The sequences and the endosomal escape mechanism of selected CPPs that are being investigated for nucleic acid delivery.
1. Introduction
Nucleic acids, including plasmid DNA (pDNA) and small interfering RNA (siRNA), are potential therapeutic macromolecules that have been widely explored for the treatment or prevention of various human diseases in the last three decades. pDNA encoding a therapeutic gene sequence can be introduced into the nuclei of the target cells to express functional proteins through transcription and translation in order to produce therapeutic effects. The therapeutic scope of pDNA includes a vast number of diseases, such as cancers (El-Aneed 2004; Yamamoto and Curiel 2005;
Johnson, Morgan et al. 2009), genetic disorders (Gaspar, Parsley et al. 2004; Aiuti, Cattaneo et al. 2009; Griesenbach and Alton 2009), infections (Yu, Poeschla et al. 1994; Hashiba, Suzuki et al. 2001; Cull, Bartlett et al. 2002) and cardiovascular diseases (Stewart, Hilton et al. 2006; Vinge, Raake et al. 2008; Henry and Satran 2012). To date, over 1600 gene therapy clinical trials have been initiated (http://www.abedia.com/wiley/phases.php; Edelstein, Abedi et al. 2007). Apart from pDNA, siRNA has recently been intensively studied as a new therapeutic agent. RNA interference (RNAi) was discovered by Fire and colleagues based on the study of
1.1. Nucleic acid delivery
In terms of delivery, therapeutic nucleic acids must be transported to their target sites (nucleus for pDNA or RISC in the cytoplasm for siRNA) before producing their biological effects. A delivery system must overcome a series of extracellular and intracellular barriers (Sanders, Rudolph et al. 2009). Nucleic acids are susceptible to endogenous nuclease degradation in the serum and the half-life of unprotected nucleic acids is approximately 10 minutes in mouse whole blood (Kawabata, Takakura et al. 1995). In addition, nucleic acids are negatively charged, hydrophilic macromolecules and are incapable of crossing the plasma membrane unassisted (Khalil, Kogure et al. 2006; Lam, Liang et al. 2012). To achieve successful transfection, an effective nucleic acid delivery system must be able to perform several functions: (i) bind or condense nucleic acids into nanoparticles, (ii) protect nucleic acids from enzymatic degradation, (iii) promote cellular uptake, (iv) release nucleic acids into the cytoplasm, (v) promote nuclear entry (for pDNA delivery) (Bally, Harvie et al. 1999). The use of a carrier system such as cationic polymers (Laga, Carlisle et al. 2012), lipids/ liposomes (Ewert, Zidovska et al. 2010) or peptides (Hassane, Saleh et al. 2010) is the most commonly investigated delivery method for clinical purposes. It was soon found that the transfection efficiency of nucleic acid delivery systems is correlated to not only the level of cellular uptake but also with their ability to escape from endosomal compartments (El Ouahabi, Thiry et al. 1997). Some nucleic acid delivery systems successfully attain high cellular uptake, but fail to achieve good transfection, partly due to their deficiency of endosomolytic property (Medina-Kauwe, Xie et al. 2005). Therefore additional measures must be adopted to promote endosomal escape of the nucleic acid delivery system.
1.2. Intracellular delivery
Viral vectors are known for their high efficiency in transferring nucleic acids into host cells as they have evolved sophisticated endosomal releasing mechanisms which take advantage of the acidic environment inside the endosomes (Cho, Kim et al. 2003). However, the clinical application of viral vectors is limited because of the strong immunogenicity and potential fatal adverse effects (Baum, Düllmann et al. 2003; Hacein-Bey-Abina, von Kalle et al. 2003; Raper, Chirmule et al. 2003; Hacein-Bey-Abina, Garrigue et al. 2008). Compared with viral vectors, non-viral vectors offer advantages of relatively low toxicity and immune response. However, the delivery efficiency of non-viral vectors is generally poor (Pérez-Martínez, Guerra et al. 2011). To enhance transfection efficiency, substantial efforts have been made to elicit effective endosomal escape. Endocytosis is the major route of cellular entry for non-viral nucleic acid delivery (Khalil, Kogure et al. 2006; Pathak, Patnaik et al. 2009). A number of endocytosis pathways are known to be involved in the uptake of non-viral gene delivery systems, including clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis and phagocytosis. However the contribution of each pathway in the internalization of non-viral vectors is not clearly understood due to the diversity of carriers (Morille, Passirani et al. 2008).
After non-viral delivery systems enter into the cells via endocytosis they are immediately transported into the endocytic vesicles. Initially, the delivery vectors are trapped in the early endosomes where the pH drops from neutral to around pH 6. Early endosomes may fuse with sorting endosomes in which the internalized content can be recycled back to the membrane and transported out of the cell by exocytosis. More often, the delivery systems are trafficked to late endosomes which are rapidly acidified to pH 5–6 by the action of the membrane-bound ATPase proton-pump. Subsequently, the late endosomes fuse with lysosomes concomitant with a further pH reduction to approximately pH 4.5 and the existence of various degradative enzymes. The low pH of lysosomes facilitates substrate denaturation and aids lysosomal hydrolases, most of which operate optimally in the range of pH 4.5-5.5 (Mellman, Fuchs et al. 1986). Nucleic acids that fail to be released from these acidic vesicles will ultimately be degraded (Pack, Hoffman et al. 2005; Khalil, Kogure et al. 2006). Therefore, the pH reduction and the enzymatic degradation process in endosomes/lysosomes is an efficiency-limiting step for successful nucleic acid delivery (Whitehead, Langer et al. 2009). Ideal vectors should release their contents from these acidic compartments at an early stage to prevent the fate of lysosomal destruction.
1.3. Endosomal escape
Various approaches have been attempted to promote early endosomal escape of non-viral gene delivery vehicles and many hypotheses have been suggested to explain these processes. The proton sponge hypothesis has been proposed for cationic polymers such as polyethylenimine (PEI) (Boussif, Lezoualc'h et al. 1995; Behr 1997) and polyamidoamine (PAMAM) dendrimers (Zhou, Wu et al. 2006). For cationic-lipid based delivery systems, the flip-flop mechanism was proposed for their endosomal escape mechanism (Xu and Szoka Jr 1996). Cell-penetrating peptides (CPPs) represent another category of promising candidates as non-viral nucleic acid carriers, for example TAT (Torchilin 2008), pep analogues (Gros, Deshayes et al. 2006), GALA (Li, Nicol et al. 2004), MPG (Simeoni, Morris et al. 2003), CADY (Konate, Crombez et al. 2010) and LAH4 derivatives (Lam, Liang et al. 2012) etc. Their mechanisms of promoting endosomal release are still controversial; it has been suggested that the opening of transient pores in the lipid bilayer of endosome is involved (Melikov and Chernomordik 2005), alternatively CPPs may undergo conformational changes in response to the acidification inside the endosomes, leading to destabilization of the endosomal membrane bilayer (Kichler, Mason et al. 2006). Last but not least, photochemical internalization (PCI) is a technique that aims to improve endosomal release. A photosensitizer is localized in the endosomal membrane and destabilizes the membrane upon illumination, triggering the release of endosomal content into cytosol (Berg, Kristian Selbo et al. 1999; Endoh and Ohtsuki 2009).
