Cationic lipid-mediated nucleic acid delivery: beyond being cationic

Chemistry and Physics of Lipids 163 (2010) 245–252

Contents lists available at ScienceDirect

Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip

Review

Cationic lipid-mediated nucleic acid delivery: beyond being cationic N. Madhusudhana Rao ∗ Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India

a r t i c l e

i n f o

Article history: Received 23 June 2009 Received in revised form 25 November 2009 Accepted 3 January 2010 Available online 12 January 2010 Keywords: Cationic lipid Transfection Nucleic acids RNAi Formulations Peptides

a b s t r a c t Realization of the potential of nucleic acids as drugs is intricately linked to their in vivo delivery. Cationic lipids demonstrated tremendous potential as safe, efficient and scalable in vitro carriers of nucleic acids. For in vivo delivery of nucleic acids, the extant two component liposomal preparations consisting of cationic lipids and nucleic acids have been largely found to be insufficient. Being a soft matter, liposomes readily respond to many physiological variables leading to complex component and morphological changes, thus confounding the efforts in a priori identification of a “competent” formulation. In the recent past many chemical moieties that provide advantage in facing the challenges of barriers in vivo, were incorporated into cationic lipids to improve the transfection efficiency. The cationic lipids, essential for DNA condensation and protection, definitely require additional components to be efficient in vivo. In addition, formulations of cationic lipid carriers with non-lipidic components, mainly peptides, have demonstrated success in in vivo transfection. The present review describes some recent successes of in vivo nucleic acid delivery by cationic lipids. © 2010 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Cationic lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Formation of cationic lipid and nucleic acids complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. The path . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extra cellular . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Cellular . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Liposomes in RNAi delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Peptides in siRNA delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Nucleic acids as drugs have become a reality. Plasmid DNA, aptamers, siRNA, antisense oligonucleotides, etc. are potential agents that can alter gene expression and thereby change a disease state. Intervention of almost all diseases could be achieved by alterations in gene expression. Gain of function, loss of function or simple supplementation of a gene can potentially alter disease burden. The primary challenge lies in the ability to deliver

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nucleic acids at the site of action, i.e. within the cell (Audouy and Hoekstra, 2001; Li and Huang, 2006; Montier et al., 2008; Niculescu-Duvaz et al., 2003; Zuhorn et al., 2007). Today the therapeutic potential of siRNA, probably the most exciting discovery of our times, waits for an efficient delivery vehicle (Aigner, 2008). Unlike small molecule drugs, nucleic acids, being anionic polymers, present an interesting challenge for their delivery (Montier et al., 2008; Wolff and Rozema, 2008). Charged polymer molecules have a ready tendency to bind electrostatically to molecules and surfaces of opposite charge. And also being natural molecules, nucleic acids are subject to degradation by a host of nucleases. These challenges demand that nucleic acids be protected and chaperoned to the target cells.

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Table 1 Examples of homopolymers of amino acids and peptides used in formulations for nucleic acid delivery. Synthetic

Sequence

NonaArginine Linear lysine polymer Branched lysine polymer LAH4 JTS-1, CADY Peptide for ocular delivery

RRRRRRRRR

Natural Transactivator of transcription (TAT) MPG Antennapedia RGD Haemagglutinin Penetratin Transportan VP22 Mu SV40 T antigen Hybrids RGD-cyclin lysine Stearylated R8 CK30PEG10 Cholesteryl oligo-d-arginine Peptide–RNA conjugate TAT-double stranded RNA binding domain Lithocolic-oleoyl-RNA

KKALLALALHHLAHLALHLALALKKA GLFEALLELLESLWELLLEA GLWRALWRLLRSLWRLLWRA GGG(ARKKAAKA)4 GGRKKRRQRRRPPQC GALFLGFLGAAGSTMGAWSQPKSKRKU RQIKIWFQNRRMKWKK GLFEAIAGFIENGWEGMIDG RQIKIWFQNRRMKWKK GWTLNSAGYLLGKINLKALAALAKISIL DAATATRGRSAARPTERPRAPARSASRPRRPVD MRRAHHRRRRASHRRMRGG PKKKRKVEDPYC

