To Be Targeted: Is the Magic Bullet Concept a Viable Option for Synthetic Nucleic Acid Therapeutics?

Abstract

Nucleic acids offer the possibility of tailor-made, individualized treatments for genetic disorders, infectious diseases, and cancer. As an alternative to viral vectors, synthetic delivery systems have a potentially improved safety profile, but often lack sufficient efficiency especially when applied in vivo. Receptor targeting of synthetic vectors can improve the specificity of the vector and increase the efficiency of nucleic acid delivery to the target site. This review covers recent concepts for targeted DNA and RNA delivery to organs like liver and lung, and also to solid cancers. Syntheses and applications of delivery systems targeted with proteins, peptides, and small molecules as ligands coupled to polymeric or lipidic nucleic acid carriers are reviewed. Therapeutic concepts for treatment of genetic and infectious diseases are explained. Systemic treatment regimens of metastasized malignancies in combination with chemotherapy and radiation have already been successfully applied in preclinical studies. In addition, a first clinical study in the human application of a targeted synthetic carrier has been performed.

Introduction

D elivery is the major bottleneck in successful implementation of therapeutic nucleic acids as medical agents. Synthetic carriers have been considered because of their flexibility in the transfer of a large variety of different sizes and types of nucleic acids, ranging from small modified oligonucleotides and aptamers to plasmid DNA (pDNA) and bacterial artificial chromosome (BAC) vectors, from small interfering RNA (siRNA) and microRNAs to chemically modified messenger RNA (mRNA) and various other forms of RNA molecules (Krützfeldt et al., 2005; Shir et al., 2006; Ulrich et al., 2006; Aagaard and Rossi, 2007; Behlke, 2008; Poeck et al., 2008; Yin et al., 2008; Kota et al., 2009; Veedu and Wengel, 2009; Frohlich and Wagner, 2010; Kormann et al., 2011).

Receptor targeting (also called “biochemical targeting”) of synthetic vector formulations was first investigated nearly 25 years ago (Wu and Wu, 1987). Since then, the positive effects of targeting ligands (i.e., usually an enhanced gene expression in surface receptor–expressing target cells) have been observed in multiple studies with nucleic acid formulations. Most findings were made in cell culture experiments in vitro; far fewer studies, however, recorded proof of receptor targeting effects in vivo. Thus, questions on the potency of the biochemical targeting strategy remain. Limitations and bottlenecks beyond simple receptor–ligand interactions have to be identified and resolved.

The myth of magic bullets has inspired pharmacists for a long time, and more recently also gene therapy scientists. However, simply decorating nanosystems with receptor-targeting ligands is far from constructing a nano-cruise missile that finds its target in a sensing and reactive mode. Receptor targeting also is not chemotaxis; ligands do not seek their receptors. It is only a biochemical recognition process once the ligand on the vector has already reached the cell-surface site. Clearly, ligand–receptor specificity and affinity, as well as multivalency (in the case of multiligand nanosystems), play an important role. Before that happens, a transport to the target tissue or cell has to occur. This more mechanical transport process, in the case of intravenous applications, is mediated by the blood flow in the circulation and, as such, is strongly influenced (and also limited) by the special architecture of the vasculature. In other types of application, other physical forces may be involved, for example, delivery by aerosol inhalation into the lung, by application of hydrodynamic pressure, by magnetic forces and/or ultrasound, or (somewhat related to the bullet concept) by the gene gun. Physical integrity and lack of inadvertent interactions with nontarget biostructures are critical in this phase of the delivery process. Bioreversible shielding of nanoparticles [for example, with polyethylene glycol (PEG)] is a major solution strategy for this delivery barrier.

Once ligand–receptor recognition happens, not only are specificity and affinity of the ligand–receptor interaction important, but also the biological type and cellular fate of the selected receptor/ligand pair. Multivalency and density of ligands on the nanoparticles, the nanoparticle size, and the presence/absence of competing physiological receptor ligands may impact the fate of the bound nanoparticle. In particular, the extent and kinetics of cellular uptake can strongly differ depending on receptor activation. Intracellular uptake processes (clathrin-mediated or caveolae-mediated endocytosis, macropinocytosis, phagocytosis, and others) can be influenced by multiligand–multireceptor cross-linkage, nanoparticle size, and cell type. Commonly, intracellular uptake and intracellular transportation are active biological processes with mechanical forces provided by various components of the cytoskeleton.

Last but not least, intracellular compartment barriers, like the escape from endolysosomes or (in the case of DNA-based gene vectors) also nuclear uptake, have to be mediated by the carrier formulation, releasing the nucleic acid in bioactive form within the proper compartment.

This communication reviews current developments of synthetic nucleic acid carriers with a special focus on nanosystems containing targeted ligands and surface shielding. Their application in biological models and therapeutic strategies are reviewed. No attempt was made to cover the abundance of previous important investigations of more than two decades, which are well documented in other reviews (Aigner, 2006; Ogris, 2006; Park et al., 2006; Li and Szoka, 2007; Behlke, 2008; De Fougerolles, 2008; Philipp et al., 2008; Schaffert and Wagner, 2008; Wu and McMillan, 2009; Guo et al., 2010; Li and Huang, 2010). In the following sections, targeted delivery to organs, such as the liver, lung, and brain, and also disease sites, including inflammation, virus-infected tissue, and tumors, are reviewed. Finally, preclinical and clinical applications of targeted, synthetic nucleic acid delivery systems are discussed. Table 1 gives an overview on the specific targeting ligands and carrier constructs applied for various target tissues as presented in the current review.

Table 1.

