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Hybrids of Nonviral Vectors for Gene Delivery Shubiao Zhang,* Yinan Zhao, Budiao Zhao, and Bing Wang SEAC-ME Key Laboratory of Biotechnology and Bioresources Utilization, College of Life Science, Dalian Nationalities University, Dalian 116600, Liaoning, China. Received June 12, 2009; Revised Manuscript Received December 27, 2009
Gene transfer has been a critical step for gene therapy. Viral vectors and nonviral vectors are commonly used for gene transfer. It has been shown that nonviral vectors have the advantage over viral ones, as they are nonimmunogenic, easy to produce, and not oncogenic. However, they have the major limitation of inefficient transfection; the hybridized usage of nonviral vectors may provide a partial solution. This article reviews the hybrids (different materials used in one delivery system) of nonviral vectors for gene delivery including hybrids between cationic lipids and helper lipids, conjugates of peptides or targeting moieties and lipids, and hybrids of cationic liposomes and polymers.
INTRODUCTION Gene therapy has been developed for treatment of both acquired and inherited diseases such as severe combined immunodeficiency (1), cystic fibrosis (2), and Parkinson’s disease (3), as well as an alternative method to traditional chemotherapy used in treating cancer (4). This approach is based on the principle of correcting the basis of diseases at their origin by delivery and subsequent expression of exogenous DNA, which encodes for a missing or defective gene product (5). Naked nucleic acids are easily degraded when used either in Vitro or in ViVo, so vectors are employed in many methods to protect nucleic acids and deliver them to targeted cells. These vectors can be generally divided into two categories, viral vectors and nonviral vectors. After several accidents (6–8), nonviral vectors gained great attention for they are nonimmunogenic, not oncogenic, easy to produce in large scale, and capable of delivering large genetic material. However, unlike viral analogues that have evolved to overcome cellular barriers and immune defense mechanisms, nonviral gene carriers consistently exhibit significantly lower transfection efficiency compared with viral ones. People have been designing and synthesizing a large number of new compounds (9, 10); at the same time, the hybridized utilization of nonviral vectors may provide a solution to the problem. We could combine cationic lipids and helper lipids, hybridize cationic lipsomes and polymers, and introduce peptides and targeting moieties into lipids to approach the requirements of gene therapy. Though many high-quality reviews have been published recently (11–13), we found it necessary to demonstrate the status of hybrids of nonviral vectors.
HYBRIDS BETWEEN CATIONIC LIPIDS AND HELPER LIPIDS Though some cationic lipids show transfection ability when used alone in cationic liposomes, in many cases helper lipids are required to be incorporated into the formulations to yield high transfection efficiency. Among helper lipids, DOPE has been researched very extensively for it often presents a super synergistic effect when used in cationic liposomes. In early study, people have realized that DOPE destabilized lipid bilayers, and it was believed to be involved in endosomal disruption (14). Then, an endocytosis model of DNA delivery *
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by cationic liposomes was proposed in which the principal function of the chase liposomes was to destabilize the endosome membrane and allow the release of DNA into the cytosol (15). When compared with other helper lipids, such as DOPC, DOPE shows superior activity. It has been proven that the replacement of DOPE with DOPC abolished most of the transfection activity of cationic liposomes (16–19). The difference in their head groups can be the reason, as DOPC contains a choline headgroup instead of the ethanolamine headgroup in DOPE (14). All the studies support DOPE as a strong destabilizer of lipid bilayers. Recent studies disclosed by Safinya et al. (20) further gave the reason in the level of phase transition. They used synchrotron small-angle X-ray scattering (SAXS) and optical microscopy to find a completely different columnar inverted hexagonal HIIC liquid-crystalline state in cationic liposome-DNA complexes. In this process, DOPE induced the phase transition from LRC to HIIC by controlling the spontaneous curvature Co ) 1/Ro of the lipid monolayer. It has been concluded that DOPE facilitates endosomal escape by forming an unstable inverted hexagonal phase at the endosomal pH that destabilizes both the complex and the endosomal membrane. Conversely, it would be expected that lipids such as dioleoylphosphatidylcholine (DOPC) would hinder the ability of cationic lipids to induce nonbilayer structure (21). Another helper lipid, cholesterol, could also promote HIIC organization as DOPE. Usage of cholesterol and its derivatives as helper lipids allowed one to attain higher transfection levels in vivo (22, 23). Reliably higher expression in many organs was revealed upon the application of cholesterol-containing liposomes (24, 25) as compared to other liposomes. Some studies indicated that in in vivo applications cholesterol was a more effective helper lipid than DOPE (26). Needless to say, the structure of cationic lipids plays a very important role in various phase structures. Safinya (27) also found that hexagonally arranged tubular lipid micelles (HIC) surrounded by DNA rods were formed though DOPC was used in the dendritic lipidbased cationic liposome. Helper lipid is indispensable for many cationic liposome formulations; the hybrids of helper lipids and cationic lipids would continue to be a research highlight.
