SEARCH

SEARCH BY CITATION

Keywords:

  • biotechnology;
  • cationic lipids;
  • DNA/oligonucleotide delivery;
  • encapsulation;
  • gene delivery;
  • gene vectors;
  • lipoplexes;
  • macromolecular drug delivery;
  • nanoparticles;
  • nonviral gene delivery

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DNA VERSUS siRNA
  5. SERUM STABILITY AND PEGYLATION
  6. ENCAPSULATION OF NUCLEIC ACIDS FOR EFFICIENT DELIVERY
  7. TARGETED DELIVERY OF NUCLEIC ACIDS
  8. BIODISTRIBUTION AND IMMUNOGENICITY
  9. PERSPECTIVES/PROSPECTIVES
  10. Acknowledgements
  11. REFERENCES

The ability to deliver nucleic acids (e.g., plasmid DNA, antisense oligonucleotides, siRNA) offers the potential to develop potent vaccines and novel therapeutics. However, nucleic acid-based therapeutics are still in their early stages as a new category of biologics. The efficacy of nucleic acids requires that these molecules be delivered to the interior of the target cell, which greatly complicates delivery strategies and compromises efficiency. Due to the safety concerns of viral vectors, synthetic vectors such as liposomes and polymers are preferred for the delivery of nucleic acid-based therapeutics. Yet, delivery efficiencies of synthetic vectors in the clinic are still too low to obtain therapeutic levels of gene expression. In this review, we focus on some key issues in the field of nucleic acid delivery such as PEGylation, encapsulation and targeted delivery and provide some perspectives for consideration in the development of improved synthetic vectors. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:38–52, 2011


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DNA VERSUS siRNA
  5. SERUM STABILITY AND PEGYLATION
  6. ENCAPSULATION OF NUCLEIC ACIDS FOR EFFICIENT DELIVERY
  7. TARGETED DELIVERY OF NUCLEIC ACIDS
  8. BIODISTRIBUTION AND IMMUNOGENICITY
  9. PERSPECTIVES/PROSPECTIVES
  10. Acknowledgements
  11. REFERENCES

The development of nucleic acid-based therapeutics has garnered tremendous interest in the past two decades as a new category of biologics. The ability to deliver nucleic acids (e.g., plasmid DNA, antisense oligonucleotides, siRNA) offers the potential to develop potent vaccines and novel therapeutics to cure many diseases that are difficult to treat effectively with traditional therapies, for example, hereditary diseases, cancer.1–4 However, the site of action of nucleic acids requires that these molecules be delivered to the interior of the target cell, which greatly complicates delivery strategies and compromises efficiency. The poor transfection efficiencies of synthetic delivery systems (nonviral vectors, i.e., complexes of polynucleotides with cationic lipids and/or polymers) has forced the majority of clinical trials to employ viral vectors despite the significant safety concerns associated with their immunogenicity and insertional mutagenesis.5, 6 Although nonviral vectors proficiently transfect cells in culture, it must be appreciated that most cell culture experiments employ rapidly dividing cells cultured in monolayers that do not accurately mimic the in vivo situation. Under these conditions, precipitation can enhance the association of the delivery system with the cell surface, which can artifactually elevate transfection rates with delivery systems that are not stable in physiological media. Conversely, delivery systems that are stable in physiological media often do not transfect efficiently in cell culture, leading to the mistaken conclusion that such systems are not worthy of further consideration. Another confounding factor with cell culture experiments is that the nuclear membrane breaks down during cell division, allowing efficient translocation of DNA into the nucleus of rapidly dividing cells that greatly facilitates transfection. These differences between cell culture and in vivo gene delivery misled several companies who employed combinatorial chemistry to create tens of thousands of novel delivery agents in the 1990s, only to screen for efficacy in cell culture. Unfortunately, these massive efforts yielded minimal insight into the structure–function relationship of delivery agents that is relevant to the in vivo situation. Similar approaches have recently been employed to develop delivery agents for siRNA, and some of the identified “lipidoids” have shown promise in nonhuman primates.7, 8 Currently, delivery efficiencies of synthetic vectors in the clinic are too low to obtain therapeutic levels of gene expression. As of December 2009, there have been 1579 gene therapy clinical trials worldwide, of which about 25% have utilized nonviral vectors.9 It should be noted that viral vectors still dominate clinical gene therapy, with the first approved gene therapy product, Gendicine, being the Recombinant Human Ad-p53.10 Besides the approved product in China, some of the viral vector-based therapeutics have progressed to phase III clinical trials,11–13 such as Rexin-G, a tumor-targeted retrovirus bearing a cytocidal cyclin G1 construct and ONYX-015, an oncolytic adenovirus. While viruses offer greater efficiency of gene delivery, it is generally agreed that synthetic vectors would be preferable due to safety concerns, and viral vectors may be more suited for ex vivo applications.14 It is clear that the strategies employed by these two classes of vectors appear to be converging,15 but the challenges associated with intracellular delivery are significant. In this review, some key issues in the field of nucleic acid delivery will be addressed for consideration in the development of improved synthetic vectors. Considering that cationic liposomes are the best studied of the nonviral delivery systems, much of the discussion will review findings with these systems, but these issues are also relevant to polymeric systems.

DNA VERSUS siRNA

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DNA VERSUS siRNA
  5. SERUM STABILITY AND PEGYLATION
  6. ENCAPSULATION OF NUCLEIC ACIDS FOR EFFICIENT DELIVERY
  7. TARGETED DELIVERY OF NUCLEIC ACIDS
  8. BIODISTRIBUTION AND IMMUNOGENICITY
  9. PERSPECTIVES/PROSPECTIVES
  10. Acknowledgements
  11. REFERENCES

Cationic liposomes and polymers are widely used as nonviral vectors both in vitro and in vivo. Both plasmid DNA and siRNA bind to cationic liposomes and polymers via electrostatic interaction between the anionic phosphodiester backbones and the positively charged group in cationic delivery agents. Indeed, it was recognized by Papahadjopoulos and coworkers16–18 that encapsulation efficiency of nucleic acids within traditional anionic liposomes was low, and thus cationic lipids were synthesized in an attempt to improve loading efficiency and promote cellular uptake. The application of this idea by Felgner et al.19 in 1987 stimulated immense interest in developing nonviral vectors for therapeutic use. At the time of this landmark paper, the mechanism of RNA interference had yet to be discovered, but cationic agents that bind DNA would be expected to interact with RNA via similar electrostatic interactions with the phosphate backbone.20, 21 However, it is important to recognize the significant structural differences between plasmid DNA and siRNA. The most obvious difference is that plasmid DNA is typically 5000 base pairs or larger whereas siRNA is typically 20–25 bp; a 200-fold difference in molecular weight. Work from our laboratory has shown that the affinity of cations for short deoxy-oligonucleotides can vary substantially by sequence, and is significantly different from that to plasmid DNA.22 Furthermore, the interaction of plasmids with some multivalent cationic agents causes a remarkable reduction in molecular volume classically referred to as “condensation.”23–25 This event is characterized by a molecular collapse that protects the DNA from nucleases and chemical degradation, but true condensation is limited to polynucleotides greater than approximately 400 bases.26 DNA condensation has been exploited by Copernicus Therapeutics to collapse single molecules of plasmid DNA into very small structures that facilitate translocation across cellular barriers.27, 28 Unfortunately, the term “condensation” is frequently misused in the delivery literature to describe the electrostatic association of nucleic acids with a delivery vehicle regardless of polynucleotide size, valency of the cation, or ability of the delivery system to affect molecular collapse. Although the complexation of poly- and oligo-nucleotides with cationic delivery vehicles typically results in nuclease resistance, condensation is rarely achieved.

In addition to molecular size, it is important to realize that DNA assumes a B conformation in physiological solutions in contrast to RNA, which assumes a less-hydrated, more compact A conformation characterized by a relatively narrow major groove and shallow minor groove. It follows that these structural differences that alter the spacing of phosphates in the backbone might also affect the interaction of cations with siRNA. This idea is consistent with experiments showing a dramatically reduced binding stoichiometry of cations for ribo- versus deoxyribo- oligonucleotides (20 bp), which may be relevant to incorporation into cationic delivery systems.29 Furthermore, a reduced binding of cationic agents to RNA could potentially explain why much higher levels of cationic agents (i.e., +/− ratio) are generally needed to deliver RNA as compared to DNA.30 Reduced interactions of RNA with cationic agents could also play a critical role in the release of siRNA from the delivery vehicle into the cytoplasm, which would influence the ultimate biological effect.

Aside from the physicochemical differences between DNA and RNA, it should be noted that plasmid DNA needs to be transported into the nucleus for gene expression, which requires crossing two biological barriers, that is, the plasma/endosomal and nuclear membranes. The endosomal release of DNA and transport into the nucleus pose additional challenges for delivery efficiency. In contrast, siRNA only needs to be transferred across the plasma membrane in order to accomplish silencing in the cytoplasm. This may explain why siRNA-induced silencing does not require endocytosis, but is thought to be mediated by direct fusion with the plasma membrane.31 Not surprisingly, it has been suggested that an optimized siRNA formulation procedure is quite different from the pDNA formulation, and that parameters such as toxicity and efficacy can be controlled by the development of specific protocols for lipid/siRNA complex formation.32 Similar results were reported in a study by Malek et al.33 using PEGylated polyethylenimine (PEI). It was shown that siRNA-mediated gene silencing is much less dependent on the N/P (PEI nitrogen/nucleic acid phosphate) ratio than is DNA transfection. These studies, in addition to the physicochemical differences noted above, strongly suggest that delivery agents and protocols need to be optimized for siRNA delivery. To date, the delivery of siRNA has predominantly utilized cationic agents that were developed for gene delivery, suggesting that delivery reagents should be specifically developed for siRNA delivery. Although novel vehicles tailored to siRNA may lead to more efficient delivery, all systems must overcome many common problems that are discussed below.

