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Chemical vectors for gene delivery: uptake and intracellular trafficking
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Chemical vectors for gene delivery: uptake and intracellular trafficking Chantal Pichon, Ludivine Billiet and Patrick Midoux Chemical vectors for non-viral gene delivery are based on engineered DNA nanoparticles produced with various range of macromolecules suitable to mimic some viral functions required for gene transfer. Many efforts have been undertaken these past years to identify cellular barriers that have to be passed for this issue. Here, we summarize the current status of knowledge on the uptake mechanism of DNA nanoparticles made with polymers and liposomes, their endosomal escape, cytosolic diffusion, and nuclear import of pDNA. Studies reported these past years regarding pDNA nanoparticles endocytosis indicated that there is no clear evident relationship between the ways of entry and the transfection efficiency. By contrast, the sequestration of pDNA in intracellular vesicles and the low number of pDNA close to the nuclear envelop are identified as the major intracellular barriers. So, intensive investigations to increase the cytosolic delivery of pDNA and its migration toward nuclear pores make sense to bring the transfection efficiency closer to that of viruses. Address Centre de Biophysique Mole´culaire CNRS UPR 4301, University of Orle´ans and Inserm, rue Charles Sadron, 45071 Orle´ans Cedex 2, France Corresponding author: Pichon, Chantal ([email protected])
Current Opinion in Biotechnology 2010, 21:640–645 This review comes from a themed issue on Tissue, cell and pathway engineering Edited by Heike Hall and Gill Geesey Available online 30th July 2010 0958-1669/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2010.07.003
Introduction Gene therapy aims to cure genetic deficiencies and a large variety of acquired diseases by the introduction of genetic material into mammalian cells. To date, viruses have demonstrated the feasibility of gene therapy and remain the best vehicles to introduce genes into cells. But with viral vectors, severe fatal adverse events of including acute immune response and insertion mutagenesis have occurred during gene therapy clinical trials raising serious safety concerns about the use of viral vectors [1,2]. Moreover, their limited size capacity, weakness of cell targeting, and several manufacturing issues have boosted efforts to search for non-viral options. Inspired by strategies used Current Opinion in Biotechnology 2010, 21:640–645
by certain viruses to enter and to transfect mammalian cells, researchers are trying to build synthetic viruses with molecules that mimic the steps allowing a virus to infect mammalian cells. So far, cationic liposomes and cationic polymers are the most studied and used chemical vectors. These carriers form electrostatic complexes with pDNA. The self assembly of pDNA with cationic polymer induces DNA condensation leading to toroid or rod DNA/polymer nanoparticles of 100 nm (so-called polyplexes) containing usually 3 or 4 pDNA molecules (Scheme 1). Electrostatic interactions between cationic liposomes and pDNA undergo topological transformation of liposomes into compact quasi-spherical vesicles of 200– 300 nm (so-called lipoplexes) containing one pDNA molecule, in which DNA and lipids adopt an ordered multilamellar structure (Scheme 1). DNA condensation provides size reduction and protection against nuclease degradation. The exact knowledge of their uptake mechanism and their intracellular trafficking could lead a rational design of efficient non-viral vectors. Figure 1 summarizes the main intracellular barriers that polyplexes and lipoplexes must face to deliver an extracellular pDNA in large amount in the nucleus. This review aims at summarizing what is known on the cell uptake mechanism, endosomal escape, cytosolic diffusion, and nuclear import of pDNA, and what could be done to increase transfection efficiency.
Uptake mechanism It is now accepted that lipoplexes and polyplexes are internalized through endocytosis. Besides the clathrindependent endocytosis considered for a long time as an exclusive uptake pathway of exogenous molecules, the past two decade investigations have revealed new pathways, so-called ‘clathrin-independent endocytosis’. It is quite difficult to associate specific pathways with a class of carriers. The endocytic pathway of a given formulation varies with the cell types and molecular composition of the cell surface, besides their global charge and size, generating conflicting data regarding the internalization mechanism used [3–5]. Kopatz et al. have indicated an initial interaction with syndecans triggering an actinmediated endocytosis event for PEI-polyplexes uptake [6]. Most of lipoplexes are internalized via the clathrindependent pathway. Indeed, the uptake and transfection efficiency of lipoplexes made with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dialkyl pyridinium surfactant (SAINT-2-), and DOTAP/1,2-dioleoylsn-glycero-3-phosphoethanolamine (DOPE) are considerably reduced by inhibitors of the clathrin-dependent www.sciencedirect.com
Chemical vectors for gene delivery: uptake and intracellular trafficking Pichon, Billiet and Midoux 641
Scheme 1
Schematic illustration of polyplex and lipoplex formation.