In order to achieve high levels of transfection, different strategies have been employed to protect nucleic acids from degradation inside endosomes and facilitate their early release from acidic compartments into the cytosol. Various endosomal escape mechanisms of non-viral vectors as well as the endosomolytic reagents which can promote endosomal release are introduced in detail here.
2. Strategies of non-viral vectors for endosomal escape
2.1. The ‘proton sponge’ hypotheses (pH-buffering effect)
There is a long history regarding the application of cationic polymers to mediate nucleic acid transfer into cells. Cationic polymers can form polyplexes with nucleic acids through electrostatic interaction. Polylysine (PLL) was one of the first cationic polymers investigated for nucleic acid delivery although it failed to display desirable transfection efficiency (Pack, Hoffman et al. 2005). Later, it was discovered that polymers that contain protonable residues at physiological pH, like polyamidoamine (PAMAM) dendrimers and lipopolyamines (Remy, Sirlin et al. 1994) successfully achieve high transfection efficiency in contrast to PLL, which does not possess buffering capacity because of the presence of the strongly charged amino groups (Haensler and Szoka Jr 1993). This pH-buffering property was soon shown to be an important feature of cationic polymers that may induce endosomal disruption and prevent nucleic acids from lysosomal degradation.
Polyethylenimine (PEI) is a synthetic cationic polymer with high amine density and various applications (Godbey, Wu et al. 1999). In 1995, Boussif
The ‘proton sponge’ phenomenon has been observed in certain cationic polymers with a high pH buffering capability over a wide pH range. These polymers usually contain protonatable secondary and/or tertiary amine groups with pKa close to endosomal/lysosomal pH. During the maturation of endosomes, the membrane-bound ATPase proton pumps actively translocate protons from the cytosol into the endosomes, leading to the acidification of endosomal compartments and activation of hydrolytic enzymes. At this stage, polymers with the ‘proton sponge’ property will become protonated and resist the acidification of endosomes (fig. 1). As a result, more protons will be continuously pumped into the endosomes with the attempt to lower the pH. The proton pumping action is followed by passive entry of chloride ions, increasing ionic concentration and leading to water influx. Eventually the osmotic pressure causes swelling and rupture of endosomes, releasing their contents to the cytosol (Boussif, Lezoualc'h et al. 1995; Behr 1997; Pack, Hoffman et al. 2005). Sonawane
To date, PEI has been demonstrated to transfect nucleic acids successfully into a broad range of cells and tissues both
On the contrary, polymeric carriers that do not possess pH buffering properties, such as chitosan and PLL, fail to achieve satisfactory nucleic acid delivery efficiencies because of their inability to induce endosomal escape even though they are capable of binding to nucleic acids and promoting cellular entry (Wagner and Kloeckner 2006). To enhance transfection efficiency, functional moieties were included into these polymeric systems for improving their buffering capacity. Histidine is one of the commonly employed molecules that was added as functional group of polymers or incorporated into peptide sequences to enhance their pH buffering capacity.
The buffering capacity of histidine is due to the presence of an imidazole ring that has a pKa around 6 and hence can be protonated in a slightly acidic pH (Midoux, Pichon et al. 2009). Midoux
2.2. Flip-flop mechanism
Lipids and liposomes, whether anionic, cationic, neutral and/or pH-sensitive, present another category of non-viral carriers that have been extensively investigated for delivering nucleic acid into mammalian cells. In general, the
The mechanism of how lipoplexes gain entry into the cells is controversial. According to literature, there are at least two routes of cellular uptake: (i) direct fusion with the plasma membrane; and (ii) endocytosis (Pedroso de Lima, Simões et al. 2001). Physicochemical properties of lipoplexes such as particle size distribution, lipid composition and charge ratio may also influence their uptake route. In order to gain a better insight of the uptake mechanism of lipid-based system, Wrobel and Collins studied the interaction between cationic liposomes and model anionic membrane as well as cultured mammalian cells. The results indicated that cell surface binding alone is insufficient for cationic liposomes to gain entry into cells via membrane fusion in the absence of endocytosis (Wrobel and Collins 1995). Zhou
To find out the intracellular fate of the lipoplexes following endocytosis, an electron microscopic study was carried out by Zabner
Neutral lipids such as the phosphatidylethanolamine (DOPE) are widely used as helper lipids in combination with cationic liposomes. It is well established that inclusion of DOPE in lipoplexes may significantly improve their transfection activity, whereas replacement of DOPE with other neutral phospholipid dioleoylphosphatidylcholine (DOPC) fails to accomplish the helper function. The role of DOPE as helper lipid is attributed to its endosomolytic activity. Zhou and Huang used transmission electron microscopy to study intracellular trafficking of cationic liposomes and found that DOPE-containing lipoplexes can destabilize the endosomal membrane (Zhou and Huang 1994) whereas the DOPC-containing lipoplexes did not show the same effect. A study carried out by Farhood
Apart from helper lipids, other approaches have been investigated to potentiate endosomal lysis and the release of nucleic acid from lipoplexes to cytosol through the ‘flip-flop’ mechanism. Simoes
2.3. Endosomal membrane fusion or destabilization mechanism
Cell-penetration peptides (CPPs) have attracted tremendous attention as non-viral nucleic acid delivery vectors in recent years. Typically cationic and/or amphipathic in nature, CPPs are short sequences of amino acids, usually 10-30 residues, claimed to have ability to cross the plasma membrane of living cells. They can facilitate the transportation of various hydrophilic macromolecules including proteins, peptides and nucleic acids into cells without the disruption of plasma membrane (Richard, Melikov et al. 2003). CPPs were originally derived from viruses, with TAT peptide being the first CPP identified and was derived from the transcription activating factor of human immunodeficiency virus 1 (HIV-l) (Brooks, Lebleu et al. 2005). Many different sequences of CPPs were soon discovered and synthetic analogues were also rapidly developed. Until now there are a number of CPPs that were documented for nucleic acid delivery.