N-terminal cystein (lysine)30 linked to PEG 10K TQIENLKEKG Fatty acids and bile acids are conjugated to 3 end of the sense strand via trans-4-hydroxyprolinol linker

Development of nucleic acid carriers for in vivo delivery has been an active research area for the last three decades (Campbell et al., 2009; Zuhorn et al., 2007). Broadly, the carriers are viral or non-viral in design. In this review, viral carriers are not discussed. Excellent reviews on viral delivery are available in literature (Mancheno-Corvo and Martin-Duque, 2006; Waehler et al., 2007). Cationic lipids were among the early non-viral carriers candidates to be investigated followed by many cationic polymers of amino acids, carbohydrates, dendrimers and, more recently, nanomaterials (Audouy and Hoekstra, 2001; Braun et al., 2005; Li and Huang, 2006; Montier et al., 2008; Niculescu-Duvaz et al., 2003; Zuhorn et al., 2007). With the demonstration of liposomes (prepared with a cationic lipid, DOTMA) mediated nucleic acid delivery, a conceptually simple strategy was realized (Felgner and Ringold, 1989). Secondly, liposomes as potential carriers of small molecule drugs, for example, doxorubicin, have been already investigated extensively (Langer and Tirrell, 2004; LaVan et al., 2003). It was felt that one could “Plug-in” liposomal technology to nucleic acid delivery albeit with cationic lipids. Publications based on cationic lipids have grown tremendously in the last decade with reports on very fascinating lipid designs and their transfection efficiencies. Several excellent reviews were published on the structure–function relations of cationic lipids (Audouy and Hoekstra, 2001; Kumar et al., 2003; Miller, 2004; Montier et al., 2008; Niidome and Huang, 2002; Smyth, 2003). Despite their tremendous potential demonstrated in in vitro studies and simplicity of their preparation, cationic lipids were always found wanting in transfection efficiencies in vivo. Unlike small molecule drugs entrapped in liposomes, nucleic acids profoundly alter the morphology of the liposome. The soft and responsive physical disposition of liposome was also a reason for their ready interaction with various components of the body leading to inability to reach the target cells for efficient delivery. Investigations into the nature of the various biological barriers for liposome-mediated transfection led to identification of several processes that limit the efficiency (Zuhorn et al., 2007). Efforts to improve the in vivo transfection efficiency of cationic liposomes have been made more complex with additional components, essentially bioinspired to overcome the cellular and organismal barriers

(Martin et al., 2005; Montier et al., 2008). This review will focus on these bioinspired entities that enhanced the liposomal transfection efficiency and also on their formulation biology.

2. The components 2.1. Cationic lipids Cationic lipids are amphiphiles with structures analogous to natural phospholipids. The difference is in the presence of a cationic charge on the molecule unlike any natural phospholipid. Cationic lipids are amphiphilic due to the presence of long hydrocarbon chains, usually two alkyl chains and the hydrophilicity is contributed by the charged group, due to quaternary nitrogen. Due to their amphiphilicity upon hydration cationic lipids self-assemble into lamellar vesicular structures as liposomes with interior aqueous phase. The similarity in the methods of phospholipid-based liposome preparations is one reason for their wide usage. Conventional liposome methods are applied to control the number of lamella and the size of the cationic liposomes (Gabizon et al., 2006; Khuller et al., 2004). Cationic lipid structures reported in literature could be described by assuming four modules in their structure (Liu et al., 2003; Nicolazzi et al., 2003; Smyth, 2003). The hydrophobic portion is largely comprised of alkyl chains of lengths ranging from 12 to 20 carbons or cholesterol (Martin et al., 2005; Wasungu and Hoekstra, 2006). The popular linkage of the alkyl groups to the rest of the molecule, i.e., via the backbone, is either an ester or an ether bond. The widely used backbone that links the hydrophobic and the head group is glycerol though amino acids and aromatic groups are also employed. The head group, i.e., the cationic module of the lipid, is a quaternary ammonium group. Very successful cationic lipids were designed using this group. In addition polyamines, guanidinium, heterocyclic, amino acid- and peptide based head groups have been tested successfully (Niculescu-Duvaz et al., 2003). Interesting departures from the above structures are the gemini surfactants and bola amphiphiles (Wasungu et al., 2006a). Detailed investigations with structural analogues of many of these modules