pathway such as chlorpromazine. However, when DOTAP/DOPE lipoplexes are coated with human serum albumin, the entry occurs via the caveolar pathway [4]. By contrast, the entry of polyplexes is more dependent on the cell type and the polymer used [7]. For instance, HepG2 cells internalized polyplexes made with histidinerich polylysine via both a clathrin-dependent pathway and macropinocytosis but only the former leads to cell transfection [7]. In these cells, gene transfer with linear polyethylenimine (lPEI) polyplexes and branched polyethylenimine (bPEI) polyplexes succeed mainly via the clathrin-dependent route, but lPEI-polyplexes being more efficient than bPEI ones [8]. These data are in line with the proton sponge effect of PEI and the rational design of histidine-rich polymers made to destabilize acid vesicles (i.e. endosomes) in order to help pDNA
Figure 1
delivery in the cytosol. PEI-polyplexes entered preferentially via a caveolar pathway than the clathrin-dependent one in HeLa cells and the transfection efficiency is equivalent. Similarly, endothelial cells that are particularly rich in caveolae, internalize dendriplexes – pDNA complexed with Superfect1 – a class of fractured polyamidoamine (PAMAM) dendrimers—through a cholesterol-dependent caveolae mediated pathway [9–11]. The internalization of alginate-chitosan polyplexes is mediated by a clathrin-dependent process in HEK293T and COS-7 cells but through caveolae in CHO cells [3]. In these cases, the PEI, PAMAM dendrimers, or chitosan proton sponge effect should not be involved to escape from caveolae. The size of lipoplexes and polyplexes could control the uptake process. Lipoplexes and polyplexes of 200 nm are taken up by the clathrin-dependent pathway, whereas aggregates larger than 500 nm by a clathrin-independent one [11,12]. As mentioned, the entry via caveolae has often been observed with polyplexes in certain cell types. Like in the case of a SV40 virus infection, this route should not be essential for the transfection efficiency [13]. This reflects only a preferred localization of polyplexes to the membrane environments with appropriate interactions. Targeting is expected to decrease the pDNA amount required to achieve an efficient transfection. Exploiting the diversity of receptors present at the cell surface, internalization and transfection activity can be enhanced by coupling ligands to DNA nanoparticles. The most popular ligands are transferrin, RGD peptides recognized by integrins of tumor vasculatures, lactose by hepatocytes, mannose by macrophages and dendritic cells, and folic acid by certain tumor cells. Receptor-mediated internalization is faster than non-specific uptake allowing cargo accumulation inside the cells. Except for that of transferrin, receptors usually convey their cargo to lysosomes. The absence of a good device to sort pDNA from this pathway is detrimental to transfection. Enhancement of transfection efficiency is often associated with the use of devices leading to disruption of endosomes indicating a direct relation between endocytosis via clathrin vesicles and transfection efficiency. However, DNA nanoparticles containing pH-sensitive molecules that enter via caveolae may also lead to transfection but connections between clathrin vesicles and caveolae have not yet been identified. Although still remaining controversial, one class of short 30 residue synthetic peptides called Cell Penetrating Peptides (CPP) seems to mediate cell entry of CPPbased nanoparticles independently to endocytosis pathway [14].
Endosomal escape Scheme of intracellular barriers to gene transfer. (1) Plasma membrane and endocytosis, (2) endosome (end) escape, (3) cytosolic migration via the microtubular network (MT) and (4) nuclear translocation. www.sciencedirect.com
After endocytosis via clathrin vesicles, cargos are confined within endosomes that either fuse with lysosomes or recycle their contents back to the cell surface. The low endosomal escape is regarded as a major limitation for Current Opinion in Biotechnology 2010, 21:640–645
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non-viral vectors. Some pH-sensitive fusogenic peptides inspired from viral fusion proteins, and undergoing conformational changes in slightly acidic conditions, were found to enhance the transfection efficiency of polyplexes and lipoplexes [15–17]. But their attachment to carriers and serum sensitivity often lead to strong reduction of efficacy. To date, melittin from honey bee venom is the widely used lytic peptide in gene delivery because of its high membrane destabilization capacity compared to the other fusogenic peptides. Melittin has been attached to either cationic polymers or cationic lipids [18,19]. But, the strong activity of mellitin for the plasma membrane at physiological pH is detrimental to the cell. Its use has been improved by masking its cationic charges with dimethylmaleic anhydride to inhibit its lytic activity at neutral pH; the lytic activity being regained after cleavage of the protecting groups at pH 5 [20]. Lysosomes swelling and membrane destabilization occur when a weak base such as chloroquine accumulates in the acidic lumen. In the same manner, cationic polymers containing unprotonated amines groups at neutral pH as PEI, PAMAM dendrimers, chitosan, or imidazole groups as histidine-rich peptides or polymers act upon protonation in endosomes as a proton sponge leading to a swelling and a rupture of these vesicles [21]. Histidinerich peptides or polymers that possess protonable imidazole groups can also induce membrane fusion and/or permeation in an acidic medium [17]. Most lipoplexes are formulated with DOPE, a neutral lipid having fusogenic behaviors increasing endosomal escape. It is admitted that DOPE induces membrane fusion inside endosome by adopting a hexagonal structure that is even more fusogenic [22]. Likewise, histidine-based and imidazole-based pH-sensitive fusogenic lipids have been developed [17]. Some of them can advantageously be used instead of DOPE. To date, there are no clear cut data reported on the quantification of DNA nanoparticle escape from endosomes to the cytosol. This requires the development of adapted and more sensitive methods.
