A non-viral delivery system uptaken by endo-lysosomes dependent pathways must be ca‐ pable of escaping endo-lysosomes. From early endosome to late endosome transport, a ma‐ turation process involving compartment acidification by proton pumps located on the endosomal membrane exists. Some non-viral vectors exhibit the ability to escape the endolysosome, called *proton sponge*, such as PEI [10, 69, 70]. PEI contains a nitrogen atom that can be protonated, and this serves to consume endosomal protons because endosomes acidify their microenvironment. As a result, an increase in endosomal chloride anion, which diffus‐ es into the endosomes with the protons, leads to an increase in osmotic pressure, thus induc‐ ing osmotic swelling [69]. Therefore, the endosome might break down and release PEI. This mode of action has been widely incorporated in recent non-viral vector designs. However, a pDAMA-based vector with endosomal buffering capacity has been reported to show no en‐ dosomal escape activity in cell-based assay, indicating that the proton sponge hypothesis may not be applicable in some cases. These findings warrant further elucidation and investi‐ gation of the mechanism of non-viral gene delivery [71].

These data demonstrate that the uptake mechanism and subsequent endocytic processing are important design parameters for gene delivery materials [76]. However, the key is con‐

Cellular Uptake Mechanism of Non-Viral Gene Delivery and Means for Improving Transfection Efficiency

http://dx.doi.org/10.5772/53353

83

Particle size is a very important factor for uptake mechanisms. In a previous study, three particles (20, 40, and 100 nm) were investigated for their uptake efficiency via CvME in en‐ dothelial cells. The results showed that the uptake efficiency levels of the 20- and 40-nm nanoparticles were 5–10 times greater than that of the 100-nm particles [6], indicating that small particles can be uptaken by CvME more efficiently compared with large ones. Howev‐ er, another study found that the uptake of microspheres with a diameter <200 nm in nonphagocytic B16 cells involved CME. With increasing size, a shift to a mechanism that relied on a caveolae-mediated pathway became apparent, which became the predominant path‐ way of entry for particles measuring 500 nm in size [77]. This can be attributed to the fact that the mechanism of CvME is cell type dependent in some cases. According to the target

CvME is a kind of receptor-mediated endocytosis pathway. As a result, some specific li‐ gands can mediate CvME via ligand–receptor binding. The insulin receptor [30], epidermal growth factor (EGF) receptor [31], transforming growth factor beta (TGFbeta) receptor [78] have been found to mediate this pathway. Another study used the cyclic Asn–Gly–Arg pep‐ tide to enhance gene transfection efficiency in CD13-positive vascular endothelial cells via CvME [79]. However, cyclic RGD ligands have been reported to facilitate CvME of thiolated c(RGDfK)-polyethylene glycol (PEG)-b-PLL micelles without high endosomal-disrupting properties and thus improve transfection efficiency [80]. The cyclic RGD peptide ligands c(RGDfK) can selectively recognize αvβ3 and αvβ5 integrin receptors on the cell surface. The receptors can mediate CvME and bypass endo-lysosomes. The αvβ3 and αvβ5 integrin receptors overexpressed on endothelial cells of tumor capillaries and neointimal tissues. As a result, the vectors with cyclic RGD peptide ligands can be used for cancer gene therapy. Cellular stress can also be used to control the cellular uptake mechanism. Heat shock and hyperosmotic shock can stimulate caveolin internalization [81]. Recent research has shown that hypertonic exposure of alveolar cells caused down-regulation of CME and fluid-phase endocytosis while stimulating CvME. An osmotic polymannitol-based gene transporter that can increase caveolae-mediated endocytosis was designed taking advantage of this mecha‐ nism [82]. The possible mechanisms have been discussed. Non-penetrating osmolytes tend to draw water from the intracellular space through an osmotic gradient, cause cell hyperton‐ ic stress accompanied by cell shrinkage. Responding the cellular hypertonic stress, phos‐ phorylation of caveolin-1 is mediated by Src-kinase. Src-kinase-mediated phosphorylation

cell type, the mechanism must be fully studied before designing a vector.

of caveolin-1 is required for caveolae budding. Finally the CvME is stimulated.

After *in vivo* administration, the non-viral delivery system can be uptaken by macrophag‐ es and then cleared. This macrophage clearance effect mainly via phagocytosis is one of the main barriers for non-viral gene delivery. Numerous methods are used to avoid phagocytosis of macrophages in vector design. Antibodies are being widely used for tar‐

trolling the uptake mechanism.

**5.3. Inhibition of phagocytosis**

For lipoplexes, the cationic liposome can interact with the anionic cytoplasmic facing mono‐ layer lipid of endosome and release the DNA from the endosome through the flip-flop mechanism [72]. 1,2-Dioleoylsn-glycero-3-phosphatidylethanolamine (DOPE), the pH-sensi‐ tive fusogenic lipid additive, is very helpful to the displacement of the anionic lipds from the cytoplasm-facingmonolayer of the endosomal membrane to the opposite direction via a flip-flop mechanism. However, the serum components are known to inactivate and destabi‐ lize the lipoplex structures that contain DOPE [73].

Viruses have the ability to destabilize the endosomal membrane, which explains why many proteins from different viruses are being used [69]. Some viruses are well known to use fusogenic peptides to cross the endosomal membrane and reach the cytosol [21]. The process by which viruses destabilize endosomal membranes in an acidification-de‐ pendent manner has been mimicked with synthetic peptides containing the amino-termi‐ nal 20-amino-acid sequence of the influenza virus HA [70]. Generally, short sequences of only 20 amino acids are needed for membrane destabilization, and they usually contain a high content of basic residues [74].

Cell-penetrating peptides (CPPs) are used to enhance endosomal escape. The HIV-1 Tat pro‐ tein is the first CPP to be discovered. It transactivates the transcription of the HIV-1 genome, has been observed to cross the plasma membrane by itself, leading to the identification of a peptide fragment (49–59 amino acids) that confers cell permeability to the protein (Tat pep‐ tide), and is one of the better characterized CPPs [75]. Most of the CPPs contain a high densi‐ ty of basic amino acids (arginines and/orlysines), which are proposed to interact with the anionic surface of the plasma membrane and enhance internalization of the peptides [75]. These peptides adopt an a -helical structure at endosomal pH leading to hydrophobic and hydrophilic faces that can interact with the endosomal membrane to cause disruption and pore formation [74].

#### **5.2. Optimization of CvME**

CvME is considered an alternative pathway that can bypass the endo-lysosomes. As gene materials will not be degraded in the lysosomal compartments, we can take advantage of CvME to improve the efficiency of transfection. For example, Nathan P. et al. targeted com‐ plexes (PEI–DNA) in CvME and CME with folic acid and transferrin, respectively; however, only vectors via CvME successfully delivered genes, as CvME is avoidant of lysosomes. These data demonstrate that the uptake mechanism and subsequent endocytic processing are important design parameters for gene delivery materials [76]. However, the key is con‐ trolling the uptake mechanism.

Particle size is a very important factor for uptake mechanisms. In a previous study, three particles (20, 40, and 100 nm) were investigated for their uptake efficiency via CvME in en‐ dothelial cells. The results showed that the uptake efficiency levels of the 20- and 40-nm nanoparticles were 5–10 times greater than that of the 100-nm particles [6], indicating that small particles can be uptaken by CvME more efficiently compared with large ones. Howev‐ er, another study found that the uptake of microspheres with a diameter <200 nm in nonphagocytic B16 cells involved CME. With increasing size, a shift to a mechanism that relied on a caveolae-mediated pathway became apparent, which became the predominant path‐ way of entry for particles measuring 500 nm in size [77]. This can be attributed to the fact that the mechanism of CvME is cell type dependent in some cases. According to the target cell type, the mechanism must be fully studied before designing a vector.

CvME is a kind of receptor-mediated endocytosis pathway. As a result, some specific li‐ gands can mediate CvME via ligand–receptor binding. The insulin receptor [30], epidermal growth factor (EGF) receptor [31], transforming growth factor beta (TGFbeta) receptor [78] have been found to mediate this pathway. Another study used the cyclic Asn–Gly–Arg pep‐ tide to enhance gene transfection efficiency in CD13-positive vascular endothelial cells via CvME [79]. However, cyclic RGD ligands have been reported to facilitate CvME of thiolated c(RGDfK)-polyethylene glycol (PEG)-b-PLL micelles without high endosomal-disrupting properties and thus improve transfection efficiency [80]. The cyclic RGD peptide ligands c(RGDfK) can selectively recognize αvβ3 and αvβ5 integrin receptors on the cell surface. The receptors can mediate CvME and bypass endo-lysosomes. The αvβ3 and αvβ5 integrin receptors overexpressed on endothelial cells of tumor capillaries and neointimal tissues. As a result, the vectors with cyclic RGD peptide ligands can be used for cancer gene therapy.

Cellular stress can also be used to control the cellular uptake mechanism. Heat shock and hyperosmotic shock can stimulate caveolin internalization [81]. Recent research has shown that hypertonic exposure of alveolar cells caused down-regulation of CME and fluid-phase endocytosis while stimulating CvME. An osmotic polymannitol-based gene transporter that can increase caveolae-mediated endocytosis was designed taking advantage of this mecha‐ nism [82]. The possible mechanisms have been discussed. Non-penetrating osmolytes tend to draw water from the intracellular space through an osmotic gradient, cause cell hyperton‐ ic stress accompanied by cell shrinkage. Responding the cellular hypertonic stress, phos‐ phorylation of caveolin-1 is mediated by Src-kinase. Src-kinase-mediated phosphorylation of caveolin-1 is required for caveolae budding. Finally the CvME is stimulated.

#### **5.3. Inhibition of phagocytosis**

lysosome, called *proton sponge*, such as PEI [10, 69, 70]. PEI contains a nitrogen atom that can be protonated, and this serves to consume endosomal protons because endosomes acidify their microenvironment. As a result, an increase in endosomal chloride anion, which diffus‐ es into the endosomes with the protons, leads to an increase in osmotic pressure, thus induc‐ ing osmotic swelling [69]. Therefore, the endosome might break down and release PEI. This mode of action has been widely incorporated in recent non-viral vector designs. However, a pDAMA-based vector with endosomal buffering capacity has been reported to show no en‐ dosomal escape activity in cell-based assay, indicating that the proton sponge hypothesis may not be applicable in some cases. These findings warrant further elucidation and investi‐

For lipoplexes, the cationic liposome can interact with the anionic cytoplasmic facing mono‐ layer lipid of endosome and release the DNA from the endosome through the flip-flop mechanism [72]. 1,2-Dioleoylsn-glycero-3-phosphatidylethanolamine (DOPE), the pH-sensi‐ tive fusogenic lipid additive, is very helpful to the displacement of the anionic lipds from the cytoplasm-facingmonolayer of the endosomal membrane to the opposite direction via a flip-flop mechanism. However, the serum components are known to inactivate and destabi‐

Viruses have the ability to destabilize the endosomal membrane, which explains why many proteins from different viruses are being used [69]. Some viruses are well known to use fusogenic peptides to cross the endosomal membrane and reach the cytosol [21]. The process by which viruses destabilize endosomal membranes in an acidification-de‐ pendent manner has been mimicked with synthetic peptides containing the amino-termi‐ nal 20-amino-acid sequence of the influenza virus HA [70]. Generally, short sequences of only 20 amino acids are needed for membrane destabilization, and they usually contain

Cell-penetrating peptides (CPPs) are used to enhance endosomal escape. The HIV-1 Tat pro‐ tein is the first CPP to be discovered. It transactivates the transcription of the HIV-1 genome, has been observed to cross the plasma membrane by itself, leading to the identification of a peptide fragment (49–59 amino acids) that confers cell permeability to the protein (Tat pep‐ tide), and is one of the better characterized CPPs [75]. Most of the CPPs contain a high densi‐ ty of basic amino acids (arginines and/orlysines), which are proposed to interact with the anionic surface of the plasma membrane and enhance internalization of the peptides [75]. These peptides adopt an a -helical structure at endosomal pH leading to hydrophobic and hydrophilic faces that can interact with the endosomal membrane to cause disruption and

CvME is considered an alternative pathway that can bypass the endo-lysosomes. As gene materials will not be degraded in the lysosomal compartments, we can take advantage of CvME to improve the efficiency of transfection. For example, Nathan P. et al. targeted com‐ plexes (PEI–DNA) in CvME and CME with folic acid and transferrin, respectively; however, only vectors via CvME successfully delivered genes, as CvME is avoidant of lysosomes.

gation of the mechanism of non-viral gene delivery [71].