N.M. Rao / Chemistry and Physics of Lipids 163 (2010) 245–252

indicated that the best efficiency is an outcome of the total structure and an optimized module need not work with other modules (Simberg et al., 2004). Excellent reviews on cationic lipids have been published cataloguing their chemical structures, phase properties, biophysical properties and structure-transfection efficiency relations (Garinot et al., 2007; Koynova et al., 2007, 2009; Middaugh and Ramsey, 2007; Xu and Anchordoquy, 2008). 2.2. Formation of cationic lipid and nucleic acids complexes The size, charge and natural chemistry of nucleic acids renders them susceptible to degradation in the biological milieu (Ma et al., 2007; Ramezani et al., 2009; Rejman et al., 2004). Eukaryotic cells do not have natural mechanisms to uptake nucleic acids; hence require facilitated transport (Hoekstra et al., 2007; Strain, 2006; Wiethoff and Middaugh, 2003). Secondly, nucleic acids being anionic have poor affinity to cell surface or endothelial glycoproteins, which are also predominantly anionic (Audouy and Hoekstra, 2001). Condensation and protection are essential to decrease their volume and also to prevent their interaction with other components to enhance their half life in the biological fluids and also for their uptake. Generally the cationic liposomes are prepared by drying initially from an organic solvent and then hydration leading to formation of vesicles. Presentation of plasmid DNA or siRNA to preformed cationic liposomes leads to complexation, which is highly cooperative. Though condensation of DNA requires that smallest charge on the cation (Koynova and MacDonald, 2005; Zhang et al., 2008) should be 3+, monocationic lipids still condense DNA efficiently because of their self assembling property due to the alignment of hydrophobic tails (Matulis et al., 2002). Consequently, the condensation was due to large increase in entropy opposed by small endothermic enthalpic change (Bloomfield, 1997; He et al., 2000). Transfection was observed to be better when the cationic liposomes were prepared with another neutral or zwitterionic lipid, for example dioleoyl phosphatidylethanolamine (DOPE) (Hirsch-Lerner et al., 2005; Hui et al., 1996; Zuhorn et al., 2002). siRNA, being small in length (usually of 22 bases), is inefficiently condensed by cationic lipid leading to unstable lipoplexes (Aigner, 2008; Tseng et al., 2009). Prior addition of cationic polymers, for example polylysine, is preferred for condensation of siRNA. The spontaneous process leads to occlusion of the nucleic acid and produces lipoplexes which are smaller than those obtained without cationic polymers. The condensation of nucleic acids invariably leads to extensive rearrangements of the vesicle components and the nucleic acids. The end product of this complexation is sensitive to the ratio of positive to negative charges and also to the sizes of the nucleic acid and the liposomes (de Lima et al., 2003). Low +/− charge complexes result in exposure of nucleic acids, thus leading to poor transfection efficiency. Small vs. large liposomes with nucleic acids led to lipoplexes of different sizes and lamellarity (Kneuer et al., 2006; Ma et al., 2007). It was also observed that extensive mixing of lipids occur during the lipoplex formation (Caracciolo et al., 2005). This spontaneous process was found to be very sensitive to the order of addition of components, buffer composition, reaction volume and duration of the process. Ionic strength and presence of serum weaken or limit the size of the lipoplex (Tranchant et al., 2004). Many methods including dynamic light scatter, electron microscopy, zeta potential, etc. have provided morphological details of the lipoplexes. Methods such as DNase sensitivity inform us on the extent of protection to the nucleic acids in a complex. Small angle X-ray diffraction data on the lipoplexes provides much more insight on the relative arrangement of lamellae and nucleic acids in a complex (Ewert et al., 2004, 2005). These results indicate that nucleic acids are arranged in parallel to each other in between the lamellae of the lipids at charge ratios where efficient transfection occurs. More importantly X-ray data also provides