SERUM STABILITY AND PEGYLATION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DNA VERSUS siRNA
  5. SERUM STABILITY AND PEGYLATION
  6. ENCAPSULATION OF NUCLEIC ACIDS FOR EFFICIENT DELIVERY
  7. TARGETED DELIVERY OF NUCLEIC ACIDS
  8. BIODISTRIBUTION AND IMMUNOGENICITY
  9. PERSPECTIVES/PROSPECTIVES
  10. Acknowledgements
  11. REFERENCES

While there are some applications for which local administration might suffice (e.g., nonresectable lesions, vaccine innoculation), most studies to date have focused on systemic delivery via intravenous injection. One major problem associated with the use of cationic delivery vehicles is their strong interaction with blood components, which can dramatically lower the transfection efficiency.34–40 Serum has been reported to exert its inhibitory effect by binding serum proteins to charged particles, which leads to structural reorganization, aggregation, and/or dissociation of the delivery vehicle.37, 41–43 The presence of nuclease in the serum can also degrade nucleic acids, resulting in a loss of supercoil content and biological activity.44 Many early studies on gene delivery focused on developing tight associations of the delivery vehicle with DNA such that nuclease resistance was imparted. However, such studies ignored the fact that these delivery systems needed to be capable of disassembly within the cell; an issue that continues to receive insufficient attention. In fact, the difficulty of extracting DNA from pure cationic lipid formulations suggests that the neutral helper lipid contributes significantly in dissociation/release. This physical effect of the helper lipid is generally not appreciated, and the role of helper lipids is typically thought to be limited to promoting hexagonal phase formation to enable endosomal escape.45 By promoting fusion with the endosomal membrane, the helper lipids also facilitate mixing with anionic lipids that allows DNA to be released from the delivery vehicle within cells.46

In the case of oligonucleotides (e.g., siRNA, antisense, aptamers) that are produced by chemical synthesis, modifications (e.g., phosphorothioates, amidates, 2′-O-methyl, locked nucleic acids) can be introduced that greatly enhance nuclease resistance.47 Because plasmid DNA is produced in bacteria, incorporation of modified bases is not possible. Although most studies on stability in blood have focused on serum proteins, it should also be recognized that delivery vehicles can bind to erythrocytes, a major constituent of blood cells, both in vitro and in vivo.48 Such interactions can result in association/fusion of the delivery vehicle with erythrocytes, which reduces transfection efficiency and accelerates removal of the delivery vehicle from the circulation via liver and spleen. In this regard, studies have shown that the helper lipid DOPE promotes fusion with erythrocytes to a much greater extent than cholesterol, and thus cholesterol is more commonly used as a helper lipid for in vivo experiments.48

The adverse effects by blood components can be circumvented in cell culture by replacing serum-containing media with serum-free media. Unfortunately, this has become a standard practice in nucleic acid delivery studies, and this media alteration is sometimes not even mentioned in the methods section of publications. Partially because of the interactions with serum proteins mentioned above, the use of serum-free media yields misleading results with regard to the transfection efficiencies that can be expected in vivo. Some researchers describe transfection rates “in serum,” but these studies typically employ media fortified with only 10% fetal bovine serum as is typically used in cell culture. Studies have clearly documented that stability and delivery efficiency are progressively compromised at increasing levels of serum, although the detrimental effects of serum are less dramatic as the serum content is increased above 50%.38, 49 Considering that approximately half of the blood volume is serum, 50% (v/v) serum is often referred to as “full-strength serum.” Similarly, delivery vehicles are often diluted with an equal volume of serum to achieve 50% (v/v) serum, and this is often described as “exposed to 100% serum.” Because the nonserum fraction of blood is occupied by cells whose volume is not available for dilution, intravenous administration exposes delivery vehicles to 100% (v/v) serum. In conducting serum stability studies, the volume in which the delivery vehicle is suspended should be considered when reporting the serum concentration to which vectors have been exposed. For practical reasons, it is difficult to perform extensive transfection and biophysical characterization studies in >50% (v/v) serum, that is, a 1:1 dilution of the delivery vehicle in serum. However, as stated above, results from such experiments can provide a more realistic estimate of in vivo performance than the serum-free conditions that are typically employed.

In order to overcome the adverse effects of serum protein binding, there is an intense effort to develop lipid- and polymer-based systems that efficiently transfer nucleic acids in the presence of serum. Among the pursuits toward this goal, the predominant strategy is to utilize PEGylated components to sterically shield the delivery vehicles from blood components.50–52 This strategy has been utilized for decades in the development of liposome-based formulations and has been shown to allow the accumulation of liposomes containing small molecule drugs in tumors.53 It is well-documented that the steric stabilization provided by PEGylation reduces particle aggregation in serum and extends circulation lifetime. It is typically assumed that the extended circulation lifetime can be attributed to the reduction or prevention of serum protein adsorption. However, there is little evidence that the presence of PEG at the surface of a vehicle actually reduces serum protein binding. Although some studies suggested that PEG on liposome surfaces can reduce specific protein interactions,54, 55 others have indicated that the clearance of liposomal formulations is not correlated with plasma protein binding to liposomes.56 In a recent study by Dos Santos et al.57 it was clearly demonstrated that the adsorption of total and specific serum proteins were not significantly affected by the presence of either PEGylated lipids or cholesterol, in spite of significant differences in their circulation lifetimes. Interestingly, liposomal formulations containing saturated phosphatidylcholine with adsorbed proteins had long circulation lifetimes. These results show that the increase of circulation lifetime cannot be directly associated with either PEGylation or protein binding, despite the observation that PEG-containing formulations typically have longer circulation lifetimes than liposomes lacking PEG. In fact, it has been suggested that the binding of proteins to PEGylated liposomes may benefit the prolonged circulation time of the vectors to some extent. Other studies have shown that PEG on the surface can result in a “dysopsonization” effect by which binding of proteins to a PEG-coated surface could reduce uptake by target cells.58–60

Regardless of the role of protein adsorption, PEGylation has a clear benefit to circulation time after intravenous administration, and thus it is commonly used for delivery studies in animal models. However, PEGylation is known to interfere with trafficking involved in intracellular delivery, and render vectors more susceptible to agitation-induced damage during processing.52, 61–65 Other studies have reported that PEGylation significantly lowers the cellular interaction and uptake of the lipid nanoparticles, resulting in reduced biological activity.66, 67 Similarly, Remaut et al.68 observed that PEGylation lowers the transfection efficiency of DOTAP/DOPE liposomes carrying nuclease stable phosphothioate ONs (PS-ONs) or nuclease sensitive phosphodiester ONs (PO-ONs). Furthermore, the authors concluded that the PEG chains inhibited the endosomal escape of the degraded ONs, which consequently caused a dramatic decrease in transfection efficiency. To overcome these shortcomings of PEGylation, strategies such as using PEG-chains that are removable in the endosomal compartment have been adopted in order to allow endosomal escape. It is known that the extent of PEGylation and the rates at which particles lose PEG shielding have significant effects on formulation pharmacokinetics.69 One of the approaches is to adopt a pH-sensitive linker between the PEG moiety and lipid. The PEG chains are then removed in the endosomal compartment due to acid-catalyzed hydrolysis.70, 71 Although this strategy could potentially be used to trigger PEG release and cell uptake in hypoxic tumors, the pH of such environments is not sufficiently low (pH ≈ 6.5) to efficiently trigger hydrolysis of the PEG chains. A more promising tactic utilizes the matrix metalloproteinase that is present at high levels in the extracellular environment of the tumor to release PEG from the vector.72 Another approach is to exchange PEG–lipid by modulating the hydrophobicity of the PEG–lipid conjugate. This can be achieved by varying the length of the alkyl chain of the lipid anchor73 because the lipid portion of the conjugate determines its affinity for the lipid delivery vehicle. This approach has been described as “programmed release,” but there is no distinct trigger to release the PEG–lipid conjugate at the target site.

It is worth pointing out that PEGylation of soluble biomolecules (e.g., peptides, small proteins, ribozymes, siRNA, antisense) differs fundamentally from the complications encountered by particulate formulations described above. While PEGylation is used to extend circulation times in both cases, the conjugation of PEG to soluble molecules is designed to increase their molecular weight above the threshold for glomerular filtration and urinary excretion. Therefore, it is critical that discussions of the merits of PEGylation distinguish between these two distinct mechanisms of extending circulation times.

ENCAPSULATION OF NUCLEIC ACIDS FOR EFFICIENT DELIVERY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DNA VERSUS siRNA
  5. SERUM STABILITY AND PEGYLATION
  6. ENCAPSULATION OF NUCLEIC ACIDS FOR EFFICIENT DELIVERY
  7. TARGETED DELIVERY OF NUCLEIC ACIDS
  8. BIODISTRIBUTION AND IMMUNOGENICITY
  9. PERSPECTIVES/PROSPECTIVES
  10. Acknowledgements
  11. REFERENCES

The landmark paper by Felgner et al.19 in 1987 demonstrated the ability of cationic agents to bind DNA, and electrostatic interactions continue to be exploited to associate nucleic acids with current delivery systems. It should be recognized that cationic agents also facilitate interactions with anionic moieties on the cell surface (e.g., proteins), and the ability of cationic lipids to target chemotherapeutics to tumor vasculature is being developed by MediGene as a commercial product.74–76 In addition to creating a positive surface charge that promotes cell and serum protein binding, cationic components are typically toxic even when biodegradable linkages are employed. It follows that the ideal gene delivery vector would not rely on cationic components to foster an interaction with the nucleic acid. Such an approach would not only avoid the problems associated with the toxicity and protein binding, but would also facilitate the release of the nucleic acid from the delivery vehicle within the cell. In order to achieve this goal, encapsulation of the payload (i.e., nucleic acids) inside the delivery vehicle is a promising strategy, which would also prevent access of nucleases to the encapsulated cargo (assuming that the delivery vehicle remains intact during delivery to the target site). If one reads the literature, it is easy to conclude that “encapsulation” is routinely achieved with virtually all delivery systems. This becomes somewhat of a semantic argument regarding the definition of “encapsulated,” and this term is now commonly used to refer to any cargo (e.g., DNA or RNA) that cannot be degraded by nucleases or separated from the delivery vehicle by washing. In this sense, nucleic acids that are associated with the delivery vehicle by whatever means (e.g., surface adsorption, electrostatic and/or hydrophobic interactions) are described as “encapsulated,” and there is no distinction made among “encapsulated,” “associated,” and “complexed.” The Merriam–Webster dictionary defines “encapsulate” as “to enclose in or as if in a capsule,” which clearly implies the lack of an interaction with the encapsulating material. This definition is consistent with the traditional use of the term in liposomal studies wherein encapsulation refers to molecules that are enclosed within lipid bilayers and reside inside the aqueous interior of liposomes. In this classic definition, the DNA or RNA would be essentially “free-floating” in the aqueous space enclosed by the liposome, not exposed to the exterior surface, and would freely diffuse away from the delivery vehicle upon disruption/fusion; highly desirable attributes for intracellular delivery. Therefore, it is impossible to achieve nucleic acid encapsulation (by the traditional definition) in delivery vehicles that employ cationic components.