82 Gene Therapy - Tools and Potential Applications

lize the lipoplex structures that contain DOPE [73].

a high content of basic residues [74].

pore formation [74].

**5.2. Optimization of CvME**

After *in vivo* administration, the non-viral delivery system can be uptaken by macrophag‐ es and then cleared. This macrophage clearance effect mainly via phagocytosis is one of the main barriers for non-viral gene delivery. Numerous methods are used to avoid phagocytosis of macrophages in vector design. Antibodies are being widely used for tar‐ geting non-viral gene delivery. However, the constant fragments can be recognized by phagocytosis and then uptaken by macrophages. Therefore, antibodies that lack constant fragments are sometimes used to help non-viral vectors avoid recognition and clearance by macrophages *in vivo* [83].

**Acknowledgements**

mission (No.10SG22).

**Author details**

**References**

651-8.

745-53.

18(1): p. 33-7.

2003. 92(2): p. 203-17.

Shengnan Xiang and Xiaoling Zhang

\*Address all correspondence to<EMAIL_ADDRESS>
(SIBS), Chinese Academy of Sciences (CAS), Shanghai, China

*peutics.* Nat Biotechnol, 2008. 26(5): p. 561-9.

*thelial cells.* ACS Nano, 2009. 3(12): p. 4110-6.

*siRNA delivery.* Nat Rev Drug Discov, 2009. 8(2): p. 129-38.

*ing liposomes as drug carriers.* Eur J Pharm Sci, 2001. 13(1): p. 85-9.

This work was supported by grants from The Ministry of Science and Technology of China (No.2011DFA30790), National Natural Science Foundation of China (No. 81190133), Chinese Academy of Sciences (No.XDA01030404, KSCX2-EW-Q-1-07), Science and Technology Com‐ mission of Shanghai Municipality (No.11QH1401600), Shanghai Municipal Education Com‐

Cellular Uptake Mechanism of Non-Viral Gene Delivery and Means for Improving Transfection Efficiency

http://dx.doi.org/10.5772/53353

85

The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) & Shanghai Institutes for Biological Sciences

[1] Gao, K. and L. Huang, *Nonviral methods for siRNA delivery.* Mol Pharm, 2009. 6(3): p.

[2] Luo, D. and W.M. Saltzman, *Synthetic DNA delivery systems.* Nat Biotechnol, 2000.

[3] Wiethoff, C.M. and C.R. Middaugh, *Barriers to nonviral gene delivery.* J Pharm Sci,

[4] Akinc, A., et al., *A combinatorial library of lipid-like materials for delivery of RNAi thera‐*

[5] Whitehead, K.A., R. Langer, and D.G. Anderson, *Knocking down barriers: advances in*

[6] Wang, Z., et al., *Size and dynamics of caveolae studied using nanoparticles in living endo‐*

[7] Harashima, H., Y. Shinohara, and H. Kiwada, *Intracellular control of gene trafficking us‐*

[8] Boussif, O., et al., *A versatile vector for gene and oligonucleotide transfer into cells in cul‐ ture and in vivo: polyethylenimine.* Proc Natl Acad Sci U S A, 1995. 92(16): p. 7297-301. [9] von Gersdorff, K., et al., *The internalization route resulting in successful gene expression depends on both cell line and polyethylenimine polyplex type.* Mol Ther, 2006. 14(5): p.

Other vectors can also be recognized by macrophages. As discussed above, some cationic polyplexes or lipoplexes will be tagged by some opsonins and then recognized *in vivo*. PE‐ Gylation is widely used to avoid the *in vivo* clearance effect by phagocytosis. The highly hy‐ drophilic nature of PEG produces a hydration shell around its conjugated partner, hence reducing intermolecular interactions and, consequently, toxicity [84]. As an effect of reduc‐ ing intermolecular interactions, PEGylation can effectively avoid phagocytosis; moreover, *in vivo* studies have reported on long circulating half-life of PEGylated vectors [85].

However, some studies have shown that PEGylation can reduce the efficiency of vectors [84] possibly because PEGylation may inhibit cellular uptake and endosomal escape of the vec‐ tors. One study compared non-PEGylated and PEGylated liposomes, with the data showing that PEGylated liposomes have poor endosomal escape capability as non-PEGylated lipo‐ somes can escape from endosome efficiently [86]. The inhibitory effects of PEGylation de‐ pend on some factors. A study about PEGylated cationic liposomes demonstrated that acidlabile PEGylation liposomes have higher transfection efficiency than acid-stable PEGylation ones, which can be ascribed to the more efficient endosomal escape activity of acid-labile PEGylation liposomes [87]. The possible mechanism involved here is that the PEG of acidlabile PEGylation liposomes can be cleaved under low pH (endosomal compartments), al‐ lowing the vector to fully interact with the endosomal membrane. So other biodegradable shielding methods should be better than classical PEGylation. According to this hypothesis, recently, an alternative to PEGylation was designed. This work reports, for the first time, the use of hydroxyethyl starch (HES) for the controlled shielding/deshielding of polyplexes. Non-viral delivery systems can be protected by HES shielding, and the HES can then be de‐ graded *in vivo*, indicating that HES shielding has less influence on the efficiency of vectors compared with PEGylation [88].

#### **6. Conclusion**

In summary, cellular uptake is the most important intracellular process. Understanding cel‐ lular uptake mechanisms is essential to determining the limits of gene delivery. Different pathways have different intracellular fates. Some vectors can enter cells via endo-lysosomal pathways. Thus, some methods have to be used to protect genes against degradation in ly‐ sosomes. Optimizing CvME can successfully deliver genes by avoiding endo-lysosomes. Each pathway has its own disadvantages, and learning how to inhibit certain pathways is significant in some cases. In conclusion, taking advantage of cellular uptake mechanisms and knowing how to control them hold considerable potential for improving the efficiency of gene delivery.

#### **Acknowledgements**

geting non-viral gene delivery. However, the constant fragments can be recognized by phagocytosis and then uptaken by macrophages. Therefore, antibodies that lack constant fragments are sometimes used to help non-viral vectors avoid recognition and clearance

Other vectors can also be recognized by macrophages. As discussed above, some cationic polyplexes or lipoplexes will be tagged by some opsonins and then recognized *in vivo*. PE‐ Gylation is widely used to avoid the *in vivo* clearance effect by phagocytosis. The highly hy‐ drophilic nature of PEG produces a hydration shell around its conjugated partner, hence reducing intermolecular interactions and, consequently, toxicity [84]. As an effect of reduc‐ ing intermolecular interactions, PEGylation can effectively avoid phagocytosis; moreover, *in*

However, some studies have shown that PEGylation can reduce the efficiency of vectors [84] possibly because PEGylation may inhibit cellular uptake and endosomal escape of the vec‐ tors. One study compared non-PEGylated and PEGylated liposomes, with the data showing that PEGylated liposomes have poor endosomal escape capability as non-PEGylated lipo‐ somes can escape from endosome efficiently [86]. The inhibitory effects of PEGylation de‐ pend on some factors. A study about PEGylated cationic liposomes demonstrated that acidlabile PEGylation liposomes have higher transfection efficiency than acid-stable PEGylation ones, which can be ascribed to the more efficient endosomal escape activity of acid-labile PEGylation liposomes [87]. The possible mechanism involved here is that the PEG of acidlabile PEGylation liposomes can be cleaved under low pH (endosomal compartments), al‐ lowing the vector to fully interact with the endosomal membrane. So other biodegradable shielding methods should be better than classical PEGylation. According to this hypothesis, recently, an alternative to PEGylation was designed. This work reports, for the first time, the use of hydroxyethyl starch (HES) for the controlled shielding/deshielding of polyplexes. Non-viral delivery systems can be protected by HES shielding, and the HES can then be de‐ graded *in vivo*, indicating that HES shielding has less influence on the efficiency of vectors

In summary, cellular uptake is the most important intracellular process. Understanding cel‐ lular uptake mechanisms is essential to determining the limits of gene delivery. Different pathways have different intracellular fates. Some vectors can enter cells via endo-lysosomal pathways. Thus, some methods have to be used to protect genes against degradation in ly‐ sosomes. Optimizing CvME can successfully deliver genes by avoiding endo-lysosomes. Each pathway has its own disadvantages, and learning how to inhibit certain pathways is significant in some cases. In conclusion, taking advantage of cellular uptake mechanisms and knowing how to control them hold considerable potential for improving the efficiency

*vivo* studies have reported on long circulating half-life of PEGylated vectors [85].

by macrophages *in vivo* [83].

84 Gene Therapy - Tools and Potential Applications

compared with PEGylation [88].

**6. Conclusion**

of gene delivery.

This work was supported by grants from The Ministry of Science and Technology of China (No.2011DFA30790), National Natural Science Foundation of China (No. 81190133), Chinese Academy of Sciences (No.XDA01030404, KSCX2-EW-Q-1-07), Science and Technology Com‐ mission of Shanghai Municipality (No.11QH1401600), Shanghai Municipal Education Com‐ mission (No.10SG22).

#### **Author details**

Shengnan Xiang and Xiaoling Zhang

\*Address all correspondence to<EMAIL_ADDRESS>
The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) & Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Shanghai, China

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[73] Sakurai, F., et al., *Effects of erythrocytes and serum proteins on lung accumulation of lipo‐ plexes containing cholesterol or DOPE as a helper lipid in the single-pass rat lung perfusion*

[74] Martin, M.E. and K.G. Rice, *Peptide-guided gene delivery.* AAPS J, 2007. 9(1): p. E18-29. [75] Endoh, T. and T. Ohtsuki, *Cellular siRNA delivery using cell-penetrating peptides modi‐*

[76] Gabrielson, N.P. and D.W. Pack, *Efficient polyethylenimine-mediated gene delivery pro‐ ceeds via a caveolar pathway in HeLa cells.* J Control Release, 2009. 136(1): p. 54-61. [77] Rejman, J., et al., *Size-dependent internalization of particles via the pathways of clathrin-*

*fied for endosomal escape.* Adv Drug Deliv Rev, 2009. 61(9): p. 704-9.

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[60] Bareford, L.M. and P.W. Swaan, *Endocytic mechanisms for targeted drug delivery.* Adv Drug Deliv Rev, 2007. 59(8): p. 748-58.