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information on the occurrences of non-lamellar structures within in the lipoplex, which are crucial for transfection efficiency. Though liposomes are formed with pure cationic lipids addition of another lipid, called colipid, was found to be essential to obtain good transfection efficiencies. DOPE is the most popular colipid in transfection formulations. Cholesterol is another popular colipid (Ramezani et al., 2009; Xu and Anchordoquy, 2008). From detailed investigations into the analogues of DOPE and other phospholipids it is clear that the presence of DOPE facilitates in cell interaction and escape from the endosomes (Hoekstra et al., 2007; Zuhorn et al., 2007). DOPE being a inverted hexagonal preferring lipid has a tendency to take the membrane away from lamellar order and destabilize it (Fletcher et al., 2006; Zuhorn et al., 2005). Such departure from lamellarity was found to be critical for fusion of lipoplex membrane with the endosomal membrane. Cholesterol, on the other hand, does not bring in similar phase alterations. It was found that some cationic lipids work exclusively with cholesterol and not with DOPE or vice versa (Banerjee et al., 1999). Often it was found that the lipoplexes containing cholesterol were found to be more efficient in in vivo applications (Dass, 2004; Kim et al., 2004). Presence of cholesterol apparently stabilizes the lipoplexes in the presence of serum. Serum has a disintegration effect on the lipoplexes; however cholesterol, probably by forming domains, reduces the interaction of proteins onto the lipoplex which is a prerequisite for disintegration (Xu and Anchordoquy, 2008). Size and charge are two parameters that are often measured to relate to transfection efficiency. Size of the lipoplex has the most critical bearing on transfection, especially in vivo transfection. However, the size of the lipoplex that is appropriate for efficient transfection has not been clearly defined yet. An early study indicated that size (200–2000 nm), uptake efficiency and transfection efficiency were strongly correlated (Ross and Hui, 1999). Later studies indicated smaller lipoplexes (<200 nm) are more efficient (Zhang et al., 2003). Large size lipoplexes may settle rapidly on the cells compared to smaller vesicles which may be diffusion limited. A careful study on endosomal uptake of fluorescent dextran beads of various sizes indicated that particles smaller than 300 nm were predominantly taken up by clathrinmediated endocytosis and particles larger than 500 nm were taken up by caveolae-mediated pathways (Rejman et al., 2004). Several publications addressed the pathway preference of lipoplex using various inhibitors, fluorescent double labels, dominant negative cell lines and colocalization (Hoekstra et al., 2007; Lechardeur et al., 2005; Prasad et al., 2005). At present it is not clear which of the endocytic pathways predominate in the lipoplex uptake. A confounding issue in these studies is the heterogeneity associated with the lipoplex size. Since the uptake of lipoplex by the cell was found to be only partial, identifying the competent size is arduous. Smaller lipoplexes, in the range of 100 nm, are preferred to transit the narrow capillaries. However, lipid with nucleic acids would spontaneously give raise to lipoplexes of sizes larger than approximately 200 nm (Campbell et al., 2009). Addition of non-lipidic condensing agents such as polyethyleneimine, polylysine and protamine sulphate before the addition of cationic lipids has been widely practised to make lipoplexes of small and homogeneous size (Elouahabi and Ruysschaert, 2005; Wagner et al., 2005). These particles called stabilized particles have been widely successful (Tyagi et al., 2006). In addition to size, another mitigating factor, a linked parameter charge density, is critical. Charge of the lipoplex determines the size. 2.3. The path Formulations for carrying nucleic acids encounter serum, tissue, cell and vesicular membrane barriers before nucleic acids elicit their action inside the cell. To overcome each of the biological bar-

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Fig. 1. Biological barriers for nucleic acid delivery and strategies to overcome the barriers.

