Designing of Tumor-Targeted HuR siRNA Nanoparticle ... - Springer Link

Chapter 17

Designing of Tumor-Targeted HuR siRNA Nanoparticle as a Therapeutic for Lung Cancer Ranganayaki Muralidharan, Anish Babu, Kanthesh Basalingappa, Meghna Mehta, Anupama Munshi, and Rajagopal Ramesh

Abstract Small interfering (si) RNA has emerged as a valuable laboratory tool not only for studying the function of a gene but also as a therapeutic for cancer treatment. siRNA-based therapies are being developed and tested against a plethora of human cancers in laboratories around the world and serve as an alternate strategy for conventional therapy, in particular for cancers that have developed resistance to therapy. Preclinical studies have convincingly demonstrated that siRNA therapy when combined with conventional therapies and small molecule inhibitors produce enhanced antitumor activity. While siRNA therapy is attractive, there are also several hurdles that need to be overcome for successfully translating to the clinic. One major hurdle is the availability of a biodegradable delivery vehicle that can efficiently protect the siRNA from rapid degradation when administered in vivo. siRNA therapy, like conventional and molecularly targeted therapies, requires specificity and targeted delivery to the rapidly growing tumor such that the tumor therapeutic efficacy is high while normal tissue toxicity is low. Recent advances made in the field of nanotechnology have led to the synthesis of nanocarriers of various compositions. These nanocarriers, often referred to as nanoparticles, can carry different payloads including siRNA. Among several nanocarriers that have been tested till date, lipid-based nanoparticles have been the most widely tested due to their excellent biocompatibility, low immunogenicity, safety, and enhanced efficacy. In the present study, we describe a lipid-based nanoparticle that is targeted toward human lung cancer cells expressing the folate R. Ramesh (*) Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA Graduate Program in Biomedical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA Department of Pathology, The Stanton Young Biomedical Research Center, Suite 1403, 975 N. E., 10th Street, Oklahoma City, OK 73104, USA e-mail: [email protected] © Springer International Publishing Switzerland 2015 V. Gandhi et al. (eds.), Multi-Targeted Approach to Treatment of Cancer, DOI 10.1007/978-3-319-12253-3_17

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receptor. The therapeutic payload carried by the nanoparticle is an siRNA that targets the human antigen R (HuR). HuR is an mRNA-binding protein that regulates the stability and translation of several oncoproteins and is overexpressed in lung cancer. Thus HuR is a druggable target and its inhibition should result in global knockdown of several oncoproteins resulting in therapeutic efficacy. The present chapter provides an overview of various lipid-based nanoformulations including tumor-specific targets for nanoparticle delivery, challenges in siRNA delivery, and molecular targets against which siRNA is developed and finally discusses about our rationally designed folate-targeted nanoparticle carrying siRNA for HuR (HuR-FNP) gene silencing and its promising potential in the treatment of lung cancer. Keywords Lung cancer • HuR • siRNA • Nanoparticles • Liposome • Metastasis • Imaging • Transferrin receptor • Folate receptor

17.1

Introduction

Worldwide, cancer has emerged as the leading cause of disease-related mortality. According to a recent report by the World Health Organization (WHO), approximately 13 million cancer-related deaths are likely to occur every year in the next couple of decades (Globocan 2012). Despite significant improvements made in cancer treatment modality, the overall 5-year survival for majority of cancers, especially for epithelial cancers such as lung cancer, is very poor. This is due to tumor heterogeneity, poor drug accumulation in the cancer cells, and development of drug resistance leading to disease relapse and metastasis. Therefore, to overcome these challenges and have improved therapeutic efficacy and clinical outcomes, there is a continued effort toward developing new therapeutic strategies in the fight against cancer. Recent advances made in the areas of molecular biology and genomic sciences have opened up new avenues for cancer therapy. RNA interference (RNAi) is one such approach which is based on the interaction of short antisense RNA sequences with target messenger RNAs (mRNAs) causing gene silencing (Hannon 2002). The short antisense RNA sequences, also referred to as small interfering RNA (siRNA), are short double-stranded (ds) RNA fragments (usually 19–23 nucleotides) that form complexes with RNA-induced silencing complexes (RISCs) upon introduction into the cell cytoplasm. These siRNA-RISC complexes are then recruited toward the target mRNAs by recognizing the complementary sequences and induce cleavage of mRNAs (Hannon 2002). Silencing of the target mRNAs result in the inhibition of expression of specific proteins that are involved in various cellular processes. In the context of cancer, RNAi aims to selectively sequence and specifically silence the expression of oncogenes that are involved in cancer cell survival, angiogenesis, invasion, and metastases thus producing a therapeutic effect (Cheng and Qin 2009). However, to realize the full potential of siRNA-based cancer therapy in vivo, it is important to develop delivery vehicles that can efficiently

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Designing of Tumor-Targeted HuR siRNA Nanoparticle as a. . .

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carry and deliver the siRNA specifically to the tumor site for producing an antitumor activity. Having such siRNA delivery vehicles will lead to innovative treatment strategies and overcome the barriers in siRNA-based therapy. Till recent, viral vectors such as lentivirus (Sun et al. 2012) and adenovirus (Kargiotis et al. 2008) were the preferred gene delivery vehicles. While viral vectors have several advantages, they also have limitations that restrict their use. These include the potential for mutagenicity and the inability to perform repeated treatments due to the induction of host-immune response against the virus (Nayak and Herzog 2010). As an alternate to virus-based therapy, nanotechnology platform has provided a variety of nanocarriers that are formulated using natural and synthetic nonviral components. These nonviral nanocarriers are less immunogenic, have improved safety profiles, and are easy to manufacture in large scale (Ku et al. 2014). Among the several nanocarriers developed and tested till date, the cationic lipid-based nanoparticles have emerged as highly attractive siRNA delivery systems for cancer treatment (Buyens et al. 2009; Ozpolat et al. 2014). To further improve the safety and efficacy of the lipid-based nanoparticles, the outer surface of the nanoparticle is often decorated with tumor-targeting moieties such that the therapeutic siRNA can be specifically delivered to the tumor (Gao et al. 2010; Yang et al. 2014). In the present chapter, we provide an overview on the various lipid-based nanoparticles that have been used for siRNA delivery, targeting moieties for ascribing tumor specificity for the nanoparticle, challenges encountered in siRNA delivery, and druggable oncotargets against which specific siRNA is developed, and finally discuss about our rationally designed folate-targeted nanoparticle carrying HuR siRNA as a therapeutic for lung cancer.