[43] Lee, H., I.K. Kim, and T.G. Park, *Intracellular trafficking and unpacking of siRNA/quan‐ tum dot-PEI complexes modified with and without cell penetrating peptide: confocal and flow*

[44] Zuhorn, I.S., R. Kalicharan, and D. Hoekstra, *Lipoplex-mediated transfection of mamma‐ lian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis.*

[45] Zhang, Y., et al., *A novel PEGylation of chitosan nanoparticles for gene delivery.* Biotech‐

[46] Chithrani, B.D., A.A. Ghazani, and W.C. Chan, *Determining the size and shape depend‐ ence of gold nanoparticle uptake into mammalian cells.* Nano Lett, 2006. 6(4): p. 662-8. [47] Chithrani, B.D. and W.C. Chan, *Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes.* Nano Lett, 2007. 7(6): p.

[48] Koping-Hoggard, M., et al., *Relationship between the physical shape and the efficiency of oligomeric chitosan as a gene delivery system in vitro and in vivo.* J Gene Med, 2003. 5(2):

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[51] Ivanov, A.I., *Pharmacological inhibition of endocytic pathways: is it specific enough to be*

[52] Tartakoff, A.M., *Perturbation of vesicular traffic with the carboxylic ionophore monensin.*

[53] Drose, S. and K. Altendorf, *Bafilomycins and concanamycins as inhibitors of V-ATPases*

[54] Vercauteren, D., et al., *The use of inhibitors to study endocytic pathways of gene carriers:*

[55] Damke, H., et al., *Induction of mutant dynamin specifically blocks endocytic coated vesicle*

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[58] Alam, M.R., et al., *Intracellular delivery of an anionic antisense oligonucleotide via receptor-*

[59] Luhmann, T., et al., *Cellular uptake and intracellular pathways of PLL-g-PEG-DNA nano‐*

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*useful?* Methods Mol Biol, 2008. 440: p. 15-33.

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*formation.* J Cell Biol, 1994. 127(4): p. 915-34.

*ing peptides.* Traffic, 2007. 8(7): p. 848-66.

*particles.* Bioconjug Chem, 2008. 19(9): p. 1907-16.

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**Chapter 5**

**Polylipid Nanoparticle,**

Additional information is available at the end of the chapter

Yahan Fan and Jian Wu

http://dx.doi.org/10.5772/54270

**1. Introduction**

**a Novel Lipid-Based Vector for Liver Gene Transfer**

Lipid nanoparticles (LNP) are invaluable carriers for drug and gene delivery, and they are classified as cationic, neutral and anionic depending on the electronic charges exist‐ ing on the surface of the vesicles [1]. These charges are originated from the charged lip‐ ids from which lipid nanoparticles are formulated. Cationic LNP are commonly used for DNA or RNA carriers due to their interaction with negatively-charged nucleotide. Both neutral and negatively-charged LNPs are used for drug delivery [2] and may be formu‐ lated as sterically stable LNPs (SSLNPs), which are amendable for cell type-specific or tissue-specific targeting delivery [3]. For liver drug delivery, tremendous efforts have been made to develop cell type-selective lipid-based drug carriers. Effective approaches in targeting hepatocytes, Kupffer cells and hepatic stellate cells have been evaluated in small animals [3, 4], and some of them may be translational to clinical application [5]. These approaches are referable when cationic LNPs are considered for cell type-selective gene delivery. A prerequisite for the success of gene therapy for liver disorders is the de‐ velopment of powerful gene carriers. Non-viral vectors have been very successful for gene transfer in an *in vitro* setting, in terms of efficiency of lipofection, applicability in variety of cell types, and amending ability of cell type-specific delivery (Fig. 1). The clini‐ cal application of LNP-mediated gene transfer has been hampered by low efficiency, in‐ stability in the bloodstream, short-term transgene expression and toxicity. These shortcomings are the bottle neck hindering the gene transfer employing LNPs as carriers for delivery of function gene(s) to solid organs, and are the challenges in moving from small to large animals of potential gene carriers and approaches, and in the translation to clinical application. However, the polylipid nanoparticles (PLNP) we have developed over the past decade represent one of the few formulations that are applicable for *in vivo*

> © 2013 Fan and Wu; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

### **Polylipid Nanoparticle, a Novel Lipid-Based Vector for Liver Gene Transfer**

Yahan Fan and Jian Wu

[78] Razani, B., et al., *Caveolin-1 regulates transforming growth factor (TGF)-beta/SMAD sig‐ naling through an interaction with the TGF-beta type I receptor.* J Biol Chem, 2001. 276(9):

[79] Liu, C., et al., *Enhanced gene transfection efficiency in CD13-positive vascular endothelial cells with targeted poly(lactic acid)-poly(ethylene glycol) nanoparticles through caveolae-*

[80] Oba, M., et al., *Polyplex micelles with cyclic RGD peptide ligands and disulfide cross-links directing to the enhanced transfection via controlled intracellular trafficking.* Mol Pharm,

[81] Kang, Y.S., Y.G. Ko, and J.S. Seo, *Caveolin internalization by heat shock or hyperosmotic*

[82] Park, T.E., et al., *Selective stimulation of caveolae-mediated endocytosis by an osmotic poly‐*

[83] Ikeda, Y. and K. Taira, *Ligand-targeted delivery of therapeutic siRNA.* Pharm Res, 2006.

[84] Fitzsimmons, R.E. and H. Uludag, *Specific effects of PEGylation on gene delivery efficacy of polyethylenimine: Interplay between PEG substitution and N/P ratio.* Acta Biomater,

[85] Chaudhari, K.R., et al., *Opsonization, biodistribution, cellular uptake and apoptosis study of PEGylated PBCA nanoparticle as potential drug delivery carrier.* Pharm Res, 2012. 29(1):

[86] Remaut, K., et al., *Pegylation of liposomes favours the endosomal degradation of the deliv‐ ered phosphodiester oligonucleotides.* J Control Release, 2007. 117(2): p. 256-66.

[87] Chan, C.L., et al., *Endosomal escape and transfection efficiency of PEGylated cationic lipo‐ some-DNA complexes prepared with an acid-labile PEG-lipid.* Biomaterials, 2012. 33(19):

[88] Noga, M., et al., *Controlled shielding and deshielding of gene delivery polyplexes using hy‐ droxyethyl starch (HES) and alpha-amylase.* J Control Release, 2012. 159(1): p. 92-103.

*mannitol-based gene transporter.* Biomaterials, 2012. 33(29): p. 7272-81.

*mediated endocytosis.* J Control Release, 2011. 151(2): p. 162-75.

p. 6727-38.

90 Gene Therapy - Tools and Potential Applications

2008. 5(6): p. 1080-92.

23(8): p. 1631-40.

2012.

p. 53-68.

p. 4928-35.

*shock.* Exp Cell Res, 2000. 255(2): p. 221-8.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54270

#### **1. Introduction**

Lipid nanoparticles (LNP) are invaluable carriers for drug and gene delivery, and they are classified as cationic, neutral and anionic depending on the electronic charges exist‐ ing on the surface of the vesicles [1]. These charges are originated from the charged lip‐ ids from which lipid nanoparticles are formulated. Cationic LNP are commonly used for DNA or RNA carriers due to their interaction with negatively-charged nucleotide. Both neutral and negatively-charged LNPs are used for drug delivery [2] and may be formu‐ lated as sterically stable LNPs (SSLNPs), which are amendable for cell type-specific or tissue-specific targeting delivery [3]. For liver drug delivery, tremendous efforts have been made to develop cell type-selective lipid-based drug carriers. Effective approaches in targeting hepatocytes, Kupffer cells and hepatic stellate cells have been evaluated in small animals [3, 4], and some of them may be translational to clinical application [5]. These approaches are referable when cationic LNPs are considered for cell type-selective gene delivery. A prerequisite for the success of gene therapy for liver disorders is the de‐ velopment of powerful gene carriers. Non-viral vectors have been very successful for gene transfer in an *in vitro* setting, in terms of efficiency of lipofection, applicability in variety of cell types, and amending ability of cell type-specific delivery (Fig. 1). The clini‐ cal application of LNP-mediated gene transfer has been hampered by low efficiency, in‐ stability in the bloodstream, short-term transgene expression and toxicity. These shortcomings are the bottle neck hindering the gene transfer employing LNPs as carriers for delivery of function gene(s) to solid organs, and are the challenges in moving from small to large animals of potential gene carriers and approaches, and in the translation to clinical application. However, the polylipid nanoparticles (PLNP) we have developed over the past decade represent one of the few formulations that are applicable for *in vivo*

© 2013 Fan and Wu; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

gene transfer [6], due to a high DNA-packaging capacity, an extremely low binding rate to serum proteins, low toxicity, and amendable synthetic approaches [7, 8]. This chapter intends to introduce the characteristics of this formulation, and to discuss our efforts in moving the non-viral gene transfer platform from small to large animals towards clinical applications.

ing cationic liposome-mediated gene transfer (NCT00004471) has been completed. A phase I trial of intratumoral epidermal growth factor receptor (EGFR) antisense DNA delivered by DC-Chol liposomes in advanced head and neck cancer, including oral squamous cell carci‐ noma (NCT00009841) and DOTAP-Chol-Fus1 liposome-mediated gene therapy for nonsmall cell lung cancer (NCT00059605] [9] were conducted respectively by University of Pittsburg and MD Anderson Cancer Center in collaboration with the National Cancer Insti‐ tute (NCI). Fus1 is a tumor suppressive gene that has been shown to be effective in sup‐ pressing the growth of original or metastatic lesions of non-small lung cancer when it is delivered locally or systemically [10]. Thus, it appears that genetic therapy using LNPs as gene carriers has the potential to be specially tailored for genetic disorders or cancers.

Polylipid Nanoparticle, a Novel Lipid-Based Vector for Liver Gene Transfer

http://dx.doi.org/10.5772/54270

93

Lipid-based gene carriers include liposomes (cationic or anionic), polymer and dendrimer nanoparticles. Cationic liposomes are capable of delivering genes to cells or tissues, and achieving maximal therapeutic efficiency with minimal adverse effects [1]. However, the use of cationic LNPs for *in vivo* DNA transfection is hindered by substantial problems; i.e. after intravenous administration, cationic LNPs bind to plasma protein and blood cells due to charge reaction. The resulting aggregates of carriers with proteins or cells block microcirculation or may be cleared rapidly [11, 12]. The common formulations for *in vivo* gene delivery are DOTMA or DOTAP-DOPE or DOTAP-cholesterol (Chol). These formulations are highly serum-reactive [6, 13]. Lungs are the major organ shown to be highly transfected probably due to the accumulation of aggregates of lipoplexes with se‐ rum proteins or blood cells when the lipoplexes are administrated intravenously [14]. For this reason, cationic LNPs were once used widely for gene delivery to the lungs; and lat‐ er for treating lung cancers and metastasis with further optimization [10, 15, 16]. LNPmediated gene delivery to the liver is more difficult than to lungs. For the development of the gene carriers, cationic LNP formulations, such as DC-Chol, DOTAP-Chol, are available for delivering genes to various tissues [17]. A few LNP formulations targeting hepatocellular carcinoma (HCC) have been developed for improving efficacies of drug therapy [18, 19]. In order to avoid the rapid clearance by the reticuloendothelial system (RES) and to increase the drug delivery through the enhanced permeability and retention (EPR) effect to a tumor site by passive targeting, novel strategies, such as reducing parti‐ cle size, minimizing rigidity of lipids, generating amphiphilic vesicles and shielding from the recognition by RES system, have been attempted in formulating lipid-based drug/ gene carriers [1, 2]. To reduce lysosomal degradation, pH-sensitive LNPs are prepared for drug or gene delivery [20]. These approaches may be instructive in the development

**2. Nanoparticle carriers for drug or gene delivery**

of LNPs for gene transfer at different stages of preclinical translation.