Cytosolic transport The motion of a plasmid DNA after its delivery into the cytosol from endocytic vesicles is another crucial step. Recent studies have shown that cytoskeleton components could be involved for the cytoplasmic motion of pDNA either free or complexed with carriers. Unspecific binding of DNA to actin, vimentin, and keratin is suggested to impair the mobility of pDNA in the cytosol [23]. By contrast, microtubules are required to efficient cytosolic transport of pDNA [24,25,26]. Recently, modulation of histone deacetylase 6 (HDAC6), and the microtubule network has been shown to increase gene expression [25]. The inhibition of HDAC6 that favors the stability of acetylated microtubules increased 10-fold the plasmid Current Opinion in Biotechnology 2010, 21:640–645
trafficking and entry in the nucleus. Thus, HDAC inhibitors might be good candidates to increase the efficiency of gene therapy by non-viral vectors. The most consistent findings for DNA particles concern the motion of PEI-polyplexes. Suh et al. demonstrated by using the Multiple Particle Tracking technique, the active motor-protein-driven transport of PEI-polyplexes on microtubules toward the nucleus in COS-7 cells [27]. Using Image Correlation Spectroscopy, an active transport of PEGylated PEI-polyplexes and bcyclodextrin polyplexes in HeLa cells occurred with linear or curvilinear trajectories that is in line with that measured for proteins moving along microtubules [28]. However, it is still not known whether these polyplexes were inside or outside vesicles during these observations. The motion of pDNA, polyplexes, and lipoplexes could be greatly improved if these particles bear a signal molecule allowing their binding to microtubules and their migration toward the nuclear envelope. Despite being proposed for years, this is still challenging and no data have yet been published. Some viruses as adenoviruses are known to migrate to the nucleus via microtubules upon their interaction with microtubule motor proteins as dynein. Knowledge about viral proteins involved in these interactions will open development of new strategies to increase the number of pDNA copies susceptible to reach the nuclear envelop once they are into the cytosol.
Nuclear import In non-dividing cells, nuclear pores regulate the passage in and out of the nucleus. Molecules, smaller than 40 kDa, diffuse passively, while larger molecules must display specific nuclear localization signals (NLS) for active transport. Nuclear entry is an inefficient process and it was estimated that only 0.1% of DNA microinjected in the cytosol was transcribed. Thus, nuclear import of pDNA is very low limiting the transfection efficiency of polyplexes and lipoplexes. In their review, Wagstaff and Jans have summarized strategies that have been explored to enhance the nuclear import of pDNA [29]. Those include the coupling of NLS peptides (from SV40 large T antigens, from the adenovirus hexon protein, importin b and the N-terminal residues of the yeast transcription factor GAL4) to either chemical vectors or DNA backbone. The gain obtained by addition of such devices in terms of gene expression and number of transfected cells was not as high as expected. The identification of six classes of other NLS peptides opens new possibilities to increase the nuclear import of pDNA [30]. In addition, snRNA m3G-CAP (m3G pyrophosphate coupled to 50 -phosphorylated 20 -O-methyloligoribonucleotides leading to m3G-capped oligonucleotides) resembling to the 50 -end of endogenous U snRNAs could be used to enhance nuclear import of pDNA nanoparticles [31]. www.sciencedirect.com
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There are emerging strategies based on the manipulation of cytosolic proteins as transcription factors and nuclear hormone receptors that shuttle between the cytoplasm and the nucleus. The capacity of the transcription factor NFkB to bind specific consensus DNA sequence and to be imported in the nucleus under specific conditions has been exploited to increase pDNA nuclear delivery [32]. For example, the insertion of the 3NF sequence (50 -CTGGGGACTTTCCAGCTGGGGACTTTCCAGCTGGGGACTTTCCAGG-30 ) comprising three 10 bp kB sites (underlined sequence) separated by a 5 bp spacer in pDNA, strongly increased pDNA nuclear import by NFkB-mediated process [33]. The number of 3NF plasmids in the nucleus of HeLa and C2C12 cells following transfection with lPEI-polyplexes or HIS-polyplexes was 6-fold higher than with 3NF free pDNA [34]. Investigations of pDNA transport through the nuclear pore have highlighted two potent