CONJUGATES OF PEPTIDES AND LIPIDS One of the most challenge things for gene delivery by cationic liposome method is the toxicity of cationic lipids originated in the cationic nature. The cationic lipids can become cytotoxic by interacting with critical enzymes such as PKC. It has been
10.1021/bc900261c 2010 American Chemical Society Published on Web 03/05/2010
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Figure 3. Chemical structure of GS.
Figure 1. Chemical structures of DOPE and DOPC. Figure 4. Chemical structure of INF7 peptide-polymer conjugates.
Figure 2. Chemical structures of DOGS and DPPES.
proven that many derivatives of cholesterol can inhibit PKC activity, which contained tertiary or quaternary nitrogen head groups (28). So, the replacement of cationic head groups has been a major trend with other more biocompatible groups, such as peptides, in recent years. The conjugates of peptides and lipids are much less toxic, while keeping the same transfection efficiency. The early examples (29) are DOGS (dioctadecylamidoglycylspermine) and DPPES (dipalmitoyl phosphatidylethanolamidospermine) in which spermine as headgroup was attached to lipids through peptide bonds. A variety of eukaryotic cell cultures also have been transfected with success for transient and stable expression. Even a single-tailed and ornithine-based cationic lipid, OLON (a cationic lipid-oleoyl ornithinate), has been proven to be less toxic and more than 10 times the transgene expression than other liposomes when used with DOPE (30). Such a combination of peptides and lipids has been reported to produce superior transfection of genes. A compound which contained cholesterol and a dipeptide consisting of glycine and sterically protected arginine has been proven to be suitable for in vitro transfection in the presence of 10% serum more efficiently than other cholesterol derivatives (31). The structure-activity study has proven that a change in peptide groups would cause very different transfection efficiency. In a structure-activity investigation, cationic lipids with alanine and β-alanine headgroups were found to be remarkably more transfection efficient than their serine headgroup counterparts, and some of the conjugates in combination with cholesterol as auxiliary lipid were much more effective than Lipofectamine 2000 (32). In a later study, they studied three novel nontoxic cationic transfection am-
phiphiles containing mono-, di-, and trihistidine functionalities in their headgroup regions in multiple cultured cells for elucidating the structure-activity relationship (33). Yosuke Obata et al (34) have proven that the lysine- or arginine-type lipids exhibited higher gene expression efficiencies than Lipofectamine 2000, against COS-7 cells. The gene expression efficiency in relation to the cationic headgroup of the lipids was as follows: lysine > arginine > histidine. Peptide-based gemini surfactants GS (Figure 3) could lead to an increase in levels of gene expression in vitro compared to well-established nonviral reagents (35). A series of new lipophilic peptides possessing a cationic tripeptide headgroup were effective nonviral vectors for gene delivery. Cytotoxity experiments performed with all the tripeptide lipophilic peptides showed no significant cytotoxicity, significantly better than Lipofectamine 2000 when exposed to chinese hamster ovarian (CHO) or NIH 3T3 cells (36). A subset of lipitoids with a repeated side chain trimer motif conjugated with dimyristoyl phosphatidyl-ethanolamine (DMPE) mediated DNA were also found to transfer cells with high efficiency (37). Some peptide headgroups could endow additional functions to the lipids, such as membrane-disturbing ability. In a hybrid molecule, the covalent coupling of an amphipathic and membranedisturbing peptide to a lipid moiety might create a stable and efficient peptide-based gene transfer system. The luciferase activity induced by the dioleoylmelittin/DNA complex was 5-500-fold higher than that induced by a cationic lipid/DNA complex, depending on the cationic lipid and the cell line (38). A membrane-disrupting peptide derived from the influenza virus was covalently linked to different polymethacrylates using N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) as a coupling agent to increase the transfection efficiency of polyplexes based on these polymers (Figure 4). In vitro transfection and toxicity were tested in COS-7 cells, and these experiments showed that the polyplexes with grafted peptides had a substantially higher transfection activity than the control polyplexes, while the toxicity remained unchanged (39). Moreover, some peptide type cationic lipids are not serumsensitive, which is crucial in in vivo applications (40–42). Dioleoylmelittin represented a new class of efficient peptidebased transfection reagents, especially suited for serum-sensitive cells, in which the presence of 10-50% fetal calf serum during dioleoylmelittin-mediated transfection enhanced 1.5-3-fold gene expression (38). The headgroups of asymmetric diether lipids have been varied by the coupling of the four naturally occurring amino acids with cationic side chains, sarginine, histidine, lysine, and tryptophan, and several formulations have
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Figure 5. Chemical structure of DAB-dendr-(NH2)8.