In the case of polymers such as poly(lactic-co-glycolic acid) (PLGA) that are used for gene delivery, cationic components are often avoided, but DNA or RNA is embedded in the polymer matrix rather than encapsulated. While this approach has many of the same advantages as encapsulation (e.g., nuclease protection, no interaction with the encapsulating material, release of naked nucleic acid), problems associated with degradation within the micro/nano spheres are significant, especially considering that nucleic acids can reside within these systems for weeks prior to release. Although this latter property could be a significant advantage of these systems, prolonged release will require that nucleic acids be stable within the polymer matrix for extended periods at body temperature. Furthermore, efficient entrapment within the polymer matrix during micro/nano sphere preparation typically requires an association with the hydrophobic polymer, which ultimately contributes to the extended release.77 For this reason, it is advantageous to formulate nucleic acids with cationic components that have nonpolar moieties which can associate with the PLA or PLGA matrix. In addition, the cationic components help to compact the DNA so that it can be embedded within nanospheres that are small enough to allow efficient cellular uptake. Some studies have been able to entrap plasmids within very small nanospheres without the use of cationic components.78 Presumably, the ability of polymers at high concentration to condense DNA contributes to their incorporation within such small delivery systems,79, 80 and the presence of a plasmid (especially at pharmaceutically acceptable loading capacities) likely has a significant effect on the polymer matrix and consequent release kinetics.

Considering the large size and polyanionic nature of DNA and RNA molecules, the conventional method of loading small molecule drugs into liposomes cannot be applied because nucleic acids are unable to cross lipid bilayers by diffusion.81 Loading of plasmid DNA has been made possible by a detergent-dialysis approach for the formation of liposomal DNA carriers known as stabilized plasmid–lipid particles (SPLP).43, 64 SPLP are formed from mixtures of plasmid and lipids (cationic and zwitterionic) by a detergent-dialysis procedure involving octyl-glucopyranoside (OGP). Details of experimental procedures for small- and large-scale production of SPLP are outlined elsewhere.82 The particles are small (about 70 nm), relatively monodisperse, and have been shown to accumulate in distal tumor sites with subsequent gene expression in mouse tumor models following intravenous injection.83 Meanwhile Maurer et al.84 developed a new formulation that utilizes an ionizable lipid [1,2-dioleoyl-3-dimethylammonium propane (DODAP)] and an ethanol-containing buffer for loading large quantities of antisense oligonucleotide in lipid vesicles. The resulting particle is known as a “stabilized antisense-lipid particle” or SALP. The SALP is a multilamellar vesicle with a small diameter (70–120 nm), which exhibits extended circulation half-life, ranging from 5 to 6 h for particles formed with PEG-CerC14 to 10 to 12 h for particles formed with PEG-CerC20.84, 85 Essentially, this same loading technology has been broadened to include other nucleic acids (SNALP—stable nucleic acid lipid particles) and has been used to successfully deliver siRNA in nonhuman primates.86, 87 This approach was originally developed by Protiva Biotherapeutics (now Tekmira) and is currently being employed by Alnylam Pharmaceuticals for some of their clinical trials on siRNA delivery. More recently, a modified SPLP method has been developed to load precondensed plasmid DNA within a lipid bilayer using a controlled mixing process.88 This method allows for the incorporation of either poly-L-lysine or poly(ethyleneimine; PEI) to condense the DNA, which results in “precondensed stable plasmid lipid particles” (pSPLPs) with small size (104 ± 3 nm) and low surface charge. Furthermore, both SPLP and PEI-pSPLP formulations exhibited significant luciferase expression in tumors and low levels of gene expression in liver and lung.88

Encapsulation of nucleic acids offers many advantages for delivery, and a process, which does not rely on cationic components would be a significant breakthrough for the field. Unfortunately, the current techniques described above still employ some cationic lipids in the formulation, and thus raise concerns about opsonization, toxicity, and release of the nucleic acid inside the cell. However, attempts to encapsulate DNA without utilizing cationic lipids have proven to be inefficient.89 One study by Bailey and Sullivan90 employed calcium to encapsulate DNA in zwitterionic lipids at reasonably high efficiencies, albeit at low DNA-to-lipid ratios. Other approaches have utilized the nucleic acid as a nucleation site for liposome formation, but have had to rely on electrostatic interactions and the use of cationic lipids.91 An interesting alternative has been investigated by Fernandez et al.92 in which association of vehicle components with the nucleic acid is accomplished by intercalation instead of electrostatics. While this clever strategy does not require cationic components, the ability of the intercalated components to dissociate from the nucleic acid may be problematic. In contrast, true encapsulation in which the nucleic acid is not bound to the vehicle would offer many advantages, and new methods of encapsulation should be explored. To this end, it should be theoretically possible to form a “shell” of neutral material around nucleic acids, however the nucleic acids would need to be sufficiently small (e.g., condensed DNA, antisense, siRNA) for the size of the resulting particle to be compatible with intracellular delivery. Hopefully, novel methods of physical encapsulation can provide opportunities for nucleic acids to be enveloped within a neutral material without the use of cationic components in the near future.

TARGETED DELIVERY OF NUCLEIC ACIDS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DNA VERSUS siRNA
  5. SERUM STABILITY AND PEGYLATION
  6. ENCAPSULATION OF NUCLEIC ACIDS FOR EFFICIENT DELIVERY
  7. TARGETED DELIVERY OF NUCLEIC ACIDS
  8. BIODISTRIBUTION AND IMMUNOGENICITY
  9. PERSPECTIVES/PROSPECTIVES
  10. Acknowledgements
  11. REFERENCES

It has been known for decades that liposomes with extended circulation times accumulate in tumors.53 In any discussion of “targeting,” it is important to distinguish between accumulation/localization at the target tissue from uptake/internalization by the target cells. Most studies simply quantify plasmid/siRNA levels in tissues without determining the extent of uptake by target cells. As mentioned above, PEGylation has been the predominant strategy employed to prolong circulation times, but the influence of PEGylation on cellular uptake and intracellular trafficking of particles compromises the ultimate delivery efficiency. Recent studies by Li et al.67 on siRNA delivery concluded that PEGylation reduces delivery efficiency in cell culture by approximately 10-fold, and in vivo studies have also observed decreased tumor accumulation with PEGylated gene delivery systems.93 Additional studies have demonstrated that enhancement of “passive accumulation” via the enhanced permeation and retention effect (EPR) requires targeting ligands to be attached to the distal end of the PEGylated components such that the ligand is projected beyond the PEG “shield” to allow binding to receptors on the target cell surface.94–96 In some cases, the conjugation of the ligand to the delivery vehicle is performed after the loading of nucleic acid, and unreacted ligand is subsequently removed. Such manipulations are suitable for academic studies, but greatly complicate commercial development.

In studies of ligand-assisted uptake, the effect of protein binding on the surface conjugated ligand is typically ignored. However, fouling of the ligand could result in inefficient/inadequate presentation to the receptor on the target cell. Since most studies employing targeting ligands also utilize PEGylation, it is assumed that protein binding is reduced to the extent that ligand fouling is not a concern. However, as discussed above, PEGylation does little to reduce serum protein binding, and thus even ligand conjugated to the distal end of PEGylated components may be fouled by adsorbed protein. The fact that many of these studies report increased cell internalization after intravenous administration suggests that ligands are still able to bind to cell receptors regardless of fouling, but the diminished capacity for binding and uptake is difficult to assess. One interesting alternative approach to utilizing PEG is to employ lipid domains that appear to resist serum protein binding, and thus may be an ideal location for presenting ligands on the particle surface.97 Preliminary studies with this approach indicate that localization of the ligand within lipid domains greatly enhances transfection rates.98

Site-specific delivery of lipoplexes requires the identification of cell surface receptors and the design of appropriate ligands for receptor-mediated endocytosis, however this strategy can traffic the delivery vehicle into the lysosomal pathway, which necessitates efficient endosomal escape. The most popular approach is to use liposomes or polymers with specific ligands such as antibodies, transferrin, folic acid, RGD peptide, anisamide, etc.67, 99–104 Studies have shown that the use of antibodies as targeting ligands are especially effective in this regard because they are large molecules that bind to specific cellular sites.105, 106 This approach clearly demonstrates the proof-of-concept that targeting ligands can be utilized to enhance the uptake of particulate delivery systems by specific cells. However, the use of proteins as targeting ligands compromises one of the primary advantages of employing synthetic delivery systems, that is, the lack of a specific immune response. Although humanized antibodies could potentially be produced to minimize ligand immunogenicity in clinical trials, it should be recognized that the general strategy of attaching delivery systems to PEGylated components that also need to be conjugated to protein ligands would be virtually impossible to realize on a commercial scale. To simply produce such an intricate delivery system, the structural integrity of the protein ligand would need to be preserved during manufacturing, conjugation, processing, and storage. In addition, the cost of the ligand, and the complications associated with conjugation, purification, etc. virtually prohibit such a strategy from commercial development. Recent studies have utilized human transferrin (approximately 80 kDa with significant secondary structure) as a targeting ligand for a cyclodextrin-based siRNA delivery system.103 Although the ability of this system to efficiently deliver siRNA is currently being tested in clinical trials by Calando Pharmaceuticals,107 it is unlikely that a protein-based ligand like transferrin would ultimately be used in a commercial drug delivery product. Furthermore, many academic studies employing protein ligands use exceedingly high levels of ligand to achieve targeted delivery. In this regard, it is common to express the level of ligand in terms of a molar ratio (e.g., lipid/transferrin = 40/1) that disguises the fact that the ligand comprises 50–90% of the delivery system on a weight basis. Although the literature typically depicts these delivery systems as elegant spherical capsules with ligand molecules protruding from the surface, a more accurate illustration would be that of many large protein molecules being randomly adhered to nucleic acid, with the cationic “vehicle” being used as an electrostatic “glue.” Thus, the particles in such preparations would be more accurately depicted as polydisperse molecular complexes of ligand and nucleic acid instead of the tightly organized, highly oriented nanoparticles they are typically portrayed to be.

The choice of spacer/linker is another consideration when developing a targeting ligand conjugate. PEGs with different molecular weight have been commonly used as a linker between the targeting ligand and its anchor.108 For example, a certain distance between the folate moiety and the lipid particles is needed for folate receptor targeting. This is believed to be due to the need for folate to enter the binding pocket of the receptor on the cell surface. Ward et al.109 reported that a folate-linked PEG800 polymer-modified pLL/DNA complex did not lead to a significant increase in transfection in vitro. Additional studies by Leanon et al. optimized the targeting activity of liposomes by modifying the length of the PEG-linker, and found that PEGs as small as a molecular weight of 1000 could function as effective linkers.110 The length of PEG1000 is estimated to be approximately 2.5 nm,111 therefore a spacer length of ≥2.5 nm might be essential for folate receptor targeting. Schaffer et al.112 also found that lengthening the crosslinker arm used to attach EGF (a 74 kDa protein) to the conjugate significantly enhances the specific binding of conjugates to the EGF receptor, which translates into more efficient gene delivery in vitro. More precisely, Handl et al. showed that an optimal linker length of 2.5 ± 1.0 nm is needed for ligand binding to its corresponding G-protein coupled receptors113. In short, studies consistently show the need for a spacer/linker of approximately 2.5 nm for proper presentation and flexibility of the ligand to facilitate binding to the target cell, regardless of whether the ligand is a large protein or a small molecule.