riers specific chemistries or physical properties are incorporated into the formulations. These strategies are employed singly or in combination with other strategies. Some of these approaches are shown in Fig. 1. 3. Extra cellular 3.1. Serum Serum probably is the most serious barrier for lipoplexmediated nucleic acid delivery (Koynova and MacDonald, 2005; Nchinda et al., 2002; Tranchant et al., 2004). Serum is a complex fluid containing various lipases, nucleases and high density lipoproteins that interact with the lipoplex and cause transfer of the liposomal contents. Hence, increasing the circulation times is critical for high transfection efficiencies. Historically, transfection studies are performed in the absence of serum or in the presence of low serum concentrations leading to poor correlations between in vitro and in vivo transfection efficiencies. Among various sera, mouse serum was identified to be closest to human serum. Preparing the liposomes with neutral or anionic surfaces was known to enhance the stability in serum. Polyethylene glycol is the most popular polyol to prolong the serum half life of the lipoplex by providing a surface that avoids binding of proteins (Masson et al., 2004). PEG with a hydrophobic moiety, usually phosphatidylethanolamine at <5 mole% of the lipoplex is the most popular component (Ahn et al., 2002; Masson et al., 2004; Mignet et al., 2008). The advantage of the long circulation times of PEG-lipoplexes is partially offset by the unfavourable interactions with the cell surface. Lipids with the PEG attached by a hydrolysable bond such as an ester are preferred so that PEG is removed from the complex in a controlled manner (Guo et al., 2003; Masson et al., 2004). Recently PE was also found to be efficient with the advantage of incorporating PEG subsequent to lipoplex formation (Li et al., 2005). In addition, cationic liposomes are known to activate the complement system. The complement

receptors on the surface of monocytes and polymorphonuclear leukocytes (PMN) have been shown to bind complement-coated particles. This property of cationic lipids was used in bringing about enhanced immune response when combines with antigens. In addition to the mitigating effects of serum, relatively high interstitial fluid pressures and the organization of the interstitial spaces have significant bearing on the building up of the lipoplex concentrations near the cells. Systemically administered lipoplexes must first contend with a glycocalyx shield of the endothelial layer (typically 50–100 nm thick) before reaching the interstitial environment (Campbell et al., 2009). The glycocalyx is a complex mesh consisting of glycolipids, glycoproteins, glycosaminoglycans, phospholipids and proteoglycans oriented on the luminal side of the vessel wall. Proteoglycans associated with the endothelial cell membrane have been shown to facilitate the uptake of a wide range of macromolecules (Erbacher et al., 1999; Kopatz et al., 2004; Wiethoff et al., 2001). The importance of proteoglycans in the uptake of DNA has been demonstrated in sulfated proteoglycandeficient HeLa cells. However, the small size, <100 nm, of the lipoplexes offers a natural advantage to the interstitial matrix of the neovasculature of the tumor tissue (Wiethoff et al., 2001). This advantage is exploited to deliver siRNA to bring about loss-offunction in the endothelial cells. Death of a single endothelial cell will eventually have a cascading affect on the survival of number of neoplastic cells (Santel et al., 2006; Tseng et al., 2009). Systemically injected lipoplexes would lead to uniform dilution of the lipoplexes in the body, which is not beneficial. Size of the lipoplex, as discussed earlier provides a natural localization in neovasculature (Aigner, 2008; Zhang et al., 2008). Similar enrichment of lipoplexes in specific tissue was attempted by conjugating small molecule ligands to lipoplexes. Such ligand-targeted lipoplexes have used many different kinds of ligands such as antibodies, receptors, peptides, vitamins, oligonucleotides, or carbohydrates (Cardoso et al., 2007, 2008; Chen and Huang, 2005; Chiu et al., 2004; Colin et al., 1998a; Hofland et al., 2002; Lee et