Receptor-Targeted Nucleic Acid Carriers and Constructs

Receptor	Targeting ligand	Carrier construct	Key findings	Reference
Liver targeting
ASGPR	Lac-α-CDE	PAMAM	i.v. pDNA delivery	Arima et al., 2010
	NAG	PEG-masked covalent polyconjugate	i.v. siRNA, silencing in liver	Rozema et al., 2007
ASGPR LDLR	NAG Endogenous apoE	LNPs (iLNPs)	Exogenous or endogenous targeting of siRNA	Akinc et al., 2010
Lung targeting
Insulin R, Lf R	Insulin, Lactoferrin	PEI	Alveolar versus bronchial epithelial cells	Elfinger et al., 2007, 2009b
β2-R	Clenbuterol	PEI	Chemical ligand	Elfinger et al., 2009a
IP₁	Iloprost	PEI	Chemical ligand, aerosol delivery	Geiger et al., 2010
Inflammation
β₇-Integrin	Monoclonal antibody	LNP protamine, hyaluronan	CyD1 siRNA against colitis	Peer et al., 2008
Tumor targeting
Bomb R	Bombesin	Lipocation, PEG	siRNA delivery	Wang et al., 2009
Folate R	Folate	PEG-PEI	Minicircle DNA delivery	Zhang et al., 2010
Folate R	Folate	LNP	HER2 siRNA, tumor growth inhibition	Yoshizawa et al., 2008
Sigma R	Anisamide	LPD	siRNA, tumor growth inhibition	Li et al., 2008a
Sigma R	Trivalent anisamide	Oligonucleotide conjugate	Antisense	Nakagawa et al., 2010
PSMA	Aptamer	Aptamer-siRNA chimera	Tumor growth inhibition	McNamara et al., 2006
Viral infections (HIV)
gp120	Aptamer	phi29 pRNA	siRNA, HIV dual function inhibition	Zhou et al., 2011
Brain tissue targeting
LRP	HDL	Vitamin E conjugated siRNA	I.C.V. infusion, BACE siRNA silencing	Uno et al., 2011
GABA_BR	RVG29	PEG-PAMAM conjugate	pDNA delivery	Liu et al., 2009
Leptin R	Leptin peptide	PEG-lysine dendrimer	pDNA delivery	Liu et al., 2010
LRP1	Angiopep	PEG-PAMAM	pDNA delivery	Ke et al., 2009

Selected recent examples are presented. For abbreviations, see text.

Liver Targeting

Hepatocyte targeting via receptor-mediated delivery by the asialoglycoprotein (ASGP) receptor (ASGPR) has been the starting point for delivery of pDNA polyplexes, in the initial studies by applying ASGP-polylysine conjugates (Wu and Wu, 1987). As the receptor recognizes trivalent galactose [or N-acetylgalactosamine (NAG)] moieties, many synthetic galactosylated and lactosylated ligands have been explored since then. For example, a recent encouraging study applied polyamidoamine (PAMAM) dendrimer conjugates with α-cyclodextrin bearing lactose (Lac-α-CDE) for intravenous pDNA delivery (Arima et al., 2010). Lac-α-CDE served two purposes: as ASGPR ligand, and as a shielding component that strongly reduces cytotoxicity. This formulation provided higher hepatocyte gene transfer activity than jetPEI-Hepatocyte and much less change in blood parameters.

ASGPR targeting has also been used for siRNA delivery by covalent coupling of siRNA with lactose via an acid-labile PEG bridge in a polyplex micelle (Oishi et al., 2005) or by attachment of siRNA as part of a dynamic polyconjugate (Rozema et al., 2007). This conjugate consisted of siRNA disulfide-linked to a membrane-active polymer masked by PEG molecules and NAG ligands in a pH-reversible fashion. After receptor-mediated uptake of the polyconjugate into endosomes, the acidic environment is expected to unmask the lytic polymer backbone by removal of PEG and targeting ligand, components that are not only not required but rather inhibitory in the subsequent escape to the cytosol. In vivo silencing in hepatocytes upon intravenous delivery was demonstrated in several models.

Low-density lipoprotein (LDL) receptor (LDLR)–mediated endocytosis has proven (with or without intention) to be a very successful entry pathway for nucleic acids into the liver. Cholesterol-conjugated siRNA and related lipophilic conjugates mediate efficient gene silencing in vivo (Soutschek et al., 2004). The efficiency depends on interaction of these conjugates with lipoprotein particles in the blood. With LDL as ligand, siRNA is primarily targeted to the liver, whereas high-density lipoprotein (HDL) enabled targeting into liver, gut, kidney, and steroidogenic organs (Wolfrum et al., 2007).

Lipid nanoparticles (LNPs) with bioreversible PEG shield have been previously found to be very effective delivery vehicles for siRNA into the liver in many species, including primates (Zimmermann et al., 2006). The mechanism was recently discovered: interestingly, apolipoprotein E (apoE) associates in the blood with these quite neutral, ionizable LNPs (iLNPs) (but not cationic LNPs) and acts as an “endogenous targeting ligand” for the LDLR (Akinc et al., 2010). Conversely, retargeting to the ASGPR was possible by incorporation of NAG as “exogenous targeting ligand” into the iLNPs. In sum, a series of data demonstrate that, for siRNA formulations in vivo, receptor targeting is possible, at least to the liver.

Lung Targeting

Lung is also an important target organ for synthetic vector development, for both genetic and acquired diseases (Hyde et al., 2008; Griesenbach and Alton, 2009; Kormann et al., 2011). Particularly attractive is the dual accessibility of the organ from both the blood and the airway sides. Recently, Rudolph and colleagues reported a series of targeted pDNA polyplexes. Adsorption of human insulin onto pDNA/polyethylenimine (PEI) nanoparticles specifically increased (up to 16-fold) gene transfer to alveolar epithelial cells (expressing insulin receptors), but not bronchial epithelial cells (without detectable receptors) (Elfinger et al., 2009b). Lactoferrin (Lf) receptors, in contrast, are present at high levels on bronchial epithelial cells, but not alveolar epithelial cells. Consistently, Lf-PEI conjugates mediated enhanced gene expression levels as compared with PEI polyplexes in bronchial epithelial cells only (Elfinger et al., 2007).

In addition to protein ligands, small established pharmacological drugs also have been used for lung tissue targeting. Clenbuterol, an agonist binding the β₂-adrenoceptor (Elfinger et al., 2009a) and prostaglandin I2 analogues iloprost (ILO) and treprostinil targeting the prostacyclin receptor IP₁ (Geiger et al., 2010) have been coupled to PEI and successfully applied for improved gene transfer of pDNA/PEI polyplexes to the lung. After aerosol delivery of pDNA with ILO-grafted PEI, gene expression in the lungs of mice was 14-fold higher than for plain PEI polyplexes.

Targeting Inflammation Sites

A monoclonal antibody to β₇-integrin was used to target specific leukocyte subsets involved in gut inflammation. Protamine-condensed cyclin D1 (CyD1) siRNA was entrapped into targeted stabilized lipid nanoparticles containing a covalent antibody–hyaluronan–dipalmitidylphosphatidylethanolamine (DPPE) conjugate (Peer et al., 2008). Systemic application of these nanoparticles in mice with experimentally induced colitis silenced CyD1 in leukocytes and reversed the colitis by suppressing leukocyte proliferation and T helper 1 cytokine expression.

Tumor-Targeting Ligands

Most efforts in developing targeted nucleic acid nanosystems have been devoted to the cancer area. For metastatic tumors, which contain unique or at least overexpressed surface receptors and antigens, systemic targeted delivery systems appear as a most logical strategy, optionally used in combination with chemotherapy or other therapies. Many different targeting ligands (antibodies, proteins, peptides, small chemical substances) and receptors have been experimentally addressed in various development phases, including classical growth factor receptors such as the transferrin (Tf) receptor or epidermal growth factor (EGF) receptor (EGFR) (Gunther et al., 2005).