With the goal of developing a commercial product, it is important to keep in mind that the components of the delivery system will need to be manufactured and assembled on a commercial scale that is very different than laboratory- or research-scale production. For this reason, many of the approaches utilized in basic research are not conducive to further development. For example, the cost of manufacturing just a relatively simple liposome product is considerable, and companies who have developed such products have needed to negotiate much lower prices from lipid manufacturers in order to achieve commercial viability. If we consider the added cost of the nucleic acid, the targeting ligand, conjugation/purification, and other molecules to enhance delivery (e.g., nuclear localization sequences, endosomolytic peptides), many of the strategies that appear promising in the literature must be viewed as academic exercises rather than viable technologies that will lead to commercial development. In particular, the cost associated with producing components (e.g., plasmid, targeting ligand) in bacteria, purifying each component, and assembling a vector with uniform physical characteristics is excessive. However, some current enzyme replacement and antibody-based therapies can cost hundreds of thousands of dollars per year, and therefore it is possible that expensive gene therapies may be commercially viable alternatives for some conditions. Regardless, components that can be chemically synthesized (e.g., siRNA, small molecule ligands) offer the best hope for regulatory approval (potentially avoiding regulation as a “biologic”) and commercial development if loading into the delivery system can be sufficiently controlled to achieve tight product specifications.

It is worth mentioning that delivery systems that are touted as “self-assembling” are often very difficult to control on a commercial scale. In practice, many of these self-assembling systems require dilute conditions to achieve reasonably uniform particle characteristics. This significantly complicates bulk manufacturing, and requires that even lab-scale preparations be concentrated prior to administration. To illustrate this point, it should be appreciated that self-assembling preparations of nonviral gene delivery vehicles typically require relatively dilute conditions (e.g., ≈0.1 mg DNA/mL) to achieve sufficiently small and uniform particle size. Therefore, many early studies in gene delivery utilized large volume injections into animal models (e.g., 1 mL bolus injection into a mouse having a blood volume of approximately 2 mL) that ultimately helped fuel speculation that nonviral gene therapy was ready for the clinic. Years later, the ability of hydrodynamic pressure to facilitate transfection is well-documented, and potentially has limited clinical application114, 115. However, this artifact was standard practice for years in the field of gene therapy, and results from studies utilizing this approach were commonly presented at conferences without mention of the extreme conditions needed to achieve such high levels of gene expression in mouse models. Many research studies still utilize this approach for target validation, promoter assessment, or effects of sequence modifications, but realistic evaluation of in vivo gene delivery efficiency requires preparations to be concentrated in order to reduce injection volumes. In this respect it is important to realize that most particle preparations are amenable to concentrating after sufficient time is allowed for self-assembly, and can even be incorporated into processes that are compatible with clinical applications, for example, rehydrating lyophilized samples with reduced volumes of diluent.116, 117

BIODISTRIBUTION AND IMMUNOGENICITY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DNA VERSUS siRNA
  5. SERUM STABILITY AND PEGYLATION
  6. ENCAPSULATION OF NUCLEIC ACIDS FOR EFFICIENT DELIVERY
  7. TARGETED DELIVERY OF NUCLEIC ACIDS
  8. BIODISTRIBUTION AND IMMUNOGENICITY
  9. PERSPECTIVES/PROSPECTIVES
  10. Acknowledgements
  11. REFERENCES

It is well known that cationic lipid- and polymer-based nucleic acid formulations accumulate predominantly in lung and liver after intravenous injection. It has been shown that cationic lipoplexes and polyplexes interact with serum proteins in the circulation, resulting in uptake by the cells of the mononuclear phagocyte system (MPS), classically known as the reticuloendothelial system.118–121 Although some studies have exploited this nonspecific accumulation for lung and liver delivery, it is clear that accumulation of the delivery vehicle in lung or liver does not necessarily indicate that uptake of the nucleic acid into cells of these organs (e.g., hepatocytes) has occurred. In fact, much of the delivery to these sites has proven to be uptake by macrophages and endothelial cells lining the blood vessel instead of the therapeutic target. Regardless, accumulation into lung and liver also presents complications for delivery into other tissues such as tumors. Considering the predominant accumulation of nanoparticles in the liver, it is surprising that studies continue to tout rapid liver accumulation as effective ligand-induced, liver targeting for hepatic diseases. One creative approach to avoiding MPS uptake has exploited drag forces to hinder engulfment by macrophages and to extend the circulation time of polymeric micelles.122

As mentioned above, PEGylation of lipid- and polymer-based formulations has been widely used to improve the circulation lifetime and reduce the lung accumulation after intravenous injection. The mechanism for this appears to be twofold: PEGylation prevents the formation of aggregates that can be trapped in pulmonary capillary beds, and also reduces the surface charge that has been linked to lung accumulation.123, 124 In this respect, it is important to mention that commonly used PEGylated lipids (e.g., DSPE-PEG) are conjugated through the cationic ethanolamine headgroup such that the zwitterionic phospholipid is converted to an anionic lipid. Therefore, the incorporation of these anionic components into a cationic lipid formulation will reduce the positive zeta potential via charge neutralization; an effect that is typically attributed only to steric stabilization by PEG. Accumulation in the liver is thought to be more closely related to particle size because the pore size of liver fenestrae is approximately 100 nm.125 Therefore nanoparticles with diameters <100 nm rapidly accumulate in the liver. In fact, it is often jokingly stated that the definition of a nanoparticle is “anything that accumulates in the liver.” In contrast, these small particles generally do not accumulate in the spleen unless aggregation occurs such that the particle size increases to >300 nm.125 In considering both surface charge and particle size, it is important to recognize that particles can be carefully prepared with the desired properties, but interaction with blood components dramatically changes these properties upon IV administration.36–38, 42 Therefore, the particle properties that are relevant for intravenous delivery are size and surface charge after blood exposure, but these properties are rarely measured and/or considered.126

Although accumulation in the lung, liver, and spleen remains a significant problem for nucleic acid delivery systems in terms of both toxicity (e.g., hepatotoxicity) and poor distribution to the target site (e.g., tumor), studies have relied on the extended circulation times provided by PEGylation to enhance tumor accumulation. However, as discussed above, PEGylation is also known to alter cellular uptake and trafficking. Attempts to overcome this drawback of PEGylation have conjugated a targeting ligand on the distal end of PEGylated lipids to further improve the uptake into target tissues. While this strategy has improved internalization at the cellular level, it has not increased the amount of material that accumulates in the tumor tissue.67, 124, 127 Instead, distribution to the tissues appears to be largely governed by the EPR effect, which is predominantly affected by particle size. From this perspective, delivery vehicles will extravasate through openings of sufficient size, as is evident from the reduced distribution to lung and liver in tumor-bearing as compared to tumor-free animals.67, 93 Further distribution within tissues (i.e., after the initial accumulation due to EPR) appears to be very dependent on particle size, and particles of 100 nm or smaller are better able to distribute throughout tumor.128 Again, because surface properties will largely determine the interaction of the drug delivery vehicle with proteins and other blood components upon injection, properties such as surface charge and hydrophobicity can have a dramatic effect on the size of the delivery vehicle that ultimately circulates and accumulates in tissues. Moreover, even the size of the particle can affect the corona of proteins that adsorb to the particle surface.129 Accordingly, more attention should be paid to particle size and surface properties after exposure to physiological fluids if significant improvements in toxicity, accumulation/localization and internalization are to be achieved. Similarly, studies that monitor accumulation and or activity (e.g., gene expression, silencing) only within the tumor ignore the significant toxicity associated with liver accumulation, which will likely limit therapeutic potential. Researchers need to be upfront about these issues, and avoid burying unflattering data (e.g., elevated liver enzymes) in supplementary material that is difficult to access (i.e., supplementary information available only online can have file sizes >50 MB that discourage downloading).

While efforts are clearly needed to improve delivery efficiency, vector safety must be assured if nucleic acid-based therapeutics are to become commercial products. It is known that although viral vectors possess higher transfection efficiency, safety concerns associated with their use in clinical trials makes the nonviral vector an attractive alternative because synthetic vectors elicit low immunogenicity compared to viral vectors.130 However, it is known that cationic lipoplexes can induce innate immune response and tissue damage after systemic injection.131–136 Intravenously injected cationic lipoplexes induce an acute inflammatory response characterized by cytokine (TNF-α and IFN-γ) release in the serum that is attributed to the unmethylated CpG motifs of the plasmid DNA used in lipoplexes and polyplexes, which is recognized by Toll-like receptors of immune cells.135, 137, 138 Removal of CpG motifs in plasmid DNA greatly reduces cytokine production,136, 139 which should be taken into account when developing a gene delivery system. In this sense, siRNA delivery offers a significant advantage over plasmid-based approaches because the nucleic acid is chemically synthesized and can be modified to reduce immunogenicity and increase nuclease resistance. In particular, studies have shown that incorporation of 2′-O-methyl modifications can virtually eliminate immunogenicity in animal models.140, 141 However, the use of CpG-containing DNA can act as a potent stimulator of the immune system that may be useful for developing improved vaccine technology. In fact, the ability of this innate immune response to shrink tumors was conclusively demonstrated a decade ago by Dow et al.,137 yet studies continue to report tumor shrinkage in response to gene delivery without considering the effects of the immune response elicited by the plasmid backbone. It is also worth noting that sequential administration of the DNA and lipid components (as opposed to a lipoplex) greatly reduces the cytokine response, suggesting that presentation of CpG motifs on a particle (and the nuclease protection associated with complexation/adsorption) is capable of enhancing the immune response.123

Furthermore, it is generally expected that PEGylation of nonviral vectors would attenuate the level of immunogenicity due to its surface shielding. Controversially, evidence from recent studies showed that intravenous administration of PEGylated liposomes causes the second dose of PEGylated liposomes (injected a few days later) to lose their long-circulating profile and accumulate extensively in liver.142–147 This phenomenon is referred to as the “accelerated blood clearance (ABC) phenomenon”142 and has been observed in mice, rats and, a rhesus monkey. Because repeated injections of PEGylated formulation are likely in the case of nucleic acid-based therapies, these findings raise concerns about the development of PEGylated formulations and their clinical application. The mechanism behind this ABC phenomenon has been proposed to involve anti-PEG IgM that is produced in the spleen in response to the first dose of PEGylated liposomes, and subsequently binds to the PEG on a second dose of these liposomes injected several days later.148 Once the PEGylated liposomes reach the spleen, they bind and crosslink to surface immunoglobulins on PEG (or PEGylated liposome)-reactive B cells in that organ, and consequently trigger the production of an anti-PEG IgM. Further investigation indicated that the ABC phenomenon is dependent on the physicochemical properties of injected liposomes as well as the time interval between injection and lipid dose.148 However, it is important to recognize that PEGylated liposome products that are currently on the market (e.g., DOXIL) have not reported any problems or reduced efficacy associated with repeated administration every few weeks.