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al., 2003; Walker et al., 2005). Bioinspired targeting of lipoplexes was initially successful with folate (Dauty et al., 2002; Hattori and Maitani, 2005; Hofland et al., 2002). Many cancers of endothelial origin were known to express excess amounts of folate receptors to provide folate for the growing cells. Given its simple chemistry folate could be easily conjugated to lipids especially to phosphatidylethanolamine, for incorporating into the formulation. Haloperidol, a small molecule ligand for Sigma receptor was shown to deliver nucleic acids into cells specifically expressing sigma receptor (Mukherjee et al., 2005). Receptors that are specifically induced in tumor cells are often targets for tumorspecific gene delivery. Her2 neu, also known as c-erbB-2, is an epidermal growth factor receptor, transferrin and CD1a, metalloendopeptidase, are few examples of importance in the context of gene delivery (Akinc et al., 2008; Li and Huang, 2008). Besides their natural ligands, many antibodies to tumor-specific proteins have been employed for tumor-specific delivery (Pagnan et al., 2000; Park et al., 1997; Xu et al., 2002). Attachment of ligands to the liposome was achieved either by directly coupling the ligand to the phospholipid or by attaching the ligand to the distal end of the PEG-lipid. The advantage of the second method is that the ligand is much more accessible for interactions with the receptors; further PEG has ability to increase the circulating half lives of the liposomes. 3.2. Cellular Lipoplexes with net positive charge and sizes less than a micron interact with the cell surface proteins and sugars of the plasma membrane, usually negatively charged, to gain an anchor on the cell surface. Once attached to the cell surface by non-specific means, their presence stimulates the endocytic pathway leading to formation of endosomal vesicles. Life cycle of endosomes is strictly defined by various extra-endosomal factors and cytoskeleton. Lipoplex-containing endosomes, predominantly due to the processes of fluid phase or receptor-mediated uptake, would mature into late endosomes and eventually fuse with the lysosomes (Hama et al., 2005; Hoekstra et al., 2007; Tachibana et al., 2002; Zhou et al., 2004). Endosomes, part of a network of processing vesicles undergo changes leading to acidification of the vesicle. Escape of lipoplexes from the endosomes is considered to be critical for efficient release of nucleic acids into the cytoplasm. The lipoplex should exit from the endosome before the fusion with lysosomes. Several insightful experiments with several formulations and phase behaviour of lipid components of the lipoplexes have revealed that electrostatic interaction of cationic lipids with the phosphatidylserine of the endosomal membrane would lead to leakage of the contents of the endosome (Bell et al., 2003; Zuhorn et al., 2005). DOPE, a classical inverse hexagonal phase preferring lipid, facilitates the formation of membrane fusion intermediates and fusion (Hattori et al., 2005), while DOPC, a classical lamellar preferring lipid, does not aid fusion. Detailed phase diagrams of cationic lipids with various colipids and charge ratios indicated that the departure from lamellar phase is critical for fusion (Ewert et al., 2004). Fusion, as would be expected, requires mixing of two opposing membranes progressing through hexagonal or cubic phase intermediates (Siegel, 1986). Saturated PE as colipid reduces transfection efficiency and also inhibits model membrane fusion. Model membrane studies with biological membrane vesicles and various cationic lipid vesicles clearly point indicating the role of inverted hexagonal phase in fusion of lipoplex with the endosomal membrane, (Bentz et al., 1987; Wasungu et al., 2006b). Recognising endosomal escape as critical for the transfection efficiency, many strategies have focused on overcoming this limitation, some of which are bioinspired. The objective of these efforts was to lyse the endosome before it reaches the lysosome. Fusion protein from influenza virus is a short amphiphilic sequence which