Noteworthy recent ligand/receptor evaluations include, for example, the peptide bombesin (Wang et al., 2009). Bombesin receptors are expressed in tumors such as small-cell lung carcinoma and gastric cancer. Wang et al. applied bombesin as ligand for siRNA delivery. In their study, siRNA was complexed with a defined lipid-modified oligocation that was prepared by solid-phase synthesis. The lipo-oligocation has endosomal pH-specific lysis activity and contains cysteine groups that stabilize the formed siRNA nanoparticle by bioreversible disulfide bonds. Nanoparticles were post-PEGylated with a reactive PEG-bombesin derivative. In vitro and in vivo gene silencing activity was reported (Wang et al., 2009).

Several recent articles evaluated folic acid for tumor targeting, as folate receptor is up-regulated in various cancers. Gene delivery was tested with PEGylated PEI/DNA polyplexes conjugated with folic acid (Cheng et al., 2009; Zhang et al., 2010). By using minicircle DNA, folate receptor-dependent gene transfer was demonstrated in cell culture. In addition, systemic delivery of the folate-labeled polyplexes resulted in preferential accumulation of transgenes in folate receptor–positive tumors. Folate was also tested as ligand for siRNA delivery, either with siRNA covalently bound to folate via a linker (Thomas et al., 2009) or as folate–PEG–distearoylphosphatidylethanolamine (DSPE) lipid conjugate in siRNA lipoplexes (Yoshizawa et al., 2008). Conjugated folate contributed to significantly higher intracellular amounts of siRNA. In vivo effects upon intratumoral injection of HER2 siRNA folate-PEG lipoplexes were observed, resulting in tumor growth inhibition of human epidermoid carcinoma (KB cells) subcutaneously implanted in immune-deficient mice.

The σ-receptor ligand anisamide is another small-molecule ligand for tumor targeting. Huang and colleagues generated lipid–protamine–calf thymus DNA (LPD) nanoparticles containing siRNA in the core and anisamide– PEG on the surface of the nanoparticles (Li et al., 2008a,b). Therapeutic effects were observed upon intravenous delivery with targeted (but not with nontargeted) siRNA nanoparticles in two lung tumor models (mouse B16F10 melanoma or human NCI-H460 xenografts). Importantly, 70–80% of injected siRNA per gram accumulated in the tumor. This was attributed to the fact that a very high density of PEG molecules could be grafted to the nanoparticle surface (Li and Huang, 2009). Each type of PEG shielding is not equal. A low-percentage superficial shielding by PEG (in a “mushroom regime” with flexible moving PEG chains) has already important consequences: it reduces the surface charge and prevents aggregation both between particles and of particles with blood cells and larger surfaces. Opsonization by serum components, however, may not be completely blocked, and blood circulation times are low. At higher densities of PEG shielding (10% in Huang's case), a densely shielded surface develops with PEG chains in a “brush regime.” This is a key requirement for nanoparticles to evade the reticuloendothelial system (RES).

σ-Receptor targeting was also explored using splice switching antisense oligonucleotide (SSO) covalently attached with mono- or trivalent anisamide (Nakagawa et al., 2010). The trivalent anisamide-SSO conjugate displayed enhanced cellular uptake and higher effectivity than the monovalent conjugate in modifying splicing of a luciferase reporter in cultured tumor cells.

Recent studies applied tumor-targeting antibodies directed against the receptor tyrosine kinases EGFR and HER2. Anti-EGFR–targeted immunoliposomes were applied for siRNA delivery into breast cancer (Gao et al., 2011). The Lieberman group (Song et al., 2005) pioneered the use of HER2 single-chain antibody-protamine fusion protein for in vivo siRNA delivery. Chitosan nanoparticles with encapsulated quantum dots (QDs) and siRNA and surface modified with HER2 antibody were successfully used for targeted delivery of HER2/neu siRNA to HER2-overexpressing SKBR3 breast cancer cells. The presence of fluorescent QDs in the formulation enabled monitoring of the delivery process (Tan et al., 2007).

Apart from antibodies, an oligopeptide MC-10 was applied for HER2 receptor targeting of plasmid DNA (Huang et al., 2010). MC-10 was conjugated with a biocompatible cationic polymer composed of oligoethylenimine cross-linked by 2-hydroxypropyl-γ-cyclodextrin. Reporter gene pDNA formulations showed targeting specificity for HER2, absence of cytotoxicity, and high gene transfer efficiency in vitro and in vivo. Antitumor effects of therapeutic interferon-α gene delivery were significantly enhanced as compared with the nontargeted control formulation or PEI (25 kDa) polyplexes.

Aptamers, i.e., nucleic acids with specific target protein–binding ability, present a chemically completely different class of targeting ligands that has been recently explored in several disease directions (see also below). Due to their nucleic acid nature, construction of chimeras with therapeutic nucleic acids appears as a promising concept. For therapy of prostate cancer, a PSMA aptamer–siRNA construct was evaluated (McNamara et al., 2006). The construct was internalized by PSMA-positive cells. In vivo the treatment reduced tumor growth in a mouse xenograft model.

Viral Infections

Various aptamer-siRNA conjugates were also tested as novel targeted anti-HIV drugs. Dual inhibitory function chimeras of anti-glycoprotein 120 (gp120) aptamer and anti-tat/rev siRNA were generated specifically binding to and being internalized into cells expressing HIV gp160 (Zhou et al., 2008). In a next step, 2′-fluoro-substituted RNA aptamers that bind to the HIV-1 gp120 protein with nanomole affinity were optimized in a molecular evolution screen (Zhou et al., 2009a). Dicer-substrate siRNA delivered by the aptamers was functionally processed into their active form, resulting in specific inhibition of HIV-1 replication and infectivity in various (including primary) cultured T cells. As a further step toward systemic delivery and anti-HIV therapy, most recently the chimeric fluoro-substituted RNA molecules were modified with an additional functional element, the phi29 “packaging RNA (pRNA)”. pRNA monomers are about 117 nucleotides in length and, in the natural phage packaging process, form a hexameric ring. They can assemble into stable dimer, trimer, or hexamer nanoparticles of 20–50 nm, which makes them useful as nanocarriers. The chimeras were found to be stable in serum and functional in target-specific gene silencing (Zhou et al., 2011).