17.2

Challenges in siRNA Delivery

While siRNA delivery and efficacy are efficient in vitro, the therapeutic potential of siRNA in vivo, however, depends on its successful delivery and targeting efficiency. In vivo delivery of naked siRNA results in enzymatic degradation by serum endonucleases as well as rapid renal clearance causing loss of its activity. For instance, the half-life of siRNA in the blood plasma is reduced to a few minutes due to its rapid degradation and clearance (Gao et al. 2009). The physicochemical characteristics of siRNA such as large molecular weight and negative charge influence its stability in the blood plasma. The stability, however, can be markedly improved by chemical modifications such as addition of 20 -methoxy (20 -OMe) and 20 -fluoro (20 -F) to the siRNA. These modifications, while demonstrating increased resistance toward plasma nucleases and enhanced stability in blood circulation, do not improve the intracellular delivery and efficacy of the siRNA (Layzer et al. 2004). Therefore, specific vehicle or nanoparticles are required to effectively deliver siRNA to the tumor while protecting it from degradation and clearance in vivo.

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Another hurdle in siRNA delivery in spite of its being contained in a delivery vehicle is the vascular endothelial barrier. Administration of the siRNA-containing nanoparticle results in it being circulated in the blood and finally reaching the tumor via the disorganized leaky vasculature found in many solid tumors. siRNAcontaining nanocarriers that are less than 500 nm in size pass the leaky vasculature and enter the tumor interstitial space and remain in the tumor milieu due to poor lymphatic drainage. This phenomenon, referred to as enhanced permeation and retention (EPR) effect in solid tumors, has been explained in detail in a recent review (Maeda 2012). The siRNA-containing nanoparticle subsequently enters the extracellular matrix (ECM) which consists of an intricate network of macromolecules such as fibrous proteins and polysaccharides. Larger nanoparticles find it difficult to pass through the dense ECM and reach the deep-seated cancer tissue. Also, it is reported that neutrally charged nanoparticles diffuse through the ECM more rapidly compared to positively and negatively charged nanoparticles (Stylianopoulos et al. 2010). Nanoparticles that successfully pass the ECM next encounter the tumor cells. Entry of an siRNA nanoparticle into a cell depends mainly on their surface charge. Positively charged particles show greater affinity toward the negatively charged cell membrane and enter the cell easily compared to the particles that are negatively charged or neutral in charge. Thus, positively charged nanoparticles such as cationic lipid-based nanoparticle and cationic polymer nanoparticle have been widely used for delivering the negatively charged siRNA (Wang et al. 2010; Lin et al. 2014). Binding of siRNA-containing nanoparticle to the cell surface is followed by clathrin-mediated or receptor-mediated endocytosis, a mechanism for cellular internalization of solid particles into the cells (Gilleron et al. 2013). Endocytosis involves the formation of endosomes in which the particles are carried and fuse with lysosomes. Unfortunately, the endosomal entrapment and lysosomal degradation of siRNA nanocarriers are the major obstacles in the successful delivery of siRNA into the cell cytoplasm. Since the therapeutic efficacy relies on an efficient silencing of the target mRNA in the cytoplasm, it is very important that the nanoparticle being used protects the siRNA from endosomal entrapment and lysosomal degradation. Thus, while developing siRNA-based therapies, it is important to ensure that the nanoparticle escapes the endosome/lysosome compartment and successfully releases the siRNA in the cytoplasm of the cell for effective silencing of the target gene. A large number of siRNA delivery vehicles have been developed that include liposomes or lipid nanoparticles (Lin et al. 2014), polymer nanocarriers (Patil and Panyam 2009), hybrid nanoparticles (Shi et al. 2014), gold and iron nanoparticles (Abigail et al. 2011), nano-sized hydrogels (Smith and Lyon 2012), porous silica nanoparticles (Li et al. 2013), and, more recently, exosomes (El-Andaloussi et al. 2012). Among the various nanoformulations developed thus far, the lipidbased nanoparticles have received the greatest attention for delivering siRNA. This is because of their ability to fuse with biomembranes, escape from endosomes, and achieve high transfection efficiency (Lin et al. 2014; Gilleron et al. 2013), the details of which are described in the section below.

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Lipid-Based Nanoparticles for siRNA Delivery

Liposome-based gene and drug delivery technology has undergone tremendous advancement over the last decade due to their biocompatibility and biodegradability (Lin et al. 2014). Liposomes are formed by the self-assembly of lipid molecules with hydrophobic and hydrophilic ends and form colloidal dispersions in aqueous environment. Liposomes have unique advantages in that they can exist as singlelayered to multilayered vesicles, are relatively easy to manipulate, can be formulated using diverse lipid compositions, can carry hydrophobic and/or hydrophilic payload(s), protect therapeutic cargo such as siRNA from degradation, and have low immunogenicity and high siRNA transfection efficiencies (Ozpolat et al. 2014; Lin et al. 2014). These unique and favorable characteristics have resulted in the extensive use of liposomes as reliable nanocarriers for gene therapeutics. When preformed, liposomes and siRNA are mixed together under ideal buffered conditions; electrostatic interactions occur between lipid and siRNA to form siRNAliposome complexes (Buyens et al. 2009). The size of the siRNA-liposome complex, however, depends upon the size of the preformed liposomes. Some investigators have created liposomes with the inner core containing cationic polymeroligonucleotide complexes. For example, Kang et al. (2014) have developed a liposome encapsulating chitosan-oligonucleotide complex (polyplex) for effective tumor delivery of therapeutic oligonucleotides (ODN). This polyplex allowed enhanced uptake and controlled release of ODN in murine melanoma cells in vitro. However, the siRNA delivery potential of such nanodelivery systems is still to be realized in in vivo tumor models. Cationic liposomes form the major class of lipid-based nanoparticles for siRNA delivery. The most commonly used cationic lipids in the nanoformulations are DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DDAB (dimethyldioctadecylammonium bromide), and DODAB (1,2-dioleoyl-3dimethylammonium propane). Cationic liposomes form complexes with siRNA (lipoplexes) via ionic interaction (Surace et al. 2009). When the positively charged liposome-siRNA complexes come in contact with a cell, they dock onto the negatively charged cell membrane and aid in a successful intracellular delivery of the siRNA. It has been shown that above 95 % of cationic liposome-siRNA complexes enter into mammalian cells via endocytotic mechanism (Lu et al. 2009). To overcome the intracellular endosome/lysosome barrier and facilitate efficient siRNA release, several strategies have been incorporated into the liposome that include the use of fusogenic lipids, stimuli-sensitive lipids, photodirected delivery of liposomal siRNA, and cell-penetrating peptide-modified lipoplexes (Dominska and Dykxhoorn 2010). Fusogenic lipids such as DOPE (dioleoylphosphatidylethanolamine) or DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) are neutral helper lipids which when mixed with cationic lipids enhance the cellular uptake and endosomal escape of siRNA. These helper lipids have the ability to form reverse hexagonal phase