Figure 6. Chemical structure of FOL-PEI-b-PEG.
been shown to transfect different cell lines with up to 10 times the efficacy of the commonly used DC-Chol/DOPE formulation (43).
CONJUGATES OF TARGETING MOIETIES AND NONVIRAL VECTORS The incorporation of targeting moieties into liposomes by direct formulation, with no covalent bond to any lipid (44); conjugated to helper lipid (45, 46), or connected directly to the cationic lipids (47, 48) can increase uptake and selectivity of liposomes for certain tissues. The targeting groups often used include folate (45, 46), galactose (47, 48), mannose (49, 50), and proteins such as antibodies (51) and transferrin (44). The hepatic asialoglycoprotein receptor could recognize and bind to β-D-galactoside terminated glycoproteins (52). By using this physical and biological feature, asialoglycoprotein receptor has been studied as a target for gene delivery into hepatocytes. Hashida et al (53) synthesized novel galactosylated cholesterol derivatives, cholesten-5-yloxy-N-(4-((1-imino-c-β-D-thiogalactosylethyl)amino)alkylformamide, for gene delivery to hepatocytes. In HepG2 cells, the liposomes containing galactosylated cholesterol derivatives showed higher transfection activities than DC-Chol liposomes based on a receptor-mediated mechanism. Later, they used galactosylated cationic liposomes to target liver cells asialo-glycoprotein receptors in vivo (48). Liu et al (54) developed an efficient route toward the synthesis of bifunctional cationic compounds with DAB-dendr-(NH2)8 (Figure 5) as the DNA binding domain and galactose ligand for binding to the asialoglycoprotein receptor to prove that the galactosyl compounds Di-Gal and Tri-Gal could effectively deliver DNA into HepG2 cells. The folate receptor (FR) is a tumor marker overexpressed in large numbers of cancer cells. As a low molecular weight ligand, folic acid has the advantages of being stable and nonimmunogenic compared to monoclonal antibodies (55), and still having a relatively high receptor affinity. Folate-targeted liposomes have been successfully used in the tumor cell specific delivery of anticancer drugs and antisense oligodeoxynucleotides in vitro
(56). In a study, folate was connected to polyethylenimine-blockpoly(ethylene glycol) to form a copolymer, FOL-PEI-b-PEG (Figure 6), which showed less toxicity in comparison with that of 25 kDa PEI and effective transfection efficiency (57). Huang et al (58, 59) developed vectors in which poly(lysine)-condensed DNA was entrapped into folate-targeted cationic liposomes (LPD). They found LPD vector to be more efficient and less cytotoxic compared to conventional cationic liposomal vectors. The physical and biological features of mannosylated vectors offer them the opportunity to deliver plasmid DNA (pDNA) to the liver NPC via mannose receptor mediated endocytosis. The application of poly(lysine) linked to mannose with naked pDNA has been reported to enhance gene expression in macrophages (60). Hashida et al. (61) have developed a novel mannosylated cholesterol derivative, Man-C4-Chol, consisting of modified cationic liposomes with mannose moieties for NPC-selective gene delivery via mannose receptors on NPC. The mannosylated cationic liposomes can deliver pDNA to liver nonparenchymal cells (61) and splenic DCs and improve immune activation in DNA vaccines (62). They also proved that DOPE could improve gene expression of the mannosylated cationic liposomes after intraportal administration into mice, and DOPE complex was accumulated in the liver more efficiently through intravenous administration (63). Targeting proteins such as antibodies or transferrin were conjugated to cationic polymers (e.g., poly(lysine) (64) or polyethylenimine 65–67) to mediate uptake of plasmid DNA and antisense oligonucleotides. Since antibodies feature a highly selective binding to their antigen, they are of particular interest as targeting moieties. PEI was modified with G250 mAb to gain targeting effect and enhanced gene delivery to tumor cells that express G250 antigen. The conjugate showed targeted gene delivery to RCC and Hela cells, even in the presence of serum, but with restricted reactivity to G250 negative cells or normal cells (51). Transferrin was covalently linked to protamine to deliver functional plasmid DNA to erythroblasts (68, 69). Hormone receptors, such as the epidermal growth factor receptor family member ErbB2, expressed on the cell surface can serve
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Figure 7. Chemical structure of PDMAPAAm-branched polymer.