Polymeric non-viral vectors have exhibited additional advantages of lower toxicity and im‐ munogenicity [21, 22]. These vectors may offer the possibility of industrial production fol‐ lowing good manufacturing practice (GMP). Amphiphilic polyethylene glycol (PEG) has been engineered as a linker, most for coupling peptides to cationic lipids. Other polymers,

**Figure 1.** *In vivo* **gene transfer mediated by viral or non-viral vectors.** Viral vectors, such as lentiviral or retroviral vectors, which lead to integration of transgene in the host genome, give rise to long-term transgene expression. How‐ ever, they may cause insertion-induced mutation that is oncogenic. Adenoviral or adeno-associated viral (AAV) vectors often yield a high level of transgene expression in host organs. However, generation of antibodies against viral com‐ ponents is still a concern. Non-viral gene transfer may be achieved by direct plasmid administration with local electro‐ poration or a hydrodynamic approach. The latter is only applicable in mice. Lipid nanoparticle (LNP)-mediated gene transfer becomes a useful approach which is often very successful for *in vitro* gene transfer. Few formulations of cati‐ onic LNPs are valuable for *in vivo* gene transfer. There has been a still demand in improving their *in vivo* stability and gene transfer efficacy.

There are a number of critical components for a potential gene therapy product to move from one step to the next in this pipeline. Promisingly, LNP-mediated gene transfection for the treatment of genetic and metabolic disorders or tumors has been moved to clinical trial phases (http://clinicaltrials.gov). A phase I pilot study of gene therapy for cystic fibrosis us‐ ing cationic liposome-mediated gene transfer (NCT00004471) has been completed. A phase I trial of intratumoral epidermal growth factor receptor (EGFR) antisense DNA delivered by DC-Chol liposomes in advanced head and neck cancer, including oral squamous cell carci‐ noma (NCT00009841) and DOTAP-Chol-Fus1 liposome-mediated gene therapy for nonsmall cell lung cancer (NCT00059605] [9] were conducted respectively by University of Pittsburg and MD Anderson Cancer Center in collaboration with the National Cancer Insti‐ tute (NCI). Fus1 is a tumor suppressive gene that has been shown to be effective in sup‐ pressing the growth of original or metastatic lesions of non-small lung cancer when it is delivered locally or systemically [10]. Thus, it appears that genetic therapy using LNPs as gene carriers has the potential to be specially tailored for genetic disorders or cancers.

#### **2. Nanoparticle carriers for drug or gene delivery**

gene transfer [6], due to a high DNA-packaging capacity, an extremely low binding rate to serum proteins, low toxicity, and amendable synthetic approaches [7, 8]. This chapter intends to introduce the characteristics of this formulation, and to discuss our efforts in moving the non-viral gene transfer platform from small to large animals towards clinical

**Figure 1.** *In vivo* **gene transfer mediated by viral or non-viral vectors.** Viral vectors, such as lentiviral or retroviral vectors, which lead to integration of transgene in the host genome, give rise to long-term transgene expression. How‐ ever, they may cause insertion-induced mutation that is oncogenic. Adenoviral or adeno-associated viral (AAV) vectors often yield a high level of transgene expression in host organs. However, generation of antibodies against viral com‐ ponents is still a concern. Non-viral gene transfer may be achieved by direct plasmid administration with local electro‐ poration or a hydrodynamic approach. The latter is only applicable in mice. Lipid nanoparticle (LNP)-mediated gene transfer becomes a useful approach which is often very successful for *in vitro* gene transfer. Few formulations of cati‐ onic LNPs are valuable for *in vivo* gene transfer. There has been a still demand in improving their *in vivo* stability and

There are a number of critical components for a potential gene therapy product to move from one step to the next in this pipeline. Promisingly, LNP-mediated gene transfection for the treatment of genetic and metabolic disorders or tumors has been moved to clinical trial phases (http://clinicaltrials.gov). A phase I pilot study of gene therapy for cystic fibrosis us‐

applications.

92 Gene Therapy - Tools and Potential Applications

gene transfer efficacy.

Lipid-based gene carriers include liposomes (cationic or anionic), polymer and dendrimer nanoparticles. Cationic liposomes are capable of delivering genes to cells or tissues, and achieving maximal therapeutic efficiency with minimal adverse effects [1]. However, the use of cationic LNPs for *in vivo* DNA transfection is hindered by substantial problems; i.e. after intravenous administration, cationic LNPs bind to plasma protein and blood cells due to charge reaction. The resulting aggregates of carriers with proteins or cells block microcirculation or may be cleared rapidly [11, 12]. The common formulations for *in vivo* gene delivery are DOTMA or DOTAP-DOPE or DOTAP-cholesterol (Chol). These formulations are highly serum-reactive [6, 13]. Lungs are the major organ shown to be highly transfected probably due to the accumulation of aggregates of lipoplexes with se‐ rum proteins or blood cells when the lipoplexes are administrated intravenously [14]. For this reason, cationic LNPs were once used widely for gene delivery to the lungs; and lat‐ er for treating lung cancers and metastasis with further optimization [10, 15, 16]. LNPmediated gene delivery to the liver is more difficult than to lungs. For the development of the gene carriers, cationic LNP formulations, such as DC-Chol, DOTAP-Chol, are available for delivering genes to various tissues [17]. A few LNP formulations targeting hepatocellular carcinoma (HCC) have been developed for improving efficacies of drug therapy [18, 19]. In order to avoid the rapid clearance by the reticuloendothelial system (RES) and to increase the drug delivery through the enhanced permeability and retention (EPR) effect to a tumor site by passive targeting, novel strategies, such as reducing parti‐ cle size, minimizing rigidity of lipids, generating amphiphilic vesicles and shielding from the recognition by RES system, have been attempted in formulating lipid-based drug/ gene carriers [1, 2]. To reduce lysosomal degradation, pH-sensitive LNPs are prepared for drug or gene delivery [20]. These approaches may be instructive in the development of LNPs for gene transfer at different stages of preclinical translation.

Polymeric non-viral vectors have exhibited additional advantages of lower toxicity and im‐ munogenicity [21, 22]. These vectors may offer the possibility of industrial production fol‐ lowing good manufacturing practice (GMP). Amphiphilic polyethylene glycol (PEG) has been engineered as a linker, most for coupling peptides to cationic lipids. Other polymers, such as dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic vector [23], poly (ethylenei‐ mine) (PEI) [24], poly (methacrylate) [25] and polyamidoamine dendrimers [26], have been demonstrated to be effective for *in vitro* gene delivery. However, striking issues still exist for cationic polymers regarding whether they are applicable for *in vivo* gene transfer to solid or‐ gans such as the liver, without significant adverse effects.

High density lipoprotein (HDL) has a high drug carry capacity, and can be recognized by HDL receptors on hepatocytes. Recombinant HDL was utilized to deliver an anti-HBV pep‐ tide (nosiheptide) to the liver, and it was shown to achieve a selective distribution in hepato‐ ma cells *in vitro* and a preferential liver distribution in rats [36]. Apolipoprotein E is cleared by hepatocytes, and it has been employed to be carriers for small interfering RNA (siRNA)

Polylipid Nanoparticle, a Novel Lipid-Based Vector for Liver Gene Transfer

http://dx.doi.org/10.5772/54270

95

Given the fact that hepatic stellate cells (HSCs) are the major cell type responsible for hepatic fibrosis, a repairing process that causes excess production of extracellular matrix compo‐ nents and deposition of fibrotic scaring in chronically injured liver [38], much attention has been focused on targeting this cell type in the last decade. A couple of cell surface molecules that are overexpressed on activated HSCs during hepatic fibrogenesis, such as insulin growth factor receptor II [39], collagen type VI and platelet-derived growth factor (PDGF) receptor β-subunit [40] are selected as the cell surface targets. Drug carriers labeled with specific peptides recognizing these cell surface molecules, such as cyclic peptide containing arginine-glycine-aspartate (RGD)-labeled sterical lipid nanoparticles [3] or Mannose-6-phos‐ phate human serum albumin (M6P/HAS) [41] exhibited HSC-selective distribution. The RGD cyclic peptide was recently used as a targeting molecule for the recognition of activat‐ ed HSCs in two animal models for early diagnosis of hepatic fibrosis with a SPECT imaging modality [42]. Using the retinol binding protein (RBP) in activated HSCs seems to be very effective in delivering siRNA against gp46 (rat homolog of human heat shock protein 47),

Targeting approaches for drug or gene delivery to other non-parenchymal cell types, such as Kupffer cells or sinusoidal endothelial cells, are summarized recently [27]. These approaches are crucial in delivering agents which are anti-inflammatory or anti-oxidants to these cell types due to the fact that Kupffer cells are pivotal in the mediation of inflammatory respons‐

Compared to drug delivery, LNP-mediated *in vivo* gene delivery is still in its development stage; and many issues that affect delivery approaches and efficacy remain to be solved. The main issues include: 1) the formation of aggregates between cationic lipids and serum pro‐ teins bearing negative charges; 2) the administration routes of LNP-DNA complexes (lipo‐ plexes); 3) intracellular trafficking from the cytoplasm to the nucleus; 4) the proliferative state of cells to be transfected; and 5) transient transgene expression for a short duration [6, 45]. Substantial efforts have been made to address these issues in our previous studies and by others [8, 46, 47]. Particularly, we polymerized an acrylamide lipid to generate a polyca‐ tionic lipid (PCL), which was able to interact with plasmid DNA effectively and form com‐ pacted complexes as demonstrated by Raman microspectral analysis [8]. PCL has a unique molecular configuration and molecular weight distribution as indicated by mass spectro‐ photometrical analysis [8]. Moreover, this lipid can be synthesized in a multiple gram quan‐

delivery to hepatocytes [37].

and inhibiting fibrosis in two animal models [43].

**4. Polylipid nanoparticle-mediated liver gene delivery**

es and subsequent fibrogenesis [44].

#### **3. Liver-specific gene delivery**

Because of our interest in gene therapy of liver disorders, we have focused our efforts on improving liver-based gene delivery. The pathogenesis of liver injury and fibrosis in‐ volves complicated interactions among different cell populations in the liver, soluble fac‐ tors, such as cytokines and reactive oxygen species (ROS), and the extracellular matrix components. In order to improve the efficacy in preventing hepatocellular injury, the use of LNPs that are capable of delivering hepatoprotective agents to the liver, selectively to hepatocytes, will increase local concentration of therapeutic agents, reduce adverse effects, and achieve maximal therapeutic efficiency. The parenchymal cell type in the liver is hep‐ atocytes, which are responsible for an array of metabolic function in the body and are of‐ ten damaged in a variety of pathological processes. The asialoglycoprotein receptor (ASGP-R) on mammalian hepatocytes provides a unique means for the development of liver-specific drug or gene carriers. The abundant receptors on hepatocytes specifically recognize the natural ligands, lectin and asialofetuin (AF), as well as those with terminal galactose or N-acetylgalactosamine residues, and hepatocytes endocytose these ligands for an intracellular degradation process [27, 28]. The use of its natural or synthetic ligands, such as galactosylated cholesterol, glycolipids or galactosylated polymers to label LNPs has achieved significant targeting efficacy to the liver [4, 28]. AF-labeled LNPs have been used for improving liver-targeting gene transfer in small animals [29], yet there have not been successful reports available in the translation to large animals, such as pigs [30]. In‐ stead, plasmid DNA was directly administrated into the hepatic vein through a catheter with a balloon closure of hepatic vein blood flow [30]. One particular attention has been drawn in terms of the use of AF-labeled drug carriers for HCC targeting. The expression of ASGP-R in HCC cells varies depending on the differentiation status of HCC cells [31]. In general, well-differentiated HCC usually expresses relatively high levels of hepatocytespecific genes, including ASGP-R; whereas poorly-differentiated HCC expresses minimal or no hepatocyte-specific genes, including ASGP-R [32]. In most cases, there exists the dra‐ matic heterogeneity of liver-specific gene expression in human HCC tissues [33], and de‐ creased expression of ASGP-R was observed in liver cancer tissue [34]. Therefore, using AF or other galactosylated or lactosylated residues to label LNPs for drug or gene deliv‐ ery may not always be effective for patients with HCC, because HCC develops on a varie‐ ty of disease backgrounds and there is a striking variation in ASGP-R expression levels in HCC from different patients. Using well-differentiated hepatoma cells, such as HepG2, Hep3B and Huh-7 cells, as an *in vitro* screening tool may not necessarily reflect targeting efficacy to tumor-specific distribution *in vivo* [35].

