Nanodelivery systems for nucleic acid therapeutics in drug resistant tumors.

Review pubs.acs.org/molecularpharmaceutics

Nanodelivery Systems for Nucleic Acid Therapeutics in Drug Resistant Tumors Arun K. Iyer,†,‡ Zhenfeng Duan,§ and Mansoor M. Amiji*,† †

Department of Pharmaceutical Sciences, School of Pharmacy, Bouvé College of Health Sciences, Northeastern University, Boston, Massachusetts 02115, United States § Department of Orthopedic Surgery, Harvard Medical School, Boston Massachusetts 02114, United States ABSTRACT: Development of intrinsic and acquired drug resistance in cancer is a significant clinical challenge for effective therapeutic outcomes. Multidrug resistance (MDR) in solid tumors is especially difficult to overcome due to the many different factors that influence clinically manifested refractory disease. Genetic profiling of MDR tumors can provide for more specific control through RNA interference (RNAi) therapy. However, there are multiple barriers to effective in vivo delivery of functional nucleic acid constructs, such as small interfering RNAs (siRNAs) and micro RNAs (miRNAs or miRs). In this review, we have briefly described the principles and mechanisms based on the RNA interference phenomenon and the barriers to its successful clinical translation. The principles of active and passive tumor targeting using nanoparticles systems are also discussed. Furthermore, illustrative examples of miRNA, siRNA, and gene−drug combination delivery using nanoparticle systems that have shown promising potentials for the treatment of diseases such as MDR cancers are covered. KEYWORDS: multifunctional nanoparticles, multidrug resistant tumors, small interfering RNA, micro RNA, tumor targeting, combination therapy

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RNA INTERFERENCE THERAPY Principles and Mechanism of Gene Silencing. RNA interference (RNAi) is a natural conserved mechanism that cells use to turn down or silence the activity of specific genes. Methods of mediating the RNAi effect involve small interfering RNAs (siRNAs) or short-hairpin RNAs (shRNA). siRNA and shRNA are double-stranded RNA molecules that induce sequence-specific degradation of homologous single-stranded message RNA (mRNA).1,2 When siRNA or shRNA is transfected into cells, with the help of endogenous RNAi machinery, it binds to and disrupts the expression of specific mRNA containing sequences. This pathway is initiated by the enzyme dicer, which cleaves long double-stranded RNA (dsRNA) into short fragments of 20−25 base pairs in length of nucleotides. One of the strands is called the guide strand, which is then incorporated into the RNA-induced silencing complex (RISC). The antisense RNA strand then guides RISC to homologous sequences to target mRNA and base pairs with a complementary sequence of mRNA, inducing cleavage by Argonaute, the catalytic component of the RISC complex. The cleaved target mRNA (mRNA) is no longer capable of translating to amino acid (protein), and therefore the mRNA is “silenced” (Figure 1). This robust silencing effect of RNAi makes it both a valuable research tool and a potential of RNAibased therapy in the clinic.3,4 In plants and insects, RNAi activity plays an important role in host cell protection against viruses and transposons. From a biological perspective, RNAi is © 2014 American Chemical Society

proving to be a very powerful technique to silence specific genes, thereby enabling the evaluation of their physiologic roles in several types of cells including humans.5 RNAi technology has several major advantages over other post-transcriptional gene silencing techniques (such as antisense or gene knockout technology), in that it is easier to deliver, requires only small doses of siRNA or shRNA to produce its silencing effect, and can inactivate a gene at almost any stage in development. Most importantly, virus-based delivery of shRNA such as lentivirusdelivered shRNAs is capable of specific, highly stable and functional silencing of targeted gene expression in various cell types and transgenic animals.6 However the utility of viral vectors for human clinical translation is questionable due to the regulatory issues concerning safety of such delivery systems. Development of alternative strategies using nonviral vectors is thus imperative. Principles and Mechanism of Micro RNA (miR) and miR Dysregulation in Cancer. The process of RNAi can be moderated by either siRNA/shRNA or microRNA (miRNA or miRs). miRs are noncoding RNA chains of 21−25 nucleotides Special Issue: Drug Delivery and Reversal of MDR Received: Revised: Accepted: Published: 2511

January 11, 2014 March 18, 2014 March 24, 2014 March 24, 2014 dx.doi.org/10.1021/mp500024p | Mol. Pharmaceutics 2014, 11, 2511−2526

Molecular Pharmaceutics

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Figure 1. siRNA and shRNA with target gene silencing. Different delivery strategies and the processing of shRNAs in the cell are shown. RNAi can be introduced by synthetic siRNA or vector-based (plasmid/lentiviral) shRNA. The shRNA is processed by Dicer into siRNA.