CPPs either form complexes with nucleic acids, through electrostatic interaction, or can be incorporated into polymeric and lipidic delivery system. In general, they can be categorized into two main classes (Futaki 2006; Patel, Zaro et al. 2007): (i) Cationic peptides that usually contain arginine and lysine residues, e.g., TAT peptide, penetratin (Derossi, Joliot et al. 1994; Derossi, Calvet et al. 1996; Muratovska and Eccles 2004) and oligoarginines (Futaki 2006); (ii) Amphipathic peptides that consist of both hydrophobic and hydrophilic segments. The amphipathicity of these peptides generates from either the primary structure or the secondary structure. Primary amphipathic peptides are sequentially made up of hydrophobic and hydrophilic residues (Fernández‐Carneado, Kogan et al. 2004; Deshayes, Morris et al. 2005), and include e.g., MPG (Simeoni, Morris et al. 2003; Veldhoen, Laufer et al. 2006), pep-1 and its analogues. Secondary amphipathic peptides adopt an amphipathic helical conformation with hydrophilic and hydrophobic regions and include e.g., HA-2 (Wagner, Plank et al. 1992; Plank, Oberhauser et al. 1994), GALA (Li, Nicol et al. 2004), KALA (Wyman, Nicol
Nevertheless, a variety of CPPs have been shown to enter cells via an endosomal pathway and induce endosomolytic activity. The majority of these membrane-destabilizing peptides were developed to mimic the endosomal disruptive properties of fusogenic sequences of viral fusion proteins. Taking the haemagglutin subunit HA2 of influenza virus as an example, this protein chain is responsible for facilitating membrane fusion. The C-terminal end is embedded in the viral membrane whereas the N-terminal end contains a fusion peptide with a sequence of hydrophobic amino acids. Once inside the endosomes, HA undergoes conformation change in response to the low pH environment and expose the highly conserved hydrophobic N-terminal region. Subsequently, this triggers the fusion of viral membrane with the endosomal membrane, leading to viral genome leakage to cytosol (Stegmann 2000). Wagner
Since the α-helical component of the HA2 appears to play a crucial role in endosomal membrane destabilization (Oehlke, Scheller et al. 1998), a series of pH-sensitive amphipathic α-helical structural motifs were designed and their structure–activity relationship were investigated. GALA is a synthetic peptide with 30 amino acid residues designed to interact with lipid bilayers at low pH. It contains a histidine and a tryptophan residue, as well as a glutamic acid-alanine-leucine-alanine (EALA) repeat. When the pH of the surrounding environment drops from 7.0 to 5.0, GALA experiences a conformational change from a random coil to an amphipathic α-helix, leading to disruption of model lipid membranes and therefore the release of entrapped aqueous content. The membrane-destabilizing character of this pH sensitive peptide in an acidic environment raises the possibility of enhancing the delivery of nucleic acid by facilitating endosomal escape (Li, Nicol et al. 2004). Haensler and Szoka Jr
The negatively charged GALA cannot bind with nucleic acid through electrostatic interaction, GALA can only be added as an additional functional component to polyplexes or lipoplexes. Newer peptides were soon developed to combine both nucleic acid binding and membrane destabilizing properties in order to produce a simpler delivery system. KALA is a modified version of GALA by partially replacing glutamic acid with lysine. It is one of the first generation peptides that is designed to bind nucleic acids as well as destabilize the endosomal membranes. Interestingly, the membrane destabilization mechanism of KALA is substantially different from that of GALA although they share similar amino acid sequence. KALA adopts α-helix conformation in a wide pH range and undergoes a pH-dependent conformational change from amphipathic α-helical to a mixture of α-helix and random coil as the pH is lowered (Wyman, Nicol et al. 1997). Apart from GALA and KALA, other amphipathic peptides that attain endosomal escape ability that is related to their α-helical structure include the Hel series peptides (Niidome, Takaji et al. 1999), INF 7 (Plank, Oberhauser et al. 1994), HGP (Kwon, Bergen et al. 2008), JTS-1 (Gottschalk, Sparrow et al. 1996), EBI (Lundberg, El-Andaloussi et al. 2007), ppTG1 (Rittner, Benavente et al. 2002) and CADY (Crombez, Aldrian-Herrada et al. 2008; Konate, Crombez et al. 2010).
TAT | GRKKRRQRRRPPQ | Unclear, endosomal escape is inefficient | (Vives 2003; Brooks, Lebleu |
Penetratin | RQIKIWFQNRRMKWKK | Unclear, endosomal escape is inefficient | (Muratovska and Eccles 2004) |
EBI | LIRLWSHLIHIWFQNRRLKWKKK | Membrane destabilization | (Lundberg, El-Andaloussi |
MPG | GALFLGFLGAAGSTMGAWSQPKKKRKV | Bypass endosomes through non-endosomal uptake | (Morris, Vidal Simeoni, Morris |
HGP | LLGRRGWEVLKYWWNLLQYWSQELC | Membrane destabilization, possibly pore formation | (Kwon, Bergen |
Pep-2 | KETWFETWFTEWSQPKKKRKV | Bypass endosomes through non-endosomal uptake | (Morris, Depollier |
HA-2 derived fusogenic peptide | GLFGAIAGFIEGGWTGMIDGWYG | Membrane fusion and destabilization | (Wagner, Plank |
H5WYG | GLFHAIAHFIHGGWHGLIHGWYG | Membrane destabilization | (Midoux, Kichler |
INF-7 | GLFEAIEGFIENGWEG MIDGWYG | Membrane fusion and destabilization | (Plank, Oberhauser et al. 1994; van Rossenberg, Sliedregt-Bol |
E5 & E5CA | GLFGAIAGFIEGGWTGMIDG GLFEAIAEFIEGGWEGLIEGCA | Membrane fusion and destabilization | (Midoux, Mendes |
JTS-1 | GLFEALLELLESLWELLLEA | Membrane destabilization | (Gottschalk, Sparrow |
ppTG1 | GLFKALLKLLKSLWKLLLKA | Membrane destabilization | (Rittner, Benavente |
GALA | WEAALAEALAEALAEHLAEALAEALEALAA | Membrane destabilization, Pore formation & flip-flop of membrane lipids | (Parente, Nir |
KALA | WEAKLAKALAKALAKHLAKALAKALKACEA | Membrane destabilization | (Wyman, Nicol |
CADY | GLWRALWRLLRSLWRLLWRA | Bypass endosomes through non-endosomal uptake | (Crombez, Aldrian-Herrada |
Peptide 46 & analogues | LARLLARLLARLLRALLRALLRAL | Membrane destabilization | (Niidome, Ohmori |
HEL peptides & analogues | KLLKLLLKLWKKLLKLLK | Membrane destabilization | (Ohmori, Niidome |
LAH4 & analogues | KKALLALALHHLAHLALHLALALKKA | Membrane destabilization | (Kichler, Leborgne |
The LAH4 peptide and its derivatives are another class of peptide that exhibits efficient gene transfer activity (Kichler, Leborgne et al. 2003; Lam, Liang et al. 2012). Peptides of the LAH4 family are synthetic cationic amphipathic peptides containing a variable number of histidine residues and hydrophobic amino acids (mainly alanines and leucines). They were initially designed to investigate the interactions that determine the alignment of membrane-associated proteins (Bechinger 1996; Vogt and Bechinger 1999).
2.4. Pore formation
Pore formation is another mechanism to explain the endosomal escape of peptide-based nucleic acid delivery systems. Parente
2.5. Photochemical internalization (PCI)
Photochemical internalization (PCI) is a light-directed delivery technology that utilizes photosensitizers to facilitate the transport of membrane impermeable macromolecules from endocytic vesicles into cytoplasm. The mechanism of PCI as an endosomal escape enhancer strategy is very straight-forward (Fig.3). Photosensitizers that are employed in the PCI technology are usually amphiphilic compounds which can bind to and localize in the plasma membrane. After being taken up by the cells through endocytosis, the photosensitizers are confined to the endosomal membranes and remain inactive until triggered by light with specific wavelengths matching their absorption spectra (Selbo, Weyergang et al. 2010). Once activated, they induce the formation of highly reactive oxygen species, mainly singlet oxygen, leading to the rupture of endosomes and lysosomes membrane. As a result, macromolecules that are trapped inside the endosomes/lysosomes can be liberated into the cytosol (Berg, Kristian Selbo et al. 1999). Photosensitizers used in clinical application are highly reactive reagents with short range of action (10-20 nm) and short life-time (0.01-0.04 μs), thus restricting the damaging effect to the production site (within the endosomal membrane) without affecting other cellular components (Moan and Berg 1991; Berg, Weyergang et al. 2010). Most of the photosensitizers do not localize to the nucleus of the cells, thereby reducing the possibility of causing any mutagenic effects (Dougherty, Henderson et al. 1998).