as target structures for nucleic acid delivery. A recombinant fusion protein of an ErbB2-specific single-chain antibody fused to protamine successfully transported plasmid DNA to ErbB2 antigen expressing cells (69, 70). Peptides containing the amino acid sequence Arg-Gly-Asp (RGD) can be used for specific targeting to integrin receptors, including the vitronectin receptor Rvβ3, which is known to be highly upregulated in certain tumors (71). Anderson et al. (72) used RGD containing peptide to coat nanoparticles; the noncovalent peptide coated nanoparticles were found to have favorable biophysical characteristics including small particle size, near-neutral ζ potential, and stability in serum. At appropriate formulation conditions, the coated nanoparticles enabled effective ligand-specific gene delivery to human primary endothelial cells in serum-containing media. Nakayama (73) also proved the fact of RGD peptide enhancing gene expression of a cationic nonviral gene carrier, poly(N,N-dimethylaminopropylacrylamide) (Figure 7), in endothelial cells. The addition of the RGD-containing peptide to the polyplex solution led to a decrease in the ζ-potential and particle size, and an increase of transfection against endothelial cells and reduction of cellular cytotoxicity.
HYBRIDS OF CATIONIC LIPOSOMES AND POLYMERS Cationic liposomes, cationic polymers, micelles, or nanoparticles are often researched for gene delivery, but none of them seem to meet the need of the theoretical study or clinical trials for gene therapy. The combination of them may provide a synergistic effect on the transfection efficiency and a promising solution to the problem frustrating us. Serum is a major hurdle for nucleic delivery of cationic liposomes, as the binding of negatively charged serum components to positively charged DNA complexes can significantly decrease transfection efficiency (74, 75). To decrease or reverse the overall particle charge and hence reduce serum opsonization, anionic polymers have been employed as an additional component of the delivery vector (76–78). Roth (79) showed that the addition of a synthetic, pH-sensitive, membrane-disrupting polyanion, poly(propylacrylic acid) (PPAA), improved the in vitro efficiency of the cationic lipid, DOTAP, with regard to oligonucleotide delivery and antisense activity. Low-molecular-weight PEIs and cationic liposomes can be combined resulting in a synergistic increase in transfection efficiency. PEI2K-DNA-Dosper complexes showed much more cellular uptake of DNA than PEI2K-DNA complexes and two times higher transfection than Dosper-DNA complexes. It has been hypothesized that Dosper improved the cellular uptake of PEI2K-DNA complexes and PEI2K improved a transfer of the complexes from lysosomes to nucleus (80). In a study, the combination of PEI and DOTAP-Chol caused more than 10fold increase in the transfection efficiency in many cells compared with using polymer or liposome alone. Minimal toxicity was also shown in these cultured cells in vitro (81).
Figure 8. PEGylated LPD through the hybridization of polyplex, liposome and PEG based lipid (This figure was adapted from Huang et al’s illustration in ref 84).
It has been reported that liposome-mediated gene transfer could be improved by natural polycations such as protamine sulfate (PS), poly(L-lysine) (PLL), and spermine (57, 82). The addition of poly(L-lysine) and protamine dramatically reduced the particle size of the complex formed between DNA and cationic liposomes and rendered DNA resistant to the nuclease activity (58). As copolymers, they can markedly enhance the transfection efficiency of several types of cationic liposomes by 2-28-fold in a number of cell lines in vitro (58) and in vivo (82). These polycations are known to form a complex with DNA and condense DNA from extended conformation to highly compact structure into 30-100 nm in size. They exhibit a synergistic effect when combined with several different types of cationic LPs in delivering plasmid DNA into several different types of cells (58). Moreover, three natural polycations, protamine sulfate (PS), poly(L-lysine), and spermine, were selected as DNA condensing active agents to enhance the in vitro and in vivo transfection activity of the cationic lipid emulsion (LE). The transfection activity of the ternary complex increased by at least 2.5-5fold in vitro cell culture system in the presence of 80% serum, as well as in vivo mouse system, as compared with LE/DNA binary complexes. More importantly, after intravenous and intranasal administrations, the in vivo transfection efficiency of the LE/PS/DNA complex was ca. 30 and 50 times higher than that of the liposome (LP)/DNA complex in spleen and lung, respectively. On the other hand, cell toxicity of the ternary complex is lower than that of binary complex (83). We have stated already that Huang et al (58, 59) developed vectors in which poly(lysine)-condensed