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(long, cylindrical, inverted micelle-like structures) and rapidly fuse with anionic membranes to release its cargo (May et al. 2000). In a different strategy, cellpenetrating peptides (fusogenic peptides) are utilized to modify the lipid carrier to aid effective cell penetration and siRNA delivery. A recent study has shown that siRNA-based gene silencing activity was improved when a pH-sensitive fusogenic peptide of 30 amino acid length called GALA (WEAALAEALAEALAEHLAEALAEALEALAA) was used to modify the siRNA nanocarrier (Hatakeyama et al. 2009). GALA has a repeating amino acid sequence of glutamine, alanine, leucine, and alanine. However, the concentration of GALA in the lipid nanocarrier was shown to be critical for achieving the best transfection efficiency (Futaki et al. 2005). Liposome- or lipid-based nanoparticle, like any other nanoparticle, faces the hurdle of rapid clearance by the reticuloendothelial system (RES) in vivo. This occurs by the interaction of the nanoparticles with serum proteins leading to the formation of large aggregates and subsequent clearance by the RES resulting in a short half-life of the nanoparticles in blood circulation. A short half-life results in poor drug accumulation in the tumor and in a diminished therapeutic effect. To overcome this limitation, polyethylene glycol (PEG) which is an inert hydrophilic polymer is often included in the lipid nanoformulation. Incorporation of PEG into the formulation imparts stealth property to the liposomes by delaying the liposomeserum protein interaction and the RES clearance thereby prolonging its circulation time in the blood (Immordino et al. 2006). Another added advantage of PEGylating the liposome is that PEG provides a wide surface area to which targeting ligand such as peptides, antibodies, and aptamers can be attached by chemical conjugation. This strategy not only renders stability to the liposome carrier but also aids in achieving tumor-targeted gene delivery. Majority of the siRNA-liposomes that are currently in preclinical or clinical stages of development are PEG modified (Bhavsar et al. 2012). While PEG modification of the liposome or lipid-based nanoparticles is advantageous, it is important to optimize the density of the PEG molecules on the surface of the liposome as enhanced hydrophilicity and neutral charge might negatively affect the interaction of the liposome with the cell membrane (Dan 2002).

17.4

Targeted Liposomes for siRNA Delivery

The rationale for developing tumor-targeted liposome or lipid-based nanoparticles is to increase the drug accumulation in the tumor while sparing the surrounding normal tissue with minimal cytotoxicity. By increasing the drug concentration inside the tumor, one could achieve a higher therapeutic index. To achieve this goal, a large number of ligands targeting different cell surface receptors that are overexpressed in cancer cells have been tested. Ligands that are often used for targeting the cell surface receptors include antibodies or antibody fragments,

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peptides, aptamers, and small molecule ligands such as transferrin, folic acid, and CD44. Antibodies offer extremely high specificity and binding affinity toward the target receptors in cancer cells. Conjugating the human epidermal growth factor receptor (EGFR/HER1) and human EGFR2 (Her2/Neu) antibodies to nanoparticles has shown specificity toward cancer cells that overexpress these receptors (Fay and Scott 2011). SiRNA delivery was highly efficient in Her2/Neu overexpressing cancer cells when anti-Her2 monoclonal antibody Fab’ fragments were conjugated with liposomes (Gao et al. 2010). In another study, Gao et al. (2012) showed highly efficient in vitro and in vivo siRNA delivery to hepatocellular carcinoma when a Fab fragment of the EGFR antibody was attached to the liposome. Similarly, lipid nanoparticles conjugated with antibodies specific to the vascular endothelial growth factor (VEGF) receptor (Yang et al. 2014) and prostate-specific membrane antigen (PSMA; Xiang et al. 2013) demonstrated increased tumor specificity and drug delivery that resulted in improved antitumor activity. RGD (Arg-Gly-Asp amino acid triplet) peptide forms another class of targeting molecule that is largely exploited for efficient cell internalization of nanoparticles or liposomes. RGD peptide binds to the integrin receptors expressed on tumor cells and tumor endothelial cells (Ruoslahti 1996). Receptor-mediated endocytosis is known to be the prominent mechanism by which RGD-liposomes are internalized into the cells (Alam et al. 2008). Sequential treatment of multidrug-resistant (MDR) MCF7/A breast cancer cells with RGD-modified liposomes carrying siRNA against the p-glycoprotein (P-gp) followed by treatment with RGD-liposome containing doxorubicin resulted in the suppression of P-gp protein expression and restoration of chemosensitivity to doxorubicin both in vitro and in vivo (Jiang et al. 2010). Thus, the reversal of drug resistance and restoration of chemosensitivity in breast cancer cells were demonstrated using the RGD-targeted nanoparticles. Aptamers are single-stranded RNA or DNA oligonucleotide fragments that have rapidly emerged as a powerful class of ligands with potential application in cancer targeted therapy (Zhang et al. 2011). Aptamers show high affinity toward the target molecules expressed on the cell surface and are generally derived by a PCR-based procedure called systematic evolution of ligands by exponential enrichment (SELEX), and the procedure is explained in detail in a recent review (Orava et al. 2010). In a recent study, Li et al. (2014a) demonstrated the potential use of aptamer-targeted liposomal delivery of siRNA for the treatment of melanoma. In the study, a nucleolin-targeted aptamer (AS1411) conjugated to PEG-liposome carrying the anti-BRAF siRNA was tested for melanoma treatment. Study results demonstrated significant silencing of BRAF gene and enhanced inhibition of melanoma tumor growth demonstrating that aptamer-mediated, tumor-targeted siRNA delivery was effective and successful. Tumor-targeted siRNA delivery via the transferrin receptor (TfR) is another area of intense investigation. TfR is overexpressed in several human cancers making it an ideal target for drug delivery. TfR-targeted nanoparticle delivery is achieved by conjugating transferrin to the liposome or lipid-based nanoparticle carrying the therapeutic siRNA. The nanoparticle on binding to the TfR gets internalized and