PCI was initially investigated for anti-tumour drug delivery. A synergistic effect of combining PCI with chemotherapeutic agents was found. PCI principally targets cellular endocytosis that may affect the distribution of molecules that are taken up by the cells via endosomal pathway. It was later employed as a tool to improve the cellular delivery of a large variety of bioactive macromolecules and nucleic acids including pDNA, siRNA and oligonucleotides (Selbo, Weyergang et al. 2010). Examples of photosensitizers that are used in non-viral nucleic acid delivery including disulfonated meso-tetraphenylporphine (TPPS2a) (Prasmickaite, Høgset et al. 2000; Kloeckner, Prasmickaite et al. 2004; Maurice-Duelli, Ndoye et al. 2004; Ndoye, Merlin et al. 2004; Ndoye, Dolivet et al. 2006; Oliveira, Fretz et al. 2007; Boe, Longva et al. 2008; Raemdonck, Naeye et al. 2009; Bøe, Sæbøe-Larssen et al. 2010), disulfonated aluminium phthalocyanine (AlPcS2a) (Berg, Prasmickaite et al. 2003; Hellum, Høgset et al. 2003; Ndoye, Dolivet et al. 2006; Yip, Weyergang et al. 2007), Zinc-phthalocyanine (Zn-Pc) dendrimer (Nishiyama, Iriyama et al. 2005; Arnida, Nishiyama et al. 2006) and 5,10,15-tri(4-acetamidophenyl)-20-mono(4-carboxyl-phenyl)porphyrin (TAMCPP) conjugated to G4 PAMAM dendrimer (Shieh, Peng et al. 2008).
To employ PCI in clinical applications, the penetration of light into the deep tissue is an important issue (Oliveira, Fretz et al. 2007). With the development of fiber optics and laser technology, the control of illumination to sites that are deep inside the human body becomes possible, e.g. gastrointestinal tract, urogenital organs, lungs, brain and pancreas (Dougherty, Henderson et al. 1998; Chatterjee, Fong et al. 2008). PCI mediated therapy can be used in many regions of the body where light delivery can be achieved and where local activation of a drug is desirable. A photosensizer is injected as a single dose prior to light activation. Parameters such as the dose of photosentizers and light, as well as the time interval between administration of photosentiziers and drugs must be carefully optimized.
2.6. Other endosomal escape mechanisms
Exogenous additives, such as chloroquine and inactivated adenovirus, have been exploited to promote endosomal escape and enhance the efficiency of nucleic acid delivery. Chloroquine is a weak base that can rapidly penetrate the plasma membrane, accumulate in acidic vesicles and increase the pH of the acidic compartment (Maxfield 1982; Mellman, Fuchs et al. 1986). Preventing endosome acidification may subsequently inhibit hydrolytic enzymes such as proteases and nucleases (Cotten, Längle-Rouault et al. 1990). Chloroquine also causes the swelling and rupture of endosomal vesicle by increasing the osmotic pressure inside the acidic compartment (Khalil, Kogure et al. 2006). Since it can neutralize acidic compartment and induce rupture of endocytic vesicles, adding chloroquine is an alternative measure to improve nucleic acid transfer (Erbacher, Roche et al. 1996).
A number of early studies found that chloroquine is able to enhance DNA transfection in various cell types (Luthman and Magnusson 1983; Cotten, Längle-Rouault
Besides chloroquine, physically coupling chemically inactivated adenovirus particles is another approach for promoting endosomal escape. This method takes the advantage of the endosomolytic activity of adenovirus to facilitate the release of nucleic acids from the endosomes (Curiel, Agarwal et al. 1991; Cotten, Wagner et al. 1992; Wagner, Zatloukal et al. 1992). Curiel
However, the application of both chloroquine and inactivated adenovirus particles are limited due to safety concern. Although chloroquine is approved by the FDA as an anti-malaria medication, it is found to be toxic to many cell types and can trigger gastrointestinal and nervous adverse effects in high dose (Pack, Putnam et al. 2000). For defective adenovirus particles, the complexity of vector production and potential immunogenicity raised by the virus components make this strategy problematic (Pack, Hoffman et al. 2005). Unless the safety issues can be solved, both methods will remain unsuitable for clinical use.
3. Conclusion
Non-viral vectors are considered to be promising vehicles for delivering therapeutic nucleic acids because of their relatively safe profile and high versatility as compared to their viral counterparts. However, the transfection efficiency of non-viral vectors is less than satisfactory for clinical purpose. The endocytosis pathway is a major route for the cellular entry of non-viral nucleic acid delivery agents. Poor endosomal escape of non-viral systems pose a major challenge for the intracellular delivery of nucleic acids. An ideal nucleic acid delivery system should fulfill several criteria: negligible toxicity, biocompatible and biodegradable, offer protection to nucleic acids from enzymatic degradation, facilitate cellular uptake, promote endosomal escape and release the nucleic acids at site of action. Elucidation of the mechanism of endosomal escape is beneficial in the development of more effective non-viral delivery vectors. However, the uptake and cytoplasmic transportation mechanisms of a variety of non-viral nucleic acid carriers still need to be investigated in more detail. In the future, with the development of cell imaging techniques such as high resolution, spinning disk live cell confocal imaging and the fluorescence correlation spectroscope, the details of intracellular trafficking of non-viral nucleic acid delivery systems will be unveiled. This will guide the future design and development of novel efficient non-viral nucleic acid delivery vectors.
References
- 1.
Aiuti A. Cattaneo F. et al. 2009 "Gene therapy for immunodeficiency due to adenosine deaminase deficiency ."360 5 447 458 . - 2.
Arnida A. Nishiyama N. et al. 2006 "Novel ternary polyplex of triblock copolymer, pDNA and anionic dendrimer phthalocyanine for photochemical enhancement of transgene expression ." 116(2):e75 EOF e77 EOF . - 3.
Arote R. Kim T. H. et al. 2007 "A biodegradable poly (ester amine) based on polycaprolactone and polyethylenimine as a gene carrier." Biomaterials28 4 735 744 . - 4.
Bøe S. Sæbøe-Larssen S. et al. 2010 "Light-Induced Gene Expression Using Messenger RNA Molecules ."20 1 1 6 . - 5.
Bally M. B. Harvie P. et al. 1999 "Biological barriers to cellular delivery of lipid-based DNA carriers ."38 3 291 315 . - 6.
Baum C. Düllmann J. et al. 2003 "Side effects of retroviral gene transfer into hematopoietic stem cells. "101 6 2099 2113 . - 7.
Bechinger B. 1996 "Towards membrane protein design: pH-sensitive topology of histidine-containing polypeptides ."263 5 768 775 . - 8.
Behr J. P. 1997 "The proton sponge: a trick to enter cells the viruses did not exploit ." Chimia International Journal for Chemistry 51(1,2):34 EOF 36 EOF . - 9.