DNA was entrapped into folate-targeted cationic liposomes (LPD). Later, they modified LPDs through different cationic liposomes wherein LPDS were used to deliver antisense oligodeoxynucleotide and siRNA (84, 85). Though they used DOTAP and N,N-distearylN-methyl-N-2[N-(N2-guanidino-L-lysinyl)] aminoethyl ammonium chloride (DSGLA), a novel non-glycerol-based cationic lipid, which contained both a guanidinium and a lysine residue as the cationic headgroup and downregulated pERK more efficiently in H460 cells than DOTAP as cationic liposomes in the process of LPD preparation, the procedure is very similar. Nucleic acids were mixed with a carrier DNA, calf thymus DNA, before complexing with protamine, a highly positively charged peptide. The resulting particles were coated with cationic liposomes to obtain LPD (liposome-polycation-DNA) nanoparticles. Ligand targeting and steric stabilization were then introduced by incubating preformed LPD nanoparticles with PEGylated ligand lipid (Figure 8). Maitani et al (86) developed cell-penetrating peptide-based gene delivery vectors. A novel oligoarginine lipid ((Arg)n-B, n
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) 4, 10) conjugated to 3,5-bis(dodecyloxy)benzamide (BDB) lipid with a poly(ethylene glycol) (PEG) spacer was synthesized. Oligoarginine lipid-coated vector was prepared by the addition of (Arg)n-B to DNA/protamine complex (PD) ((Arg)n-B-PD). This result suggested that negatively charged tetraarginine conjugated-PEG lipid-coated PD is a promising gene delivery vector for intratumoral injection. In another study (87), polyamidoamine (PAMAM) dendrimer/ DNA complexes encapsulated in a water-soluble polymer, polyR,β-[N-(2-hydroxyethyl)-L-aspartamide) (PHEA), were supported on a cholic acid functionalized star poly(DL-lactide) film with a fast degradation rate to mediate localized gene delivery. The star poly(DL-lactide) supported PHEA encapsulated PAMAM/ DNA complexes could effectively mediate transfection to indicate that the existence of PHEA played an important role in the efficient transfection. pH-sensitive fusogenic polymermodified liposomes exhibited higher transfection activity toward DC2.4 cells than some commercial reagents and hence may be useful as a gene vector for DCs (88). Recently, hybrids of nonviral and viral carriers have been extended to deliver genes, though they have mainly been used for drug delivery systems (89, 90). Bionanocapsules (BNCs) consisting of hepatitis B virus (HBV) surface antigen (HBsAg) and lipid bilayer and displaying a human hepatocyte-recognizing molecule (pre-S1 peptide) were approximately 50 nm hollow particles which have been used as an HB vaccine (91, 92). BNCs could be produced efficiently in recombinant yeast and therefore were free of other components of HBV (93). On the basis of these studies, Kuroda et al (94) developed a new BNC conjugated with liposomes. The conjugate could deliver large materials (100 nm fluorescence-labeled polystyrene beads and N30 kbp plasmids) to human hepatocytes specifically in vitro and in vivo. The transfection efficiency of the BNC-liposome complex was significantly higher than that of the original BNC.
CONCLUDING REMARKS Though the past decade has seen tremendous progress in the design and synthesis of nonviral vectors for gene delivery as well as application of a battery of techniques, the hybrids of nonviral vectors provide another route to overcome the low transfection efficiency of nonviral vectors. Cationic lipids themselves are rarely used, as they do not show effective transfection without the addition of helper lipids such as DOPE. The combination between cationic lipids and helper lipids represents the most successful formulations for gene delivery. However, this may not meet the requirements of in vivo transfection, because of the lack of targeting ability. To promote the uptake of vector-DNA complexes by specific cell types, vectors are usually conjugated with a number of cell targeting agents, particularly folate for cancer cell targeting and galactose for hepatic cell targeting. It has been proven that tertiary amine and quaternary ammonium containing vectors show toxicity to some extent; the conjugates of peptides and lipids are being explored to resolve this problem. They show relatively low toxicity, while retaining acceptable transfection efficiency. The combined utilization of cationic liposomes and other nonviral vectors may provide the most exciting solution for gene delivery, for many advantages could concentrate on the delivery systems. Besides the above-mentioned hybrids, other new formulations are being explored, such as the combination of nonviral and viral carriers.
ACKNOWLEDGMENT The study was supported by Program for New Century Excellent Talents in University (NCET-08-0654) and the National Natural Science Foundation of China (20876027).
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