PERSPECTIVES/PROSPECTIVES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DNA VERSUS siRNA
  5. SERUM STABILITY AND PEGYLATION
  6. ENCAPSULATION OF NUCLEIC ACIDS FOR EFFICIENT DELIVERY
  7. TARGETED DELIVERY OF NUCLEIC ACIDS
  8. BIODISTRIBUTION AND IMMUNOGENICITY
  9. PERSPECTIVES/PROSPECTIVES
  10. Acknowledgements
  11. REFERENCES

Nucleic acid-based therapeutics as a new category of biologics is still in its early stages. Lessons have been learned in the frantic course of gene therapy research during the 1990s when the potential of gene therapy was hyped as the ultimate panacea, which actually made the barriers to success even greater.149 From the FDA database (www.clinicaltrials.gov), there are 80 clinical trials (completed and active) of antisense technology, and 70 clinical trials of plasmid DNA-based therapeutics; most of the latter are vaccine therapy. In fact, two of these plasmid therapeutics are in late-stage phase III clinical trials (Collategene by AnGes Inc and Allovectin-7 by Vical Inc). Since the demonstration of RNA interference in mammalian cells in 2001, siRNA has become the predominant focus for development, and has rapidly progressed to clinical trials. Starting from 2005, there are already 14 clinical trials of siRNA-based therapeutics. It should be recognized that therapeutics based on synthetic oligonucleotides (i.e., antisense and siRNA) are significantly more amenable to development than is plasmid DNA, and the ability to silence specific genes has applications to a wide variety of diseases. However, it was just over a decade ago when gene therapy was in a similar spotlight, and the inability to demonstrate clear efficacy in clinical trials suggests that the hype outpaced the science. We must learn from this experience, and honestly present both promising and problematic issues associated with nucleic acid delivery in the scientific literature.150 To this end, it is counterproductive to present data in an unconventional way to make the results appear more promising. For example, presenting nucleic acid accumulation on a per tumor basis can be misleading if tumor sizes are abnormally large. Conversely, accumulation in very small tumors is misleading if presented as “percent injected dose per gram” because the percent in the tumor is multiplied by a large number (the inverse of a relatively small tumor weight) in order to be presented on a “per gram” basis. Direct comparison of such values to other tissues (e.g., liver, lung) obscures the fact that a large percentage of the injected dose (>90%) accumulates in organs with weights many-fold greater than that of the tumor. Instead, researchers in the field should present their data in a consistent, straightforward manner that does not disguise the problems with current technologies. Clearly, many of the issues revolve around delivery,151 and novel technologies are needed to avoid MPS uptake, accumulate therapeutic levels of nucleic acid at the target site, facilitate distribution within the target tissue, and stimulate internalization by target cells.

It is also worth noting again that PEGylated liposomes have been linked to ABC and an immune response after repeated injection.148 Considering these issues, as well as the decreased cellular interactions and altered trafficking noted above, it would be beneficial to develop alternative strategies to PEGylation for increasing circulation lifetimes. Unfortunately, PEGylation has become the standard method for achieving prolonged circulation, and surface modification by alternative hydrophilic polymers (e.g., poloxamers) are also likely to result in a decreased interaction with the target cell surface and altered trafficking. As discussed above, PEGylation does not appear to reduce protein binding, and thus the mechanism by which PEGylation increases circulation time is unclear. It has been suggested that PEGylation serves to shield the surface charge, and thereby reduce rates of liposome clearance.152 Alternatively, PEGylation could prevent particle–particle interactions that result in rapid clearance in liver and lung, thereby leading to prolonged circulation times.57 In this context, it is interesting that high levels of cholesterol in lipid-based formulations reduce aggregation in serum and result in longer circulation lifetimes.38, 41 Furthermore, work from our lab has shown that high cholesterol formulations accumulate in tumors to levels greater than PEGylated formulations.93 This approach has the potential advantage of avoiding the problems associated with PEGylation, and other alternative strategies for increasing circulation times should be investigated.

In addition to the issues associated with serum stability, targeting, and biodistribution, the significant hurdles to developing a regulated, marketable, pharmaceutical product should not be ignored. It is important to recognize that the late-stage plasmid therapeutics mentioned above are relatively simple products in which development is not complicated by elaborate multi-component delivery systems. While the added cost associated with producing a delivery system may be a significant barrier to commercial development, it must be recognized that dosing and stability also represent formidable obstacles. For example, animal dosing of nucleic acids is typically in the 0.2–1 mg/kg range, translating to a single dose in the tens of milligrams for an adult human. Also, these doses are often administered at least weekly in animal studies, raising concerns about the expense and toxicity surrounding long-term clinical deployment. Furthermore, the stability issues associated with particulate delivery systems are substantial, and more work on the stability of nucleic acid formulations on pharmaceutically relevant timescales is clearly warranted, especially since the stability of RNA and many nucleic acid modifications are virtually unknown.67, 153, 154 In particular, the prospect of developing a two-vial formulation in which vectors are assembled by mixing in the pharmacy is wholly unrealistic considering the significant effects that mixing parameters (e.g., speed, order of mixing, dilution, ionic strength) are known to have on vector properties. To this end, we reiterate that many of the current academic studies on nucleic acid delivery must be viewed as “proof of concept” (even if they advance to clinical trials), with the realization that such elaborate molecular assemblies are incompatible with scale-up and commercial development. Progression to marketed pharmaceutical products will require understanding the mechanisms exploited by the intricate delivery systems currently extolled in the literature, and identifying more robust vehicles with fewer components that can duplicate their delivery performance. In this regard, conjugate/polymeric delivery systems offer a significant advantage by reducing the number of components while endowing the delivery vehicle with multiple functionalities.104, 151, 155–157

