**2. Intracellular barriers and strategies to improve transfection efficiency**

A key factor conditioning transfection efficiency is the ability of the gene delivery system to overcome the intracellular and extracellular barriers. In this section, we will describe the main intracellular barriers that gene delivery systems must overcome to reach an efficient transfec‐ tion and the different strategies used for this purpose. Intracellular barriers involve all the obstacles that a gene delivery system must overcome from cell surface association to nuclear entry in target cells. The knowledge of the molecular features that command all these processes for the non-viral vectors and the overcoming of these hurdles are mandatory issues that need to be deeply considered in order to design efficient gene delivery methods. In this section, we will review the cellular uptake pathways and intracellular trafficking of non-viral vectors and we will discuss the existing methods to enhance the endosomal escape and the nuclear entry, which are the principal strategies to achieve an efficient transfection.

### **2.1. Cellular uptake pathways**

Cell surface association is the first intracellular barrier that non-viral gene delivery platforms need to overcome and it can directly influence the next intracellular fates of the non-viral complexes [17]. Cell-binding interactions of non-viral vectors can be receptor-independent or receptor-mediated. Receptor-independent cell surface association occurs by electrophilic attraction between the positively charged non-viral complexes (i.e. cationic lipoplexes and cationic polyplexes) and the negatively charged cell surface proteoglycans [18]. This binding method can efficiently transfect many cell types *in vitro*, but therapeutic potential *in vivo* requires additional refinement. In fact, in order to specifically deliver a gene into a target tissue *in vivo*, non-specific cell binding would require very high and potentially toxic doses of the non-viral vector. The addition of cell-specific ligands or antibodies to the vectors reduces this problem, allowing the use of lower and safer vector doses and promoting tissue targeting [18]. For instance, transferrin (Tf), which is an iron-transporting protein, has been used to achieve brain delivery in view that the Tf receptor is expressed in neurons and in the capillary endothelial cells of the brain-blood-barrier (BBB) [19]. Ligand choice not only depends on the cell type being targeted, but it is also important to consider the type of cell entry pathway that will be induced after ligation. As discussed in the following section, the endocytic pathway used by the vector can depend on the targeting ligand.

Once bound to the cell surface, non-viral vectors need to cross the plasma membrane to enter the cell and initiate the intracellular trafficking to enter the nucleus. The cellular uptake of macromolecules and solutes into membrane-bound vesicles derived by the invagination and pinching off of pieces of the plasma membrane is known as endocytosis [20]. There are four principal endocytic pathways: clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), phagocytosis and macropinocytosis [17,21]. These endocytic pathways are described below.

#### *2.1.1. Clathrin-mediated endocytosis*

Clathrin-mediated endocytosis (CME) is a highly regulated and energy-dependent process, and it constitutes the major and best characterized endocytic pathway [20]. The first step in CME is the strong binding of a ligand to a specific cell surface receptor. This triggers the localized accumulation of clathrin structures on the cytoplasmic surface of the plasma membrane, which helps to deform the membrane into a coated pit with a size about 100-150 nm [22]. As the clathrin lattice formation continues, the coated pits become deeply invaginated and they finally pinch off from the plasma membrane to form intracellular clathrin-coated vesicles (CCVs) [20]. The clathrin coats then depolymerize, resulting in early endosomes. A kind of GTPase named dynamin is necessary for the vesicle fission from the plasma membrane [23]. Cholesterol seems to be also important for CCV formation because its depletion impedes the coated pits to pinch off from the plasma membrane [24].

In the next step of the CME pathway, the endocyted vesicles internalized from the plasma membrane are integrated into late endosomes and those then deliver their cargos to lysosomes [25]. During maturation from early to late endosomes, proton pumps located on the endosome membrane produce the acidification of the compartment, and there is a further reduction to pH 5 in the progression from late endosomes to lysosomes [20]. The acid pH in endosomes seems to cause the dissociation of the ligands from their receptors. Most authors state that, in the absence of an endosomal escape mechanism, non-viral vector/DNA complexes are retained and degraded in the lysosomes due to the acid environment and the enzymatic activity in these compartments. The final result is that DNA molecules have little or almost no access to the nucleus [20].