mechanisms. When a specific consensus sequence that binds to transcription factors is inserted in pDNA, the interaction of importin b1 with the classical NLS peptide of transcription factors is necessary for the nuclear import [35]. It has been recently shown that in the absence of such a classical NLS sequence, transportin (karyopherin b2) plays a specific role in this transport. Transportin binds to a glycine-rich NLS known as M9 NLS. It has been found to mediate the nuclear entry of DNA with the help of histones acting as an adaptor [36]. Of note, transportin was also found to bind to adenoviral terminal protein VII and to mediate the import of adenoviral DNA [37]. Thus, the transportin mechanism could be prominent in DNA import. Comparatively, the nuclear import of pDNA/polymer complexes is still illknown. FRET analyses of transfection made with PEI, HIS and chitosan have led to the conclusion that most of nuclear pDNA was still condensed with the polymer [34,38,39]. This raises the question of the passage of polyplexes through the nuclear pore complex. Nucleopores can only be expanded up to 39 nm that is considerably smaller than the current polyplexes size (70–300 nm). Nevertheless, the fact that Tat peptide can mediate the import of 90 nm beads into the nucleus of digitonin permeabilized HeLa cells suggests the possible expansion of nuclear pores [40]. Further investigations dealing with the location of both pDNA and the polymer in the nucleopores as well as their interactions with components of the nuclear pore complex on both sides of the nuclear envelop will contribute to elucidate this question. By contrast, lipoplexes were never observed in the nucleus indicating that pDNA enters probably as free pDNA [38]. This is in line with recent FRET investigations revealing that after 9 h incubation; more than 50% lipoplexes were dissociated in the cytoplasm [41]. It cannot be excluded that fusion between the nuclear envelop and liposomes could occur in this process. Indeed, it has been reported several years ago that liposomes interact and may fuse with the nuclear envelop www.sciencedirect.com
[42,43]. Recently, the role of DNA-phosphatidylcholine liposomes-Mg2+ was shown to be involved in the formation of some cell structures at the first stage of a nuclear envelope and pore complex assembly [44]. Recent quantification studies show that the gene expression of LipofectamineTM lipoplexes was more efficient than that of PEI-polyplexes despite a similar amount of plasmid found in the nucleus [45,46]. This difference could be ascribed to the condensation state of pDNA.
Conclusions Considerable efforts have been done to delineate the uptake and intracellular trafficking of polyplexes, lipoplexes, and pDNA. It appears that several routes are used for the entry of DNA complexes, each leading to transfection depending on the cell type and the vector. Compared to adenoviruses, the lower transfection efficiency of lipoplexes principally arises from differences in nuclear transcription and translation efficiencies rather than in the intracellular trafficking [47,48]. In the case of polyplexes, there is a high sequestration in the endosomes that limits the amount of pDNA reaching nuclear envelope. Indeed, 90% of PEI-polyplexes are colocalized with late endosomes/lysosomes at 2 h post-transfection versus only 5% of adenoviruses [49]. Recent knowledge on the nuclear import mechanism shows that this step could not be the major bottleneck for transfection if pDNA bears a NLS signal recognized by karyopherins. One of the major limitations is certainly the cytosolic migration of DNA complexes to the perinuclear space to increase the amount of pDNA available to translocate through the nuclear pore. Indeed, adenoviruses exhibit higher diffusion than PEI-polyplexes owing to their motion along microtubules toward the nucleus once in the cytosol. Therefore, future investigations to increase the endosomal escape and cytosolic migration of polyplexes toward the nuclear pores make sense to bring their transfection efficiency closer to that of adenoviruses. Lastly, there is a need to know the positioning of pDNA once inside the nucleus because transcription factories are only active in distinct subnuclear domains. In perspective, automation and high throughput combinatorial chemistry will be helpful for rational sorting of devices based on structure–function relationship. It must be kept in mind that devices generated to bypass the different barriers have to be assembled on the same multifunctional vehicle but must work independently and sequentially.