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delivers the payload inside the cell. Transferrin can be exploited therapeutically in two ways: (1) by blocking the function of the TfR thereby inhibiting cell growth and (2) by enhancing the delivery of therapeutics via TfR. Targeted delivery of oligonucleotides (ODN) or siRNA via TfR is a valuable approach in cancer gene therapeutics. Delivery of an siRNA targeted to c-Jun and contained in a transferrin-conjugated DOTAP-cholesterol (DOTAP-Chol) liposome showed enhanced gene silencing effect in TfR overexpressing hepatocellular carcinoma and glioma cell lines (Cardoso et al. 2007). Mendonça et al. (2010a) using TfR-targeted liposome showed that BCR-ABL siRNA effectively knocked down the target gene in myeloid leukemia cells. The same group also demonstrated that the TfR-targeted liposome system can be exploited for co-encapsulating an anticancer drug and the siRNA for the treatment of chronic myeloid leukemia (Mendonça et al. 2010b). Another receptor that is widely tested for tumor-targeted gene and drug delivery is the folic acid receptor (FR). Folic acid (folate) is the natural ligand for FR. Studies have shown that FR expression is overexpressed in many types of solid tumors of epithelial origin. FRs are glycoproteins, which exist in several isoforms of which the isoforms FRα and FRβ are both anchored in the cell membrane by a glycosylphosphatidylinositol domain (Høier-Madsen et al. 2008). Similar to the uptake process of the vitamin folate, FRs mediate the cellular internalization of folate conjugates via FR-mediated endocytosis making this process more specific. The use of folic acid as targeting agent has several advantages which include its smaller size, accessibility for modification, stability in a wide range of pH and at elevated temperatures, and nontoxicity to healthy tissues and organs. The expression level of FRα on tumors has a prognostic value, since FRα expression was shown to correlate with patient survival (O’Shannessy et al. 2012). Similarly, high FRα expression correlated with better survival in patients diagnosed with non-small cell lung cancer (Iwakiri et al. 2008). All of these features make folic acid a reliable ligand for targeting FRα-expressing cancer cells. Folateconjugated liposome delivery of N-myc-targeted siRNA in a metastatic neuroblastoma mouse model showed that the siRNA specifically is distributed in the neuroblastoma tumors and caused the silencing of N-myc at both mRNA and protein levels (Zhu et al. 2013). In the same study, the folate-targeted liposome was shown to be superior in producing antitumor activity in vivo compared to the nontargeted liposome and traditional transfection reagent. In another study, the treatment of K562 leukemia tumors with folate-PEG-liposome containing siRNA targeted toward the antiapoptotic protein, survivin, greatly reduced survivin protein expression and inhibited tumor growth when compared to control groups (Li et al. 2014b). It is important to note that a few of the targeted liposome-siRNA systems have entered into clinical trials emphasizing the progress made in liposome- or lipidbased siRNA therapeutics. For example, Atu 027, a therapeutic liposome-siRNA complex designed for cancer gene silencing therapy, carries a chemically stabilized siRNA for targeted suppression of PKN3 gene in cancer cells. It is currently being tested for the treatment of advanced solid tumors and pancreatic cancer metastasis (https://clinicaltrials.gov). Similarly, a lipid nanoparticle formulation (TKM 080301) containing siRNA for polo-like kinase 1 (PLK-1) gene is under clinical

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investigation for the treatment of hepatocellular carcinoma, neuroendocrine tumors, and adrenocortical carcinoma in humans (https://clinicaltrials.gov). These studies indicate the growing potential for targeted liposomal siRNA-based therapeutics in cancer treatment. Our laboratory has taken tremendous effort in developing biocompatible, targeted liposomal carriers for siRNA-based therapeutics for clinical application, especially for lung cancer therapy. In previous studies, we have shown that the nontargeted cationic DOTAP-Chol lipid nanoparticle can deliver a broad spectrum of tumor suppressor genes (p53, FUS1, and MDA7/IL-24) to lung tumors (Shanker et al. 2011). At present, our investigations are focused on improving the rationally designed DOTAP-Chol lipid nanoparticle for tumor-targeted delivery of siRNA for lung cancer treatment; the details of which are described in the latter section of this article.

17.5

siRNA Therapeutic Targets in Cancer

A number of molecular targets have been identified for siRNA-based silencing and therapy for cancer. These targets include genes involved in apoptosis, angiogenesis, cell survival, metastasis, multidrug resistance, and cell-cell communication. An excellent summary of cancer-associated genes targeted by siRNA is provided in some recent reviews (Devi 2006; Huang et al. 2008). A few examples of molecular targets in cancer against which siRNA therapy has been developed include BCL2, BAX, pyruvate kinase isozyme M2 (PKM2), hypoxia-inducible factor 1(HIF1)-α, claudin-3, and ATP-binding cassette (Li et al. 2012). Certain key signaling pathways in cancer cells have also been explored as specific molecular targets for siRNA-based therapy. For example, STAT3 (signal transducers and activators of transcription) cytoplasmic proteins, involved in the JAK/STAT pathway, act as signal messengers and transcription factors and participate in normal cellular response to growth factors (cytokines). Silencing of STAT3 using siRNA has been shown to induce Fas-mediated apoptosis by activating the caspase cascade in breast cancer cells (Kunigal et al. 2009) and increase chemosensitivity to paclitaxel in lung cancer cells (Su et al. 2012). Another target for siRNA-based cancer therapy is the vascular endothelial growth factor (VEGF). VEGF has been shown to play a major role in tumor angiogenesis and tumor cell survival. Therefore, siRNAmediated inhibition of VEGF should halt tumor cell survival and angiogenesis resulting in antitumor activity. In a recent in-human clinical trial and first of its kind, RNAi-based cancer therapy targeting VEGF and kinesin spindle proteins (KSP) has been initiated (Tabernero et al. 2013). Study reports showed the safety, pharmacokinetics, and mechanism of action of siRNA-based therapeutics in cancer patients. While there are several studies targeting oncogenes and pro-survival genes, we in our laboratory have focused our studies on targeting an mRNA-binding protein called human antigen R (HuR) in lung cancer. HuR is overexpressed in lung cancer and other solid tumors and has been identified as a possible druggable target for cancer treatment (Latorre et al. 2012; Tanaka et al. 2006a).

