state and size of uncondensed pDNA (81, 82). Use of cationic condensing agents such as the core protein Vll of adenovirus type 2 or histones that contain inherent NLS have been shown to help overcome such issues (83, 84). However, more success has been derived from the conjugation of NLS to polycation binding proteins, thus reducing interference from cargo. Yi et al. (85) reported a 200-fold enhancement in transfection efficiency of Tat conjugated to the NLS PKKKRKV-NH2 (PV) compared to Tat/DNA complexes alone. Furthermore, complexes formed by non-specific electrostatic interaction (Tat/PV/DNA) showed no significant enhancement in transfection. Therefore with respect to peptide design it is better to covalently attach the NLS to the peptide and also ensure availability of the NLS to the importin proteins within the cytosol.
The phrase “from needle to nucleus” is one that has been coined as the ideal in vector development for gene therapy. In reality however, the process is a stepwise one, with numerous hurdles that must first be overcome before the nucleus of target cells is reached. The purpose of this article is to identify peptides that can be utilized to help advance this agenda.
One criticism of peptide-based gene therapy might be the failure, thus far, to identify a single peptide sequence independently capable of highly efficient gene delivery. Progress therefore, relies on a multifaceted approach to nanoparticle design; one that involves collaboration across various non-viral disciplines and one that is based on systematically addressing the biological barriers faced. Such a philosophy has led to the development of a variety of peptide-enhanced, multifunctional nanoparticle systems, some of which have been referred to herein. Examples include the amelioration of polymers, lipids, micelles, chitosan and MENDs with peptides for improved gene delivery. For a comprehensive analysis of multifunctional non-viral vectors in gene therapy the reader is referred to a recent review by Wang et al. (86).
Therefore, whether required for targeting, nucleic acid condensation, membrane destabilization, endosomal escape or nuclear localization, peptides offer a wealth of promise when incorporated into multifunctional system designs such as these.
Friedmann T, Roblin R. Gene therapy for human disease. Science 1972;175:949–55.Google Scholar
Park S, Lee SJ, Chung H, Her S, Choi Y, Kim K, et al. Cellular uptake pathway and drug release characteristics of drug-encapsulated glycol chitosan nanoparticles in live cells. Microsc Res Tech 2010;73:857–65.CrossrefGoogle Scholar
Gong P, Shi B, Zhang P, Hu D, Zheng M, Zheng C, et al. DNase-activatable fluorescence probes visualizing the degradation of exogenous DNA in living cells. Nanoscale 2012;4:2454–62.CrossrefGoogle Scholar
Rejman J, Oberle V, Zuhorn IS, Hoekstra D. Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J 2004;377:159–69.Google Scholar
Gratton SEA, Ropp PA, Pohlhaus PD, Luft JC, Madden VJ, Napier ME, et al. The effect of particle design on cellular internalization pathways. Proc Natl Acad Sci USA 2008;105:11613–8.CrossrefGoogle Scholar
Kwoh D, Coffin C, Lollo CP, Jovenal J, Banaszczyk MG, Mullen P, et al. Stabilization of poly-L-lysine/DNA polyplexes for in vivo gene delivery to the liver. Biochim Biophys Acta 1999;1444:171–90.Google Scholar
Murray KD, Etheridge CJ, Shah SI, Matthews DA, Russell W, Gurling HM, et al. Enhanced cationic liposome-mediated transfection using the DNA-binding peptide mu (mu) from the adenovirus core. Gene Ther [Internet] 2001;8:453–60. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11313824.
Cahill K. Molecular electroporation and the transduction of oligoarginines. Phys Biol [Internet] 2009 Jan [cited 2014 May 9];7:16001. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20009189.
Tunnemann G, Ter-Avetisyan G, Martin RM, Stockl M, Herrmann A, Cardoso MC. Live-cell analysis of cell penetration ability and toxicity of oligo-arginines. J Pept Sci 2008;14:469–76.CrossrefGoogle Scholar
Mann A, Shujla V, Khanduri R, Dabral S, Singh H, Ganguli M. Linear short histidine and cysteine modified arginine peptides constitute a potential class of DNA delivery agents. Mol Pharm 2014;11:683–96.CrossrefGoogle Scholar
Midoux P, Pichon C, Yaouanc J-J, Jaffrès P-A. Chemical vectors for gene delivery: a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers. Br J Pharmacol 2009;157:166–78.Google Scholar
Hosokawa K, Sung MT. Isolation and characterization of an extremely basic protein from adenovirus type 5. J Virol 1976;17:924–34.Google Scholar
McCarthy HO, Zholobenko AV, Wang Y, Canine B, Robson T, Hirst DG, et al. Evaluation of a multi-functional nanocarrier for targeted breast cancer iNOS gene therapy. Int J Pharm [Internet] 2011 Mar 28 [cited 2014 Oct 29];405:196–202. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21134429.