Berg K. Dietze A. et al. 2005 "Site-specific drug delivery by photochemical internalization enhances the antitumor effect of bleomycin ."11 23 8476 8485 . - 10.
Berg K. Kristian P. Selbo et. al 1999 "Photochemical Internalization ." Cancer Research59 6 1180 1183 . - 11.
Berg K. Weyergang A. et al. 2010 "The Potential of Photochemical Internalization (PCI) for the Cytosolic Delivery of Nanomedicines" in Organelle-Specific Pharmaceutical Nanotechnology: John Wiley & Sons, Inc.311 322 . - 12.
Bernstein E. Caudy A. A. et al. 2001 "Role for a bidentate ribonuclease in the initiation step of RNA interference. "409 6818 363 365 . - 13.
Boe S. Longva A. S. et al. 2008 "Evaluation of various polyethylenimine formulations for light-controlled gene silencing using small interfering RNA molecules ."18 2 123 132 . - 14.
Boletta A. Benigni A. et al. 1997 "Nonviral gene delivery to the rat kidney with polyethylenimine ."8 10 1243 1251 . - 15.
Boussif O. Lezoualc’h F. et al. 1995 "A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine ." 92(16):7297 EOF 7301 EOF . - 16.
Brooks H. Lebleu B. et al. 2005 "Tat peptide-mediated cellular delivery: back to basics. "57 4 559 577 . - 17.
Chang K. L. Higuchi Y. et al. 2010 "Efficient gene transfection by histidine-modified chitosan through enhancement of endosomal escape ."21 6 1087 1095 . - 18.
Chatterjee D. K. Fong L. S. et al. 2008 "Nanoparticles in photodynamic therapy: An emerging paradigm ."60 15 1627 1637 . - 19.
Cho Y. W. Kim J. D. et al. 2003 "Polycation gene delivery systems: escape from endosomes to cytosol ."55 6 721 734 . - 20.
Chollet P. Favrot M. C. et al. 2002 "Side‐effects of a systemic injection of linear polyethylenimine-DNA complexes." The Journal of Gene Medicine4 1 84 91 . - 21.
Coll J. L. Chollet P. et al. 1999 "In vivo delivery to tumors of DNA complexed with linear polyethylenimine ."10 10 1659 1666 . - 22.
Cotten M. Längle-Rouault F. et al. 1990 "Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels ." 87(11):4033 EOF 4037 EOF . - 23.
Cotten M. Wagner E. et al. 1992 "High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles ." 89(13):6094 EOF 6098 EOF . - 24.
Crombez L. Aldrian-Herrada G. et al. 2008 "A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells ."17 1 95 103 . - 25.
Cull V. S. Bartlett E. J. et al. 2002 "Type I interferon gene therapy protects against cytomegalovirus‐induced myocarditis." Immunology106 3 428 437 . - 26.
Curiel D. T. Agarwal S. et al. 1991 "Adenovirus enhancement of transferrin-polylysine-mediated gene delivery ." 88(19):8850 EOF 8854 EOF . - 27.
Davis M. E. Zuckerman J. E. et al. 2010 "Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles ."464 7291 1067 1070 . - 28.
Derossi D. Calvet S. et al. 1996 "Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. " Journal of Biological Chemistry271 30 18188 18193 . - 29.
Derossi D. Joliot A. H. et al. 1994 "The third helix of the Antennapedia homeodomain translocates through biological membranes. " Journal of Biological Chemistry269 14 10444 10450 . - 30.
Deshayes S. Morris M. et al. 2005 "Cell-penetrating peptides: tools for intracellular delivery of therapeutics ."62 16 1839 1849 . - 31.
Deshayes S. Morris M. et al. 2008 "Delivery of proteins and nucleic acids using a non-covalent peptide-based strategy. " 60(4-5):537 EOF 47 EOF . - 32.
De Vincenzo J. Cehelsky J. E. et al. 2008 "Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV) ."77 3 225 231 . - 33.
De Vincenzo J. Lambkin-Williams R. et al. 2010 "A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus ." Proceedings of the National Academy of Sciences 107(19): 8800. - 34.
Dougherty T. J. Henderson B. W. et al. 1998 "Photodynamic therapy." Journal of the National Cancer Institute90 12 889 905 . - 35.
Edelstein M. L. Abedi M. R. et al. 2007 "Gene therapy clinical trials worldwide to 2007--an update. "9 10 833 842 . - 36.
El -Aneed A. 2004 "An overview of current delivery systems in cancer gene therapy ."94 1 1 14 . - 37.
El Ouahabi A. Thiry M. et al. 1997 "The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids. "414 2 187 192 . - 38.
Elbashir S. M. Lendeckel W. et al. 2001 "RNA interference is mediated by 21-and 22-nucleotide RNAs." 15 2 188 200 . - 39.
Endoh T. Ohtsuki T. 2009 "Cellular siRNA delivery using cell-penetrating peptides modified for endosomal escape ."61 9 704 709 . - 40.
Erbacher P. Roche A. C. et al. 1996 "Putative role of chloroquine in gene transfer into a human hepatoma cell line by DNA/lactosylated polylysine complexes. "225 1 186 194 . - 41.
Ewert K. Zidovska A. et al. 2010 "Cationic Liposome-Nucleic Acid Complexes for Gene Delivery and Silencing: Pathways and Mechanisms for Plasmid DNA and siRNA ." Nucleic Acid Transfection:191 226 . - 42.
Farhood H. Serbina N. et al. 1995 "The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. " Biochimica et Biophysica Acta (BBA)- Biomembranes 1235: (2)289 EOF 95 EOF - 43.
Fattal E. Nir S. et al. 1994 "Pore-forming peptides induce rapid phospholipid flip-flop in membranes. "33 21 6721 6731 . - 44.
Felgner P. L. Gadek T. R. et al. 1987 "Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure ." 84(21):7413 EOF 7417 EOF . - 45.
Fernández‐ Carneado. J. Kogan M. J. et al. 2004 "Amphipathic peptides and drug delivery." Peptide Science76 2 196 203 . - 46.
Fire A. Xu S. Q. et al. 1998 "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. "391 6669 806 811 . - 47.
Fischer D. Bieber T. et al. 1999 "A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity ."16 8 1273 1279 . - 48.
Fischer P. M. Krausz E. et al. 2001 "Cellular delivery of impermeable effector molecules in the form of conjugates with peptides capable of mediating membrane translocation. "12 6 825 841 . - 49.
Fominaya J. Gasset M. et al. 2000 "An optimized amphiphilic cationic peptide as an efficient non‐viral gene delivery vector." The Journal of Gene Medicine2 6 455 464 . - 50.
Funhoff A. M. van Nostrum C. F. et al. 2005 "Cationic polymethacrylates with covalently linked membrane destabilizing peptides as gene delivery vectors ."101 1 233 246 . - 51.
Futaki S. 2005 "Membrane-permeable arginine-rich peptides and the translocation mechanisms. "57 4 547 558 . - 52.
Futaki S. 2006 "Oligoarginine vectors for intracellular delivery: Design and cellular‐uptake mechanisms." Peptide Science84 3 241 249 . - 53.
Gaspar H. B. Parsley K. L. et al. 2004 "Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. " The364 9452 2181 2187 . - 54.