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undergoes conformational change at acidic pH, this was the natural mechanism of the virus to escape the endosome (Vaughan and Dean, 2006; Verma et al., 2005). Conjugation of the fusion protein to liposome brought about similar destabilization of the endosome and enhanced the transfection efficiency. Presence of pH titratable moieties, which undergo protonation at low pH, such as histidines, could cause destabilization of the endosomal membrane. Similar to fusion protein, poly-(amidoamine)s undergo a large conformational change from a relatively coiled hydrophobic structure at neutral pH to a relaxed hydrophilic structure at acidic pH, and may be thus exploited as endosomolytic agents (Criscione et al., 2009; Waite et al., 2009). Cationic polyethyleneimine can induce non-leaky fusion at pH 7, and is even being investigated as a gene delivery vesicle on its own (Eliyahu et al., 2005). Another class of bioinspired lipids are lipids sensitive to the redox state. Cytoplasm the milieu for the nucleic acid for gene silencing or the nuclear uptake is reductive in nature due to the presence of glutathione, thioredoxine and glutaredoxine (Li et al., 2004; Tang and Hughes, 1998, 1999). Redox state labile structure in the lipoplex may lead to disaggregation of the lipoplex. Disulfide is the simplest redox sensitive group DOGSDSO and CHDTAEA are disulfide-containing ornithine conjugates which could be reduced by dithiothretol and possess 50-fold higher transfection compared to non-cleavable lipid (Tang and Hughes, 1999). Shermann investigated the effect of position of the disulfide bond on transfection and reported that the presence of disulfide between the cationic group and the head was poor for transfection. Disulfide linking the fatty acid chains to the head was more effective (Leblond et al., 2007). 3.3. Liposomes in RNAi delivery RNAi-mediated gene silencing vis a vis disease management, arguably, is an exciting therapeutic procedure (Aigner, 2008; Tseng et al., 2009; Whitehead et al., 2009). The biology of gene silencing is understood in sufficient details to support development of therapeutics based on siRNA. A few siRNA based protocols are in Phase 2 trials (http://www.rnaiweb.com/RNAi/ RNAi Web Resources/RNAi Therapy Clinical Trials/). The limitations experienced by liposome-mediated gene and drug delivery are also encountered with siRNA. From the formulation point of view it is important to recognise that due to its small size the condensation of siRNA by cationic lipid directly was found to be insufficient and thereby led to unstable liposomes. Hence pre-condensation of the siRNA by polymeric cations such as polyethyleneimine and polylysine was found to be beneficial. SiRNA-mediated gene silencing occurs at post transcriptional level in the cytoplasm. Hence, stable in vivo delivery of siRNA was achieved by complexing siRNA targeted to HBV RNA with liposomes. The lipoplexes called stabilized nucleic acid particles (SNAP) of DSPC:Cholesterol:PEG-C/DMA:DLIN DMA were prepared by ethanol dialysis method. Similarly, apo-lipoprotein B targeted siRNA in SNAP resulted in more than 90% silencing after intravenous injection. In another SNAP formulation targeting polymerase gene of Ebola virus completely protected cynomologus monkeys after an intravenous injection. In another successfully targeted in vivo delivery to suppress tumor suppression anti-transferrin receptor single chain body was coupled to liposomes for gene therapy (Xu et al., 2001). Realization of silencing effects of siRNA requires that it should profitably overcome barriers on systemic administration. Issues involving siRNA and plasmid DNA delivery are qualitatively similar. However the site of action for siRNA is the cytoplasm; thus nuclear delivery is not required for silencing. In RNA silencing both shRNA and siRNA are used. DNA based shRNA was discovered to long lasting silencing. Transferrin conjugated DOTAP: cholesterol lipoplexes efficiently silenced luciferase in the primary cultures of cortical neurons paving of

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the possibility of gene silencing in brain (Cardoso et al., 2008). Similarly folate conjugated to DSPE-PEG and in combination with cholesterylcarboxyamidomethylene-N-hydroxylamine efficiently delivered siRNA in to human nasopharyngeal KB cells (Yoshizawa et al., 2008). In an interesting combinatorial method, 1200 lipid carriers were synthesised by an automated modular chemistry and tested in mice, rats and non-human primates. Several lipidoids, products of the combinatorial method, were found to be very efficient in gene silencing (Zimmermann et al., 2006). Immunolipoplexes with monoclonal antibodies for insulin and ligands for transferrin receptor were found to be efficient for crossing the blood–brain barrier and siRNA delivery to the brain (Boado, 2007). When lipoplexes were compared with polyplexes in siRNA delivery, it was found to be that the polyplexes (PEI) were comparably efficient, however they did not permit development (Hassani et al., 2005). The ability to silence the cellular genes in a non-specific way was observed upon administering siRNA with several commercially available lipid formulations (Spagnou et al., 2004). 3.4. Peptides in siRNA delivery Conceptually cationic lipid-mediated nucleic acid delivery is probably simplest of all the non-viral strategies for nucleic acid delivery. While being a simple strategy cationic lipid-mediated delivery is also recognised to be inefficient due to many sequestering processes in the biological milieu. All signal and material transduction processes in cells are facilitated by peptides or proteins. The recurring theme in these peptides is the presence of cationic amino acids in their sequences. Specific sequences of proteins, not the entire protein, are sufficient to recognise a transport process in a membrane. Oligopeptides or peptides, inspired by their biological functionalities, have been incorporated in the lipoplexes. RGD and GALA peptide are such examples (Colin et al., 1998). The peptides could be either synthesised chemically or produced (Rajagopalan et al., 2007; Xavier et al., 2009) heterologously if they are longer peptides. The advantage of heterologous production would be that a large number of combinatorial peptides could be generated using corresponding gene sequences in one construct. With the discovery of properties cell penetrating peptide (CPP) of HIV-1 TAT many peptides were characterized viz. HSV-1 protein VP22, transportan, MPG, model amphipathic peptide, poly arginine, etc., which can facilitate the transport of the “cargo” molecules along with it (Mann et al., 2008; Wolff and Rozema, 2008; Xavier et al., 2009) (Table 1). Similarly many peptide sequences in viral protein and other proteins, such as histones, were recognised to condense nucleic acids efficiently. Several peptide sequences have recently been identified which have the potential to cross the plasma membrane by virtue of unique protein transduction domains (PTDs). Attempts in the last several years have led to the use of polycationic, precondensing agents to compact DNA prior to adding cationic liposomes. Combinations of PTDs with DNA-binding motifs such as were found to be very efficient in enhancing liposomal transfection (Xavier et al., 2009). Though these peptides could condense the nucleic acids and protect them from DNases, their ability to bring about transfection was not significant. However, when the lipoplexes were made with nucleic acids pre-condensed with these peptides, the transfection efficiency tremendously improved (Rajagopalan et al., 2007). Dynamic polyconjugates described by Wolff combined the synthetic endosomolytic moieties (for example polybutyl and amino vinyl ether (PBAVE)) with liver targeting ligand (for example galactose) to a polymer using acid-labile carboxylated dimethyl maleate cross linking. To this polymer siRNA is attached via a thiol bond. These formulations provide longer serum stability (polymer), tissue targeting (galactose), endosomolysis (PBAVE) and efficient cytoplasmic release of siRNA (thiol) (Rozema et al., 2007). Thus peptides