Akita H, Masuda T, Nishio T, Niikura K, Ijiro K, Harashima H. Improving in vivo hepatic transfection activity by controlling intra- cellular trafficking: the function of GALA and maltotriose. Mol Pharm 2011;8:1436–42.CrossrefGoogle Scholar
Noh SM, Park MO, Shim G, Han SE, Lee HY, Huh JH, et al. Pegylated poly-l-arginine derivatives of chitosan for effective delivery of siRNA. J Control Release [Internet] 2010 Jul 14 [cited 2014 May 3];145:159–64. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20385182.
Mishra S, Webster P, Davis ME. PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles. Eur J Cell Biol 2004;83:97–111.CrossrefGoogle Scholar
Zhu L, Perche F, Wang T, Torchilin VP. Matrix metalloproteinase 2-sensitive multifunctional polymeric micelles for tumor-specific co-delivery of siRNA and hydrophobic drugs. Biomaterials 2014;35:4213–22.CrossrefGoogle Scholar
Bechara C, Sagan S. Cell-penetrating peptides: 20 years later, where do we stand? FEBS Lett 2013;587:1693–702.Google Scholar
Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc Natl Acad Sci USA 2000;97:13003–8.CrossrefGoogle Scholar
Åmand HL, Fant K, Nordén B, Esbjörner EK. Stimulated endocytosis in penetratin uptake: Effect of arginine and lysine. Biochem Biophys Res Commun 2008;371:621–5.Google Scholar
Ma Y, Gong C, Ma Y, Fan F, Luo M, Yang F, et al. Direct cytosolic delivery of cargoes in vivo by a chimera consisting of D- and L-arginine residues. J Control Release [Internet]. 2012 Sep 10 [cited 2014 May 3];162:286–94. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22824782.
Rothbard JB, Jessop TC, Wender PA. Adaptive translocation: the role of hydrogen bonding and membrane potential in the uptake of guanidinium-rich transporters into cells. Adv Drug Deliv Rev 2005;57:495–504.CrossrefGoogle Scholar
Deshayes S, Plenat T, Aldrian-Herrada G, Divita G, Grimmellec CD, Heitz F. Primary amphipathic cell-penetrating peptides: structural requirements and interactions with model membranes. Biochemistry 2004;43:7698–706.CrossrefGoogle Scholar
Ohmori N, Niidome T, Kiyota T, Lee S, Sugihara G, Wada A, et al. Importance of hydrophobic region in amphiphilic structures of a -helical peptides for their gene transfer-ability into cells. Biochem Biophys Res Commun 1998;245:259–65.Google Scholar
Derossi D, Calvet S, Trembleau A, Brunissen A, Chassaing G, Prochiantz A. Cell Internalization of the Third Helix of the Antennapedia Homeodomain Is Receptor-independent. J Biol Chem 1996;271:18188–93.Google Scholar
Lécorché P, Walrant A, Burlina F, Dutot L, Sagan S, Mallet J-M, et al. Cellular uptake and biophysical properties of galactose and/or tryptophan containing cell-penetrating peptides. Biochim Biophys Acta 2012;1818:448–57.Google Scholar
Dom G, Shaw-Jackson C, Matis C, Bouffioux O, Picard J, Prochiantz A. Cellular uptake of Antennapedia Penetratin peptides is a two-step process in which phase transfer precedes a tryptophan-dependent translocation. Nucleic Acids Res 2003;31:556–61.CrossrefGoogle Scholar
Jafari M, Karunaratne DN, Sweeting CM, Chen P. Modification of a designed amphipathic cell-penetrating peptide and its effect on solubility, secondary structure, and uptake efficiency. Biochemistry 2013;52:3428–35.CrossrefGoogle Scholar
Subrizi A, Tuominen E, Bunker A, Róg T, Antopolsky M, Urtti A. Tat (48–60) peptide amino acid sequence is not unique in its cell penetrating properties and cell-surface glycosaminoglycans inhibit its cellular uptake. J Control Release 2012;158:277–85.Google Scholar
Rydberg HA, Matson M, Åmand HL, Esbjorner EK, Norden B. Effects of tryptophan content and backbone spacing on the uptake efficiency of cell-penetrating peptides. Biochemistry 2012;51:5531–9.CrossrefGoogle Scholar
Maiolo JR, Ferrer M, Ottinger EA. Effects of cargo molecules on the cellular uptake of arginine-rich cell-penetrating peptides. Biochim Biophys Acta 2005;1712:161–72.Google Scholar
Tünnemann G, Martin RM, Haupt S, Patsch C, Edenhofer F, Cardoso MC. Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells. FASEB J 2006;20:1775–84.CrossrefGoogle Scholar