Acknowledgements Authors would like to acknowledge Pr Michel Monsigny (Emeritus Professor of Biochemistry, University of Orleans) for helpful discussion and critically reading this manuscript. We are grateful to Association Franc¸aise contre les Myopathies, Vaincre la Mucoviscidose and Ligue Nationale contre le Cancer for grant supports. Current Opinion in Biotechnology 2010, 21:640–645
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Current Opinion in Biotechnology 2010, 21:640–645
Chemical vectors for gene delivery: uptake and intracellular trafficking Chantal Pichon, Ludivine Billiet and Patrick Midoux Chemical vectors for non-viral gene delivery are based on engineered DNA nanoparticles produced with various range of macromolecules suitable to mimic some viral functions required for gene transfer. Many efforts have been undertaken these past years to identify cellular barriers that have to be passed for this issue. Here, we summarize the current status of knowledge on the uptake mechanism of DNA nanoparticles made with polymers and liposomes, their endosomal escape, cytosolic diffusion, and nuclear import of pDNA. Studies reported these past years regarding pDNA nanoparticles endocytosis indicated that there is no clear evident relationship between the ways of entry and the transfection efficiency. By contrast, the sequestration of pDNA in intracellular vesicles and the low number of pDNA close to the nuclear envelop are identified as the major intracellular barriers. So, intensive investigations to increase the cytosolic delivery of pDNA and its migration toward nuclear pores make sense to bring the transfection efficiency closer to that of viruses. Address Centre de Biophysique Mole´culaire CNRS UPR 4301, University of Orle´ans and Inserm, rue Charles Sadron, 45071 Orle´ans Cedex 2, France Corresponding author: Pichon, Chantal ([email protected])
Current Opinion in Biotechnology 2010, 21:640–645 This review comes from a themed issue on Tissue, cell and pathway engineering Edited by Heike Hall and Gill Geesey Available online 30th July 2010 0958-1669/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2010.07.003
Introduction Gene therapy aims to cure genetic deficiencies and a large variety of acquired diseases by the introduction of genetic material into mammalian cells. To date, viruses have demonstrated the feasibility of gene therapy and remain the best vehicles to introduce genes into cells. But with viral vectors, severe fatal adverse events of including acute immune response and insertion mutagenesis have occurred during gene therapy clinical trials raising serious safety concerns about the use of viral vectors [1,2]. Moreover, their limited size capacity, weakness of cell targeting, and several manufacturing issues have boosted efforts to search for non-viral options. Inspired by strategies used Current Opinion in Biotechnology 2010, 21:640–645
by certain viruses to enter and to transfect mammalian cells, researchers are trying to build synthetic viruses with molecules that mimic the steps allowing a virus to infect mammalian cells. So far, cationic liposomes and cationic polymers are the most studied and used chemical vectors. These carriers form electrostatic complexes with pDNA. The self assembly of pDNA with cationic polymer induces DNA condensation leading to toroid or rod DNA/polymer nanoparticles of 100 nm (so-called polyplexes) containing usually 3 or 4 pDNA molecules (Scheme 1). Electrostatic interactions between cationic liposomes and pDNA undergo topological transformation of liposomes into compact quasi-spherical vesicles of 200– 300 nm (so-called lipoplexes) containing one pDNA molecule, in which DNA and lipids adopt an ordered multilamellar structure (Scheme 1). DNA condensation provides size reduction and protection against nuclease degradation. The exact knowledge of their uptake mechanism and their intracellular trafficking could lead a rational design of efficient non-viral vectors. Figure 1 summarizes the main intracellular barriers that polyplexes and lipoplexes must face to deliver an extracellular pDNA in large amount in the nucleus. This review aims at summarizing what is known on the cell uptake mechanism, endosomal escape, cytosolic diffusion, and nuclear import of pDNA, and what could be done to increase transfection efficiency.
Uptake mechanism It is now accepted that lipoplexes and polyplexes are internalized through endocytosis. Besides the clathrindependent endocytosis considered for a long time as an exclusive uptake pathway of exogenous molecules, the past two decade investigations have revealed new pathways, so-called ‘clathrin-independent endocytosis’. It is quite difficult to associate specific pathways with a class of carriers. The endocytic pathway of a given formulation varies with the cell types and molecular composition of the cell surface, besides their global charge and size, generating conflicting data regarding the internalization mechanism used [3–5]. Kopatz et al. have indicated an initial interaction with syndecans triggering an actinmediated endocytosis event for PEI-polyplexes uptake [6]. Most of lipoplexes are internalized via the clathrindependent pathway. Indeed, the uptake and transfection efficiency of lipoplexes made with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dialkyl pyridinium surfactant (SAINT-2-), and DOTAP/1,2-dioleoylsn-glycero-3-phosphoethanolamine (DOPE) are considerably reduced by inhibitors of the clathrin-dependent www.sciencedirect.com
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Scheme 1
Schematic illustration of polyplex and lipoplex formation.