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17.6


HuR as Molecular Target for siRNA-Based Cancer Therapy

HuR, a ubiquitously expressed member of the embryonic lethal abnormal vision (ELAV)-like protein gene family, is one of the well-characterized RNA-binding proteins. HuR is a nucleocytoplasmic protein that is predominantly localized in the nucleus. HuR is activated under various stimuli and when activated binds to the AU-rich elements (AREs) that are present at the 30 and/or 50 untranslated region (UTR) of several mRNAs. The HuR-mRNA complex is then transported from the nucleus to the cytoplasm where it stabilizes the mRNA from degradation and enhances protein translation (Brennan and Steitz 2001). HuR has been shown to regulate cell proliferation, cell survival, angiogenesis, metastasis, and immune functions (Abdelmohsen et al. 2007; Galban et al. 2008; Abdelmohsen and Gorospe 2010). HuR binds to several mRNAs that have AREs at the 30 UTR and whose protein products are often overexpressed in cancer. mRNAs that have 30 UTR and are bound by HuR include those of cyclins, oncogenes, antiapoptotic genes, and growth factor genes. For example, HuR associates with mRNAs encoding for cyclin D1 and cyclin E1 resulting in aberrant alteration in the cell cycle phases and cell division (Kim and Gorospe 2008). HuR also suppresses p27 protein expression by binding to the ARE region present at the 50 end of the p27 promoter and thus preventing the cells from transitioning from the G phase to the S phase of the cell cycle (Millard et al. 2000; Wang et al. 2013). HuR has been shown to enhance the expression of HIF-1α and VEGF under hypoxic condition (Galban et al. 2008) and increase the stability of COX-2 mRNA aiding in increased tumor vasculature (Kurosu et al. 2011). HuR has also been shown to promote the expression of the antiapoptotic protein Bcl-2 and repress the production of c-Myc, thus having an antiapoptotic effect on cancer cells (Wang et al. 2013). Clinical studies have shown that HuR is overexpressed in several human cancers including lung cancer (Tanaka et al. 2006a, b; Barbisan et al. 2009; Latorre et al. 2012; Lauriola et al. 2012). Additionally, HuR overexpression was shown to be a poor prognostic marker and inversely correlated with the survival of patients diagnosed with lung cancer (Tanaka et al. 2006a, b). HuR expression was also shown to correlate with therapy resistance (Hostetter et al. 2008). Given the plethora of information about HuR and its ability to regulate multiple mRNAs whose protein products are overexpressed in cancer, we hypothesized that HuR is a druggable target and that siRNA-mediated inhibition of HuR will knock down several HuR-regulated oncoproteins resulting in a pronounced antitumor activity. To test our hypothesis, we developed a tumor-targeted lipid nanoparticle carrying HuR siRNA and tested its efficacy in vitro and in vivo using lung cancer as a model. Tumor targeting was achieved by coating the nanoparticle with folic acid that targets the folate receptor. Details on the design of the nanoparticle and its ability to deliver HuR siRNA to lung cancer cells are provided in the section described below.

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Folate-Targeted Lipid Nanoparticles for HuR siRNA Delivery in Lung Cancer

Studies from our laboratory have previously shown that the cationic DOTAP-Chol lipid nanoparticle was efficient in delivering tumor suppressor genes to human lung cancer cells both in vitro and in vivo (Shanker et al. 2011). A Phase I clinical trial was also conducted using our nanoparticle formulation for treating human lung cancer patients with a tumor suppressor gene (Lu et al. 2012). Given our ability to demonstrate the utility of the DOTAP-Chol nanoparticle for cancer therapy both in preclinical and clinical studies, we in the present study have used the same nanoparticle formulation for conducting HuR siRNA-based studies. What sets this study apart from our previous studies is that we have utilized a folate receptor (FR)-targeted nanoparticle in the present study. The rationale to target FRα is due to the fact that FRα is overexpressed in human lung cancer cell lines (data not shown). Thus, we designed an FRα-targeted lipid-based nanoparticle for delivering HuR siRNA and tested its efficacy against human lung cancer cells both in vitro and in vivo. The FRα-targeted lipid nanoparticle (FNP) was synthesized using the following strategy. The DOTAP-Chol lipid nanoparticle was synthesized as previously described (Ramesh et al. 2001). DSPE-PEG-folate (Avanti Polar Lipids, Alabaster, AL) ligand was subsequently conjugated to the DOTAP-Chol lipid nanoparticle by post-insertion technique. The resulting nanoparticle was labeled as FNP and used for characterization and functional studies. PEG-5000 was used in the formulation as pegylation was previously shown to prolong the in vivo half-life of the nanoparticle (Shiokawa et al. 2005). The schematic diagram of FNP carrying siRNA is shown in Fig. 17.1a. Optimization studies showed that FNP containing 0.03 mol% of DSPE-PEG-folate had a small size (200–300 nm) and a positive charge of +4.3 mV. Encapsulation efficiency studies using scrambled siRNA showed that more than 70 % of siRNA was entrapped inside the nanoparticle and protected from serum degradation. Analysis of the siRNA-containing FNP by transmission electron microscopy (TEM) showed that the particles were of uniform size and shape with no observable aggregation (Fig. 17.1b). In vitro studies confirmed that the nanoparticle efficiently and specifically targeted the FRα-overexpressing H1299 lung cancer cells compared to low FRα-expressing A549 cells (data not shown). Addition of excess free folate to the tissue culture medium resulted in the blocking of the FNP uptake by H1299 tumor cells demonstrating a receptor-mediated uptake of the nanoparticles. Based on these study results, we next initiated functional studies using HuR siRNA. FNP containing HuR siRNA was labeled as HuR-FNP, while FNP containing scrambled (control) siRNA was labeled as C-FNP. Cell proliferation assay showed that HuR-FNP treatment markedly inhibited H1299 lung cancer cell proliferation (31.3 % inhibition) at 48 h after treatment when compared to control cells that did not receive any treatment. In contrast, C-FNP treatment of H1299 cells resulted in only 7.3 % inhibition of cell proliferation compared to control cells (Fig. 17.2a). Cell cycle analysis revealed

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Fig. 17.1 (a) Schematic diagram showing folate-targeted nanoparticle (FNP) containing siRNA. (b) Transmission electron microscopy showing that HuR-FNPs are uniform in size and shape and do not show aggregation

Fig. 17.2 (a) H1299 lung cancer cells were treated with C-FNP or HuR-FNP. Cells that did not receive any treatment served as control. Cell proliferation was determined at 48 h after treatment. HuR-FNP treatment showed marked inhibition of cell proliferation when compared to C-FNP. (b) Western blot showed HuR-FNP treatment reduced expression of HuR protein and HuR-regulated oncoproteins at 48 h after treatment. Beta actin was used as loading control

HuR-NP-induced G1 phase cell cycle arrest in H1299 cells compared to C-NP and untreated control cells. Molecular analysis showed marked reduction in HuR protein expression in HuR-FNP-treated H1299 cells compared to C-FNP-treated cells (Fig. 17.2b). Associated with HuR knockdown was the global reduction in the expression of several HuR-regulated proteins which included cyclin D1, cyclin E, and Bcl-2 (Fig. 17.2b). In contrast, HuR inhibition resulted in the upregulation of