High density lipoprotein (HDL) has a high drug carry capacity, and can be recognized by HDL receptors on hepatocytes. Recombinant HDL was utilized to deliver an anti-HBV pep‐ tide (nosiheptide) to the liver, and it was shown to achieve a selective distribution in hepato‐ ma cells *in vitro* and a preferential liver distribution in rats [36]. Apolipoprotein E is cleared by hepatocytes, and it has been employed to be carriers for small interfering RNA (siRNA) delivery to hepatocytes [37].

such as dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic vector [23], poly (ethylenei‐ mine) (PEI) [24], poly (methacrylate) [25] and polyamidoamine dendrimers [26], have been demonstrated to be effective for *in vitro* gene delivery. However, striking issues still exist for cationic polymers regarding whether they are applicable for *in vivo* gene transfer to solid or‐

Because of our interest in gene therapy of liver disorders, we have focused our efforts on improving liver-based gene delivery. The pathogenesis of liver injury and fibrosis in‐ volves complicated interactions among different cell populations in the liver, soluble fac‐ tors, such as cytokines and reactive oxygen species (ROS), and the extracellular matrix components. In order to improve the efficacy in preventing hepatocellular injury, the use of LNPs that are capable of delivering hepatoprotective agents to the liver, selectively to hepatocytes, will increase local concentration of therapeutic agents, reduce adverse effects, and achieve maximal therapeutic efficiency. The parenchymal cell type in the liver is hep‐ atocytes, which are responsible for an array of metabolic function in the body and are of‐ ten damaged in a variety of pathological processes. The asialoglycoprotein receptor (ASGP-R) on mammalian hepatocytes provides a unique means for the development of liver-specific drug or gene carriers. The abundant receptors on hepatocytes specifically recognize the natural ligands, lectin and asialofetuin (AF), as well as those with terminal galactose or N-acetylgalactosamine residues, and hepatocytes endocytose these ligands for an intracellular degradation process [27, 28]. The use of its natural or synthetic ligands, such as galactosylated cholesterol, glycolipids or galactosylated polymers to label LNPs has achieved significant targeting efficacy to the liver [4, 28]. AF-labeled LNPs have been used for improving liver-targeting gene transfer in small animals [29], yet there have not been successful reports available in the translation to large animals, such as pigs [30]. In‐ stead, plasmid DNA was directly administrated into the hepatic vein through a catheter with a balloon closure of hepatic vein blood flow [30]. One particular attention has been drawn in terms of the use of AF-labeled drug carriers for HCC targeting. The expression of ASGP-R in HCC cells varies depending on the differentiation status of HCC cells [31]. In general, well-differentiated HCC usually expresses relatively high levels of hepatocytespecific genes, including ASGP-R; whereas poorly-differentiated HCC expresses minimal or no hepatocyte-specific genes, including ASGP-R [32]. In most cases, there exists the dra‐ matic heterogeneity of liver-specific gene expression in human HCC tissues [33], and de‐ creased expression of ASGP-R was observed in liver cancer tissue [34]. Therefore, using AF or other galactosylated or lactosylated residues to label LNPs for drug or gene deliv‐ ery may not always be effective for patients with HCC, because HCC develops on a varie‐ ty of disease backgrounds and there is a striking variation in ASGP-R expression levels in HCC from different patients. Using well-differentiated hepatoma cells, such as HepG2, Hep3B and Huh-7 cells, as an *in vitro* screening tool may not necessarily reflect targeting

gans such as the liver, without significant adverse effects.

efficacy to tumor-specific distribution *in vivo* [35].

**3. Liver-specific gene delivery**

94 Gene Therapy - Tools and Potential Applications

Given the fact that hepatic stellate cells (HSCs) are the major cell type responsible for hepatic fibrosis, a repairing process that causes excess production of extracellular matrix compo‐ nents and deposition of fibrotic scaring in chronically injured liver [38], much attention has been focused on targeting this cell type in the last decade. A couple of cell surface molecules that are overexpressed on activated HSCs during hepatic fibrogenesis, such as insulin growth factor receptor II [39], collagen type VI and platelet-derived growth factor (PDGF) receptor β-subunit [40] are selected as the cell surface targets. Drug carriers labeled with specific peptides recognizing these cell surface molecules, such as cyclic peptide containing arginine-glycine-aspartate (RGD)-labeled sterical lipid nanoparticles [3] or Mannose-6-phos‐ phate human serum albumin (M6P/HAS) [41] exhibited HSC-selective distribution. The RGD cyclic peptide was recently used as a targeting molecule for the recognition of activat‐ ed HSCs in two animal models for early diagnosis of hepatic fibrosis with a SPECT imaging modality [42]. Using the retinol binding protein (RBP) in activated HSCs seems to be very effective in delivering siRNA against gp46 (rat homolog of human heat shock protein 47), and inhibiting fibrosis in two animal models [43].

Targeting approaches for drug or gene delivery to other non-parenchymal cell types, such as Kupffer cells or sinusoidal endothelial cells, are summarized recently [27]. These approaches are crucial in delivering agents which are anti-inflammatory or anti-oxidants to these cell types due to the fact that Kupffer cells are pivotal in the mediation of inflammatory respons‐ es and subsequent fibrogenesis [44].

#### **4. Polylipid nanoparticle-mediated liver gene delivery**

Compared to drug delivery, LNP-mediated *in vivo* gene delivery is still in its development stage; and many issues that affect delivery approaches and efficacy remain to be solved. The main issues include: 1) the formation of aggregates between cationic lipids and serum pro‐ teins bearing negative charges; 2) the administration routes of LNP-DNA complexes (lipo‐ plexes); 3) intracellular trafficking from the cytoplasm to the nucleus; 4) the proliferative state of cells to be transfected; and 5) transient transgene expression for a short duration [6, 45]. Substantial efforts have been made to address these issues in our previous studies and by others [8, 46, 47]. Particularly, we polymerized an acrylamide lipid to generate a polyca‐ tionic lipid (PCL), which was able to interact with plasmid DNA effectively and form com‐ pacted complexes as demonstrated by Raman microspectral analysis [8]. PCL has a unique molecular configuration and molecular weight distribution as indicated by mass spectro‐ photometrical analysis [8]. Moreover, this lipid can be synthesized in a multiple gram quan‐ tity in a laboratory, and the synthetic approach is amendable for industrial production at a quantity sufficient enough for large animal use [8]. PLNP was formulated with a neutral lip‐ id, cholesterol. The PLNP size was reduced to approximately 100 nm in diameter [7], and the Zeta potential of PLNP was decreased to neutral by neutralizing extra-positive charges with excess plasmid DNA [8]. Not only was this formulation of PLNP non-toxic, but it also displayed transfection efficiency equivalent to other commercially available transfection agents, such as Lipofectamine in hepatoma cell lines [7]. Moreover, high-resolution fluores‐ cent deconvolution microscopy documented that PLNP-mediated gene transfection led to earlier GFP expression in hepatoma cells than Lipofectamine [8]. The unique feature of this formulation is that it is extremely serum-resistant, and exposure to cell culture medium con‐ taining 50% fetal bovine serum for 24 hours did not affect its size significantly. PLNP react‐ ed up to 30-fold less with serum proteins or blood cells after intravenous administration in comparison with DOTAP-DOPE or DOTAP-Chol formulations [6]. This feature makes PLNP formulation particularly useful for *in vivo* gene transfer. In the subsequent studies, we have proved that it is very effective in the transfer of reporter genes or function genes to normal mouse livers as demonstrated in Fig. 2 by bioluminescent imaging of firefly lucifer‐ ase gene expression 24 hours after portal vein injection of PLNP-plasmid DNA complexes (polyplexes) or preclinical models [48, 49].

We also developed an approach to promote normal hepatocytes to proliferate *in situ* without partial hepatectomy, which favors the transgene expression by lipofection but is not accepta‐ ble for clinical application [6]. Furthermore, placing an indwelling catheter in the portal vein allows repeated administration of polyplexes for sustained transgene expression [6]. All these efforts render our formulation of PLNP distinct from other lipid-based nanoparticles. Our animal experiments have clearly demonstrated that PLNP is characterized as extremely stable in the bloodstream, and highly effective in liver-based gene transfer when polyplexes are administrated through the portal vein [6, 17]. In comparison with other commonly used lipid formulations of nanoparticles, our formulation possesses the notable advantages essen‐

**Characteristics PCL PLNP Lipofectamine DOTAP-Chol**

Irrelevant 125±54 358 ±85 110±20

Irrelevant >10E7 >10E7 >10E7

10±3% (>5%)

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97

Low Low Obvious 20-30-fold higher than

11±3.5% (>5%)

PLNP

*In vitro* transfection *In vivo* transfection

Cationic lipid Yes LNP LNP LNP

Size changes(50%FBS) Irrelevant 100±20nm 2206 ± 311 nm 1050±100 nm

*In vivo* stability Irrelevant Stable Not determined Instability

*In vitro* or *in vivo* transfection

The content in this table was summarized according to our previous publications [6-8]. FBS = fetal bovine serum. RLU =

In order to demonstrate that our PLNP formulation is effective in delivering functional genes to the liver, we established a liver injury model in mice caused by the treatment with D-galactosamine (D-Gal) and lipopolysaccharide (LPS). This combination of D-Gal/LPS treatment resulted in a profound acute liver injury characterized by massive liver cell death through apoptosis, elevation of serum alanine aminotransferase (ALT), significant oxidant stress, depletion of the reduced form of glutathione and enhanced lipid peroxidation [50]. In

tial for *in vivo* gene delivery as illustrated in Table 1.

Cytotoxicity (LDH release) Low or none Normal

PLNP

**5. Preclinical trials for proof of the concept**

**Table 1.** Comparison of common transfection agents for *in vitro* and *in vivo* application

Usage Raw material for

relative light unit. LDH = lactate dehydrogenase.

Particle size (nm)

(in RLU)

protein

*In vitro* transfection efficiency Luciferase activity

Binding rate to serum

**Figure 2. PLNP-mediated gene transfer into mice through portal vein injection.** One day after the intravenous injec‐ tion of polyplexes with pNDLux.2 plasmid encoding the firefly luciferase gene, the animal was imaged by CCD camera. The expression of luciferase was clearly shown in the liver area, demonstrating the effectiveness of this delivery approach and the applicability of a non-invasive imaging modality in the determination of transgene expression in animals.