Some authors suggest that in some cases, depending on the formulation of the non-viral vector, the CME pathway might be the most suitable to achieve a high transfection efficiency because the lysosomal activity facilitates the cytosolic release of nanoparticles and enhances the nuclear entry of DNA [26]. Depending on the composition of the vector, the most appropriate inter‐ nalization mechanism may be modulated [26]. Therefore, it is crucial to have a comprehensive understanding of the cellular internalization pathways of non-viral gene delivery systems.

#### *2.1.2. Caveolae-mediated endocytosis*

**2. Intracellular barriers and strategies to improve transfection efficiency**

A key factor conditioning transfection efficiency is the ability of the gene delivery system to overcome the intracellular and extracellular barriers. In this section, we will describe the main intracellular barriers that gene delivery systems must overcome to reach an efficient transfec‐ tion and the different strategies used for this purpose. Intracellular barriers involve all the obstacles that a gene delivery system must overcome from cell surface association to nuclear entry in target cells. The knowledge of the molecular features that command all these processes for the non-viral vectors and the overcoming of these hurdles are mandatory issues that need to be deeply considered in order to design efficient gene delivery methods. In this section, we will review the cellular uptake pathways and intracellular trafficking of non-viral vectors and we will discuss the existing methods to enhance the endosomal escape and the nuclear entry,

Cell surface association is the first intracellular barrier that non-viral gene delivery platforms need to overcome and it can directly influence the next intracellular fates of the non-viral complexes [17]. Cell-binding interactions of non-viral vectors can be receptor-independent or receptor-mediated. Receptor-independent cell surface association occurs by electrophilic attraction between the positively charged non-viral complexes (i.e. cationic lipoplexes and cationic polyplexes) and the negatively charged cell surface proteoglycans [18]. This binding method can efficiently transfect many cell types *in vitro*, but therapeutic potential *in vivo* requires additional refinement. In fact, in order to specifically deliver a gene into a target tissue *in vivo*, non-specific cell binding would require very high and potentially toxic doses of the non-viral vector. The addition of cell-specific ligands or antibodies to the vectors reduces this problem, allowing the use of lower and safer vector doses and promoting tissue targeting [18]. For instance, transferrin (Tf), which is an iron-transporting protein, has been used to achieve brain delivery in view that the Tf receptor is expressed in neurons and in the capillary endothelial cells of the brain-blood-barrier (BBB) [19]. Ligand choice not only depends on the cell type being targeted, but it is also important to consider the type of cell entry pathway that will be induced after ligation. As discussed in the following section, the endocytic pathway

Once bound to the cell surface, non-viral vectors need to cross the plasma membrane to enter the cell and initiate the intracellular trafficking to enter the nucleus. The cellular uptake of macromolecules and solutes into membrane-bound vesicles derived by the invagination and pinching off of pieces of the plasma membrane is known as endocytosis [20]. There are four principal endocytic pathways: clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), phagocytosis and macropinocytosis [17,21]. These endocytic pathways

Clathrin-mediated endocytosis (CME) is a highly regulated and energy-dependent process, and it constitutes the major and best characterized endocytic pathway [20]. The first step in

which are the principal strategies to achieve an efficient transfection.

used by the vector can depend on the targeting ligand.

**2.1. Cellular uptake pathways**

152 Gene Therapy - Principles and Challenges

are described below.

*2.1.1. Clathrin-mediated endocytosis*

Caveolae-mediated endocytosis (CvME) begins in membrane microdomains called caveolae, which are small, hydrophobic, and cholesterol- and sphingolipid-rich smooth invaginations [17,20]. As well as CME, CvME is a type of receptor-mediated and dynamin-dependent pathway in which cholesterol also plays an important role [27]. The main difference between the clathrin- and the caveolae-mediated pathways is that in CvME there are no endosomes. Instead, internalized molecules go into intracellular vesicles called caveosomes, which do not fuse with lysosomes and, therefore, the potential degradation process of the DNA is avoided in this pathway [17]. In fact, CvME is generally considered a non-acidic and non-digestive internalization pathway, meaning that the internalized molecules can be directly transported into their intracellular target sites without being degraded in lysosomes [17,28]. Nevertheless, this issue is still under debate because some authors have recently reported that sometimes caveosomes join the classical endocytic pathway, in which they eventually fuse with lysosomes [29]. Therefore, in this regard further evidence is needed in order to understand the relation‐ ship between caveosomes and lysosomes.