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p27 protein expression (data not shown). The upregulation of p27 protein expression is not surprising as a previous study demonstrated that the 50 end of p27 promoter is rich in AU sequence and HuR binds to this site and suppresses p27 transcriptional activity (Millard et al. 2000). By inhibiting HuR, the p27 promoter activity is repressed, and hence increased p27 protein expression is observed in the H1299 lung cancer cell line. Since our in vitro studies established that the HuR-FNP exhibited specificity toward FRα-positive lung tumor cells resulting in antitumor activity, we next conducted pilot in vivo studies to determine if a similar antitumor activity was observed. For this purpose, subcutaneous lung tumor xenografts were established in nude mice by injecting H1299 lung tumor cells expressing the luciferase (luc) marker gene. The expression of luciferase by the tumor cells allows for monitoring tumor growth by noninvasive molecular imaging. When the tumors were about 50– 100 mm in size, mice were injected intravenously with HuR-FNP via the tail vein. The HuR siRNA was fluorescently tagged with DY647 dye such that we could noninvasively monitor the nanoparticle biodistribution and accumulation in the tumor and other organs over time using the IVIS Spectrum (PerkinElmer, Waltham, MA) molecular imaging system. As shown in Fig. 17.3a, colocalization of fluorescently tagged HuR siRNA within the luciferase-positive H1299 tumor showed efficient accumulation of the particles in the tumor. The results also revealed that the FNPs were selectively internalized by the tumors compared to the other

Fig. 17.3 (a) Nude mice bearing subcutaneous H1299 lung tumors and stably expressing luciferase were injected with HuR-FNP intravenously via the tail vein. HuR siRNA contained in the FNP was tagged with fluorescent dye DY647. Mice were imaged using IVIS Spectrum imaging system (PerkinElmer). Colocalization of HuR-FNP in the tumors was observed at 24 h after HuR-FNP injection. Arrows indicate HuR-FNP accumulation in the luciferase-positive H1299 tumors. (b) HuR-FNP-treated tumors showed reduced HuR, cyclin D1, cyclin E, and COX-2 protein expression compared to control tumors. Beta actin was used as loading control

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surrounding organs. From our preliminary study results, we can speculate that the nonspecific uptake of the particles by RES is significantly reduced and that pegylation of the NPs facilitated in reducing the RES-mediated clearance of the NPs. To evaluate whether the accumulation of HuR-FNP in the tumor caused HuR gene silencing, tumors were harvested and analyzed for HuR protein expression. Western blotting showed that HuR protein expression was significantly reduced in tumors harvested from mice that received HuR-FNP treatment compared to HuR expression in tumor harvested from control mice that did not receive any treatment (Fig. 17.3b). Associated with the reduction in HuR protein expression was the reduction in cyclin D, cyclin E, and COX2 protein expression in the tumors from HuR-FNP-treated mice. Our preliminary in vitro and in vivo study results demonstrated that lung tumor-targeted delivery of HuR siRNA using FNPs suppresses cell proliferation and induces cell cycle arrest by global knockdown of HuR-regulated proteins resulting in an antitumor activity. While our study results are interesting, it is to be noted that additional in vitro and in vivo studies are warranted. In vivo studies that are currently underway in the laboratory are focused on determining HuR-FNP biodistribution, pharmacokinetics, and efficacy. The results of these ongoing studies are expected to be available in the future. Conclusions The application of siRNA technology for cancer therapy is exciting and offers an alternate cancer treatment strategy in situations where conventional therapy has failed. While several new nanoformulations are being tested, lipidbased nanoparticles continue to hold their position in drug and gene delivery with several of them being tested in the clinic. While several advantages exist in using lipid-based nanoparticles, there is still room for improvements that can be made especially in areas of size, charge, half-life, etc. The use of appropriate tumor-targeting ligand is another important aspect to consider. While several ligands have been identified for tumor-targeted siRNA delivery, it is pertinent that we use the most appropriate ligand relevant to a particular cancer type or histology for drug delivery. The use of such strategies will provide better therapeutic outcomes. In the present chapter, we have given a brief account of siRNA-based gene silencing using tumor-targeted nanoparticles for cancer treatment. As a proof of concept, we have demonstrated that the delivery of HuR siRNA using FNP produced an inhibitory effect not only on HuR but also on HuR-regulated proteins. While targeting HuR alone is interesting, it is important to realize that combinatorial therapy or cocktail therapy might be more efficacious in reducing the tumor burden. Finally, incorporating molecular imaging agents in conjunction with HuR siRNA into FNP will provide an opportunity to monitor response to therapy in real time and facilitate important treatment (continued)

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decisions based on response to treatment. Finally, it is important to consider the challenges in producing clinical-grade FNP in large scale under good manufacturing practice (GMP) and ensuring that the FNPs meet the Federal Drug Administration (FDA) guidelines for approval in clinical testing.

Acknowledgments This study was supported by the National Cancer Institute grant R01 CA167516 (RR), by the National Institutes of Health (NIH) grant P20 GM103639-02 (AM) from the COBRE Program of NIH, by the Oklahoma Tobacco Research Center (OTRC) seed grant (RR), and by the Jim and Christy Everest Endowed Chair in Cancer Developmental Therapeutics (RR). We thank the Stephenson Cancer Center at the University of Oklahoma, Oklahoma City, OK, and the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103639 for the use of Molecular Imaging Core, which provided small animal imaging service. R.R. is an Oklahoma TSET research scholar and holds the Jim and Christy Everest Endowed Chair in Cancer Developmental Therapeutics.