We also developed an approach to promote normal hepatocytes to proliferate *in situ* without partial hepatectomy, which favors the transgene expression by lipofection but is not accepta‐ ble for clinical application [6]. Furthermore, placing an indwelling catheter in the portal vein allows repeated administration of polyplexes for sustained transgene expression [6]. All these efforts render our formulation of PLNP distinct from other lipid-based nanoparticles. Our animal experiments have clearly demonstrated that PLNP is characterized as extremely stable in the bloodstream, and highly effective in liver-based gene transfer when polyplexes are administrated through the portal vein [6, 17]. In comparison with other commonly used lipid formulations of nanoparticles, our formulation possesses the notable advantages essen‐ tial for *in vivo* gene delivery as illustrated in Table 1.

The content in this table was summarized according to our previous publications [6-8]. FBS = fetal bovine serum. RLU = relative light unit. LDH = lactate dehydrogenase.

**Table 1.** Comparison of common transfection agents for *in vitro* and *in vivo* application

#### **5. Preclinical trials for proof of the concept**

tity in a laboratory, and the synthetic approach is amendable for industrial production at a quantity sufficient enough for large animal use [8]. PLNP was formulated with a neutral lip‐ id, cholesterol. The PLNP size was reduced to approximately 100 nm in diameter [7], and the Zeta potential of PLNP was decreased to neutral by neutralizing extra-positive charges with excess plasmid DNA [8]. Not only was this formulation of PLNP non-toxic, but it also displayed transfection efficiency equivalent to other commercially available transfection agents, such as Lipofectamine in hepatoma cell lines [7]. Moreover, high-resolution fluores‐ cent deconvolution microscopy documented that PLNP-mediated gene transfection led to earlier GFP expression in hepatoma cells than Lipofectamine [8]. The unique feature of this formulation is that it is extremely serum-resistant, and exposure to cell culture medium con‐ taining 50% fetal bovine serum for 24 hours did not affect its size significantly. PLNP react‐ ed up to 30-fold less with serum proteins or blood cells after intravenous administration in comparison with DOTAP-DOPE or DOTAP-Chol formulations [6]. This feature makes PLNP formulation particularly useful for *in vivo* gene transfer. In the subsequent studies, we have proved that it is very effective in the transfer of reporter genes or function genes to normal mouse livers as demonstrated in Fig. 2 by bioluminescent imaging of firefly lucifer‐ ase gene expression 24 hours after portal vein injection of PLNP-plasmid DNA complexes

**Figure 2. PLNP-mediated gene transfer into mice through portal vein injection.** One day after the intravenous injec‐ tion of polyplexes with pNDLux.2 plasmid encoding the firefly luciferase gene, the animal was imaged by CCD camera. The expression of luciferase was clearly shown in the liver area, demonstrating the effectiveness of this delivery approach and

the applicability of a non-invasive imaging modality in the determination of transgene expression in animals.

(polyplexes) or preclinical models [48, 49].

96 Gene Therapy - Tools and Potential Applications

In order to demonstrate that our PLNP formulation is effective in delivering functional genes to the liver, we established a liver injury model in mice caused by the treatment with D-galactosamine (D-Gal) and lipopolysaccharide (LPS). This combination of D-Gal/LPS treatment resulted in a profound acute liver injury characterized by massive liver cell death through apoptosis, elevation of serum alanine aminotransferase (ALT), significant oxidant stress, depletion of the reduced form of glutathione and enhanced lipid peroxidation [50]. In separate studies we have demonstrated that anti-oxidant enzyme such as extracellular su‐ peroxide dismutase (EC-SOD), SOD mimetics (MnTBAP) and catalase are effective in the prevention of hepatic toxicity caused by xenobiotics in primary hepatocytes or hepatoma cells [51-53], and they improved recipient survival and graft function and growth after small-for-size liver transplantation in rats [54]. Therefore, we chose the human EC-SOD gene as a functional gene to prove the feasibility. The EC-SOD gene product was exclusively secreted into the extracellular space and functions as an ROS scavenger. ROS are generated in both intracellular and extracellular spaces, and superoxide anions and hydrogen peroxide (H2O2) are able to cross the plasmatic membrane to enter the extracellular space [17]. It was found that two days after portal vein injection of EC-SOD polyplexes, liver EC-SOD gene expression was increased approximately 50-fold compared to the group receiving injection of control plasmid polyplexes, and serum SOD activity was increased accordingly. On the other hand, serum ALT was reduced to nearly one third in mice receiving EC-SOD polyplex injection compared to those with D-Gal/LPS challenge, along with improved liver histology, restored glutathione levels and decreased lipid peroxidation [48]. The findings of this pre‐ clinical trial confirmed the effectiveness of PLNP-mediated EC-SOD gene delivery to the liv‐ er, and that the delivery protected the mice from oxidant stress-associated liver injury. The results also indicate that this anti-oxidant gene delivery approach could be useful in attenu‐ ating xenobiotics or drug metabolite-induced toxicity to the liver.

**6. Challenges in scaling-up and moving towards clinical applications**

Our preclinical studies were performed in mice, and there are certainly a number of issues to face when this anti-oxidant gene therapy approach is considered to be evaluated in mid‐ dle or large size animals such as rabbits, dogs, monkeys or pigs. The first issue is to scale-up, which includes the plasmid DNA generation, synthesis of PCL in a quantity, and formula‐ tion of PLNP at a volume sufficient enough for the use in large animals. More challenges exist regarding how to stimulate liver cells to proliferate in large animals and deliver poly‐ plexes locally to the liver. Using a catheter through the femoral vein or jugular vein for ret‐ rograde administration into hepatic vein or passing into the portal vein for administration similar to the transjugular intrahepatic portosystemic shunt (TIPS) procedure, which is used to lower portal hypertension in cirrhotic patients, should be feasible in large animals when angiography and the administration are performed by an experienced specialist with the availability of angiographic devices. The latter method was used to administer adenoviral vector in baboons [59]. One trial of plasmid DNA injection into the hepatic vein by blocking the hepatic vein out-flow with an inflated balloon achieved high gene expression levels in selected pig liver lobes [30]. Safety concerns include amount of polyplexes to be administrat‐ ed locally and the effects of the plasmid DNA, PLNP and polyplexes on the liver as well as systematically. LNP-mediated gene transfer is usually transient; therefore, there will be less concern for long-term effects of the transgene products on the host. However, immune reac‐ tion to human gene products in animals may occur if the transgene products are produced at sustained levels for a long period of time. It is preventable by administration of immuno‐ suppressive agents, such as FK506. Moreover, innate immunity to plasmid DNA with bacte‐ rial unmethylated CG dinucleotide (CpG) can be eliminated by using CpG-free plasmid [60].

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99

An additional concern is to establish a liver injury model to evaluate the effect of anti-oxi‐ dant gene transfer by PLNP in large animals. For pigs, exposure to a loading dose of 0.25 g/kg, maintaining the blood concentration of acetaminophen at 350-450 mg/dl, and adapting enteric maintenance dose of 1,000-3,000 mg/hour resulted in the onset of acute liver failure (prothrombin time value <30%) within 32±4.4 hours, and further mortality in 15.8±2.4 hours [61]. A large dose of acetaminophen intake causes significant oxidant stress and acute liver injury due to its metabolism and generation of an interactive metabolite, n-acetyl-p-benzo‐ quinone imine (NAPQI), which binds to the cytoplasmic membrane, leads to lipid peroxida‐ tion, depletion of antioxidants, such as glutathione, and results in hepatic injury. Not only will the delivery of antioxidant genes with PLNP in a pig model of liver injury assess the therapeutic efficacy, but also take advantage of a regenerative response to the injury for high transgene expression. Alternatively, small size graft liver transplantation (SSGLT) at ≤50% graft volume could be performed in rabbits or pigs to mimic living donor liver transplanta‐ tion in humans. Significant oxidant stress-associated injury and regenerative response in the small size grafts will be the best fit for the high transgene expression and ROS scavenging property of the gene product. Therefore, SSGLT may be considered to be a valuable model for evaluating the feasibility and efficacy of anti-oxidant gene transfer for small-for-size-as‐

sociated graft failure in a transplant setting.

Ischemia/reperfusion (I/R)-associated donor organ damage is inevitable in all solid organ transplantation, and is caused by enhanced oxidant stress with release of inflammatory cytokines, such as tumor growth factor-α (TNF-α) and interleukin 2 (IL-2). Although the precise molecular mechanism of the I/R-associated liver injury remains to be investigated, enhanced oxidant stress with release of superoxide anions or H2O2, depletion of the re‐ duced form of glutathione and increased lipid peroxidation has been the key element in the pathogenesis in orthotopic liver transplantation (OLT) or small size liver graft trans‐ plantation (SSLGT) [54-56]. Thus, it is rational to use of antioxidant gene transfer to mini‐ mize oxidant stress and improve the donor organ quality and function after the implantation. We delivered either EC-SOD, catalase gene or in combination, using the same approach as described above. Two days after the delivery, the transgene expression was increased for 10-50-fold, with increased SOD or catalase activity in the mouse liver. This delivery led to a marked decrease in superoxide anion levels and H2O2 release along with a decrease in serum ALT levels, liver lipid peroxidation and dramatic improvement of liver histology [49]. This study was positively commented by two well-known hepatol‐ ogists from Europe as an editorial, quoting "beyond a proof of the principle, the study could be the basis for studies with larger animals and may help bridge the gap between the basic understanding of pathophysiologic processes in animal models towards a practi‐ cal clinical application in liver transplantation" [57]. The findings are especially applicable in living donor liver transplantation, for which small or margin donor livers were used for transplantation. Much more pronounced oxidant stress, a higher rate of graft failure, and retarded graft growth are found in small size liver transplantation than OLT [54, 58]. The margin grafts with small size or steatosis and fibrotic deposition are often used for transplantation in clinics due to severe shortage of donor organs.

#### **6. Challenges in scaling-up and moving towards clinical applications**

separate studies we have demonstrated that anti-oxidant enzyme such as extracellular su‐ peroxide dismutase (EC-SOD), SOD mimetics (MnTBAP) and catalase are effective in the prevention of hepatic toxicity caused by xenobiotics in primary hepatocytes or hepatoma cells [51-53], and they improved recipient survival and graft function and growth after small-for-size liver transplantation in rats [54]. Therefore, we chose the human EC-SOD gene as a functional gene to prove the feasibility. The EC-SOD gene product was exclusively secreted into the extracellular space and functions as an ROS scavenger. ROS are generated in both intracellular and extracellular spaces, and superoxide anions and hydrogen peroxide (H2O2) are able to cross the plasmatic membrane to enter the extracellular space [17]. It was found that two days after portal vein injection of EC-SOD polyplexes, liver EC-SOD gene expression was increased approximately 50-fold compared to the group receiving injection of control plasmid polyplexes, and serum SOD activity was increased accordingly. On the other hand, serum ALT was reduced to nearly one third in mice receiving EC-SOD polyplex injection compared to those with D-Gal/LPS challenge, along with improved liver histology, restored glutathione levels and decreased lipid peroxidation [48]. The findings of this pre‐ clinical trial confirmed the effectiveness of PLNP-mediated EC-SOD gene delivery to the liv‐ er, and that the delivery protected the mice from oxidant stress-associated liver injury. The results also indicate that this anti-oxidant gene delivery approach could be useful in attenu‐

Ischemia/reperfusion (I/R)-associated donor organ damage is inevitable in all solid organ transplantation, and is caused by enhanced oxidant stress with release of inflammatory cytokines, such as tumor growth factor-α (TNF-α) and interleukin 2 (IL-2). Although the precise molecular mechanism of the I/R-associated liver injury remains to be investigated, enhanced oxidant stress with release of superoxide anions or H2O2, depletion of the re‐ duced form of glutathione and increased lipid peroxidation has been the key element in the pathogenesis in orthotopic liver transplantation (OLT) or small size liver graft trans‐ plantation (SSLGT) [54-56]. Thus, it is rational to use of antioxidant gene transfer to mini‐ mize oxidant stress and improve the donor organ quality and function after the implantation. We delivered either EC-SOD, catalase gene or in combination, using the same approach as described above. Two days after the delivery, the transgene expression was increased for 10-50-fold, with increased SOD or catalase activity in the mouse liver. This delivery led to a marked decrease in superoxide anion levels and H2O2 release along with a decrease in serum ALT levels, liver lipid peroxidation and dramatic improvement of liver histology [49]. This study was positively commented by two well-known hepatol‐ ogists from Europe as an editorial, quoting "beyond a proof of the principle, the study could be the basis for studies with larger animals and may help bridge the gap between the basic understanding of pathophysiologic processes in animal models towards a practi‐ cal clinical application in liver transplantation" [57]. The findings are especially applicable in living donor liver transplantation, for which small or margin donor livers were used for transplantation. Much more pronounced oxidant stress, a higher rate of graft failure, and retarded graft growth are found in small size liver transplantation than OLT [54, 58]. The margin grafts with small size or steatosis and fibrotic deposition are often used for

ating xenobiotics or drug metabolite-induced toxicity to the liver.