#### *2.1.3. Phagocytosis*

Phagocytosis is a special type of endocytic pathway that is primarily used by professional phagocytes such as macrophages, monocytes, neutrophils and dendritic cells, although other cells might use it too [17]. Cup-like membrane extensions larger than 1 µm mediate the phagocytic pathway, and it is usually employed by cells to internalize large particles such as bacteria or dead cells, although large lipoplexes and polyplexes can also be internalized through this pathway [17].

Phagocytosis usually involves three steps that are common to all molecules internalized through this pathway, including non-viral vectors. First, non-viral vector/DNA complexes are recognized by opsonins and, therefore, opsonized, in the bloodstream. Second, the opsonized complexes bind to the surface of macrophages through the interaction between macrophage receptors and the constant fragment of particle-adsorbed immunoglobulins [17]. Antibodies lacking the constant fragment can be employed in non-viral gene delivery systems to prevent their recognition and clearance by macrophages *in vivo* [30].

Finally, the union of the molecule or non-viral/DNA complex to the macrophage receptors activates Rho-family GTPases, which trigger actin assembly and cell surface extension formation [17]. The complexes are ingested by the macrophages when the surface extension zippers up around them [20]. The vesicles internalized in the cells though the phagocytic pathway are called phagosomes and they usually have a diameter of 0.5–1.0 µm [20]. The phagosomes carrying the internalized complexes form mature phagolysosomes when they fuse with lysosomes, where the complexes undergo an acidification process [20]. In view of the fact that the intracellular fate of the phagocytic pathway is the fusion with lysosomes, the nucleic acids carried in the non-viral vector complexes will probably be degraded in this internalization pathway [31].

#### *2.1.4. Macropinocytosis*

Macropinocytosis is an internalization pathway based on fluid-phase endocytosis, since it nonspecifically takes up a large amount of fluid-phase contents [17]. Similarly to the phagocytic pathway, macropinocytosis also happens through the formation of actin-directed membrane protuberances. Nevertheless, here the protrusions do not zipper up the ligand-coated particle. Alternatively, as shown in Figure 5, in the macropinocytic pathway the protuberances fuse with the plasma membrane. Macropinosomes are also different from clathrin-coated vesicles (CCVs) and caveosomes, since macropinosomes have no coat structures and, even if they are heterogeneous in size, they use to be larger than 0.2 µm in diameter [32]. As well as the other endocytic pathways, macropinocytosis also depends on small GTPase proteins since they are necessary for the vesicle fission from the plasma membrane [17].

The connection between macropinosomes and lysosomes remains still unknown. In some studies, early macropinosomes have been reported to show the same markers as early endosomes, and late macropinosomes have been reported to present lysosome markers [17]. However, macropinosomes have been shown to present different intracellular fates depending on the cell type, even if the explanation of this event remains unclear, and they do not always fuse with lysosomes [17].

#### **2.2. Endosomal escape mechanisms**

*2.1.3. Phagocytosis*

through this pathway [17].

154 Gene Therapy - Principles and Challenges

internalization pathway [31].

*2.1.4. Macropinocytosis*

fuse with lysosomes [17].