pathway such as chlorpromazine. However, when DOTAP/DOPE lipoplexes are coated with human serum albumin, the entry occurs via the caveolar pathway [4]. By contrast, the entry of polyplexes is more dependent on the cell type and the polymer used [7]. For instance, HepG2 cells internalized polyplexes made with histidinerich polylysine via both a clathrin-dependent pathway and macropinocytosis but only the former leads to cell transfection [7]. In these cells, gene transfer with linear polyethylenimine (lPEI) polyplexes and branched polyethylenimine (bPEI) polyplexes succeed mainly via the clathrin-dependent route, but lPEI-polyplexes being more efficient than bPEI ones [8]. These data are in line with the proton sponge effect of PEI and the rational design of histidine-rich polymers made to destabilize acid vesicles (i.e. endosomes) in order to help pDNA
Figure 1
delivery in the cytosol. PEI-polyplexes entered preferentially via a caveolar pathway than the clathrin-dependent one in HeLa cells and the transfection efficiency is equivalent. Similarly, endothelial cells that are particularly rich in caveolae, internalize dendriplexes – pDNA complexed with Superfect1 – a class of fractured polyamidoamine (PAMAM) dendrimers—through a cholesterol-dependent caveolae mediated pathway [9–11]. The internalization of alginate-chitosan polyplexes is mediated by a clathrin-dependent process in HEK293T and COS-7 cells but through caveolae in CHO cells [3]. In these cases, the PEI, PAMAM dendrimers, or chitosan proton sponge effect should not be involved to escape from caveolae. The size of lipoplexes and polyplexes could control the uptake process. Lipoplexes and polyplexes of 200 nm are taken up by the clathrin-dependent pathway, whereas aggregates larger than 500 nm by a clathrin-independent one [11,12]. As mentioned, the entry via caveolae has often been observed with polyplexes in certain cell types. Like in the case of a SV40 virus infection, this route should not be essential for the transfection efficiency [13]. This reflects only a preferred localization of polyplexes to the membrane environments with appropriate interactions. Targeting is expected to decrease the pDNA amount required to achieve an efficient transfection. Exploiting the diversity of receptors present at the cell surface, internalization and transfection activity can be enhanced by coupling ligands to DNA nanoparticles. The most popular ligands are transferrin, RGD peptides recognized by integrins of tumor vasculatures, lactose by hepatocytes, mannose by macrophages and dendritic cells, and folic acid by certain tumor cells. Receptor-mediated internalization is faster than non-specific uptake allowing cargo accumulation inside the cells. Except for that of transferrin, receptors usually convey their cargo to lysosomes. The absence of a good device to sort pDNA from this pathway is detrimental to transfection. Enhancement of transfection efficiency is often associated with the use of devices leading to disruption of endosomes indicating a direct relation between endocytosis via clathrin vesicles and transfection efficiency. However, DNA nanoparticles containing pH-sensitive molecules that enter via caveolae may also lead to transfection but connections between clathrin vesicles and caveolae have not yet been identified. Although still remaining controversial, one class of short 30 residue synthetic peptides called Cell Penetrating Peptides (CPP) seems to mediate cell entry of CPPbased nanoparticles independently to endocytosis pathway [14].
Endosomal escape Scheme of intracellular barriers to gene transfer. (1) Plasma membrane and endocytosis, (2) endosome (end) escape, (3) cytosolic migration via the microtubular network (MT) and (4) nuclear translocation. www.sciencedirect.com
After endocytosis via clathrin vesicles, cargos are confined within endosomes that either fuse with lysosomes or recycle their contents back to the cell surface. The low endosomal escape is regarded as a major limitation for Current Opinion in Biotechnology 2010, 21:640–645
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non-viral vectors. Some pH-sensitive fusogenic peptides inspired from viral fusion proteins, and undergoing conformational changes in slightly acidic conditions, were found to enhance the transfection efficiency of polyplexes and lipoplexes [15–17]. But their attachment to carriers and serum sensitivity often lead to strong reduction of efficacy. To date, melittin from honey bee venom is the widely used lytic peptide in gene delivery because of its high membrane destabilization capacity compared to the other fusogenic peptides. Melittin has been attached to either cationic polymers or cationic lipids [18,19]. But, the strong activity of mellitin for the plasma membrane at physiological pH is detrimental to the cell. Its use has been improved by masking its cationic charges with dimethylmaleic anhydride to inhibit its lytic activity at neutral pH; the lytic activity being regained after cleavage of the protecting groups at pH 5 [20]. Lysosomes swelling and membrane destabilization occur when a weak base such as chloroquine accumulates in the acidic lumen. In the same manner, cationic polymers containing unprotonated amines groups at neutral pH as PEI, PAMAM dendrimers, chitosan, or imidazole groups as histidine-rich peptides or polymers act upon protonation in endosomes as a proton sponge leading to a swelling and a rupture of these vesicles [21]. Histidinerich peptides or polymers that possess protonable imidazole groups can also induce membrane fusion and/or permeation in an acidic medium [17]. Most lipoplexes are formulated with DOPE, a neutral lipid having fusogenic behaviors increasing endosomal escape. It is admitted that DOPE induces membrane fusion inside endosome by adopting a hexagonal structure that is even more fusogenic [22]. Likewise, histidine-based and imidazole-based pH-sensitive fusogenic lipids have been developed [17]. Some of them can advantageously be used instead of DOPE. To date, there are no clear cut data reported on the quantification of DNA nanoparticle escape from endosomes to the cytosol. This requires the development of adapted and more sensitive methods.