and environment sensitive bonds provide additional opportunities to design efficient and target specific formulations for nucleic acid delivery. 4. Summary Science and technology of cationic lipids-mediated nucleic acid delivery is absolutely critical for realizing siRNA as a drug. By far, among nucleic acid carriers, cationic liposomes are probably the safest, simplest, scalable and modulable carriers. Extensive investigations into the chemistry and physics of cationic lipids and their transfection efficiencies points at the following generalities: A. Supramolecular processes in lipoplex formulation are equally important for transfection as the chemical nature of the cationic lipid. Many cationic lipids that were poor in a particular combination could be made efficient by variation in formulations. B. Safety of a carrier has same emphasis as efficiency for in vivo nucleic acid delivery. Incorporation of biodegradability into the structure in otherwise efficient structures has helped in reducing the toxicity. C. Ternary complexes of cationic lipid, colipid and nucleic acid was found to be limiting for in vivo applications. Formulations that condense nucleic acid efficiently and thereby change the size and stability of a lipoplex are increasingly becoming popular. D. The abundant empirical knowledge on drug liposomes offers many more tested options to enhance the lipoplex efficiency. Targeting ligands or camouflaging chemical entities for enhanced serum stability and tissue specificity have given encouraging results. E. Membrane phase of lipoplex is crucial for transfection. Either excess rigidification or fluidization of membrane has negative impact on the efficiency. A poorly defined condition of metastability of lipoplex membrane is recurringly found to be important for productive interactions with various biological components. F. Peptide conjugates or additives to liposomal formulations are proving to be versatile and are providing the chemical complexity required to cross cellular and organismal barriers. G. Use of nanomaterials has provided another dimension in lipoplex formulations. Designs of nanostructures that possess controlled and tunable release of nucleic acids have become a possibility. H. The palette of cationic lipids is large and has excellent examples of highly efficient in vitro transfection agents. This success should also be achieved with in vivo transfections. The gap is due to our incomplete understanding of the various cellular and tissue barriers and their interactions with lipoplexes. I. We urgently require methods to monitor and estimate the pre-transcription or pre-silencing processes encountered by a lipoplex within the cell to optimize lipoplex design. The design of lipoplexes has transitioned from binary system to a multi-component system that would incorporate carbohydrate, proteins, peptide and nanomaterials for efficient transfections in vivo. Acknowledgements I thank Dr. Vijaya Gopal for the support during the preparation of the manuscript. The efforts were supported by a CSIR Network grant (NWP0035). References Ahn, C.H., Chae, S.Y., Bae, Y.H., Kim, S.W., 2002. Biodegradable poly(ethylenimine) for plasmid DNA delivery. J. Control. Release 80, 273–282.

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