Phagocytosis is a special type of endocytic pathway that is primarily used by professional phagocytes such as macrophages, monocytes, neutrophils and dendritic cells, although other cells might use it too [17]. Cup-like membrane extensions larger than 1 µm mediate the phagocytic pathway, and it is usually employed by cells to internalize large particles such as bacteria or dead cells, although large lipoplexes and polyplexes can also be internalized

Phagocytosis usually involves three steps that are common to all molecules internalized through this pathway, including non-viral vectors. First, non-viral vector/DNA complexes are recognized by opsonins and, therefore, opsonized, in the bloodstream. Second, the opsonized complexes bind to the surface of macrophages through the interaction between macrophage receptors and the constant fragment of particle-adsorbed immunoglobulins [17]. Antibodies lacking the constant fragment can be employed in non-viral gene delivery systems to prevent

Finally, the union of the molecule or non-viral/DNA complex to the macrophage receptors activates Rho-family GTPases, which trigger actin assembly and cell surface extension formation [17]. The complexes are ingested by the macrophages when the surface extension zippers up around them [20]. The vesicles internalized in the cells though the phagocytic pathway are called phagosomes and they usually have a diameter of 0.5–1.0 µm [20]. The phagosomes carrying the internalized complexes form mature phagolysosomes when they fuse with lysosomes, where the complexes undergo an acidification process [20]. In view of the fact that the intracellular fate of the phagocytic pathway is the fusion with lysosomes, the nucleic acids carried in the non-viral vector complexes will probably be degraded in this

Macropinocytosis is an internalization pathway based on fluid-phase endocytosis, since it nonspecifically takes up a large amount of fluid-phase contents [17]. Similarly to the phagocytic pathway, macropinocytosis also happens through the formation of actin-directed membrane protuberances. Nevertheless, here the protrusions do not zipper up the ligand-coated particle. Alternatively, as shown in Figure 5, in the macropinocytic pathway the protuberances fuse with the plasma membrane. Macropinosomes are also different from clathrin-coated vesicles (CCVs) and caveosomes, since macropinosomes have no coat structures and, even if they are heterogeneous in size, they use to be larger than 0.2 µm in diameter [32]. As well as the other endocytic pathways, macropinocytosis also depends on small GTPase proteins since they are

The connection between macropinosomes and lysosomes remains still unknown. In some studies, early macropinosomes have been reported to show the same markers as early endosomes, and late macropinosomes have been reported to present lysosome markers [17]. However, macropinosomes have been shown to present different intracellular fates depending on the cell type, even if the explanation of this event remains unclear, and they do not always

their recognition and clearance by macrophages *in vivo* [30].

necessary for the vesicle fission from the plasma membrane [17].

As mentioned before, most non-viral vectors are internalized in the cells mainly through the clathrin-mediated endocytic pathway. The major problem here is the intracellular fate of the endosomes that fuse with lysosomes and this can potentially lead to the degradation of the nucleic acids. In order to avoid this effect, while taking advantage of the CME pathway for cellular uptake, several attempts have been made to promote the early endosomal escape of non-viral gene delivery systems.

Many pathogens, mainly viruses and bacteria, have evolved different mechanisms to promote endosomal escape when internalized in cells. Several endosomal escape agents derive from virus (i.e. haemagglutinin protein of influenza virus) and bacteria (i.e. diphtheria toxin), and some derive from plants (i.e, ricin), human (i.e. fibroblast growth factors) or animals (i.e. melittin form bee venom) too [33]. The understanding of the mechanism used by pathogens allows to design and to ameliorate endosomal escape strategies applicable to non-viral gene delivery systems. Nowadays, several synthetic peptides with specific sequences and length are designed (i.e. the amphiphatic Sweet Arrow Peptide), as well as specific chemical agents (i.e. the polymer polyethylenimine PEI) for endosomal escape induction [33]. In the following paragraphs, the principal endosomal escape mechanisms are described.

#### *2.2.1. Pore formation in the endosomal membrane*

Pore formation is based on the interplay between a membrane tension that enlarges the pore and a line tension that closes the pore. Some peptides have a high affinity for the edge of the pore, and binding of those peptides to the edge of the pore produces a reduction of the line tension [33].

Some studies have reported that the union of cationic amphiphilic peptides to the lipid bilayer produces a strong internal membrane tension able to create pores in the lipid membrane [33].