Brain Tissue Targeting

Targeting the brain presents a dual challenge because of the blood–brain barrier (BBB). Two basic strategies have to be considered: targeting of cells within the brain after local infusion, and systemic delivery and crossing of the BBB. Similar to liver targeting, lipoprotein receptor–mediated uptake has been used for delivery of siRNA into neurons. Uno et al. combined HDL with vitamin E (α-tocopherol)–conjugated siRNA (Toc-siRNA) and applied the formulation by intracerebroventricular (i.c.v.) infusion (Uno et al., 2011). Very efficient and specific knockdown of a target gene (BACE1) was observed in the cerebral cortex and hippocampus. Nonconjugated siRNA was nonfunctional, and the silencing efficiency was significantly decreased with Toc-siRNA without HDL or in lipoprotein-receptor knockout mice.

Recent novel targeting ligands in systemic delivery include a peptide ligand (RVG29) derived from rabies virus glycoprotein, supposed to bind the nicotinic acetylcholine receptor (nAchR) on neuronal cells. In one approach, the peptide was C-terminally extended with nine arginine residues to enable siRNA binding (Kumar et al., 2007). After intravenous injection of siRNA polyplexes, transvascular delivery to the brain and efficient gene silencing were reported. Jiang and colleagues (Liu et al., 2009) applied the same peptide ligand for brain-targeted delivery of pDNA. RVG29-PEG-PAMAM conjugates were applied for pDNA polyplex formation, and were found to be taken up by brain capillary endothelial cells by endocytosis. The process was competitively inhibited by free RVG29 and γ-aminobutyric acid (GABA), but not by nAchR agonists/antagonists, indicating that RVG29 probably relates to the GABA_B receptor (GABA_BR) besides the nAchR as reported previously. In vivo imaging showed preferential accumulation of the polyplexes in brain. Gene expression of the targeted polyplexes in brain was significantly higher than that of nontargeted polyplexes. The same laboratory also successfully evaluated two other brain-targeting transcytotic peptide ligands for gene delivery, a leptin-derived peptide (Liu et al., 2010) and Angiopep (Ke et al., 2009) targeting the LDLR-related protein LRP1, both in the form of PEG-modified dendritic polymer conjugates.

Targeting Using Ligand Combinations

The choice of targeting ligand can influence cell binding, nanoparticle internalization, and cell activation. Many natural viruses use more than one cell-surface molecule for entry into their host cell. For example, adenovirus serotypes 2 and 5 at first bind to the coxsackievirus and adenovirus receptor (CAR) via an exposed region of their fiber knob, followed by subsequent binding to integrins by their RGD domain at the penton base (the less exposed domain where the fiber is anchored within the adenovirus particle). Also in the artificial context, two (or more) different targeting ligands might be combined in an effective way. Ligands with unique cell-binding characteristics but low internalization rate might be combined with coligands mediating rapid and efficient endocytosis. “Dual targeting” options have been developed for nanoparticle-mediated imaging, chemotherapy, and gene therapy strategies (Li et al., 2008c; Quan et al., 2009; Kluza et al., 2010). For example, synergistic dual targeting characteristics were observed with DNA polyplexes containing two different peptide ligands in transfections of prostate cancer cells (Nie et al., 2011). PEGylated PEI/DNA polyplexes were decorated with RGD peptide ligands for integrin targeting and peptide B6 as ligand for the Tf receptor. In a series of flow cytometry experiments, cell association and cell internalization with and without ligand competition were evaluated. RGD (attached via a longer 3.4-kDa PEG spacer) was found to play the major role in cell-surface binding, whereas B6 (attached via a shorter 2-kDa PEG spacer) had the major role in intracellular uptake.

Systemic Delivery, Passive Plus Active Tumor Targeting

Although targeting ligands have proven positive effects on bioactivity in their target cells in vitro and also in vivo, far less influence was observed with regard to biodistribution of the formulation, at least in the case of systemic tumor studies. In general, systemically applied gene vectors face several hurdles before successfully reaching their target cells. Only when properly protected against unwanted interactions with blood components (complement system, blood cells, antibodies, etc.) and cells of the RESs (tissue macrophages), premature clearance from the blood stream can be avoided. Size restrictions exist, for example, in liver cell targeting, where the fenestrations in liver (100–200 nm in size, depending on species) limit accessibility of hepatocytes. Initially small gene carriers can grow in size after injection by aggregation or decoration with blood components, and may be subsequently cleared by liver macrophages (Kupffer cells). For systemic tumor-targeting approaches, such effects should be avoided. The well-known EPR effect (enhanced permeability and retention effect) will allow the influx of macromolecular drugs, including gene carriers, into the tumor tissue, as long as they have a certain circulation time in blood and are not secreted via the kidneys (Maeda, 2010). In the case of solid tumor tissue, the leakiness of tumor endothelium and the incomplete lymphatic drainage can be harnessed for this so-called passive targeting approach. Depending of the site of the targeted receptor, the pharmacokinetics of ligand-decorated particles can be influenced in principle. A receptor expressed at the apical site of endothelial cells, like integrins, will be immediately accessible for intravenously injected particles, whereas receptors at the basolateral site are only accessible after bypassing one or several cell layers. Only a few studies were carried out on the pharmacokinetics of targeted versus nontargeted nucleic acid formulations, and how ligand targeting potentially influences clearance (Bartlett et al., 2007; Zhou et al., 2009b). Such studies are carried out either by measuring the blood concentration of the nucleic acid delivered after repeated blood collection or by endpoint measurements. More recently, methods for live imaging, such as near-infrared spectroscopy (Zintchenko et al., 2009), positron emission tomography (PET), PET/computed tomography (Hatanaka et al., 2010), or other combinations of imaging modalities (Medarova et al., 2007), have been applied to study the pharmacokinetics of gene carriers within the living organism. Within a multimodal imaging approach, biodistribution and siRNA-mediated antitumor activity were followed in living animals with nanoparticles targeted to the MUC-1 antigen in subcutaneously implanted breast carcinoma tumors (Kumar et al., 2010). Targeting peptide EPPT, the near-infrared fluorescent dye Cy5.5, and siRNA duplexes directed against the antiapoptotic gene product survivin were all covalently coupled to an aminodextran-coated iron oxide core. After systemic delivery, nanoparticles accumulated in tumor tissue as followed by near-infrared imaging and magnetic resonance imaging, and the siRNA delivered led to tumor cell apoptosis and reduced tumor growth.