References Abdelmohsen K, Gorospe M (2010) Posttranscriptional regulation of cancer traits by HuR. Wiley Interdiscip Rev RNA 1:214–229 Abdelmohsen K, Lal A, Kim HH et al (2007) Posttranscriptional orchestration of an anti-apoptotic program by HuR. Cell Cycle 6:1288–1292 Abigail KR, Jean L, Langer R et al (2011) Five years of siRNA delivery: spotlight on gold nanoparticles. Small 7:1932–1937 Alam MR, Dixit V, Kang H et al (2008) Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis. Nucleic Acids Res 36:2764–2776 Barbisan F, Mazzucchelli R, Santinelli A et al (2009) Overexpression of ELAV-like protein HuR is associated with increased COX-2 expression in atrophy, high-grade prostatic intraepithelial neoplasia, and incidental prostate cancer in cystoprostatectomies. Eur Urol 56:105–112 Bhavsar D, Subramanian K, Sethuraman S et al (2012) Translational siRNA therapeutics using liposomal carriers: prospects & challenges. Curr Gene Ther 12:315–332 Brennan CM, Steitz JA (2001) HuR and mRNA stability. Cell Mol Life Sci 58:266–277 Buyens K, Demeester J, De Smedt SS et al (2009) Elucidating the encapsulation of short interfering RNA in PEGylated cationic liposomes. Langmuir 25:4886–4891 Cardoso AL, Simo˜es S, de Almeida LP et al (2007) siRNA delivery by a transferrin-associated lipid-based vector: a non-viral strategy to mediate gene silencing. J Gene Med 9:170–183 Cheng K, Qin B (2009) RNA interference for cancer therapy. In: Lu Y, Mahato RI (eds) Pharmaceutical perspectives of cancer therapeutics. Springer, New York, pp 399–440 Dan N (2002) Effect of liposome charge and PEG polymer layer thickness on cell–liposome electrostatic interactions. Biochim Biophys Acta 1564:343–348 Devi GR (2006) siRNA-based approaches in cancer therapy. Cancer Gene Ther 13:819–829 Dominska M, Dykxhoorn DM (2010) Breaking down the barriers: siRNA delivery and endosome escape. J Cell Sci 123:1183–1189 El-Andaloussi S, Lee Y, Lakhal-Littleton S et al (2012) Exosome-mediated delivery of siRNA in vitro and in vivo. Nat Protoc 7:2112–2126 Fay F, Scott CJ (2011) Antibody-targeted nanoparticles for cancer therapy. Immunotherapy 3:381–394

292


Futaki S, Masui Y, Nakase I (2005) Unique features of a pH-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes. J Gene Med 7:1450–1458 Galban S, Kuwano Y, Pullmann R Jr et al (2008) RNA-binding proteins HuR and PTB promote the translation of hypoxia-inducible factor 1 alpha. Mol Cell Biol 28:93–107 Gao S, Dagnaes-Hansen F, Nielsen E et al (2009) The effect of chemical modification and nanoparticle formulation on stability and biodistribution of siRNA in mice. Mol Ther 17:1225–1233 Gao J, Sun J, Li H (2010) Lyophilized HER2-specific PEGylated immunoliposomes for active siRNA gene silencing. Biomaterials 31:2655–2664 Gao J, Yu Y, Zhang Y (2012) EGFR-specific PEGylated immunoliposomes for active siRNA delivery in hepatocellular carcinoma. Biomaterials 33:270–282 Gilleron J, Querbes W, Zeigerer A et al (2013) Image-based analysis of lipid nanoparticlemediated siRNA delivery, intracellular trafficking and endosomal escape. Nat Biotechnol 31:638–646 Globocan 2012 (2012) IARC, World Health Organisation. http://www.iarc.fr/en/media-centre/pr/ 2014/pdfs/pr224_E.pdf. Accessed 10 Jun 2014 Hannon GJ (2002) RNA interference. Nature 418:244–251 Hatakeyama H, Ito E, Akita HA (2009) pH-sensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silencing of siRNA-containing nanoparticles in vitro and in vivo. J Control Release 139:127–132 Høier-Madsen M, Holm J, Hansen SI (2008) Alpha isoforms of soluble and membrane-linked folate-binding protein in human blood. Biosci Rep 28:153–160 Hostetter C, Licata LA, Witkiewicz A et al (2008) Cytoplasmic accumulation of RNA binding protein HuR is central to tamoxifen resistance in estrogen receptor positive breast cancer cells. Cancer Biol Ther 7:1496–1506 Huang C, Li M, Chen C, Yao Q (2008) Small interfering RNA therapy in cancer: mechanism, potential targets, and clinical applications. Expert Opin Ther Targets 12:637–645 Immordino ML, Dosio F, Cattel L (2006) Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential. Int J Nanomedicine 1:297–315 Iwakiri S, Sonobe M, Nagai S et al (2008) Expression status of folate receptor alpha is significantly correlated with prognosis in non-small-cell lung cancers. Ann Surg Oncol 15:889–899 Jiang J, Yang SJ, Wang JC et al (2010) Sequential treatment of drug-resistant tumors with RGD-modified liposomes containing siRNA or doxorubicin. Eur J Pharm Biopharm 76:170– 178 Kang JH, Battogtokh G, Ko YT (2014) Folate-targeted liposome encapsulating chitosan/oligonucleotide polyplexes for tumor targeting. AAPS Pharm Sci Tech. doi:10.1208/s12240-0140136-5, Epub ahead of print Kargiotis O, Chetty C, Gondi CS et al (2008) Adenovirus-mediated transfer of siRNA against MMP-2 mRNA results in impaired invasion and tumor-induced angiogenesis, induces apoptosis in vitro and inhibits tumor growth in vivo in glioblastoma. Oncogene 27:4830–4840 Kim HH, Gorospe M (2008) Phosphorylated HuR shuttles in cycle. Cell Cycle 7:3124–3126 Ku SH, Kim K, Choi K (2014) Tumor-targeting multifunctional nanoparticles for siRNA delivery: recent advances in cancer therapy. Adv Healthcare Mater. doi:10.1002/adhm.201300607 Kunigal S, Lakka SS, Sodadasu PK et al (2009) Stat3-siRNA induces Fas-mediated apoptosis in vitro and in vivo in breast cancer. Int J Oncol 34:1209–1220 Kurosu T, Ohga N, Hida Y et al (2011) HuR keeps an angiogenic switch on by stabilising mRNA of VEGF and COX-2 in tumour endothelium. Br J Cancer 104:819–829. doi:10. 1038/bjc.2011.20 Latorre E, Tebaldi T, Viero G et al (2012) Downregulation of HuR as a new mechanism of doxorubicin resistance in breast cancer cells. Mol Cancer 11:13. doi:10.1186/1476-4598-11-13 Lauriola L, Granone P, Ramella S et al (2012) Expression of the RNA-binding protein HuR and its clinical significance in human stage I and II lung adenocarcinoma. Histol Histopathol 27:617– 626