98 Gene Therapy - Tools and Potential Applications

transplantation in clinics due to severe shortage of donor organs.

Our preclinical studies were performed in mice, and there are certainly a number of issues to face when this anti-oxidant gene therapy approach is considered to be evaluated in mid‐ dle or large size animals such as rabbits, dogs, monkeys or pigs. The first issue is to scale-up, which includes the plasmid DNA generation, synthesis of PCL in a quantity, and formula‐ tion of PLNP at a volume sufficient enough for the use in large animals. More challenges exist regarding how to stimulate liver cells to proliferate in large animals and deliver poly‐ plexes locally to the liver. Using a catheter through the femoral vein or jugular vein for ret‐ rograde administration into hepatic vein or passing into the portal vein for administration similar to the transjugular intrahepatic portosystemic shunt (TIPS) procedure, which is used to lower portal hypertension in cirrhotic patients, should be feasible in large animals when angiography and the administration are performed by an experienced specialist with the availability of angiographic devices. The latter method was used to administer adenoviral vector in baboons [59]. One trial of plasmid DNA injection into the hepatic vein by blocking the hepatic vein out-flow with an inflated balloon achieved high gene expression levels in selected pig liver lobes [30]. Safety concerns include amount of polyplexes to be administrat‐ ed locally and the effects of the plasmid DNA, PLNP and polyplexes on the liver as well as systematically. LNP-mediated gene transfer is usually transient; therefore, there will be less concern for long-term effects of the transgene products on the host. However, immune reac‐ tion to human gene products in animals may occur if the transgene products are produced at sustained levels for a long period of time. It is preventable by administration of immuno‐ suppressive agents, such as FK506. Moreover, innate immunity to plasmid DNA with bacte‐ rial unmethylated CG dinucleotide (CpG) can be eliminated by using CpG-free plasmid [60].

An additional concern is to establish a liver injury model to evaluate the effect of anti-oxi‐ dant gene transfer by PLNP in large animals. For pigs, exposure to a loading dose of 0.25 g/kg, maintaining the blood concentration of acetaminophen at 350-450 mg/dl, and adapting enteric maintenance dose of 1,000-3,000 mg/hour resulted in the onset of acute liver failure (prothrombin time value <30%) within 32±4.4 hours, and further mortality in 15.8±2.4 hours [61]. A large dose of acetaminophen intake causes significant oxidant stress and acute liver injury due to its metabolism and generation of an interactive metabolite, n-acetyl-p-benzo‐ quinone imine (NAPQI), which binds to the cytoplasmic membrane, leads to lipid peroxida‐ tion, depletion of antioxidants, such as glutathione, and results in hepatic injury. Not only will the delivery of antioxidant genes with PLNP in a pig model of liver injury assess the therapeutic efficacy, but also take advantage of a regenerative response to the injury for high transgene expression. Alternatively, small size graft liver transplantation (SSGLT) at ≤50% graft volume could be performed in rabbits or pigs to mimic living donor liver transplanta‐ tion in humans. Significant oxidant stress-associated injury and regenerative response in the small size grafts will be the best fit for the high transgene expression and ROS scavenging property of the gene product. Therefore, SSGLT may be considered to be a valuable model for evaluating the feasibility and efficacy of anti-oxidant gene transfer for small-for-size-as‐ sociated graft failure in a transplant setting.

In summary, moving promising PLNP-mediated antioxidant gene transfer from small ani‐ mals to large animals may face more challenges than discussed above, and it is even more challenging when further considering for clinical use, in terms of safety concern and admin‐ istrative approval. Fig. 3 provides a schematic illustration of the roadmap from bench to bedsides of a potential biological therapy. The reality is that with limited funding opportu‐ nities from governmental or private agencies, to cope with multi-facet challenges at a large scale, it is less likely to reach the final goal in a short term. Attracting financial investments and taking advantages of cutting-edging technologies and vast resources from biopharma‐ ceutical companies may advance this process in a fast pace. In this context, the net benefits would be the early clinical application of this promising antioxidant gene transfer in pa‐ tients with critical needs and the financial return from the investment. We would foresee such a movement occurring in the near future.

vectors, such as lipid nanoparticles (LNPs) possess their own drawbacks when they are con‐ sidered for *in vivo* use. One prominent issue is the interaction of cationic LNPs with serum protein and blood cells, and this causes a series of issues, such as instability of the lipoplexes or polyplexes and adverse effects to the host, including non-preferential distribution, embo‐ lism of the aggregates of lipoplex-protein or blood cells, and inflammatory responses. For these reasons, many gene transfer agents are very effective in cell culture; whereas they have less applicability *in vivo*. Up to date, only a few formulations of cationic LNPs have proved to be effective and safe in animals and have reached the stage of clinical trials, such as DO‐ TAP-Chol and DC-Chol. Our PLNP formulation has a superior stability profile, and dis‐ played much less reactivity to serum proteins and blood cells when compared to other commercially available formulations. At the same time, it has proved to be the most effec‐ tive liver-based gene transfer agent [6]. Two preclinical trials with different models of oxi‐ dant-stress-associated liver injury have demonstrated the effectiveness of the anti-oxidant gene delivery in the liver, and the efficacy of the gene delivery in minimizing oxidant-stress, attenuating liver cell death, and improving liver histology [48, 49]. Further efforts have been made to move this promising PLNP-mediated anti-oxidant gene transfer technology from bench to bedside. The strategies in pushing this movement towards clinical trials include: 1) Scaling-up of the polycationic lipid production and generation of PLNPs; 2) Generation of specific antioxidant gene plasmids in a GMP facility at the standard for clinical use; 3) Estab‐ lishing large animal models for safety and efficacy assessment; and 4) Preparation for ob‐ taining administrative approval of clinical application. Although the clinical translation of this potential technology will need tremendous efforts, we anticipate that this technology will eventually reach to patients with critical needs as a novel therapy. Potential indications which may benefit from this therapy range from alcohol or drug toxicity to living donor liv‐ er transplantation with a margin graft. This technology is also applicable in oxidant stressassociated disorders in other systems, such as ischemic cardiac, pulmonary, brain or renal damage, etc. [17]. With the combination of our extensive expertise in drug and gene deliv‐ ery, advanced knowledge and skills in liver injury, fibrosis, transplant and cancer research and practice, in addition to the engine of financial investment from various sources, such as venture capital and governmental support in entrepreneurship, we are optimistic to foresee the benefits of this technology in indicated patients in a near future. Nevertheless, the road to reach this goal will not be smooth, and various challenges demand powerful solutions.

Polylipid Nanoparticle, a Novel Lipid-Based Vector for Liver Gene Transfer

http://dx.doi.org/10.5772/54270

101

The studies presented in the chapter were supported by the UC Davis Health System Award, American Liver Foundation Liver Scholar Award, UC Davis Technology Transfer Award, and the National Institute of Diabetes, Digestive and Kidney Diseases (DK069939) to JW. The commercialization and translation to clinical application of this technology is sup‐ ported by the Nanjing Municipal Innovative Technology Award (321 Plan). Yahan Fan is the

recipient of China Scholarship Council Award<PHONE_NUMBER>03).

**Acknowledgements**

**Figure 3. Translation roadmap of a potential gene therapy platform from bench to bedside.** This illustration summarizes the major steps in moving a potential gene transfer approach from laboratory research to clinical trials. The actual actions could be more complicated than described. However, for the guarantee of patient safety, each new therapeutic agent must be well characterized, and evaluated in preclinical settings, and then move to large animals for feasibility assessment. The balance between therapeutic benefits and potential risks of an innovative therapy plat‐ form always leans on the patient safety as the first priority.

#### **7. Conclusion and prospectives**

Non-viral vector-mediated gene transfer has less concern in terms of integration-associated long-term transgene expression and insertion-induced mutation. In general, non-viral vector elicits minimal immune responses in contrast to adenoviral vectors [17]. However, non-viral vectors, such as lipid nanoparticles (LNPs) possess their own drawbacks when they are con‐ sidered for *in vivo* use. One prominent issue is the interaction of cationic LNPs with serum protein and blood cells, and this causes a series of issues, such as instability of the lipoplexes or polyplexes and adverse effects to the host, including non-preferential distribution, embo‐ lism of the aggregates of lipoplex-protein or blood cells, and inflammatory responses. For these reasons, many gene transfer agents are very effective in cell culture; whereas they have less applicability *in vivo*. Up to date, only a few formulations of cationic LNPs have proved to be effective and safe in animals and have reached the stage of clinical trials, such as DO‐ TAP-Chol and DC-Chol. Our PLNP formulation has a superior stability profile, and dis‐ played much less reactivity to serum proteins and blood cells when compared to other commercially available formulations. At the same time, it has proved to be the most effec‐ tive liver-based gene transfer agent [6]. Two preclinical trials with different models of oxi‐ dant-stress-associated liver injury have demonstrated the effectiveness of the anti-oxidant gene delivery in the liver, and the efficacy of the gene delivery in minimizing oxidant-stress, attenuating liver cell death, and improving liver histology [48, 49]. Further efforts have been made to move this promising PLNP-mediated anti-oxidant gene transfer technology from bench to bedside. The strategies in pushing this movement towards clinical trials include: 1) Scaling-up of the polycationic lipid production and generation of PLNPs; 2) Generation of specific antioxidant gene plasmids in a GMP facility at the standard for clinical use; 3) Estab‐ lishing large animal models for safety and efficacy assessment; and 4) Preparation for ob‐ taining administrative approval of clinical application. Although the clinical translation of this potential technology will need tremendous efforts, we anticipate that this technology will eventually reach to patients with critical needs as a novel therapy. Potential indications which may benefit from this therapy range from alcohol or drug toxicity to living donor liv‐ er transplantation with a margin graft. This technology is also applicable in oxidant stressassociated disorders in other systems, such as ischemic cardiac, pulmonary, brain or renal damage, etc. [17]. With the combination of our extensive expertise in drug and gene deliv‐ ery, advanced knowledge and skills in liver injury, fibrosis, transplant and cancer research and practice, in addition to the engine of financial investment from various sources, such as venture capital and governmental support in entrepreneurship, we are optimistic to foresee the benefits of this technology in indicated patients in a near future. Nevertheless, the road to reach this goal will not be smooth, and various challenges demand powerful solutions.