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DNA VERSUS siRNA
  5. SERUM STABILITY AND PEGYLATION
  6. ENCAPSULATION OF NUCLEIC ACIDS FOR EFFICIENT DELIVERY
  7. TARGETED DELIVERY OF NUCLEIC ACIDS
  8. BIODISTRIBUTION AND IMMUNOGENICITY
  9. PERSPECTIVES/PROSPECTIVES
  10. Acknowledgements
  11. REFERENCES
  • 1
    Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, Selz F, Hue C, Certain S, Casanova JL, Bousso P, Deist FL, Fischer A. 2000. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288: 669672.
  • 2
    Hyde SC, Southern KW, Gileadi U, Fitzjohn EM, Mofford KA, Waddell BE, Gooi HC, Goddard CA, Hannavy K, Smyth SE, Egan JJ, Sorgi FL, Huang L, Cuthbert AW, Evans MJ, Colledge WH, Higgins CF, Webb AK, Gill DR. 2000. Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis. Gene Ther 7: 11561165.
  • 3
    Nabel GJ, Nabel EG, Yang ZY, Fox BA, Plautz GE, Gao X, Huang L, Shu S, Gordon D, Chang AE. 1993. Direct gene transfer with DNA-liposome complexes in melanoma: Expression, biologic activity, and lack of toxicity in humans. Proc Natl Acad Sci USA 90: 1130711311.
  • 4
    Restifo NP, Ying H, Hwang L, Leitner WW. 2000. The promise of nucleic acid vaccines. Gene Ther 7: 8992.
  • 5
    Dewey RA, Morrissey G, Cowsill CM, Stone D, Bolognani F, Dodd NJ, Southgate TD, Klatzmann D, Lassmann H, Castro MG, Lowenstein PR. 1999. Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: Implications for clinical trials. Nat Med 5: 12561263.
  • 6
    Fox JL. 2000. Gene-therapy death prompts broad civil lawsuit. Nat Biotechnol 18: 1136.
  • 7
    Akinc A, Zumbuehl A, Goldberg M, Leshchiner ES, Busini V, Hossain N, Bacallado SA, Nguyen DN, Fuller J, Alvarez R, Borodovsky A, Borland T, Constien R, de Fougerolles A, Dorkin JR, Narayanannair Jayaprakash K, Jayaraman M, John M, Koteliansky V, Manoharan M, Nechev L, Qin J, Racie T, Raitcheva D, Rajeev KG, Sah DW, Soutschek J, Toudjarska I, Vornlocher HP, Zimmermann TS, Langer R, Anderson DG. 2008. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat Biotechnol 26: 561569.
  • 8
    Akinc A, Goldberg M, Qin J, Dorkin JR, Gamba-Vitalo C, Maier M, Jayaprakash KN, Jayaraman M, Rajeev KG, Manoharan M, Koteliansky V, Rohl I, Leshchiner ES, Langer R, Anderson DG. 2009. Development of lipidoid-siRNA formulations for systemic delivery to the liver. Mol Ther 17: 872879.
  • 9
    2009. Gene therapy clinical trials world wide. J Gene Med http://www.wiley.co.uk/genetherapy/clinical/.
  • 10
    Garber K. 2006. China approves world's first oncolytic virus therapy for cancer treatment. J Natl Cancer Inst 98: 298300.
  • 11
    Gordon EM, Hall FL. 2010. Rexin-G, a targeted genetic medicine for cancer. Expert Opin Biol Ther 10: 819832.
  • 12
    Rainov NG. 2000. A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme. Hum Gene Ther 11: 23892401.
  • 13
    Kirn D. 2001. Oncolytic virotherapy for cancer with the adenovirus dl1520 (Onyx-015): Results of phase I and II trials. Expert Opin Biol Ther 1: 525538.
  • 14
    Cartier N, Hacein-Bey-Abina S, Bartholomae CC, Veres G, Schmidt M, Kutschera I, Vidaud M, Abel U, Dal-Cortivo L, Caccavelli L, Mahlaoui N, Kiermer V, Mittelstaedt D, Bellesme C, Lahlou N, Lefrere F, Blanche S, Audit M, Payen E, Leboulch P, l'Homme B, Bougneres P, Von Kalle C, Fischer A, Cavazzana-Calvo M, Aubourg P. 2009. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326: 818823.
  • 15
    Wagner E. 2008. Converging paths of viral and non-viral vector engineering. Mol Ther 16: 12.
  • 16
    Fraley R, Subramani S, Berg P, Papahadjopoulos D. 1980. Introduction of liposome-encapsulated SV40 DNA into cells. J Biol Chem 255: 1043110435.
  • 17
    Fraley R, Straubinger RM, Rule G, Springer EL, Papahadjopoulos D. 1981. Liposome-mediated delivery of deoxyribonucleic acid to cells: Enhanced efficiency of delivery related to lipid composition and incubation conditions. Biochemistry 20: 69786987.
  • 18
    Straubinger RM, Papahadjopoulos D. 1983. Liposomes as carriers for intracellular delivery of nucleic acids. Methods Enzymol 101: 512527.
  • 19
    Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M. 1987. Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA 84: 74137417.
  • 20
    Fraley RT, Dellaporta SL, Papahadjopoulos D. 1982. Liposome-mediated delivery of tobacco mosaic virus RNA into tobacco protoplasts: A sensitive assay for monitoring liposome-protoplast interactions. Proc Natl Acad Sci USA 79: 18591863.
  • 21
    Malone RW, Felgner PL, Verma IM. 1989. Cationic liposome-mediated RNA transfection. Proc Natl Acad Sci USA 86: 60776081.
  • 22
    Patel MM, Anchordoquy TJ. 2006. Ability of spermine to differentiate between DNA sequences—Preferential stabilization of A-tracts. Biophys Chem 122: 515.
  • 23
    Hansma HG, Golan R, Hsieh W, Lollo CP, Mullen-Ley P, Kwoh D. 1998. DNA condensation for gene therapy as monitored by atomic force microscopy. Nucleic Acids Res 26: 24812487.
  • 24
    Golan R, Pietrasanta LI, Hsieh W, Hansma HG. 1999. DNA toroids: Stages in condensation. Biochemistry 38: 1406914076.
  • 25
    Patel MM, Anchordoquy TJ. 2005. Contribution of hydrophobicity to thermodynamics of ligand-DNA binding and DNA collapse. Biophys J 88: 20892103.
  • 26
    Bloomfield VA. 1991. Condensation of DNA by multivalent cations: Considerations on mechanism. Biopolymers 31: 14711481.
  • 27
    Perales JC, Ferkol T, Molas M, Hanson RW. 1994. An evaluation of receptor-mediated gene transfer using synthetic DNA-ligand complexes. Eur J Biochem 226: 255266.
  • 28
    Perales JC, Ferkol T, Beegen H, Ratnoff OD, Hanson RW. 1994. Gene transfer in vivo: Sustained expression and regulation of genes introduced into the liver by receptor-targeted uptake. Proc Natl Acad Sci USA 91: 40864090.
  • 29
    Patel MM, Anchordoquy TJ. 2010. Significant differences in binding stoichiometry of spermine to RNA than to DNA oligonucleotides. in preparation
  • 30
    Bouxsein NF, McAllister CS, Ewert KK, Samuel CE, Safinya CR. 2007. Structure and gene silencing activities of monovalent and pentavalent cationic lipid vectors complexed with siRNA. Biochemistry 46: 47854792.
  • 31
    Lu JJ, Langer R, Chen J. 2009. A novel mechanism is involved in cationic lipid-mediated functional siRNA delivery. Mol Pharm 6: 763771.
  • 32
    Spagnou S, Miller AD, Keller M. 2004. Lipidic carriers of siRNA: Differences in the formulation, cellular uptake, and delivery with plasmid DNA. Biochemistry 43: 1334813356.
  • 33
    Malek A, Czubayko F, Aigner A. 2008. PEG grafting of polyethylenimine (PEI) exerts different effects on DNA transfection and siRNA-induced gene targeting efficacy. J Drug Target 16: 124139.
  • 34
    Zelphati O, Uyechi LS, Barron LG, Szoka FC, Jr. 1998. Effect of serum components on the physico-chemical properties of cationic lipid/oligonucleotide complexes and on their interactions with cells. Biochim Biophys Acta 1390: 119133.
  • 35
    Thierry AR, Rabinovich P, Peng B, Mahan LC, Bryant JL, Gallo RC. 1997. Characterization of liposome-mediated gene delivery: Expression, stability and pharmacokinetics of plasmid DNA. Gene Ther 4: 226237.
  • 36
    Yang JP, Huang L. 1997. Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA. Gene Ther 4: 950960.
  • 37
    Yang JP, Huang L. 1998. Time-dependent maturation of cationic liposome-DNA complex for serum resistance. Gene Ther 5: 380387.
  • 38
    Zhang Y, Anchordoquy TJ. 2004. The role of lipid charge density in the serum stability of cationic lipid/DNA complexes. Biochim Biophys Acta 1663: 143157.
  • 39
    Ogris M, Brunner S, Schuller S, Kircheis R, Wagner E. 1999. PEGylated DNA/transferrin-PEI complexes: Reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther 6: 595605.
  • 40
    Plank C, Mechtler K, Szoka FC, Jr., Wagner E. 1996. Activation of the complement system by synthetic DNA complexes: A potential barrier for intravenous gene delivery. Hum Gene Ther 7: 14371446.
  • 41
    Crook K, Stevenson BJ, Dubouchet M, Porteous DJ. 1998. Inclusion of cholesterol in DOTAP transfection complexes increases the delivery of DNA to cells in vitro in the presence of serum. Gene Ther 5: 137143.
  • 42
    Li S, Tseng WC, Stolz DB, Wu SP, Watkins SC, Huang L. 1999. Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: Implications for intravenous lipofection. Gene Ther 6: 585594.
  • 43
    Wheeler JJ, Palmer L, Ossanlou M, MacLachlan I, Graham RW, Zhang YP, Hope MJ, Scherrer P, Cullis PR. 1999. Stabilized plasmid-lipid particles: Construction and characterization. Gene Ther 6: 271281.
  • 44
    Wolff JA, Dowty ME, Jiao S, Repetto G, Berg RK, Ludtke JJ, Williams P, Slautterback DB. 1992. Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle. J Cell Sci 103: 12491259.
  • 45
    Hafez IM, Maurer N, Cullis PR. 2001. On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids. Gene Ther 8: 11881196.
  • 46
    Xu Y, Szoka FC, Jr. 1996. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 35: 56165623.
  • 47
    Juliano R, Bauman J, Kang H, Ming X. 2009. Biological barriers to therapy with antisense and siRNA oligonucleotides. Mol Pharm 6: 686695.
  • 48
    Sakurai F, Nishioka T, Saito H, Baba T, Okuda A, Matsumoto O, Taga T, Yamashita F, Takakura Y, Hashida M. 2001. Interaction between DNA-cationic liposome complexes and erythrocytes is an important factor in systemic gene transfer via the intravenous route in mice: The role of the neutral helper lipid. Gene Ther 8: 677686.
  • 49
    Smyth Templeton N. 2003. Cationic liposomes as in vivo delivery vehicles. Curr Med Chem 10: 12791287.
  • 50
    Torchilin VP, Omelyanenko VG, Papisov MI, Bogdanov AA, Jr., Trubetskoy VS, Herron JN, Gentry CA. 1994. Poly(ethylene glycol) on the liposome surface: On the mechanism of polymer-coated liposome longevity. Biochim Biophys Acta 1195: 1120.
  • 51
    Torchilin VP, Shtilman MI, Trubetskoy VS, Whiteman K, Milstein AM, Torchilin VP, Omelyanenko VG, Papisov MI, Bogdanov AA, Jr., Trubetskoy VS, Herron JN, Gentry CA. 1994. Amphiphilic vinyl polymers effectively prolong liposome circulation time in vivo. Biochim Biophys Acta 1195: 181184.
  • 52
    Harvie P, Wong FM, Bally MB. 2000. Use of poly(ethylene glycol)-lipid conjugates to regulate the surface attributes and transfection activity of lipid-DNA particles. J Pharm Sci 89: 652663.
  • 53
    Gabizon A, Papahadjopoulos D. 1988. Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors. Proc Natl Acad Sci USA 85: 69496953.
  • 54