Cytosolic transport The motion of a plasmid DNA after its delivery into the cytosol from endocytic vesicles is another crucial step. Recent studies have shown that cytoskeleton components could be involved for the cytoplasmic motion of pDNA either free or complexed with carriers. Unspecific binding of DNA to actin, vimentin, and keratin is suggested to impair the mobility of pDNA in the cytosol [23]. By contrast, microtubules are required to efficient cytosolic transport of pDNA [24,25,26]. Recently, modulation of histone deacetylase 6 (HDAC6), and the microtubule network has been shown to increase gene expression [25]. The inhibition of HDAC6 that favors the stability of acetylated microtubules increased 10-fold the plasmid Current Opinion in Biotechnology 2010, 21:640–645
trafficking and entry in the nucleus. Thus, HDAC inhibitors might be good candidates to increase the efficiency of gene therapy by non-viral vectors. The most consistent findings for DNA particles concern the motion of PEI-polyplexes. Suh et al. demonstrated by using the Multiple Particle Tracking technique, the active motor-protein-driven transport of PEI-polyplexes on microtubules toward the nucleus in COS-7 cells [27]. Using Image Correlation Spectroscopy, an active transport of PEGylated PEI-polyplexes and bcyclodextrin polyplexes in HeLa cells occurred with linear or curvilinear trajectories that is in line with that measured for proteins moving along microtubules [28]. However, it is still not known whether these polyplexes were inside or outside vesicles during these observations. The motion of pDNA, polyplexes, and lipoplexes could be greatly improved if these particles bear a signal molecule allowing their binding to microtubules and their migration toward the nuclear envelope. Despite being proposed for years, this is still challenging and no data have yet been published. Some viruses as adenoviruses are known to migrate to the nucleus via microtubules upon their interaction with microtubule motor proteins as dynein. Knowledge about viral proteins involved in these interactions will open development of new strategies to increase the number of pDNA copies susceptible to reach the nuclear envelop once they are into the cytosol.
Nuclear import In non-dividing cells, nuclear pores regulate the passage in and out of the nucleus. Molecules, smaller than 40 kDa, diffuse passively, while larger molecules must display specific nuclear localization signals (NLS) for active transport. Nuclear entry is an inefficient process and it was estimated that only 0.1% of DNA microinjected in the cytosol was transcribed. Thus, nuclear import of pDNA is very low limiting the transfection efficiency of polyplexes and lipoplexes. In their review, Wagstaff and Jans have summarized strategies that have been explored to enhance the nuclear import of pDNA [29]. Those include the coupling of NLS peptides (from SV40 large T antigens, from the adenovirus hexon protein, importin b and the N-terminal residues of the yeast transcription factor GAL4) to either chemical vectors or DNA backbone. The gain obtained by addition of such devices in terms of gene expression and number of transfected cells was not as high as expected. The identification of six classes of other NLS peptides opens new possibilities to increase the nuclear import of pDNA [30]. In addition, snRNA m3G-CAP (m3G pyrophosphate coupled to 50 -phosphorylated 20 -O-methyloligoribonucleotides leading to m3G-capped oligonucleotides) resembling to the 50 -end of endogenous U snRNAs could be used to enhance nuclear import of pDNA nanoparticles [31]. www.sciencedirect.com
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There are emerging strategies based on the manipulation of cytosolic proteins as transcription factors and nuclear hormone receptors that shuttle between the cytoplasm and the nucleus. The capacity of the transcription factor NFkB to bind specific consensus DNA sequence and to be imported in the nucleus under specific conditions has been exploited to increase pDNA nuclear delivery [32]. For example, the insertion of the 3NF sequence (50 -CTGGGGACTTTCCAGCTGGGGACTTTCCAGCTGGGGACTTTCCAGG-30 ) comprising three 10 bp kB sites (underlined sequence) separated by a 5 bp spacer in pDNA, strongly increased pDNA nuclear import by NFkB-mediated process [33]. The number of 3NF plasmids in the nucleus of HeLa and C2C12 cells following transfection with lPEI-polyplexes or HIS-polyplexes was 6-fold higher than with 3NF free pDNA [34]. Investigations of pDNA transport through the nuclear pore have highlighted two potent mechanisms. When a specific consensus sequence that binds to transcription factors is inserted in pDNA, the interaction of importin b1 with the classical NLS peptide of transcription factors is necessary for the nuclear