Barany-Wallje E, Gaur J, Lundberg P, Langel U, Grasland A. Differential membrane perturbation caused by the cell penetrating peptide Tp10 depending on attached cargo. FEBS Lett 2007;581:2389–93.Google Scholar
Vasconcelos L, Madani F, Arukuusk P, Pärnaste L, Gräslund A, Langel U. Effects of cargo molecules on membrane perturbation caused by transportan10 based cell-penetrating peptides. Biochim Biophys Acta 2014;1838:3118–29.Google Scholar
Amand HL, Rydberg HA, Fornander LH, Lincoln P, Nordén B, Esbjörner EK. Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans. Biochim Biophys Acta 2012;1818:2669–78.Google Scholar
Poon GM, Gariepy J. Cell-surface proteoglycans as molecular portals for cationic peptide and polymer entry into cells. Biochem Soc Trans 2007;35:788–93.Google Scholar
Naik RJ, Chandra P, Mann A, Ganguli M. Exogenous and cell surface glycosaminoglycans alter DNA delivery efficiency of arginine and lysine homopeptides in distinctly different ways. J Biol Chem 2011;286:18982–93.Google Scholar
Naik RJ, Chatterjee A, Ganguli M. Different roles of cell surface and exogenous glycosaminoglycans in controlling gene delivery by arginine-rich peptides with varied distribution of arginines. Biochim Biophys Acta 2013;1828:1484–93.Google Scholar
Epand RF, Macosko JC, Russell CJ, Shin YK, Epand RM. The ectodomain of HA2 of influenza virus promotes rapid pH dependent membrane fusion. J Mol Biol 1999;286:489–503.Google Scholar
Durrer P, Galli C, Hoenke S, Corti C, Glück R, Vorherr T, et al. H + -induced membrane insertion of influenza virus hemagglutinin involves the HA2 amino-terminal fusion peptide but not the coiled coil region. J Biol Chem 1996;271:13417–21.Google Scholar
Wang Y, Mangipudi SS, Canine BF, Hatefi A. A designer biomimetic vector with a chimeric architecture for targeted gene transfer. J Control Release 2009;137:46–53.Google Scholar
Khalil IA, Hayashi Y, Mizuno R, Harashima H. Octaarginine- and pH sensitive fusogenic peptide-modified nanoparticles for liver gene delivery. J Control Release 2011;156:374–80.Google Scholar
Wyman TB, Nicol F, Zelphati O, Scaria PV, Plank C, Szoka FC. Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry 1997;36:3008–17.CrossrefGoogle Scholar
McCarthy HO, McCaffrey J, McCrudden CM, Zholobenko A, Ali AA, McBride JW, et al. Development and characterization of self-assembling nanoparticles using a bio-inspired amphipathic peptide for gene delivery. J Control Release [Internet] 2014 Sep 10 [cited 2014 Oct 14];189:141–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24995949.
Nouri FS, Wang X, Dorrani M, Karjoo Z, Hatefi A. A recombinant biopolymeric platform for reliable evaluation of the activity of pH-responsive amphiphile fusogenic peptides. Biomacromolecules [Internet] 2013;14:2033–40. Available from: http://pubs.acs.org/doi/abs/10.1021/bm400380s.Crossref
Plank C, Oberhauser B, Mechtler K, Koch C, Wagner E. The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems. J Biol Chem 1994;269:12918–24.Google Scholar
Midoux P, Kichler A, Boutin V, Maurizot JC, Monsigny M. Membrane permeabilization and efficient gene transfer by a peptide containing several histidines. Bioconjug Chem 1998;9:260–7.CrossrefGoogle Scholar
Funhoff AM, van Nostrum CF, Koning GA, Schuurmans-Nieuwenbroek NME, Crommelin DJ, Hennink WE. Endosomal escape of polymeric gene delivery complexes is not always enhanced by polymers buffering at low pH. Biomacromolecules 2004;5:32–9.CrossrefGoogle Scholar
Aa MAEM Van Der, Mastrobattista E, Oosting RS, Hennink WE, Koning GA, Crommelin DJA. Expert review the nuclear pore complex: the gateway to successful nonviral gene delivery. Pharm Res 2006;23:447–59.Google Scholar
Brunner S, Sauer T, Carotta S, Cotten M, Saltik M, Wagner E. Nonviral transfer technology cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther 2000;7:401–7.CrossrefGoogle Scholar
Kim B-K, Kang H, Doh K-O, Lee S-H, Park J-W, Lee S-J, et al. Homodimeric SV40 NLS peptide formed by disulfide bond as enhancer for gene delivery. Bioorg Med Chem Lett [Internet]. Elsevier Ltd; 2012 Sep 1 [cited 2014 May 3];22:5415–8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22871581.