    Chiu GN, Bally MB, Mayer LD. 2001. Selective protein interactions with phosphatidylserine containing liposomes alter the steric stabilization properties of poly(ethylene glycol). Biochim Biophys Acta 1510: 5669.
  • 55
    Li WM, Mayer LD, Bally MB. 2002. Prevention of antibody-mediated elimination of ligand-targeted liposomes by using poly(ethylene glycol)-modified lipids. J Pharmacol Exp Ther 300: 976983.
  • 56
    Allen C, Dos Santos N, Gallagher R, Chiu GN, Shu Y, Li WM, Johnstone SA, Janoff AS, Mayer LD, Webb MS, Bally MB. 2002. Controlling the physical behavior and biological performance of liposome formulations through use of surface grafted poly(ethylene glycol). Biosci Rep 22: 225250.
  • 57
    Dos Santos N, Allen C, Doppen AM, Anantha M, Cox KA, Gallagher RC, Karlsson G, Edwards K, Kenner G, Samuels L, Webb MS, Bally MB. 2007. Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: Relating plasma circulation lifetimes to protein binding. Biochim Biophys Acta 1768: 13671377.
  • 58
    Moghimi SM, Muir IS, Illum L, Davis SS, Kolb-Bachofen V. 1993. Coating particles with a block co-polymer (poloxamine-908) suppresses opsonization but permits the activity of dysopsonins in the serum. Biochim Biophys Acta 1179: 157165.
  • 59
    Moghimi SM, Patel HM. 1989. Serum opsonins and phagocytosis of saturated and unsaturated phospholipid liposomes. Biochim Biophys Acta 984: 384387.
  • 60
    Johnstone SA, Masin D, Mayer L, Bally MB. 2001. Surface-associated serum proteins inhibit the uptake of phosphatidylserine and poly(ethylene glycol) liposomes by mouse macrophages. Biochim Biophys Acta 1513: 2537.
  • 61
    Hyvonen Z, Ronkko S, Toppinen MR, Jaaskelainen I, Plotniece A, Urtti A. 2004. Dioleoyl phosphatidylethanolamine and PEG-lipid conjugates modify DNA delivery mediated by 1,4-dihydropyridine amphiphiles. J Control Release 99: 177190.
  • 62
    Monck MA, Mori A, Lee D, Tam P, Wheeler JJ, Cullis PR, Scherrer P. 2000. Stabilized plasmid-lipid particles: Pharmacokinetics and plasmid delivery to distal tumors following intravenous injection. J Drug Target 7: 439452.
  • 63
    Shi F, Wasungu L, Nomden A, Stuart MC, Polushkin E, Engberts JB, Hoekstra D. 2002. Interference of poly(ethylene glycol)-lipid analogues with cationic-lipid-mediated delivery of oligonucleotides; role of lipid exchangeability and non-lamellar transitions. Biochem J 366: 333341.
  • 64
    Tam P, Monck M, Lee D, Ludkovski O, Leng EC, Clow K, Stark H, Scherrer P, Graham RW, Cullis PR. 2000. Stabilized plasmid-lipid particles for systemic gene therapy. Gene Ther 7: 18671874.
  • 65
    Armstrong TK, Girouard LG, Anchordoquy TJ. 2002. Effects of PEGylation on the preservation of cationic lipid/DNA complexes during freeze-thawing and lyophilization. J Pharm Sci 91: 25492558.
  • 66
    Deshpande MC, Davies MC, Garnett MC, Williams PM, Armitage D, Bailey L, Vamvakaki M, Armes SP, Stolnik S. 2004. The effect of poly(ethylene glycol) molecular architecture on cellular interaction and uptake of DNA complexes. J Control Release 97: 143156.
  • 67
    Li SD, Chono S, Huang L. 2008. Efficient gene silencing in metastatic tumor by siRNA formulated in surface-modified nanoparticles. J Control Release 126: 7784.
  • 68
    Remaut K, Lucas B, Braeckmans K, Demeester J, De Smedt SC. 2007. Pegylation of liposomes favours the endosomal degradation of the delivered phosphodiester oligonucleotides. J Control Release 117: 256266.
  • 69
    Adlakha-Hutcheon G, Bally MB, Shew CR, Madden TD. 1999. Controlled destabilization of a liposomal drug delivery system enhances mitoxantrone antitumor activity. Nat Biotechnol 17: 775779.
  • 70
    Li W, Huang Z, MacKay JA, Grube S, Szoka FC, Jr. 2005. Low-pH-sensitive poly(ethylene glycol; PEG)-stabilized plasmid nanolipoparticles: Effects of PEG chain length, lipid composition and assembly conditions on gene delivery. J Gene Med 7: 6779.
  • 71
    Shin J, Shum P, Thompson DH. 2003. Acid-triggered release via dePEGylation of DOPE liposomes containing acid-labile vinyl ether PEG-lipids. J Control Release 91: 187200.
  • 72
    Hatakeyama H, Akita H, Kogure K, Oishi M, Nagasaki Y, Kihira Y, Ueno M, Kobayashi H, Kikuchi H, Harashima H. 2007. Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid. Gene Ther 14: 6877.
  • 73
    Webb MS, Saxon D, Wong FM, Lim HJ, Wang Z, Bally MB, Choi LS, Cullis PR, Mayer LD. 1998. Comparison of different hydrophobic anchors conjugated to poly(ethylene glycol): Effects on the pharmacokinetics of liposomal vincristine. Biochim Biophys Acta 1372: 272282.
  • 74
    Schuch G. 2005. EndoTAG-1. MediGene. Curr Opin Investig Drugs 6: 12591265.
  • 75
    Eichhorn ME, Luedemann S, Strieth S, Papyan A, Ruhstorfer H, Haas H, Michaelis U, Sauer B, Teifel M, Enders G, Brix G, Jauch KW, Bruns CJ, Dellian M. 2007. Cationic lipid complexed camptothecin (EndoTAG-2) improves antitumoral efficacy by tumor vascular targeting. Cancer Biol Ther 6: 920929.
  • 76
    Eichhorn ME, Ischenko I, Luedemann S, Strieth S, Papyan A, Werner A, Bohnenkamp H, Guenzi E, Preissler G, Michaelis U, Jauch KW, Bruns CJ, Dellian M. 2010. Vascular targeting by EndoTAG-1 enhances therapeutic efficacy of conventional chemotherapy in lung and pancreatic cancer. Int J Cancer 126: 12351245.
  • 77
    Allison SD. 2008. Effect of structural relaxation on the preparation and drug release behavior of poly(lactic-co-glycolic)acid microparticle drug delivery systems. J Pharm Sci 97: 20222035.
  • 78
    Prabha S, Zhou WZ, Panyam J, Labhasetwar V. 2002. Size-dependency of nanoparticle-mediated gene transfection: Studies with fractionated nanoparticles. Int J Pharm 244: 105115.
  • 79
    Lerman LS. 1971. A transition to a compact form of DNA in polymer solutions. Proc Natl Acad Sci USA 68: 18861890.
  • 80
    Evdokimov YM, Platonov AL, Tikhonenko AS, Varshavsky YM. 1972. A compact form of double-stranded DNA in solution. FEBS Lett 23: 180184.
  • 81
    Madden TD, Harrigan PR, Tai LC, Bally MB, Mayer LD, Redelmeier TE, Loughrey HC, Tilcock CP, Reinish LW, Cullis PR. 1990. The accumulation of drugs within large unilamellar vesicles exhibiting a proton gradient: A survey. Chem Phys Lipids 53: 3746.
  • 82
    Fenske DB, MacLachlan I, Cullis PR. 2002. Stabilized plasmid-lipid particles: A systemic gene therapy vector. Methods Enzymol 346: 3671.
  • 83
    Fenske DB, MacLachlan I, Cullis PR. 2001. Long-circulating vectors for the systemic delivery of genes. Curr Opin Mol Ther 3: 153158.
  • 84
    Maurer N, Wong KF, Stark H, Louie L, McIntosh D, Wong T, Scherrer P, Semple SC, Cullis PR. 2001. Spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanol-destabilized cationic liposomes. Biophys J 80: 23102326.
  • 85
    Semple SC, Klimuk SK, Harasym TO, Dos Santos N, Ansell SM, Wong KF, Maurer N, Stark H, Cullis PR, Hope MJ, Scherrer P. 2001. Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: Formation of novel small multilamellar vesicle structures. Biochim Biophys Acta 1510: 152166.
  • 86
    Zimmermann TS, Lee AC, Akinc A, Bramlage B, Bumcrot D, Fedoruk MN, Harborth J, Heyes JA, Jeffs LB, John M, Judge AD, Lam K, McClintock K, Nechev LV, Palmer LR, Racie T, Rohl I, Seiffert S, Shanmugam S, Sood V, Soutschek J, Toudjarska I, Wheat AJ, Yaworski E, Zedalis W, Koteliansky V, Manoharan M, Vornlocher HP, MacLachlan I. 2006. RNAi-mediated gene silencing in non-human primates. Nature 441: 111114.
  • 87
    Frank-Kamenetsky M, Grefhorst A, Anderson NN, Racie TS, Bramlage B, Akinc A, Butler D, Charisse K, Dorkin R, Fan Y, Gamba-Vitalo C, Hadwiger P, Jayaraman M, John M, Jayaprakash KN, Maier M, Nechev L, Rajeev KG, Read T, Rohl I, Soutschek J, Tan P, Wong J, Wang G, Zimmermann T, de Fougerolles A, Vornlocher HP, Langer R, Anderson DG, Manoharan M, Koteliansky V, Horton JD, Fitzgerald K. 2008. Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates. Proc Natl Acad Sci USA 105: 1191511920.
  • 88
    Heyes J, Palmer L, Chan K, Giesbrecht C, Jeffs L, MacLachlan I. 2007. Lipid encapsulation enables the effective systemic delivery of polyplex plasmid DNA. Mol Ther 15: 713720.
  • 89
    Jeffs LB, Palmer LR, Ambegia EG, Giesbrecht C, Ewanick S, MacLachlan I. 2005. A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA. Pharm Res 22: 362372.
  • 90
    Bailey AL, Sullivan SM. 2000. Efficient encapsulation of DNA plasmids in small neutral liposomes induced by ethanol and calcium. Biochim Biophys Acta 1468: 239252.
  • 91
    Gao K, Huang L. 2009. Nonviral methods for siRNA delivery. Mol Pharm 6: 651658.
  • 92
    Fernandez CAK, Baumhover N, Rice K. 2009. Gene delivery using polyacridne-PEG-peptides. Annual Meeting of American Association of Pharmaceutical Scientists poster T3050.
  • 93
    Zhang Y, Bradshaw-Pierce EL, Delille A, Gustafson DL, Anchordoquy TJ. 2008. In vivo comparative study of lipid/DNA complexes with different in vitro serum stability: Effects on biodistribution and tumor accumulation. J Pharm Sci 97: 237250.
  • 94
    Woodle MC, Scaria P, Ganesh S, Subramanian K, Titmas R, Cheng C, Yang J, Pan Y, Weng K, Gu C, Torkelson S. 2001. Sterically stabilized polyplex: Ligand-mediated activity. J Control Release 74: 309311.
  • 95
    Ogris M, Walker G, Blessing T, Kircheis R, Wolschek M, Wagner E. 2003. Tumor-targeted gene therapy: Strategies for the preparation of ligand-polyethylene glycol-polyethylenimine/DNA complexes. J Control Release 91: 173181.
  • 96
    Verbaan FJ, Oussoren C, Snel CJ, Crommelin DJ, Hennink WE, Storm G. 2004. Steric stabilization of poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes mediates prolonged circulation and tumor targeting in mice. J Gene Med 6: 6475.
  • 97
    Xu L, Anchordoquy TJ. 2008. Cholesterol domains in cationic lipid/DNA complexes improve transfection. Biochim Biophys Acta 1778: 21772181.
  • 98
    Xu L, Anchordoquy TJ. 2010. Effect of cholesterol nanodomains on the targeting of lipid-based gene delivery in cultured cells. Mol Pharm In press.
  • 99
    Xu L, Huang CC, Huang W, Tang WH, Rait A, Yin YZ, Cruz I, Xiang LM, Pirollo KF, Chang EH. 2002. Systemic tumor-targeted gene delivery by anti-transferrin receptor scFv-immunoliposomes. Mol Cancer Ther 1: 337346.
  • 100
    Bruckheimer E, Harvie P, Orthel J, Dutzar B, Furstoss K, Mebel E, Anklesaria P, Paul R. 2004. In vivo efficacy of folate-targeted lipid-protamine-DNA (LPD-PEG-Folate) complexes in an immunocompetent syngeneic model for breast adenocarcinoma. Cancer Gene Ther 11: 128134.
  • 101
    Harvie P, Dutzar B, Galbraith T, Cudmore S, O'Mahony D, Anklesaria P, Paul R. 2003. Targeting of lipid-protamine-DNA (LPD) lipopolyplexes using RGD motifs. J Liposome Res 13: 231247.