#### **Acknowledgements**

In summary, moving promising PLNP-mediated antioxidant gene transfer from small ani‐ mals to large animals may face more challenges than discussed above, and it is even more challenging when further considering for clinical use, in terms of safety concern and admin‐ istrative approval. Fig. 3 provides a schematic illustration of the roadmap from bench to bedsides of a potential biological therapy. The reality is that with limited funding opportu‐ nities from governmental or private agencies, to cope with multi-facet challenges at a large scale, it is less likely to reach the final goal in a short term. Attracting financial investments and taking advantages of cutting-edging technologies and vast resources from biopharma‐ ceutical companies may advance this process in a fast pace. In this context, the net benefits would be the early clinical application of this promising antioxidant gene transfer in pa‐ tients with critical needs and the financial return from the investment. We would foresee

**Figure 3. Translation roadmap of a potential gene therapy platform from bench to bedside.** This illustration summarizes the major steps in moving a potential gene transfer approach from laboratory research to clinical trials. The actual actions could be more complicated than described. However, for the guarantee of patient safety, each new therapeutic agent must be well characterized, and evaluated in preclinical settings, and then move to large animals for feasibility assessment. The balance between therapeutic benefits and potential risks of an innovative therapy plat‐

Non-viral vector-mediated gene transfer has less concern in terms of integration-associated long-term transgene expression and insertion-induced mutation. In general, non-viral vector elicits minimal immune responses in contrast to adenoviral vectors [17]. However, non-viral

such a movement occurring in the near future.

100 Gene Therapy - Tools and Potential Applications

form always leans on the patient safety as the first priority.

**7. Conclusion and prospectives**

The studies presented in the chapter were supported by the UC Davis Health System Award, American Liver Foundation Liver Scholar Award, UC Davis Technology Transfer Award, and the National Institute of Diabetes, Digestive and Kidney Diseases (DK069939) to JW. The commercialization and translation to clinical application of this technology is sup‐ ported by the Nanjing Municipal Innovative Technology Award (321 Plan). Yahan Fan is the recipient of China Scholarship Council Award<PHONE_NUMBER>03).

#### **Abbreviations used in the chapter**

ASGP-R = asialoglycoprotein receptor; DOTAP = (dioleoyloxy)-3-(trimethylamonio) pro‐ pane; DOPE = L-a dioleoyl phosphatidylethanolamine; EC-SOD = extracellular superoxide dismutase; HCC = hepatocellular carcinoma; LDLT = Living donor liver transplantation; LNP = lipid nanoparticles; OLT = orthotopic liver transplantation; PEG = polyethylene gly‐ col; PLNP = polylipid nanoparticles; polyplex = PLNP-plasmid DNA complex; RES = reticu‐ loendothelial system.

[7] Wu J, Lizarzaburu ME, Kurth MJ, Liu L, Wege H, Zern MA, Nantz MH. Cationic lip‐ id polymerization as a novel approach for constructing new DNA delivery agents.

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103

[8] Nyunt MT, Dicus CW, Cui YY, Yappert MC, Huser TR, Nantz MH, Wu J. Physicochemical characterization of polylipid nanoparticles for gene delivery to the liver. Bi‐

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#### **Author details**

Yahan Fan1,2\* and Jian Wu1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Dept. of Internal Medicine, Division of Gastroenterology & Hepatology, University of California, Davis Medical Center, Sacramento, CA, USA

2 Dept. of Internal Medicine, Division of Gastroenterology, Xinqiao Hospital, The Third Military Medical University, Chongqin, P. R. China

#### **References**

[7] Wu J, Lizarzaburu ME, Kurth MJ, Liu L, Wege H, Zern MA, Nantz MH. Cationic lip‐ id polymerization as a novel approach for constructing new DNA delivery agents. Bioconjug Chem 2001;12:251-257.

**Abbreviations used in the chapter**

102 Gene Therapy - Tools and Potential Applications

\*Address all correspondence to<EMAIL_ADDRESS>
Military Medical University, Chongqin, P. R. China

California, Davis Medical Center, Sacramento, CA, USA

Expert Opin Investig Drugs 1998;7:1795-1817.

Hemorheol Microcirc 2011;49:271-278.

loendothelial system.

**Author details**

**References**

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Yahan Fan1,2\* and Jian Wu1

ASGP-R = asialoglycoprotein receptor; DOTAP = (dioleoyloxy)-3-(trimethylamonio) pro‐ pane; DOPE = L-a dioleoyl phosphatidylethanolamine; EC-SOD = extracellular superoxide dismutase; HCC = hepatocellular carcinoma; LDLT = Living donor liver transplantation; LNP = lipid nanoparticles; OLT = orthotopic liver transplantation; PEG = polyethylene gly‐ col; PLNP = polylipid nanoparticles; polyplex = PLNP-plasmid DNA complex; RES = reticu‐

1 Dept. of Internal Medicine, Division of Gastroenterology & Hepatology, University of

2 Dept. of Internal Medicine, Division of Gastroenterology, Xinqiao Hospital, The Third

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**Chapter 6**

**DNA Electrotransfer: An Effective Tool for Gene**

The concept of gene therapy was first introduced in the mid-80s, and is based on the deliv‐ ery of genetic material (DNA or RNA) in the nucleus of patient cells, so that it is expressed

**•** Correcting defective function by supplying a functional gene to the cells, thereby directly

**•** Transferring a gene encoding a therapeutic protein, in order to treat, prevent or slow the

**•** Introducing antisense DNA inhibiting the formation of a protein or the replication of a vi‐

Originally developed for monogenic diseases, and therefore associated with the compensa‐ tion of genes whose alteration is responsible for diseases, the concept of gene therapy has rapidly expanded to the use of DNA as a new type of drug. Therefore, gene therapy leads to indications which are far beyond the case of genetic diseases, since a DNA drug can, in prin‐ ciple, replace any medication which will control protein synthesis. Gene therapy seems an alternative choice to fight against diseases currently treated imperfectly, or not treated with

In addition, gene therapy has many advantages compared to the administration of recombi‐ nant proteins. Indeed, recombinant proteins are costly and their elimination from the blood

> © 2013 Burgain-Chain and Scherman; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

Aurore Burgain-Chain and Daniel Scherman

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52528

and produces a therapeutic effect.

Different approaches can be considered:

progression of certain diseases.

conventional pharmaceutical approaches.

properly cited.

addressing the cause of a genetic disease.

**•** Introducing a gene leading to the death of a diseased cell

**1. Introduction**

rus

**Therapy**

## **DNA Electrotransfer: An Effective Tool for Gene Therapy**

Aurore Burgain-Chain and Daniel Scherman

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52528

**1. Introduction**

The concept of gene therapy was first introduced in the mid-80s, and is based on the deliv‐ ery of genetic material (DNA or RNA) in the nucleus of patient cells, so that it is expressed and produces a therapeutic effect.

Different approaches can be considered:

Originally developed for monogenic diseases, and therefore associated with the compensa‐ tion of genes whose alteration is responsible for diseases, the concept of gene therapy has rapidly expanded to the use of DNA as a new type of drug. Therefore, gene therapy leads to indications which are far beyond the case of genetic diseases, since a DNA drug can, in prin‐ ciple, replace any medication which will control protein synthesis. Gene therapy seems an alternative choice to fight against diseases currently treated imperfectly, or not treated with conventional pharmaceutical approaches.

In addition, gene therapy has many advantages compared to the administration of recombi‐ nant proteins. Indeed, recombinant proteins are costly and their elimination from the blood

© 2013 Burgain-Chain and Scherman; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

flow is fast, while gene therapy leads to a long-term and potentially regulated production of a therapeutic protein. Gene therapy also allows the localized expression of the transgene, avoiding any risk associated with the presence of a systemic exogenous protein.

lization and permeabilization of the plasma membrane of suspended cells, thus promoting the entry of exogenous DNA into these cells. Two years later [3], the confirmation of this re‐ sult opened the way for the development of electroporation (or electropermeabilization) into bacterial [4], fungal [5] vegetal or animal cells. This method is routinely used now. The opti‐ mization of electrical parameters is critical to allow transient permeabilization, together with

DNA Electrotransfer: An Effective Tool for Gene Therapy

http://dx.doi.org/10.5772/52528

111

In initial studies, *in vivo* DNA electrotransfer has been tested in the skin in 1991, by the use of exponentially decaying electrical pulses, and in 1996 in the liver using trains of short 100 µs pulses [7]. In 1998, four independent teams showed the effectiveness of electrotransfer using pulses of long duration (5-50ms): in skeletal muscle, our team in collaboration with that of Luis Mir [8] and Aihara [9], in tumors, Rols *et al.* [10] and in liver Suzuki *et al.* [11]. *In vivo* DNA electrotransfer has now been successfully used in a broad range of target tissues and organs including for example : arteries [12], skin [13], tendon [14], bladder [15], cornea

Electropermeabilization can also be used to deliver chemical drugs into the cells: e.g. electro‐ chemotherapy in tumors, with the use of bleomycin, developed since 1991 [20]. Several clini‐ cal trials are underway [21], primarily for the treatment of subcutaneous or skin tumors [22, 23] and recently for the treatment of breast cancer with cisplatin [24] (For a review see [25]).

The technique of electroporation for the transfer of nucleic acids has been used since the 80s, however its exact mechanism is not yet completely elucidated [26, 27]. At the cell level, it seems that two phenomena occur: first the permeabilization of the cell to small molecules, probably due to a destabilization of the cell membrane, and secondly the transport of DNA

The lipid bi-layer of the plasma membrane separates two solutions with very high ionic con‐ ductivity: the cytoplasm and the extracellular medium. Typically, at rest, the membrane po‐ tential difference (ΔVm0) is around -70mV. When an electric field is applied to a cell, the resulting current induces an accumulation of electric charges at the cell membrane which leads to a variation of thistransmembrane potential. And if the transmembrane potential ex‐ ceeds a certain threshold value, the cell membrane is disorganized and structural changes

Shall the cell be considered a hollow sphere where the thickness of the membrane is negligi‐ ble vis-à-vis the cell radius, then the transmembrane potential difference ΔVm induced by

a satisfactory cell survival rate [6].

by electrophoresis.

**3.1. Permeabilization**

[16], the retinal cells [17], spinal cord [18]and brain [19].

**3. Mechanism of electrotransfer at the cell level**

occur. That is a necessary condition for an effective gene transfer [28].

an electric field is, as described by Schwann's equation:

The main limitation of current gene therapy is the development of effective gene transfer. Indeed, in order to reach the cell nucleus, the therapeutic gene has to cross several biological barriers. Therefore, the success of any gene therapy requires the development of efficient and appropriate methods and vectors for introducing the gene of interest into target cells. The ideal vehicle for gene transfer must have the following properties: (1) specificity to tar‐ get cells, (2) localized gene delivery, (3) resistance to metabolic degradation and/or attack by the immune system, (4) minimum side effects, and (5) eventually controlled temporal trans‐ gene expression [1].

Many methods of *in vivo* gene transfer exist and are generally classified into two main cate‐ gories: viral and non viral. Viruses are very efficient vehicles for gene transfer; however their use is limited by high production costs and safety concerns, such as immune response, possible pathogen reversion, mutagenesis and carcinogenesis. Considering these limitations, the delivery of therapeutic genes to target cells by non viral approaches may be of great val‐ ue for the development of gene therapy. Among these approaches, *in vivo* electroporation, also called *in vivo* electropermeabilization or *in vivo* electrotransfer, has proven to be one of the simplest and most efficient methods for gene therapy, while at the same time being safe, cheap, and easy to perform.