DNA nanoparticles formed with polylysine and stabilized by the hydrophilic, multivalent polymer multivalent N-(2-hydroxypropyl)methacrylamide (HPMA) were cleared from the bloodstream exhibiting two compartment model characteristics (fast alpha clearance, slower beta clearance) (Zhou et al., 2009b). HPMA stabilization was responsible for an enhanced circulation time, as uncoated particles were rapidly eliminated within seconds. Interestingly, incorporation of the α_vβ₃-integrin ligand RGD did not significantly influence the clearance kinetics in tumor-bearing mice. Here, intact pDNA was quantified in blood plasma after repeated blood drawings. Bartlett et al. applied Tf receptor–targeted, PEGylated cyclodextrin–based siRNA particles in a subcutaneous neuroblastoma model (murine Neuro2A neuroblastoma), where PET imaging of ⁶⁴Cu-labeled particles revealed no differences in terms of biodistribution and tumor accumulation (Bartlett et al., 2007). Nevertheless, the Tf receptor–targeted formulation was more efficient in mediating siRNA-induced target knockdown. The authors explained this effect by improved cellular uptake of targeted particles by tumor cells. One has to keep in mind that the type and density of shielding strongly influence the fate in systemic circulation. Low-density shielding may already be beneficial for preventing physical aggregation between particles or with erythrocytes, but high-density shielding will be required for long-term circulation in blood. We recently showed that a rather short PEG chain of 2 kDa is already sufficient to prevent aggregation-mediated transgene expression in lung tissue of systemically applied polyplexes based on linear PEI (Klutz et al., 2011), although the polyplexes were exhibiting a net positive surface charge. When carrying the EGFR-binding peptide GE11 on the distal end of the PEG chain, subcutaneously implanted, EGFR-overexpressing HUH7 hepatoma tumors expressed significantly higher transgene levels compared with the untargeted formulation.

Targeted Nucleic Acid Therapy for Cancer—Preclinical and Clinical Studies

Nucleic acids especially have the potential to act as highly specific, personalized medicines in cancer therapy. A high level of selectivity can be achieved by the DNA delivery of therapeutically active proteins, where also (more or less) tumor-specific promoter elements can limit the transgene expression to tumor cells. In the case of siRNA, specific knockdown of targets overexpressed in tumors also helps to obtain selectivity. In any case, the side effects of nucleic acid–based therapeutics should not be neglected, as, for example, plasmid vectors, but also siRNA molecules, may induce the innate immune system via Toll-like receptor activation, leading to inflammatory reactions and inactivation of the delivered nucleic acids (Hodges et al., 2004; Samuel-Abraham and Leonard, 2010). Still, one has to keep in mind that, in the case of transgene expression or siRNA treatment, in theory every single tumor cell has to express the transferred DNA or process the siRNA delivered. Therefore, potent bystander effects are important for successful tumor therapies to affect also nontransfected tumor cells in the near vicinity of transfected cells. Targeted delivery is of high importance, helping to allow efficient action at the target site and, at the same time, to reduce the total dose applied, thus minimizing accumulation at nontarget sites. Both pDNA- and siRNA-based therapies are often combined with chemotherapy regimens. A “double magic bullet” was designed in the laboratory of Leaf Huang, packing siRNA and doxorubicin into the same targeted nanoparticle (Chen et al., 2010). The peptide sequence NGR (asparagine-glycine-arginine) binds specifically to aminopeptidase-N, which is overexpressed in several tumor types and in tumor endothelium. NGR-containing nanoparticles containing anti-c-myc siRNA and doxorubicin were significantly more efficient in reducing tumor growth (subcutaneous HT1080 human sarcoma) than similar ones containing the control peptide ARA after three systemic injections.

Synthetic, double-stranded RNA [polyinosine/polycytosine (polyIC)] is a well-known compound activating the innate immune system. The antitumoral mechanism of polyIC is characterized by its action via several pathways: by interacting with the Toll-like receptor 3, it induces the secretion of inflammatory cytokines and activates phospholipase A, leading to apoptotic cell death. After a promising phase I clinical study, a phase II study combining intramuscular application of polyIC/polylysine polyplexes with temozolomide treatment revealed that polyIC application distant to the tumor only induced cytotoxic cytokines working in a paracrine fashion, whereas the direct antitumoral action of polyIC could not be unleashed (Butowski et al., 2009). We have recently developed a targeted delivery system for polyIC, where polyplexes are formed with a conjugate based on branched PEI for polyIC condensation, the endosomolytic peptide melittin, and recombinant murine EGF as ligand to target the polyplex to EGFR-overexpressing glioblastoma (Shir et al., 2006). Intratumoral injection into glioblastoma xenografts in mice with an EGFR-targeted polyIC formulation successfully eradicated the implanted tumors, pointing at the importance of efficient delivery into tumor cells (Shir et al., 2006). More recently, such EGFR-targeted polyIC polyplexes were also able to eradicate disseminated tumors (A549 human lung adenocarcinoma) in NOD-SCID mice after systemic polyplex application in combination with human peripheral blood monocytes (Shir et al., 2011). For the polyIC polyplexes, efficient endosomal release significantly improved the antitumoral effect. Two cytosolic enzymes, namely, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated antigen 5 (MDA-5), are activated by polyIC, and this activation leads to highly effective induction of apoptosis and subsequent cell killing (Besch et al., 2009). In another study, efficient treatment of subcutaneous lung adenocarcinoma A431 was enabled with polyIC polyplexes based on linear PEI and recombinant murine EGF (Schaffert et al., 2011). Due to the improved endosomolytic properties of linear PEI, no additional endosomolytic compound was necessary.

For plasmid-based approaches, several concepts have been pursued to achieve potent bystander effects, such as gene-directed enzyme prodrug therapy: the tumor-targeted expression of a specific enzyme converts an initially inactive prodrug into the cytotoxic drug directly at the tumor site (Schellmann et al., 2010). Another approach is a combination of radiotherapy with gene therapy. The sodium iodide symporter (NIS) is a transmembrane protein highly expressed in thyroidal cells and responsible for iodine accumulation in the thyroid. As NIS is not expressed at significant levels in tumors except of thyroidal origin, targeting NIS transgene expression into NIS-negative tumor cells in combination with radioiodine has the potential of selective tumor imaging, but also tumor eradication (Baril et al., 2010). Also, depending on the radionuclide used, a potent bystander effect can be achieved, for example, by the β-emitter ¹³¹I, or other radionuclides transported by NIS, like rhenium or astatine. Initially, viral vectors were used for this approach in several advanced clinical studies. We recently applied this concept in a preclinical murine tumor model, applying untargeted NIS gene–containing polyplexes formed with a biodegradable polycation, leading to reduced tumor growth and prolonged survival (Klutz et al., 2009). More recently, EGFR-targeted NIS gene delivery was achieved with a vector based on linear PEI coupled to the EGFR-targeting peptide GE11 (Klutz et al., 2011). The GE11 peptide has been identified by the phage display technique and allowed tumor-selective reporter gene expression in a subcutaneous hepatoma tumor (Li et al., 2005). Our studies clearly showed that EGFR targeting led to significantly higher NIS activity in HUH7 tumors when compared with an untargeted formulation. After four cycles of systemically applied NIS polyplexes and subsequent ¹³¹I treatment, tumor growth was significantly reduced and prolonged survival achieved (Klutz et al., 2011). In contrast to natural ligands, peptidic ligands offer the advantage of convenient chemical synthesis, a low molecular weight, and potentially reduced immunogenicity. Also, receptor activation after ligand binding can bear an additional risk, especially when growth factors are used as ligands. We recently showed that recombinant murine EGF coupled to linear PEI activated EGFR, leading to phosphorylation of downstream targets Akt and Erk and subsequent overall receptor internalization (Schäfer et al., 2011). When the GE11 peptide (see above) was used as ligand, EGFR activation was avoided, although transfection efficiency was similar or even elevated when compared with EGF.