  • 102
    Mamot C, Drummond DC, Noble CO, Kallab V, Guo Z, Hong K, Kirpotin DB, Park JW. 2005. Epidermal growth factor receptor-targeted immunoliposomes significantly enhance the efficacy of multiple anticancer drugs in vivo. Cancer Res 65: 1163111638.
  • 103
    Davis ME. 2009. The first targeted delivery of siRNA in humans via a self-assembling, cyclodextrin polymer-based nanoparticle: From concept to clinic. Mol Pharm 6: 659668.
  • 104
    Fernandez CA, Rice KG. 2009. Engineered nanoscaled polyplex gene delivery systems. Mol Pharm 6: 12771289.
  • 105
    Lukyanov AN, Gao Z, Torchilin VP. 2003. Micelles from polyethylene glycol/phosphatidylethanolamine conjugates for tumor drug delivery. J Control Release 91: 97102.
  • 106
    Torchilin V. 2004. Polymeric immunomicelles: Carriers of choice for targeted delivery of water-insoluble pharmaceuticals. Drug Deliv Technol 4.
  • 107
    Davis ME, Zuckerman JE, Choi CH, Seligson D, Tolcher A, Alabi CA, Yen Y, Heidel JD, Ribas A. 2010. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464: 10671070.
  • 108
    Hattori Y, Maitani Y. 2005. Folate-linked lipid-based nanoparticle for targeted gene delivery. Curr Drug Deliv 2: 243252.
  • 109
    Ward CM, Pechar M, Oupicky D, Ulbrich K, Seymour LW. 2002. Modification of pLL/DNA complexes with a multivalent hydrophilic polymer permits folate-mediated targeting in vitro and prolonged plasma circulation in vivo. J Gene Med 4: 536547.
  • 110
    Leamon CP, Cooper SR, Hardee GE. 2003. Folate-liposome-mediated antisense oligodeoxynucleotide targeting to cancer cells: Evaluation in vitro and in vivo. Bioconjug Chem 14: 738747.
  • 111
    Knoll D, Hermans J. 1983. Polymer-protein interactions. Comparison of experiment and excluded volume theory. J Biol Chem 258: 57105715.
  • 112
    Schaffer DV, Lauffenburger DA. 1998. Optimization of cell surface binding enhances efficiency and specificity of molecular conjugate gene delivery. J Biol Chem 273: 2800428009.
  • 113
    Handl HL, Sankaranarayanan R, Josan JS, Vagner J, Mash EA, Gillies RJ, Hruby VJ. 2007. Synthesis and evaluation of bivalent NDP-alpha-MSH(7) peptide ligands for binding to the human melanocortin receptor 4 (hMC4R). Bioconjug Chem 18: 11011109.
  • 114
    Sebestyen MG, Budker VG, Budker T, Subbotin VM, Zhang G, Monahan SD, Lewis DL, Wong SC, Hagstrom JE, Wolff JA. 2006. Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules. J Gene Med 8: 852873.
  • 115
    Lewis DL, Wolff JA. 2007. Systemic siRNA delivery via hydrodynamic intravascular injection. Adv Drug Deliv Rev 59: 115123.
  • 116
    Anchordoquy TJ, Armstrong TK, Molina MC. 2005. Low molecular weight dextrans stabilize nonviral vectors during lyophilization at low osmolalities: Concentrating suspensions by rehydration to reduced volumes. J Pharm Sci 94: 12261236.
  • 117
    Zillies JC, Zwiorek K, Hoffmann F, Vollmar A, Anchordoquy TJ, Winter G, Coester C. 2008. Formulation development of freeze-dried oligonucleotide-loaded gelatin nanoparticles. Eur J Pharm Biopharm 70: 514521.
  • 118
    Patel HM. 1992. Serum opsonins and liposomes: Their interaction and opsonophagocytosis. Crit Rev Ther Drug Carrier Syst 9: 3990.
  • 119
    Cullis PR, Chonn A, Semple SC. 1998. Interactions of liposomes and lipid-based carrier systems with blood proteins: Relation to clearance behaviour in vivo. Adv Drug Deliv Rev 32: 317.
  • 120
    Kamps JA, Scherphof GL. 1998. Receptor versus non-receptor mediated clearance of liposomes. Adv Drug Deliv Rev 32: 8197.
  • 121
    Ishida T, Harashima H, Kiwada H. 2001. Interactions of liposomes with cells in vitro and in vivo: Opsonins and receptors. Curr Drug Metab 2: 397409.
  • 122
    Geng Y, Discher DE. 2005. Hydrolytic degradation of poly(ethylene oxide)-block-polycaprolactone worm micelles. J Am Chem Soc 127: 1278012781.
  • 123
    Zhang JS, Liu F, Huang L. 2005. Implications of pharmacokinetic behavior of lipoplex for its inflammatory toxicity. Adv Drug Deliv Rev 57: 689698.
  • 124
    Li SD, Huang L. 2008. Pharmacokinetics and biodistribution of nanoparticles. Mol Pharm 5: 496504.
  • 125
    Liu D, Mori A, Huang L. 1992. Role of liposome size and RES blockade in controlling biodistribution and tumor uptake of GM1-containing liposomes. Biochim Biophys Acta 1104: 95101.
  • 126
    Li SD, Huang L. 2008. Targeted delivery of siRNA by nonviral vectors: Lessons learned from recent advances. Curr Opin Investig Drugs 9: 13171323.
  • 127
    Kirpotin DB, Drummond DC, Shao Y, Shalaby MR, Hong K, Nielsen UB, Marks JD, Benz CC, Park JW. 2006. Antibody targeting of long-circulating lipidic nanoparticles does not increase tumor localization but does increase internalization in animal models. Cancer Res 66: 67326740.
  • 128
    Perrault SD, Walkey C, Jennings T, Fischer HC, Chan WC. 2009. Mediating tumor targeting efficiency of nanoparticles through design. Nano Lett 9: 19091915.
  • 129
    Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson KA. 2008. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc Natl Acad Sci USA 105: 1426514270.
  • 130
    Li SD, Huang L. 2007. Non-viral is superior to viral gene delivery. J Control Release 123: 181183.
  • 131
    Li S, Wu SP, Whitmore M, Loeffert EJ, Wang L, Watkins SC, Pitt BR, Huang L. 1999. Effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors. Am J Physiol 276: L796L804.
  • 132
    Whitmore M, Li S, Huang L. 1999. LPD lipopolyplex initiates a potent cytokine response and inhibits tumor growth. Gene Ther 6: 18671875.
  • 133
    Sakurai F, Terada T, Yasuda K, Yamashita F, Takakura Y, Hashida M. 2002. The role of tissue macrophages in the induction of proinflammatory cytokine production following intravenous injection of lipoplexes. Gene Ther 9: 11201126.
  • 134
    Liu F, Shollenberger LM, Huang L. 2004. Non-immunostimulatory nonviral vectors. FASEB J 18: 17791781.
  • 135
    Zhao H, Hemmi H, Akira S, Cheng SH, Scheule RK, Yew NS. 2004. Contribution of Toll-like receptor 9 signaling to the acute inflammatory response to nonviral vectors. Mol Ther 9: 241248.
  • 136
    Sakurai H, Sakurai F, Kawabata K, Sasaki T, Koizumi N, Huang H, Tashiro K, Kurachi S, Nakagawa S, Mizuguchi H. 2007. Comparison of gene expression efficiency and innate immune response induced by Ad vector and lipoplex. J Control Release 117: 430437.
  • 137
    Dow SW, Fradkin LG, Liggitt DH, Willson AP, Heath TD, Potter TA. 1999. Lipid-DNA complexes induce potent activation of innate immune responses and antitumor activity when administered intravenously. J Immunol 163: 15521561.
  • 138
    Yasuda K, Ogawa Y, Yamane I, Nishikawa M, Takakura Y. 2005. Macrophage activation by a DNA/cationic liposome complex requires endosomal acidification and TLR9-dependent and -independent pathways. J Leukoc Biol 77: 7179.
  • 139
    Hyde SC, Pringle IA, Abdullah S, Lawton AE, Davies LA, Varathalingam A, Nunez-Alonso G, Green AM, Bazzani RP, Sumner-Jones SG, Chan M, Li H, Yew NS, Cheng SH, Boyd AC, Davies JC, Griesenbach U, Porteous DJ, Sheppard DN, Munkonge FM, Alton EW, Gill DR. 2008. CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression. Nat Biotechnol 26: 549551.
  • 140
    Judge AD, Bola G, Lee AC, MacLachlan I. 2006. Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo. Mol Ther 13: 494505.
  • 141
    Judge A, MacLachlan I. 2008. Overcoming the innate immune response to small interfering RNA. Hum Gene Ther 19: 111124.
  • 142
    Dams ET, Laverman P, Oyen WJ, Storm G, Scherphof GL, van Der Meer JW, Corstens FH, Boerman OC. 2000. Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes. J Pharmacol Exp Ther 292: 10711079.
  • 143
    Laverman P, Carstens MG, Boerman OC, Dams ET, Oyen WJ, van Rooijen N, Corstens FH, Storm G. 2001. Factors affecting the accelerated blood clearance of polyethylene glycol-liposomes upon repeated injection. J Pharmacol Exp Ther 298: 607612.
  • 144
    Semple SC, Harasym TO, Clow KA, Ansell SM, Klimuk SK, Hope MJ. 2005. Immunogenicity and rapid blood clearance of liposomes containing polyethylene glycol-lipid conjugates and nucleic Acid. J Pharmacol Exp Ther 312: 10201026.
  • 145
    Ishida T, Ichihara M, Wang X, Yamamoto K, Kimura J, Majima E, Kiwada H. 2006. Injection of PEGylated liposomes in rats elicits PEG-specific IgM, which is responsible for rapid elimination of a second dose of PEGylated liposomes. J Control Release 112: 1525.
  • 146
    Ishida T, Wang X, Shimizu T, Nawata K, Kiwada H. 2007. PEGylated liposomes elicit an anti-PEG IgM response in a T cell-independent manner. J Control Release 122: 349355.
  • 147
    Wang X, Ishida T, Kiwada H. 2007. Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes. J Control Release 119: 236244.
  • 148
    Ishida T, Kiwada H. 2008. Accelerated blood clearance (ABC) phenomenon upon repeated injection of PEGylated liposomes. Int J Pharm 354: 5662.
  • 149
    2009. Gene therapy deserves a fresh chance. Nature 461: 1173.
  • 150
    2009. Mind the spin. Nature 461: 1174.
  • 151
    Baker M. 2010. RNA interference: Homing in on delivery. Nature 464: 12251228.
  • 152
    Levchenko TS, Rammohan R, Lukyanov AN, Whiteman KR, Torchilin VP. 2002. Liposome clearance in mice: The effect of a separate and combined presence of surface charge and polymer coating. Int J Pharm 240: 95102.
  • 153
    Anchordoquy TJ, Armstrong TK, Molina MdC, Allison SD, Zhang Y, Patel MM, Lentz YK, Koe GS. 2004. Formulation considerations for DNA-based therapeutics. In: LuDR, ØieS, editors. Cellular drug delivery: Principle and practice, Totowa, NJ: Humana Press.
  • 154
    de Fougerolles AR. 2008. Delivery vehicles for small interfering RNA in vivo. Hum Gene Ther 19: 125132.
  • 155
    Soutschek J, Akinc A, Bramlage B, Charisse K, Constien R, Donoghue M, Elbashir S, Geick A, Hadwiger P, Harborth J, John M, Kesavan V, Lavine G, Pandey RK, Racie T, Rajeev KG, Rohl I, Toudjarska I, Wang G, Wuschko S, Bumcrot D, Koteliansky V, Limmer S, Manoharan M, Vornlocher HP. 2004. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432: 173178.
  • 156
    Rozema DB, Lewis DL, Wakefield DH, Wong SC, Klein JJ, Roesch PL, Bertin SL, Reppen TW, Chu Q, Blokhin AV, Hagstrom JE, Wolff JA. 2007. Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes. Proc Natl Acad Sci USA 104: 1298212987.
  • 157
    Meyer M, Dohmen C, Philipp A, Kiener D, Maiwald G, Scheu C, Ogris M, Wagner E. 2009. Synthesis and biological evaluation of a bioresponsive and endosomolytic siRNA-polymer conjugate. Mol Pharm 6: 752762.