Godbey W. Wu K. K. et al. 1999 "Poly (ethylenimine) and its role in gene delivery." Journal of Controlled Release 60(2-3): 149-160. - 55.
Gottschalk S. Sparrow J. et al. 1996 "A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells. " 3(5):448 EOF 57 EOF . - 56.
(1998). "Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system." Gene Therapy 5(5): 712.Goula D. Remy J. et al. - 57.
Griesenbach U. Alton E. W. F. W. 2009 "Cystic fibrosis gene therapy: successes, failures and hopes for the future ."3 4 363 371 . - 58.
Gros E. Deshayes S. et al. 2006 "A non-covalent peptide-based strategy for protein and peptide nucleic acid transduction. " (BBA)-Biomembranes1758 3 384 393 . - 59.
Hacein-Bey-Abina S. Garrigue A. et al. 2008 "Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1 ." The 118(9):3132 EOF 3142 EOF . - 60.
Hacein-Bey-Abina S. von C. Kalle et. al 2003 "A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. " New England Journal of Medicine348 3 255 256 . - 61.
Haensler J. Szoka F. C. Jr 1993 "Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. "4 5 372 379 . - 62.
Hammond S. M. Boettcher S. et al. 2001 "Argonaute2, a link between genetic and biochemical analyses of RNAi ." 293(5532):1146 EOF 1150 EOF . - 63.
Hannon G. J. Rossi J. J. 2004 "Unlocking the potential of the human genome with RNA interference. "431 7006 371 378 . - 64.
Hashiba T. Suzuki M. et al. 2001 "Adenovirus-mediated transfer of heme oxygenase-1 cDNA attenuates severe lung injury induced by the influenza virus in mice. " 8(19):1499 EOF 507 EOF . - 65.
Hassane F. S. Saleh A. F. et al. 2010 "Cell penetrating peptides: overview and applications to the delivery of oligonucleotides ."67 5 715 726 . - 66.
Hellum M. Høgset A. et al. 2003 "Photochemically enhanced gene delivery with cationic lipid formulations ." Photochemical & Photobiological Science2 4 407 411 . - 67.
Henry T. D. Satran D. 2012 "Therapeutic angiogenesis ." Coronary Artery Disease:67 74 . - 68.
Hirschberg H. Zhang M. J. et al. 2009 "Targeted delivery of bleomycin to the brain using photo-chemical internalization of Clostridium perfringens epsilon prototoxin ."95 3 317 329 . - 69.
http://www.abedia.com/wiley/phases.php. date of access: 22/03/2012’ - 70.
Hui S. W. Langner M. et al. 1996 "The role of helper lipids in cationic liposome-mediated gene transfer. "71 2 590 599 . - 71.
Johnson L. A. Morgan R. A. et al. 2009 "Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen ."114 3 535 546 . - 72.
Kaiser P. K. Symons R. C. et al. 2010 "RNAi-based treatment for neovascular age-related macular degeneration by Sirna-027 ."150 1 33 39 - 73.
Kawabata K. Takakura Y. et al. 1995 "The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake. "12 6 825 830 . - 74.
Ketting R. F. Fischer S. E. J. et al. 2001 "Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. " 15(20):2654 EOF 9 EOF . - 75.
Khalil I. A. Kogure K. et al. 2006 "Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. "58 1 32 45 . - 76.
Kichler A. Leborgne C. et al. 2003 "Histidine-rich amphipathic peptide antibiotics promote efficient delivery of DNA into mammalian cells ." 100(4):1564 EOF 1568 EOF . - 77.
Kichler A. Mason A. et al. 2006 "Cationic amphipathic histidine-rich peptides for gene delivery. " (BBA)-Biomembranes1758 3 301 307 . - 78.
Klink D. T. Chao S. et al. 2001 "Nuclear translocation of lactosylated poly-L-lysine/cDNA complex in cystic fibrosis airway epithelial cells ."3 6 831 841 . - 79.
Kloeckner J. Prasmickaite L. et al. 2004 "Photochemically enhanced gene delivery of EGF receptor-targeted DNA polyplexes ."12 4 205 213 . - 80.
Konate K. Crombez L. et al. 2010 "Insight into the cellular uptake mechanism of a secondary amphipathic cell-penetrating peptide for siRNA delivery ."49 16 3393 3402 . - 81.
Kunath K. von A. Harpe et. al 2003 "Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine ."89 1 113 125 . - 82.
Kwon E. J. Bergen J. M. et al. 2008 "Application of an HIV gp41-derived peptide for enhanced intracellular trafficking of synthetic gene and siRNA delivery vehicles ."19 4 920 927 . - 83.
Laga R. Carlisle R. et al. 2012 "Polymer coatings for delivery of nucleic acid therapeutics." Journal of Controlled Release (doi:10.1016/j.jconrel.2012.02.013). - 84.
Lam J. K. W. Liang W. et al. 2012 "Pulmonary delivery of therapeutic siRNA ."64 1 1 15 . - 85.
Lam J. K. W. Liang W. et al. 2012 "Effective endogenous gene silencing mediated by pH responsive peptides proceeds via multiple pathways ."158 2 293 303 . - 86.
Leachman S. A. Hickerson R. P. et al. 2009 "First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin disorder ."18 2 442 446 . - 87.
Lee Y. J. Johnson G. et al. 2011 "A HA2-Fusion tag limits the endosomal release of its protein cargo despite causing endosomal lysis. " (BBA)-General Subjects1810 752 758 . - 88.
Legendre J. Y. Szoka F. C. Jr 1992 "Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: comparison with cationic liposomes. "9 10 1235 1242 . - 89.
Lehto T. Abes R. et al. 2010 "Delivery of nucleic acids with a stearylated (RxR)4 peptide using a non-covalent co-incubation strategy. "141 1 42 51 . - 90.
Li W. Nicol F. et al. 2004 "GALA: a designed synthetic pH-responsive amphipathic peptide with applications in drug and gene delivery. "56 7 967 985 . - 91.
Lundberg P. El -Andaloussi S. et al. 2007 "Delivery of short interfering RNA using endosomolytic cell-penetrating peptides ."21 11 2664 2671 . - 92.
Lundin P. Johansson H. et al. 2008 "Distinct uptake routes of cell-penetrating peptide conjugates ."19 12 2535 2542 . - 93.
Luthman H. Magnusson G. 1983 "High efficiency polyoma DNA transfection of chloroquine treated cells. " 11(5):1295 EOF 308 EOF . - 94.
Mason A. J. Martinez A. et al. 2006 "The antibiotic and DNA-transfecting peptide LAH4 selectively associates with, and disorders, anionic lipids in mixed membranes ."20 2 320 322 . - 95.
Maurice-Duelli A. Ndoye A. et al. 2004 "Enhanced cell growth inhibition following PTEN nonviral gene transfer using polyethylenimine and photochemical internalization in endometrial cancer cells. "3 5 459 465 . - 96.
Maxfield F. R. 1982 "Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts ."95 2 676 681 . - 97.
Medina-Kauwe L. Xie J. et al. 2005 "Intracellular trafficking of nonviral vectors. "12 24 1734 1751 . - 98.
Melikov K. Chernomordik L. 2005 "Arginine-rich cell penetrating peptides: from endosomal uptake to nuclear delivery. " Cellular and Molecular Life Sciences62 23 2739 2749 . - 99.