Apart from numerous preclinical studies, so far only one receptor-targeted, nonviral nucleic acid delivery system has been applied systemically in human subjects (Davis et al., 2010). Preclinical safety studies were carried out in cynomolgus monkeys, where intravenously applied siRNA polyplexes based on PEGylated, cationized cyclodextrin coupled to Tf were well tolerated up to a dose of 9 mg/kg (Heidel et al., 2007b). At an elevated dose of 27 mg/kg, rising creatinase levels in blood indicated kidney toxicity, and increased cytokine levels after repeated administration pointed at mild signs of immunogenicity of the vector, although only low levels of anti-human Tf antibodies were detected. Ribonucleotide reductase is up-regulated in proliferating cancer cells and, hence, is a valid target for cancer therapies. An siRNA duplex was identified directed against the M2 subunit of ribonucleotide reductase 2 (RRM2), leading to optimal antitumoral activity in a murine xenograft model; it also showed antiproliferative effects in tumor cells from mice, primate, and humans (Heidel et al., 2007a). In a clinical phase I study, melanoma patients with metastatic melanoma refractory to standard therapies were treated with four infusions of siRNA polyplexes at doses of 18, 24, and 30 mg/m² (Davis et al., 2010). Fluorescently labeled siRNA was detected in tumor biopsies, and concentration correlated with the dose applied. No adverse reactions were observed in any of the three patients treated; functionality of polyplexes in tumors was proven by analyzing specific cleavage of RRM2 mRNA and increased apoptosis rate found in tumor biopsies after siRNA treatment.

Conclusion and Outlook

Biochemical targeting of synthetic nucleic acid vectors does not provide magic bullets, but can considerably improve their effectiveness. Several advanced preclinical studies as well as a first clinical study prove their potential for the treatment of diseases where standard treatments are not applicable. To proceed further into advanced clinical studies with the aim to produce approved nucleic acid medicines, the development of up-scalable, reproducible production, and formulation processes will be of utmost importance.

Footnotes

Acknowledgments

Funding of the authors' work in the reviewed research area by DFG grants OG63/4-1 and SFB 824 (to M.O.), DFG Cluster of Excellence “Nanosystems Initiative Munich,” and the BMBF Biotech cluster m⁴ project T12 (to E.W.) is gratefully acknowledged.

Author Disclosure Statement

No competing financial interests exist.

References

Aagaard

, Rossi

J.J.

2007. RNAi therapeutics: principles, prospects and challenges. Adv. Drug Deliv. Rev., 59:75–86.

Aigner

2006. Gene silencing through RNA interference (RNAi) in vivo: strategies based on the direct application of siRNAs. J. Biotechnol., 124:12–25.

Akinc

, Querbes

, De

et al. 2010. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol. Ther., 18:1357–1364.

Arima

, Yamashita

, Mori

et al. 2010. In vitro and in vivo gene delivery mediated by lactosylated dendrimer/alpha-cyclodextrin conjugates (G2) into hepatocytes. J. Control. Release, 146:106–117.

Baril

, Martin-Duque

, Vassaux

2010. Visualization of gene expression in the live subject using the Na/I symporter as a reporter gene: applications in biotherapy. Br. J. Pharmacol., 159:761–771.

Bartlett

D.W.

, Su

, Hildebrandt

I.J.

et al. 2007. Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging. Proc. Natl. Acad. Sci. U.S.A., 104:15549–15554.

Behlke

M.A.

2008. Chemical modification of siRNAs for in vivo use. Oligonucleotides, 18:305–319.

Besch

, Poeck

, Hohenauer

et al. 2009. Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells. J. Clin. Invest., 119:2399–2411.

Butowski

, Lamborn

K.R.

, Lee

B.L.

et al. 2009. A North American brain tumor consortium phase II study of poly-ICLC for adult patients with recurrent anaplastic gliomas. J. Neurooncol., 91:183–189.

10.

Chen

Y.C.

, Wu

J.Z.J.

, Huang

2010. Nanoparticles targeted with NGR motif deliver c-myc siRNA and doxorubicin for anticancer therapy. Mol. Ther., 18:828–834.

11.

Cheng

, Zhu

J.L.

, Zeng

et al. 2009. Targeted gene delivery mediated by folate-polyethylenimine-block-poly(ethylene glycol) with receptor selectivity. Bioconjug. Chem., 20:481–487.

12.

Davis

M.E.

, Zuckerman

J.E.

, Choi

C.H.

et al. 2010. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature, 464:1067–1070.

13.

De Fougerolles

A.R.

2008. Delivery vehicles for small interfering RNA in vivo. Hum. Gene Ther., 19:125–132.

14.

Elfinger

, Maucksch

, Rudolph

2007. Characterization of lactoferrin as a targeting ligand for nonviral gene delivery to airway epithelial cells. Biomaterials, 28:3448–3455.

15.

Elfinger

, Geiger

, Hasenpusch

et al. 2009a. Targeting of the beta(2)-adrenoceptor increases nonviral gene delivery to pulmonary epithelial cells in vitro and lungs in vivo. J. Control. Release, 135:234–241.

16.

Elfinger

, Pfeifer

, Uezguen

et al. 2009b. Self-assembly of ternary insulin-polyethylenimine (PEI)-DNA nanoparticles for enhanced gene delivery and expression in alveolar epithelial cells. Biomacromolecules, 10:2912–2920.

17.

Frohlich

, Wagner

2010. Peptide- and polymer-based delivery of therapeutic RNA. Soft Matter, 6:226–234.

18.

Gao

, Liu

, Xia

et al. 2011. The promotion of siRNA delivery to breast cancer overexpressing epidermal growth factor receptor through anti-EGFR antibody conjugation by immunoliposomes. Biomaterials, 32:3459–3470.

19.

Geiger

, Aneja

M.K.