#### *2.2.2. pH-buffering effect (the proton sponge effect)*

In this endosomal escape mechanism, the low pH of the endosomal environment leads to the protonation of the entrapped agents with a high buffering capacity. Protonation causes an influx of ions (H+ and Cl- ) and water into endosomes, resulting in osmotic swelling and endosome rupture [33].

The proton-sponge effect has been observed in certain cationic polymers with a high H+ buffering capacity over a wide pH range [34]. These polymers usually contain protonable secondary or tertiary amine groups with pKa close to endosomal/lysosomal pH. As explained before, during the maturation of endosomes, the membrane-bound ATPase proton pumps actively translocate protons from the cytosol into endosomes, causing the acidification of the endosomal compartments. At this point, cationic polymers with high buffering capacity become protonated and resist the acidification of endosomes, which results in more protons pumped into the endosome in an attempt to decrease the pH [34]. The proton pumping action is followed by passive chloride ions entry, increasing ionic concentration and, consequently, water influx [34]. The high osmotic pressure produces the swelling and the rupture of endosomes, releasing their contents to the cytosol [34]. Histidine-rich molecules show a buffering effect upon protonation [33] and histidine can be included in non-viral vectors to enhance transfection efficiency by facilitating endosomal escape.

#### *2.2.3. The flip-flop mechanism*

This endosomal escape mechanism can be useful for endocytozed lipoplexes. Lipoplexes are endocytozed and become entrapped inside the early endosomes. There is an electrostatic interaction between the cationic lipoplexes and the anionic lipids of the endosomal membrane [34]. The anionic lipids of the endosomal membrane laterally diffuse into the lipoplexes and form charge-neutralized ion pair with cationic lipids of the lipoplexes, resulting in the nucleic acids being displaced from the lipoplexes and released in the cytoplasm [34].

#### *2.2.4. Fusion in the endosomal membrane*

This mechanism of endosomal escape is based on the destabilization of the endosomal membrane by water soluble and partly hydrophobic, and/or polybasic peptides known as cellpenetration peptides or CPPs. CPPs were originally derived from viruses, and they constitute short sequences of amino acids (10-30 residues) that use to be cationic and/or amphiphatic [34]. The main features of CPPs are their abilities to penetrate the cell membrane at low molecular concentrations without causing significant membrane damage and to internalize electrostati‐ cally or covalently bound biologically active cargoes (including proteins, peptides and nucleic acids) with high efficiency and low toxicity [35]. CPPs either form complexes with nucleic acids, through electrostatic interaction, or can be incorporated into polymeric and lipidic delivery systems [34]. To date, the internalization mechanism of CPPs still remains contro‐ versial, since there is evidence for both energy-independent and endocytic processes for cellular uptake of CPPs. Nowadays, it is generally accepted that endocytosis is the major internalization mechanism for most CPPs. However, it seems plausible that several CPPs utilize two or more cellular uptake pathways depending on the experimental conditions [35]. Further research would be needed in order to elucidate the exact uptake mechanisms and to identify the precise factors influencing these processes.

There are different criteria to categorize CPPs into different families. In general, CPPs can be classified into two categories [36]: (i) Cationic peptides that usually contain arginine and lysine residues; and (ii) amphiphatic peptides that consist of both hydrophobic and hydrophilic segments. Two examples of CPPs currently used to improve transfection efficiency of nonviral gene delivery platforms are the transcriptional activator protein or TAT (which belongs to the first category and was the first CPP identified, derived from the transcription activating factor of human immunodeficiency virus 1 (HIV-1)) [33,37] and the Sweet Arrow Peptide or SAP (which belongs to the second category and is a proline-rich amphipathic peptide of synthetic origin) [38].