import [35]. It has been recently shown that in the absence of such a classical NLS sequence, transportin (karyopherin b2) plays a specific role in this transport. Transportin binds to a glycine-rich NLS known as M9 NLS. It has been found to mediate the nuclear entry of DNA with the help of histones acting as an adaptor [36]. Of note, transportin was also found to bind to adenoviral terminal protein VII and to mediate the import of adenoviral DNA [37]. Thus, the transportin mechanism could be prominent in DNA import. Comparatively, the nuclear import of pDNA/polymer complexes is still illknown. FRET analyses of transfection made with PEI, HIS and chitosan have led to the conclusion that most of nuclear pDNA was still condensed with the polymer [34,38,39]. This raises the question of the passage of polyplexes through the nuclear pore complex. Nucleopores can only be expanded up to 39 nm that is considerably smaller than the current polyplexes size (70–300 nm). Nevertheless, the fact that Tat peptide can mediate the import of 90 nm beads into the nucleus of digitonin permeabilized HeLa cells suggests the possible expansion of nuclear pores [40]. Further investigations dealing with the location of both pDNA and the polymer in the nucleopores as well as their interactions with components of the nuclear pore complex on both sides of the nuclear envelop will contribute to elucidate this question. By contrast, lipoplexes were never observed in the nucleus indicating that pDNA enters probably as free pDNA [38]. This is in line with recent FRET investigations revealing that after 9 h incubation; more than 50% lipoplexes were dissociated in the cytoplasm [41]. It cannot be excluded that fusion between the nuclear envelop and liposomes could occur in this process. Indeed, it has been reported several years ago that liposomes interact and may fuse with the nuclear envelop www.sciencedirect.com
[42,43]. Recently, the role of DNA-phosphatidylcholine liposomes-Mg2+ was shown to be involved in the formation of some cell structures at the first stage of a nuclear envelope and pore complex assembly [44]. Recent quantification studies show that the gene expression of LipofectamineTM lipoplexes was more efficient than that of PEI-polyplexes despite a similar amount of plasmid found in the nucleus [45,46]. This difference could be ascribed to the condensation state of pDNA.
Conclusions Considerable efforts have been done to delineate the uptake and intracellular trafficking of polyplexes, lipoplexes, and pDNA. It appears that several routes are used for the entry of DNA complexes, each leading to transfection depending on the cell type and the vector. Compared to adenoviruses, the lower transfection efficiency of lipoplexes principally arises from differences in nuclear transcription and translation efficiencies rather than in the intracellular trafficking [47,48]. In the case of polyplexes, there is a high sequestration in the endosomes that limits the amount of pDNA reaching nuclear envelope. Indeed, 90% of PEI-polyplexes are colocalized with late endosomes/lysosomes at 2 h post-transfection versus only 5% of adenoviruses [49]. Recent knowledge on the nuclear import mechanism shows that this step could not be the major bottleneck for transfection if pDNA bears a NLS signal recognized by karyopherins. One of the major limitations is certainly the cytosolic migration of DNA complexes to the perinuclear space to increase the amount of pDNA available to translocate through the nuclear pore. Indeed, adenoviruses exhibit higher diffusion than PEI-polyplexes owing to their motion along microtubules toward the nucleus once in the cytosol. Therefore, future investigations to increase the endosomal escape and cytosolic migration of polyplexes toward the nuclear pores make sense to bring their transfection efficiency closer to that of adenoviruses. Lastly, there is a need to know the positioning of pDNA once inside the nucleus because transcription factories are only active in distinct subnuclear domains. In perspective, automation and high throughput combinatorial chemistry will be helpful for rational sorting of devices based on structure–function relationship. It must be kept in mind that devices generated to bypass the different barriers have to be assembled on the same multifunctional vehicle but must work independently and sequentially.
Acknowledgements Authors would like to acknowledge Pr Michel Monsigny (Emeritus Professor of Biochemistry, University of Orleans) for helpful discussion and critically reading this manuscript. We are grateful to Association Franc¸aise contre les Myopathies, Vaincre la Mucoviscidose and Ligue Nationale contre le Cancer for grant supports. Current Opinion in Biotechnology 2010, 21:640–645
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