, Hasenpusch

et al. 2010. Targeting of the prostacyclin specific IP1 receptor in lungs with molecular conjugates comprising prostaglandin I2 analogues. Biomaterials, 31:2903–2911.

20.

Griesenbach

, Alton

E.W.

2009. Gene transfer to the lung: lessons learned from more than 2 decades of CF gene therapy. Adv. Drug Deliv. Rev., 61:128–139.

21.

Gunther

, Wagner

, Ogris

2005. Specific targets in tumor tissue for the delivery of therapeutic genes. Curr. Med. Chem. Anticancer Agents, 5:157–171.

22.

Guo

, Coban

, Snead

N.M.

et al. 2010. Engineering RNA for targeted siRNA delivery and medical application. Adv. Drug Deliv. Rev., 62:650–666.

23.

Hatanaka

, Asai

, Koide

et al. 2010. Development of double-stranded siRNA labeling method using positron emitter and its in vivo trafficking analyzed by positron emission tomography. Bioconjug. Chem., 21:756–763.

24.

Heidel

J.D.

, Liu

J.Y.

, Yen

et al. 2007a. Potent siRNA inhibitors of ribonucleotide reductase subunit RRM2 reduce cell proliferation in vitro and in vivo. Clin. Cancer Res., 13; 2207–2215.

25.

Heidel

J.D.

, Yu

, Liu

J.Y.

et al. 2007b. Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA. Proc. Natl. Acad. Sci. U.S.A., 104:5715–5721.

26.

Hodges

B.L.

, Taylor

K.M.

, Joseph

M.F.

et al. 2004. Long-term transgene expression from plasmid DNA gene therapy vectors is negatively affected by CpG dinucleotides. Mol. Ther., 10:269–278.

27.

Huang

, Yu

, Tang

et al. 2010. Low molecular weight polyethylenimine cross-linked by 2-hydroxypropyl-gamma-cyclodextrin coupled to peptide targeting HER2 as a gene delivery vector. Biomaterials, 31:1830–1838.

28.

Hyde

S.C.

, Pringle

I.A.

, Abdullah

et al. 2008. CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression. Nat. Biotechnol., 26:549–551.

29.

, Shao

, Huang

et al. 2009. Gene delivery targeted to the brain using an Angiopep-conjugated polyethyleneglycol-modified polyamidoamine dendrimer. Biomaterials, 30:6976–6985.

30.

Klutz

, Russ

, Willhauck

M.J.

et al. 2009. Targeted radioiodine therapy of neuroblastoma tumors following systemic nonviral delivery of the sodium iodide symporter gene. Clin. Cancer Res., 15:6079–6086.

31.

Klutz

, Schaffert

, Willhauck

M.J.

et al. 2011. Epidermal growth factor receptor-targeted ¹³¹I-therapy of liver cancer following systemic delivery of the sodium iodide symporter gene. Mol. Ther., 19:676–685.

32.

Kluza

, van der Schaft

D.W.

, Hautvast

P.A.

et al. 2010. Synergistic targeting of alphavbeta3 integrin and galectin-1 with heteromultivalent paramagnetic liposomes for combined MR imaging and treatment of angiogenesis. Nano Lett., 10:52–58.

33.

Kormann

M.S.

, Hasenpusch

, Aneja

M.K.

et al. 2011. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat. Biotechnol., 29:154–157.

34.

Kota

, Chivukula

R.R.

, O'Donnell

K.A.

et al. 2009. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell, 137:1005–1017.

35.

Krützfeldt

, Rajewsky

, Braich

et al. 2005. Silencing of microRNAs in vivo with 'antagomirs' Nature, 438:685–689.

36.

Kumar

, Yigit

, Dai

G.P.

et al. 2010. Image-guided breast tumor therapy using a small interfering RNA nanodrug. Cancer Res., 70:7553–7561.

37.

Kumar

, Wu

, McBride

J.L.

et al. 2007. Transvascular delivery of small interfering RNA to the central nervous system. Nature, 448:39–43.

38.

, Huang

2010. Targeted delivery of RNAi therapeutics for cancer therapy. Nanomedicine (Lond), 5:1483–1486.

39.

S.D.

, Huang

2009. Nanoparticles evading the reticuloendothelial system: role of the supported bilayer. Biochim. Biophys. Acta, 1788:2259–2266.

40.

S.D.

, Chen

Y.C.

, Hackett

M.J.

, Huang

2008a. Tumor-targeted delivery of siRNA by self-assembled nanoparticles. Mol. Ther., 16:163–169.

41.

S.D.

, Chono

, Huang

2008b. Efficient oncogene silencing and metastasis inhibition via systemic delivery of siRNA. Mol. Ther., 16:942–946.

42.

, Szoka

F.C.

Jr . 2007. Lipid-based nanoparticles for nucleic acid delivery. Pharm. Res, 24:438–449.

43.

, Zhao

, Wu

et al. 2005. Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics. FASEB J., 19:1978–1985.

44.

Z.B.

, Wu

, Chen

et al. 2008c. ¹⁸F-labeled BBN-RGD heterodimer for prostate cancer imaging. J. Nucl. Med., 49:453–461.

45.

Liu

, Huang

, Han

et al. 2009. Brain-targeting gene delivery and cellular internalization mechanisms for modified rabies virus glycoprotein RVG29 nanoparticles. Biomaterials, 30:4195–4202.

46.

Liu

, Li

, Shao

et al. 2010. A leptin derived 30-amino-acid peptide modified pegylated poly-L-lysine dendrigraft for brain targeted gene delivery. Biomaterials, 31:5246–5257.

47.

Maeda

2010. Tumor-selective delivery of macromolecular drugs via the EPR effect: background and future prospects. Bioconjug. Chem., 21:797–802.

48.

McNamara

J.O.

2nd , Andrechek

E.R.

, Wang

et al. 2006. Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nat. Biotechnol., 24:1005–1015.

49.

Medarova

, Pham

, Farrar

et al. 2007. In vivo imaging of siRNA delivery and silencing in tumors. Nat. Med., 13:372–377.

50.

Nakagawa

, Ming

, Huang

, Juliano

R.L.

2010. Targeted intracellular delivery of antisense oligonucleotides via conjugation with small-molecule ligands. J. Am. Chem. Soc., 132:8848–8849.

51.

Nie

, Schaffert

, Rodl

et al. 2011. Dual-targeted polyplexes: one step towards a synthetic virus for cancer gene therapy. J. Control. Release[Epub ahead of print]

52.

Ogris

2006. Nucleic acid based therapeutics for tumor therapy. Anticancer Agents Med. Chem., 6:563–570.

53.