Pinol-Roma S, Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature. 1992;355:730–2.Google Scholar
Canine BF, Wang Y, Hatefi A. Biosynthesis and characterization of a novel genetically engineered polymer for targeted gene transfer to cancer cells. J Control Release 2009;138:188–96.Google Scholar
Gu L, Tsuji T, Jarboui MA, Yeo GP, Sheehy N, Hall WW, et al. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: novel insights into the regulation of Rev nuclear import. Retrovirology [Internet]. BioMed Central Ltd; 2011 Jan [cited 2014 Oct 31];8:17. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3062594&tool=pmcentrez&rendertype=abstract.
Dahl E, Kristiansen G, Gottlob K, Klaman I, Ebner E, Hinzmann B, et al. Molecular profiling of laser-microdissected matched tumor and normal breast tissue identifies karyopherin α 2 as a potential novel prognostic marker in breast cancer. Clin Cancer Res 2006;3950–60.CrossrefGoogle Scholar
Kim I, Kim D, Han S, Chin M, Nam H, Cho H, et al. Genes: structure and regulation: truncated form of importin α identified in breast cancer cell inhibits nuclear import of p53. J Biol Chem 2000;275:23139–45.Google Scholar
Lechardeur D, Sohn K, Haardt M, Joshi PB, Monck M, Graham RW, et al. Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Ther 1999;482–97.CrossrefGoogle Scholar
Dauty E, Verkman AS. Actin cytoskeleton as the principal determinant of size-dependent DNA mobility in cytoplasm: a new barrier for non-viral gene delivery. J Biol Chem [Internet] 2005 Mar 4 [cited 2014 Oct 31];280:7823–8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15632160.
Lee TWR, Lawrence FJ, Dauksaite V, Akusja G, Blair GE, Matthews DA. Precursor of human adenovirus core polypeptide Mu targets the nucleolus and modulates the expression of E2 proteins. J Gen Virol 2004;85:185–96.Google Scholar
Wagstaff KM, Glover DJ, Tremethick DJ, Jans DA. Histone-mediated transduction as an efficient means for gene delivery. Mol Ther 2007;15:721–31.Google Scholar
Yi W-J, Yang J, Li C, Wang H-Y, Liu C-W, Tao L, et al. Enhanced nuclear import and transfection efficiency of TAT peptide-based gene delivery systems modified by additional nuclear localization signals. Bioconjug Chem [Internet]. 2012 Jan 18;23:125–34. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22148643.
Wang T, Upponi JR, Torchilin VP. Design of multifunctional non-viral gene vectors to overcome physiological barriers: dilemmas and strategies. Int J Pharm 2012;427:3–20.Google Scholar
About the article
Stephen Patrick Loughran
Stephen Loughran was awarded a Masters in Pharmacy (1st Class) by Queens University Belfast in 2011. He is currently in his second year of a PhD research project which focuses on the design of multifunctional peptide vectors capable of delivering microRNA to treat metastatic prostate cancer.
Cian Michael McCrudden
Cian McCrudden was awarded a BSc (Hons) degree in Biomedical Sciences (which included a year’s research placement in the Pharmacology Department in the University of Nevada’s Medical School) and a Masters in Research by the University of Ulster, and received his PhD in peptide pharmacology from Queen’s University, Belfast. Since 2007, Dr McCrudden has gained extensive postdoctoral experience in cancer pharmacology, and since 2012 has been characterizing the potential of a DNA delivery peptide for the inducible nitric oxide synthase gene therapy treatment of metastatic breast and prostate cancer.
Helen Olga McCarthy
Helen O. McCarthy is a Reader in Experimental Therapeutics in the School of Pharmacy, Queen’s University Belfast. Her research is focused on cancer gene therapy for metastatic breast and prostate cancer and the development of bio-inspired non-viral systems for macromolecular delivery. She is particularly interested in creating multi-functional delivery systems designed to overcome all the cellular barriers to gene delivery. To that end she has created novel peptide delivery systems for oligonucleotide delivery and is applying these for systemic therapies using a number of genes including inducible nitric oxide synthase. She is also utilizing these delivery systems for DNA vaccination for prostate cancer and cervical cancer. She has received funding from Cancer Research UK, Breast Cancer Campaign, Prostate Cancer UK, Royal Pharmaceutical Society of Great Britain, The Royal Society, Medical Research Council, Invest Northern Ireland, National Science Foundation and Touchlight Genetics.
Published Online: 2015-03-21
Published in Print: 2015-04-01