Mellman I. Fuchs R. et al. 1986 "Acidification of the endocytic and exocytic pathways. "55 1 663 700 . - 100.
Mello C. C. Conte D. 2004 "Revealing the world of RNA interference. "431 7006 338 342 . - 101.
Merkel O. M. Beyerle A. et al. 2009 "Nonviral siRNA Delivery to the Lung: Investigation of PEG− PEI Polyplexes and Their In Vivo Performance." Molecular Pharmaceutics6 4 1246 1260 . - 102.
Midoux P. Kichler A. et al. 1998 "Membrane permeabilization and efficient gene transfer by a peptide containing several histidines. "9 2 260 267 . - 103.
Midoux P. Mendes C. et al. 1993 "Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells. "21 4 871 878 . - 104.
Midoux P. Monsigny M. 1999 "Efficient gene transfer by histidylated polylysine/pDNA complexes. "10 3 406 411 . - 105.
Midoux P. Pichon C. et al. 2009 "Chemical vectors for gene delivery: a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers ."157 2 166 178 . - 106.
Min S. H. Lee D. C. et al. 2006 "A composite gene delivery system consisting of polyethylenimine and an amphipathic peptide KALA. "8 12 1425 1434 . - 107.
Moan J. Berg K. 1991 "The photodegradation of porphyrins in cells can be used to estimate the lifetime of singlet oxygen. "53 4 549 553 . - 108.
Moreira C. Oliveira H. et al. 2009 "Improving chitosan-mediated gene transfer by the introduction of intracellular buffering moieties into the chitosan backbone ."5 8 2995 3006 . - 109.
Morille M. Passirani C. et al. 2008 "Progress in developing cationic vectors for non-viral systemic gene therapy against cancer ." 29(24-25):3477 EOF 3496 EOF . - 110.
Morris M. Vidal P. et al. 1997 "A new peptide vector for efficient delivery of oligonucleotides into mammalian cells. "25 14 2730 2736 . - 111.
Morris M. C. Chaloin L. et al. 2000 "Translocating peptides and proteins and their use for gene delivery ."11 5 461 466 . - 112.
Morris M. C. Depollier J. et al. 2001 "A peptide carrier for the delivery of biologically active proteins into mammalian cells. "19 12 1173 1176 . - 113.
Muratovska A. Eccles M. R. 2004 "Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells. " 558(1-3):63 EOF 8 EOF . - 114.
Navarro-Quiroga I. Antonio J. González-Barrios et. al 2002 "Improved neurotensin-vector-mediated gene transfer by the coupling of hemagglutinin HA2 fusogenic peptide and Vp1 SV40 nuclear localization signal. " Molecular 105(1-2):86 EOF 97 EOF . - 115.
Ndoye A. Dolivet G. et al. 2006 "Eradication of ." 13(6): 1156-1162.53 head and neck squamous cell carcinoma xenografts using nonviral p53 gene therapy and photochemical internalization - 116.
Ndoye A. Merlin J. L. et al. 2004 "Enhanced gene transfer and cell death following53 gene transfer using photochemical internalisation of glucosylated PEI‐DNA complexes." The Journal of Gene Medicine 6(8): 884-894. - 117.
Niidome T. Ohmori N. et al. 1997 "Binding of cationic alpha-helical peptides to plasmid DNA and their gene transfer abilities into cells. " Journal of Biological Chemistry272 24 15307 15312 . - 118.
Niidome T. Takaji K. et al. 1999 "Chain length of cationic alpha-helical peptide sufficient for gene delivery into cells. "10 5 773 780 . - 119.
Nishiyama N. Iriyama A. et al. 2005 "Light-induced gene transfer from packaged DNA enveloped in a dendrimeric photosensitizer. "4 12 934 941 . - 120.
Norum O. J. Gaustad J. V. et al. 2009 "Photochemical internalization of bleomycin is superior to photodynamic therapy due to the therapeutic effect in the tumor periphery ."85 3 740 749 . - 121.
Numata K. Kaplan D. L. 2010 "Silk-based gene carriers with cell membrane destabilizing peptides ."11 3189 3195 . - 122.
Oehlke J. Scheller A. et al. 1998 "Cellular uptake of an [alpha]-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocyticall y." (BBA)-Biomembranes 1414(1-2):127 EOF 39 EOF . - 123.
Ohmori N. Niidome T. et al. 1998 "Importance of Hydrophobic Region in Amphiphilic Structures of α-Helical Peptides for Their Gene Transfer-Ability into Cells." Biochemical and Biophysical Research Communications245 1 259 265 . - 124.
Oliveira S. Fretz M. M. et al. 2007 "Photochemical internalization enhances silencing of epidermal growth factor receptor through improved endosomal escape of siRNA. " (BBA)- Biomembranes1768 5 1211 1217 . - 125.
Oliveira S. Hogset A. et al. 2008 "Delivery of siRNA to the target cell cytoplasm: photochemical internalization facilitates endosomal escape and improves silencing efficiency, in vitro and in vivo ."14 34 3686 3697 . - 126.
Pérez-Martínez F. Guerra J. et al. 2011 "Barriers to Non-Viral Vector-Mediated Gene Delivery in the Nervous System. "28 8 1843 1858 . - 127.
Pack D. W. Hoffman A. S. et al. 2005 "Design and development of polymers for gene delivery. " Drug Discovery4 7 581 593 . - 128.
Pack D. W. Putnam D. et al. 2000 "Design of imidazole‐containing endosomolytic biopolymers for gene delivery." Biotechnology and Bioengineering67 2 217 223 . - 129.
Parente R. A. Nir S. et al. 1990 "Mechanism of leakage of phospholipid vesicle contents induced by the peptide GALA. "29 37 8720 8728 . - 130.
Patel L. N. Zaro J. L. et al. 2007 "Cell penetrating peptides: intracellular pathways and pharmaceutical perspectives ."24 11 1977 1992 . - 131.
Pathak A. Patnaik S. et al. 2009 "Recent trends in non‐viral vector‐mediated gene delivery." Biotechnology Journal4 11 1559 1572 . - 132.
Pedroso de Lima. M. C. Simões S. et al. 2001 "Cationic lipid-DNA complexes in gene delivery: from biophysics to biological applications ." 47(2-3):277 EOF -294. - 133.
Pfeifer C. Hasenpusch G. et al. 2011 "Dry powder aerosols of polyethylenimine (PEI)-based gene vectors mediate efficient gene delivery to the lung." Journal of Controlled Release154 1 69 76 . - 134.
Pichon C. Freulon I. et al. 1997 "Cytosolic and nuclear delivery of oligonucleotides mediated by an amphiphilic anionic peptide ."7 4 335 343 . - 135.
Pichon C. Gonçalves C. et al. 2001 "Histidine-rich peptides and polymers for nucleic acids delivery. "53 1 75 94 . - 136.
Pires P. Simões S. et al. 1999 "Interaction of cationic liposomes and their DNA complexes with monocytic leukemia cells. " (BBA)-Biomembranes1418 1 71 84 . - 137.
Plank C. Oberhauser B. et al. 1994 "The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems. " Journal of Biological Chemistry269 17 12918 12924 . - 138.