#### *2.2.5. Photochemical disruption of the endosomal membrane*

Photochemical internalization (PCI) is a light-directed delivery technology that utilizes photosensitizers to facilitate the transport of membrane-impermeable macromolecules from endocytic vesicles into the cytoplasm [34]. Photosensitizers are usually amphiphilic com‐ pounds that can bind and localize in the plasma membrane. In this mechanism, photosensi‐ tizers bind to and localize in the plasma membrane, and they can be taken up by endocytosis together with the non-viral gene delivery systems. Photosensitizers are confined to the endosomal membrane and remain inactive until they are triggered by light with specific wavelengths matching their absorption spectra [39]. Once activated, they induce the formation of highly reactive oxygen species, causing the rupture of endosomes' and lysosomes' mem‐ brane. As a result, macromolecules that are trapped inside the endosomes/lysosomes can be liberated into the cytosol [34].

In general, the enhancement of endosomal escape is believed to be a crucial factor in non-viralvector-basedDNAdeliveryplatforms.Different strategies for endosomal escape havedifferent characteristics. A safe endosomal escape agent applicable in the clinic should have low immunogenicity and toxicity, high efficiency, ease of use and production, modular attach‐ ment of targeting ligands and the potential for cost-effective large-scale manufacture [33].

#### **2.3. Nuclear import**

water influx [34]. The high osmotic pressure produces the swelling and the rupture of endosomes, releasing their contents to the cytosol [34]. Histidine-rich molecules show a buffering effect upon protonation [33] and histidine can be included in non-viral vectors to

This endosomal escape mechanism can be useful for endocytozed lipoplexes. Lipoplexes are endocytozed and become entrapped inside the early endosomes. There is an electrostatic interaction between the cationic lipoplexes and the anionic lipids of the endosomal membrane [34]. The anionic lipids of the endosomal membrane laterally diffuse into the lipoplexes and form charge-neutralized ion pair with cationic lipids of the lipoplexes, resulting in the nucleic

This mechanism of endosomal escape is based on the destabilization of the endosomal membrane by water soluble and partly hydrophobic, and/or polybasic peptides known as cellpenetration peptides or CPPs. CPPs were originally derived from viruses, and they constitute short sequences of amino acids (10-30 residues) that use to be cationic and/or amphiphatic [34]. The main features of CPPs are their abilities to penetrate the cell membrane at low molecular concentrations without causing significant membrane damage and to internalize electrostati‐ cally or covalently bound biologically active cargoes (including proteins, peptides and nucleic acids) with high efficiency and low toxicity [35]. CPPs either form complexes with nucleic acids, through electrostatic interaction, or can be incorporated into polymeric and lipidic delivery systems [34]. To date, the internalization mechanism of CPPs still remains contro‐ versial, since there is evidence for both energy-independent and endocytic processes for cellular uptake of CPPs. Nowadays, it is generally accepted that endocytosis is the major internalization mechanism for most CPPs. However, it seems plausible that several CPPs utilize two or more cellular uptake pathways depending on the experimental conditions [35]. Further research would be needed in order to elucidate the exact uptake mechanisms and to

There are different criteria to categorize CPPs into different families. In general, CPPs can be classified into two categories [36]: (i) Cationic peptides that usually contain arginine and lysine residues; and (ii) amphiphatic peptides that consist of both hydrophobic and hydrophilic segments. Two examples of CPPs currently used to improve transfection efficiency of nonviral gene delivery platforms are the transcriptional activator protein or TAT (which belongs to the first category and was the first CPP identified, derived from the transcription activating factor of human immunodeficiency virus 1 (HIV-1)) [33,37] and the Sweet Arrow Peptide or SAP (which belongs to the second category and is a proline-rich amphipathic peptide of

acids being displaced from the lipoplexes and released in the cytoplasm [34].

enhance transfection efficiency by facilitating endosomal escape.

*2.2.3. The flip-flop mechanism*

156 Gene Therapy - Principles and Challenges

*2.2.4. Fusion in the endosomal membrane*

identify the precise factors influencing these processes.

synthetic origin) [38].

In the previous section, we have seen several strategies suitable for non-viral gene delivery to avoid endosomal degradation of the DNA and to enhance its release to the cytoplasm. However, in order to achieve an effective transfection, the DNA molecules have to enter the nucleus. Here, we will discuss the principal strategies to transport DNA to the nucleus once released in the cytoplasm.