17


293

Layzer JM, McCaffrey AP, Tanner AK (2004) In vivo activity of nuclease-resistant siRNAs. RNA 10:766–771 Li YT, Chua MJ, Kunnath AP et al (2012) Reversing multidrug resistance in breast cancer cells by silencing ABC transporter genes with nanoparticle-facilitated delivery of target siRNAs. Int J Nanomedicine 7:2473–2481 Li X, Chen Y, Wang M et al (2013) A mesoporous silica nanoparticle-PEI-fusogenic peptide system for siRNA delivery in cancer therapy. Biomaterials 34:1391–1401 Li L, Hou J, Liu X et al (2014a) Nucleolin-targeting liposomes guided by aptamer AS1411 for the delivery of siRNA for the treatment of malignant melanomas. Biomaterials 35:3840–3850 Li J, Yue M, Shi X et al (2014b) Evaluation of anti-cancer activity of Survivin siRNA delivered by folate receptor-targeted polyethylene glycol liposomes in K562-bearing xenograft mice. Biomed Eng Appl Basis Commun 26. doi:10.4015/S1016237214500264 Lin Q, Chen J, Zhang Z et al (2014) Lipid-based nanoparticles in the systemic delivery of siRNA. Nanomedicine (Lond) 9:105–120 Lu JJ, Langer R, Chen J (2009) A novel mechanism is involved in cationic lipid mediated functional siRNA delivery. Mol Pharm 6:763–771 Lu C, Stewart DJ, Lee JJ et al (2012) Phase I clinical trial of systemically administered TUSC2 (FUS1)-nanoparticles mediating functional gene transfer in humans. PLoS One 7:e34833 Maeda H (2012) Macromolecular therapeutics in cancer treatment: the EPR effect and beyond. J Control Release 164:138–144 May S, Harries D, Ben-Shaul A (2000) The phase behavior of cationic lipid-DNA complexes. Biophys J 78:1681–1697 Mendonça LS, Firmino F, Moreira JN et al (2010a) Transferrin receptor targeted liposomes encapsulating anti-BCR-ABL siRNA or as ODN for chronic myeloid leukemia treatment. Bioconjug Chem 21:157–168 Mendonça LS, Moreira JN, de Lima MC (2010b) Co-encapsulation of anti-BCR-ABL siRNA and imatinib mesylate in transferring receptor-targeted sterically stabilized liposomes for chronic myeloid leukemia treatment. Biotechnol Bioeng 107:884–893 Millard SS, Vidal A, Markus M et al (2000) A U-rich element in the 50 untranslated region is necessary for the translation of p27 mRNA. Mol Cell Biol 20:5947–5959 Orava EW, Cicmil N, Garie´py J (2010) Delivering cargoes into cancer cells using DNA aptamers targeting internalized surface portals. Biochim Biophys Acta 1798:2190–2200 O’Shannessy DJ, Yu G, Smale R et al (2012) Folate receptor alpha expression in lung cancer: diagnostic and prognostic significance. Oncotarget 3:414–425 Ozpolat B, Sood AK, Lopez-Berestein G (2014) Liposomal siRNA nanocarriers for cancer therapy. Adv Drug Deliv Rev 66:110–116 Patil Y, Panyam J (2009) Polymeric nanoparticles for siRNA delivery and gene silencing. Int J Pharm 367:2195–2203 Ramesh R, Saeki T, Templeton NS et al (2001) Successful treatment of primary and disseminated human lung cancers by systemic delivery of tumor suppressor genes using an improved liposome vector. Mol Ther 3:337–350 Ruoslahti E (1996) RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 12:697–715 Shanker M, Jin J, Branch CD et al (2011) Tumor suppressor gene-based nanotherapy: from test tube to the clinic. J Drug Deliv 2011:465845. doi:10.1155/2011/465845 Shi J, Xu Y, Xu X et al (2014) Hybrid lipid-polymer nanoparticles for sustained siRNA delivery and gene silencing. Nanomedicine 10(5):897–900. doi:10.1016/j.nano.2014.03.006, pii: S1549-9634(14)00124-5 Shiokawa T, Hattori Y, Kawano K et al (2005) Effect of polyethylene glycol linker chain length of folate-linked microemulsions loading Aclacinomycin A on targeting ability and antitumor effect in vitro and in vivo. Clin Cancer Res 11:2018–2025 Smith MH, Lyon LA (2012) Multifunctional nanogels for siRNA delivery. Acc Chem Res 45:985– 993

294


Stylianopoulos T, Poh MZ, Insin N et al (2010) Diffusion of particles in the extracellular matrix: the effect of repulsive electrostatic interactions. Biophys J 99:1342–1349 Su WP, Cheng FY, Shieh DB et al (2012) PLGA nanoparticles codeliver paclitaxel and Stat3 siRNA to overcome cellular resistance in lung cancer cells. Int J Nanomedicine 7:4269–4283 Sun P, Yu H, Zhang WQ et al (2012) Lentivirus-mediated siRNA targeting VEGF inhibits gastric cancer growth in vivo. Oncol Rep 28:1687–1692 Surace C, Arpicco S, Dufay¨-Wojcicki A et al (2009) Lipoplexes targeting the CD44 hyaluronic acid receptor for efficient transfection of breast cancer cells. Mol Pharm 6:1062–1073 Tabernero J, Shapiro GI, LoRusso PM et al (2013) First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discov 3:406–417 Tanaka F, Ogawa E, Shanker M et al (2006a) Expression of HuR, a mRNA-binding protein in non-small cell lung cancer (NSCLC) correlates with cox-2, MVD and tumor progression. In: Abstracts of Proc Amer Assoc Cancer Res, Washington, DC, 1–9 Apr 2006 Tanaka F, Ogawa E, Wada H et al (2006b) Clinical significance of HuR expression, an mRNAbinding protein in non-small cell lung cancer (NSCLC). J Clin Oncol 24:10098 Wang J, Lu Z, Wientjes MG et al (2010) Delivery of siRNA therapeutics: barriers and carriers. AAPS J 12:492–503 Wang J, Guo Y, Chu H et al (2013) Multiple functions of the RNA-binding protein HuR in cancer progression, treatment responses and prognosis. Int J Mol Sci 14:10015–10041 Xiang B, Dong DW, Shi NQ (2013) PSA-responsive and PSMA-mediated multifunctional liposomes for targeted therapy of prostate cancer. Biomaterials 34:6976–6991 Yang ZZ, Li JQ, Wang ZZ et al (2014) Tumor-targeting dual peptides-modified cationic liposomes for delivery of siRNA and docetaxel to gliomas. Biomaterials 35:5226–5239 Zhang Y, Hong H, Cai W (2011) Tumor-targeted drug delivery with aptamers. Curr Med Chem 18:4185–4194 Zhu Q, Feng C, Liao W et al (2013) Target delivery of MYCN siRNA by folate-nanoliposomes delivery system in a metastatic neuroblastoma model. Cancer Cell Int 13:65. doi:10.1186/ 1475-2867-13-65