Prasmickaite L. Høgset A. et al. 2000 "Role of endosomes in gene transfection mediated by photochemical internalisation (PCI). "2 6 477 488 . - 139.
Raemdonck K. Naeye B. et al. 2009 "Biodegradable dextran nanogels for RNA interference: focusing on endosomal escape and intracellular siRNA delivery ."19 9 1406 1415 . - 140.
Raper S. E. Chirmule N. et al. 2003 "Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. " 80(1-2):148 EOF 58 EOF . - 141.
Remy J. S. Sirlin C. et al. 1994 "Gene transfer with a series of lipophilic DNA-binding molecules. "5 6 647 654 . - 142.
Richard J. P. Melikov K. et al. 2003 "Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. " Journal of Biological Chemistry278 1 585 590 . - 143.
Rittner K. Benavente A. et al. 2002 "New basic membrane-destabilizing peptides for plasmid-based gene delivery in vitro and in vivo ."5 2 104 114 . - 144.
Sanders N. Rudolph C. et al. 2009 "Extracellular barriers in respiratory gene therapy ."61 2 115 127 . - 145.
Sasaki, K., K. Kogure, et al. (2008). "An artificial virus-like nano carrier system: enhanced endosomal escape of nanoparticles via synergistic action of pH-sensitive fusogenic peptide derivatives." Analytical and Bioanalytical Chemistry 391(8): 2717-2727. - 146.
Selbo P. K. Sivam G. et al. 2001 "In vivo documentation of photochemical internalization, a novel approach to site specific cancer therapy. "92 5 761 766 . - 147.
Selbo P. K. Weyergang A. et al. 2010 "Photochemical internalization provides time- and space-controlled endolysosomal escape of therapeutic molecules ."148 1 2 12 . - 148.
Shieh M. J. Peng C. L. et al. 2008 "Non-toxic phototriggered gene transfection by PAMAM-porphyrin conjugates ."129 3 200 206 . - 149.
Shim M. S. Kwon Y. J. 2010 "Efficient and targeted delivery of siRNA in vivo ."277 4814 4827 . - 150.
Simeoni F. Morris M. C. et al. 2003 "Insight into the mechanism of the peptide‐based gene delivery system MPG: implications for delivery of siRNA into mammalian cells." Nucleic Acids Research31 11 2717 2724 . - 151.
Simoes S. Slepushkin V. et al. 1999 "Mechanisms of gene transfer mediated by lipoplexes associated with targeting ligands or pH-sensitive peptides. " 6(11):1798 EOF 807 EOF . - 152.
Sonawane N. D. Szoka F. C. et al. 2003 "Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes ."278 45 44826 44831 . - 153.
Stegmann T. 2000 "Membrane fusion mechanisms: the influenza hemagglutinin paradigm and its implications for intracellular fusion ."1 8 598 604 . - 154.
Stewart D. Hilton J. et al. 2006 "Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF121 (AdVEGF121) versus maximum medical treatment." 13 21 1503 1511 . - 155.
Sui G. Soohoo C. et al. 2002 "A DNA vector-based RNAi technology to suppress gene expression in mammalian cells ."99 8 5515 5520 . - 156.
Torchilin V. P. 2008 "Tat peptide-mediated intracellular delivery of pharmaceutical nanocarriers ."60 4 548 558 . - 157.
Urban-Klein B. Werth S. et al. 2004 "RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo." Gene Therapy12 5 461 466 . - 158.
van Rossenberg S. M. W. Sliedregt-Bol K. M. et al. 2002 "Targeted lysosome disruptive elements for improvement of parenchymal liver cell-specific gene delivery ."277 48 45803 45810 . - 159.
Veldhoen S. Laufer S. D. et al. 2006 "Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect ."34 22 6561 6573 . - 160.
Vinge L. E. Raake P. W. et al. 2008 "Gene therapy in heart failure ."102 12 1458 1470 . - 161.
Vivès E. Brodin P. et al. 1997 "A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. " Journal of Biological Chemistry272 25 16010 16017 . - 162.
Vives E. 2003 "Cellular utake of the Tat peptide: an endocytosis mechanism following ionic interactions ."16 5 265 271 . - 163.
Vlasov G. Lesina E. et al. 2005 "Optimization of transfection properties of DNA-lysine dendrimer complexes ."31 2 153 159 . - 164.
Vliegenthart J. Knollen W. et al. 1999 "Enhanced efficiency of lactosylated poly-L-lysine-mediated gene transfer into cystic fibrosis airway epithelial cells. "20 5 1081 1086 . - 165.
Vogt T. C. B. Bechinger B. 1999 "The interactions of histidine-containing amphipathic helical peptide antibiotics with lipid bilayers." Journal of Biological Chemist ry 274(41):29115 EOF 21 EOF . - 166.
Wagner E. Kloeckner J. 2006 "Gene delivery using polymer therapeutics ." Polymer Therapeutics I:135 173 . - 167.
Wagner E. Plank C. et al. 1992 "Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle ."89 17 7934 7938 . - 168.
Wagner E. Zatloukal K. et al. 1992 "Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes ."89 13 6099 6103 . - 169.
Whitehead K. A. Langer R. et al. 2009 "Knocking down barriers: advances in siRNA delivery ."8 2 129 138 . - 170.
Wrobel I. Collins D. 1995 "Fusion of cationic liposomes with mammalian cells occurs after endocytosis. " (BBA)-Biomembranes1235 2 296 304 . - 171.
Wyman T. B. Nicol F. et al. 1997 "Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers."36 10 3008 3017 . - 172.
Xu Y. Szoka F. C. Jr 1996 "Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. "35 18 5616 5623 . - 173.
Yamamoto M. Curiel D. T. 2005 "Cancer gene therapy." Technology in Cancer Research & Treatment4 4 315 330 . - 174.
Yang S. R. Lee H. J. et al. 2006 "Histidine-conjugated poly (amino acid) derivatives for the novel endosomolytic delivery carrier of doxorubicin." Journal of Controlled Release114 1 60 68 . - 175.
Yip W. L. Weyergang A. et al. 2007 "Targeted delivery and enhanced cytotoxicity of cetuximab-saporin by photochemical internalization in EGFR-positive cancer cells. "4 2 241 251 . - 176.
Yu M. Poeschla E. et al. 1994 "Progress towards gene therapy for HIV infection. "1 1 13 26 . - 177.
Zabner J. Fasbender A. J. et al. 1995 "Cellular and molecular barriers to gene transfer by a cationic lipid ."270 32 18997 19007 . - 178.
Zelphati O. Szoka F. C. 1996 "Intracellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids. "13 9 1367 1372 . - 179.
Zelphati O. Szoka F. C. 1996 "Mechanism of oligonucleotide release from cationic liposomes ." 93(21):11493 EOF 11498 EOF . - 180.
Zhou J. Wu J. et al. 2006 "PAMAM dendrimers for efficient siRNA delivery and potent gene silencing ." (22):2362 EOF 2364 EOF . - 181.
Zhou X. Huang L. 1994 "DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action. " (BBA)-Biomembranes1189 2 195 203 . - 182.
Zuhorn I. S. Bakowsky U. et al. 2005 "Nonbilayer phase of lipoplex-membrane mixture determines endosomal escape of genetic cargo and transfection efficiency. " Molecular Therapy11 5 801 810 .