To enter the nucleus, molecules must pass through nuclear pore complexes (NPCs), which are multimeric structures with a central channel of 9 nm that prevents molecules with a molecular weight higher than 45 kDa from passively diffusing into the nucleus [40]. In the case of naked DNA, molecules smaller than 300 bp can passively diffuse into the nucleus, but larger DNA molecules, even when condensed by a non-viral vector, are excluded from the nucleus except when cells are undergoing mitosis [40-42]. During cell division, the integrity of the nuclear membrane is lost, which allows the nuclear entry of DNA-vector complexes within the daughter cells [21,40]. This is the case in the *in vitro* transfection with dividing cells, but *in vivo* transfection often targets slow dividing or terminally differentiated cells [21,40]. There‐ fore, the nuclear envelope cannot be neglected in *in vivo* situations, and there is considerable interest in improving the nuclear import efficiency of non-viral vectors [21,40].

Classically, proteins that are destined for the nucleus contain a nuclear localization signal (NLS), which is abundant in basic amino acids and it can be recognized by cytoplasmic proteins known as importins [17] that mediate energy-dependent transport through the NPC [40,43]. The same approach can be used to enhance non-viral gene delivery to the nucleus [21]. An NLS-containing vector can be added to the DNA-vector formulation or an NLS sequence can be directly bound to the DNA in order to promote its transport to the nucleus by the importins [40]. In addition, highly basic polymers such as polylysine and protamine, the highly basic sequence of which resembles typical NLS sequences, have been used as potential agents to enhance nuclear targeting when complexed with DNA [40].

**Figure 5.** Cell entry pathways and intracellular trafficking of non-viral gene delivery systems. A) Phagocytosis. B) Cla‐ thrin-mediated endocytosis (CME). C) Caveolae-mediated endocytosis (CvME). D) Macropinocytosis. Internalized vec‐ tor/DNA complexes following the CME pathway escape from endosomes and can be transported into the nucleus by the importins if they contain an NLS. When vector and DNA dissociate from each other, the transgene is expressed in the target cells. Yellow filaments represent actin; red chain represents clathrin coat; green filaments represent caveolin dimers; orange little circles represent the small GTPase dynein; blue filaments represent opsonins. (Adapted from [23; 24])

Finally, it should be considered that, once inside the nucleus, the non-viral vector itself may constitute a barrier to transgene expression. In fact, the agent used to condense the DNA could potentially interfere with the access of the cellular transcription machinery to the transgene promoter, thereby reducing or preventing its expression [40]. Still, premature release of DNA from the vector may expose the DNA to enzymatic degradation before expression can occur [40,44]. For liposomal-based vectors, DNA displacement from the vector seems to be connected to endosomal escape, driven by the anionic lipids of the endosomal membrane that neutralize the charge of the cationic lipids in the liposomal formulation [40,45,46]. In contrast, polycation/ DNA complexes appear to release from each other in the nucleus through exchange of the polycations in the complexes with the protein components of the surrounding proteins [40,47,48]. However, it seems plausible that additional mechanisms other than competitive charge interactions may be involved in the dissociation of DNA from polycations, and a deeper understanding of chromatin remodelling mechanisms may shed further light on this issue.

In summary, non-viral vectors for DNA delivery systems must overcome several intracellular barriers from cell-surface association to nuclear entry and DNA release. Depending on the cell type and on the cellular internalization pathway, some intracellular barriers may differ. Most non-viral vectors are taken up in cells through the CME pathway, which presents some problems such as the acid environment of endosomal and lysosomal compartments and the risk of DNA degradation. Therefore, several endosomal escape agents and mechanisms are currently being studied to avoid DNA degradation and to enhance its cytosolic release. In addition, DNA molecules have to enter the nucleus for the transgene expression. The most employed strategy for this purpose is the incorporation of an NLS in the vector-DNA complex. Finally, once inside the nucleus or earlier during the endocytic pathway, the vector-DNA complex needs to dissociate in order to allow the transcriptional machinery of the cell to access the transgene promoter. Figure 5 summarizes the most relevant aspects described in this section.