Oishi

, Nagasaki

, Itaka

et al. 2005. Lactosylated poly(ethylene glycol)-siRNA conjugate through acid-labile beta-thiopropionate linkage to construct pH-sensitive polyion complex micelles achieving enhanced gene silencing in hepatoma cells. J. Am. Chem. Soc., 127:1624–1625.

54.

Park

T.G.

, Jeong

J.H.

, Im

S.W.

2006. Current status of polymeric gene delivery systems. Adv. Drug Deliv. Rev., 58:467–486.

55.

Peer

, Park

E.J.

, Morishita

et al. 2008. Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target. Science, 319:627–630.

56.

Philipp

, Meyer

, Wagner

2008. Extracellular targeting of synthetic therapeutic nucleic acid formulations. Curr. Gene Ther., 8:324–334.

57.

Poeck

, Besch

, Maihoefer

et al. 2008. 5′-Triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma. Nat. Med., 14:1256–1263.

58.

Quan

C.Y.

, Chang

, Wei

et al. 2009. Dual targeting of a thermosensitive nanogel conjugated with transferrin and RGD-containing peptide for effective cell uptake and drug release. Nanotechnology, 20:335101.

59.

Rozema

D.B.

, Lewis

D.L.

, Wakefield

D.H.

et al. 2007. Dynamic polyconjugates for targeted in vivo delivery of siRNA to hepatocytes. Proc. Natl. Acad. Sci. U.S.A., 104:12982–12987.

60.

Samuel-Abraham

, Leonard

J.N.

2010. Staying on message: design principles for controlling nonspecific responses to siRNA. FEBS J., 277:4828–4836.

61.

Schäfer

, Pahnke

, Schaffert

et al. 2011. By-passing the yin and yang relation of epidermal growth factor receptor (EGFR) mediated delivery: a fully synthetic, EGFR targeted gene transfer system avoiding receptor activation. Hum. Gene Ther.[Epub ahead of print]

62.

Schaffert

, Wagner

2008. Gene therapy progress and prospects: synthetic polymer-based systems. Gene Ther., 15:1131–1138.

63.

Schaffert

, Kiss

, Rodl

et al. 2011. Poly(I:C)-mediated tumor growth suppression in EGF-receptor overexpressing tumors using EGF-polyethylene glycol-linear polyethylenimine as carrier. Pharm. Res., 28:731–741.

64.

Schellmann

, Deckert

P.M.

, Bachran

et al. 2010. Targeted enzyme prodrug therapies. Mini Rev. Med. Chem., 10:887–904.

65.

Shir

, Ogris

, Wagner

, Levitzki

2006. EGF receptor-targeted synthetic double-stranded RNA eliminates glioblastoma, breast cancer, and adenocarcinoma tumors in mice. PLoS Med., 3:e6.

66.

Shir

, Ogris

, Roedl

et al. 2011. EGFR-homing dsRNA activates cancer-targeted immune response and eliminates disseminated EGFR-overexpressing tumors in mice. Clin. Cancer Res., 17:1033–1043.

67.

Song

, Zhu

, Lee

S.K.

et al. 2005. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol., 23:709–717.

68.

Soutschek

, Akinc

, Bramlage

et al. 2004. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature, 432:173–178.

69.

Tan

W.B.

, Jiang

, Zhang

2007. Quantum-dot based nanoparticles for targeted silencing of HER2/neu gene via RNA interference. Biomaterials, 28:1565–1571.

70.

Thomas

, Kularatne

S.A.

, Qi

et al. 2009. Ligand-targeted delivery of small interfering RNAs to malignant cells and tissues. Ann. N.Y. Acad. Sci., 1175:32–39.

71.

Ulrich

, Trujillo

C.A.

, Nery

A.A.

et al. 2006. DNA and RNA aptamers: from tools for basic research towards therapeutic applications. Comb. Chem. High Throughput Screen., 9:619–632.

72.

Uno

, Piao

, Miyata

et al. 2011. High-density lipoprotein facilitates in vivo delivery of α-tocopherol–conjugated short-interfering RNA to the brain. Hum. Gene Ther., 22:711–719.

73.

Veedu

R.N.

, Wengel

2009. Locked nucleic acid as a novel class of therapeutic agents. RNA Biol., 6:321–323.

74.

Wang

X.L.

, Xu

, Lu

Z.R.

2009. A peptide-targeted delivery system with pH-sensitive amphiphilic cell membrane disruption for efficient receptor-mediated siRNA delivery. J. Control. Release, 134:207–213.

75.

Wolfrum

, Shi

, Jayaprakash

K.N.

et al. 2007. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat. Biotechnol., 25:1149–1157.

76.

G.Y.

, Wu

C.H.

1987. Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J. Biol. Chem., 262:4429–4432.

77.

S.Y.

, McMillan

N.A.

2009. Lipidic systems for in vivo siRNA delivery. AAPS J., 11:639–652.

78.

Yin

, Lu

, Wood

2008. Effective exon skipping and restoration of dystrophin expression by peptide nucleic acid antisense oligonucleotides in mdx mice. Mol. Ther., 16:38–45.

79.

Yoshizawa

, Hattori

, Hakoshima

et al. 2008. Folate-linked lipid-based nanoparticles for synthetic siRNA delivery in KB tumor xenografts. Eur. J. Pharm. Biopharm., 70:718–725.

80.

Zhang

, Gao

, Jiang

et al. 2010. Targeted minicircle DNA delivery using folate-poly(ethylene glycol)-polyethylenimine as non-viral carrier. Biomaterials, 31:6075–6086.

81.

Zhou

, Li

et al. 2008. Novel dual inhibitory function aptamer-siRNA delivery system for HIV-1 therapy. Mol. Ther., 16:1481–1489.

82.

Zhou

, Swiderski

, Li

et al. 2009a. Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells. Nucleic Acids Res., 37:3094–3109.

83.

Zhou

, Shu

, Guo

et al. 2011. Dual functional RNA nanoparticles containing phi29 motor pRNA and anti-gp120 aptamer for cell-type specific delivery and HIV-1 Inhibition. Methods.

84.

Zhou

Q.H.

, Wu

, Manickam

D.S.

, Oupicky

2009b. Evaluation of pharmacokinetics of bioreducible gene delivery vectors by real-time PCR. Pharm. Res., 26:1581–1589.

85.

Zimmermann

T.S.

, Lee

A.C.

, Akinc

et al. 2006. RNAi-mediated gene silencing in non-human primates. Nature, 441:111–114.

86.

Zintchenko

, Susha

A.S.

, Concia

et al. 2009. Drug nanocarriers labeled with near-infrared-emitting quantum dots (quantoplexes): imaging fast dynamics of distribution in living animals. Mol. Ther., 17:1849–1856.