Figure 2. Mechanisms of miRNA(miR). miRNA (miR) genes are transcribed by RNA polymerase II (Pol II) to generate the primary transcripts (pri-miRNAs). The following step is mediated by the Drosha complex that generates ∼70 nucleotide (nt) precursor-miRs. On export from the nucleus, the cytoplasmic RNase III Dicer catalyzes the second processing step to produce miR duplexes. Dicer and Argonaute mediate the processing of pre-miR, and the miR mature strand is then assembled into the RNA-induced silencing complex (RISC) and targets the complementary mRNA 3UTR’ sequences for gene regulation.

that regulate gene expression, influencing many cellular functions such as proliferation, differentiation, apoptosis,

oncogenesis, and drug sensitivity in normal or malignant cells.7−9 There are some differences between siRNA/shRNA 2512

dx.doi.org/10.1021/mp500024p | Mol. Pharmaceutics 2014, 11, 2511−2526


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while minimizing any undesirable off-target effects. While the use of siRNA/miRs has been effective for short-term gene inhibition in mammalian cell lines, its use for stable long-term transcriptional knockdown of target genes has been problematic.10,18,19 Similar to other nucleic acid constructs, siRNA/miRs have stability and delivery problems for clinical applications. Both siRNAs and miRs have poor cellular membrane permeability and limited stability in vivo and intracellular delivery in tumor cells upon systemic administration.20 The translation of RNAi/ miR based cancer therapeutics to the clinic is hindered by several challenges associated with the delivery system, including susceptibility to rapid degradation by nucleases; rapid blood clearance, off-target effects, insufficient circulation half-life due to phagocytosis, and transient and poor biodistribution in the tumor tissue; cellular uptake; and inability to escape endosomes and release into the cytosolic compartment for an RNAi/miRmediated effect.12,20 In order to translate RNAi/miRs from an experimental approach to a clinical-viable therapeutic strategy that can benefit cancer patients, specific and efficient delivery systems are needed. Currently, plasmid and viral-based vectors are being used for siRNA/shRNA and miR delivery. Despite their high transduction efficiency, viral delivery approaches face serious challenges.21 Several gene therapy trials based on viral delivery systems have produced adverse effects questioning their safety.22,23 In addition, high costs for producing large amounts of viral stocks for clinical use and limited quantities of nucleic acids that can be packaged for therapy also limit the applications of viral delivery systems. It is, therefore, important to develop safe and effective nonviral RNAi/miR delivery systems. In recent years, in order to overcome these challenges, nanotechnology has been applied to facilitate delivery of RNAi/ miR in different model systems. Nanotechnology based delivery systems provide unique advantages of protecting the labile payload, ease of systemic administration, tumor-specific and intracellular delivery, enhancing residence, and overcoming drug resistance. Some examples are discussed in latter sections.

and miR. Both are processed inside the cell by the enzyme called Dicer and incorporated into a complex of RISC (Figure 2). Although siRNA/shRNA is considered exogenous doublestranded RNA that is taken up by cells, or enters via vectors like plasmids or viruses (lentivirus or adenovirus), miR is singlestranded RNA synthesized endogenously (made inside the cell). Another difference is that, in mammalian cells, siRNA or shRNA typically binds perfectly to its mRNA target, whereas miR can inhibit translation of many different parts of mRNA sequences because of its imperfect pairing.10,11 The importance and varied functions of miRs are demonstrated by diverse phenotypes, including different diseases that arise when miRs are improperly expressed. The association of miR dysregulation with various disease phenotypes has given rise to the idea that selective modulation of miRs could alter the course of disease such as cancer. miRs are known to undergo genetic alterations such as amplification, deletion, and epigenetic silencing, which can ultimately activate oncogenes and inactivate tumor suppressors in cancer cells.12,13 Certain miRs are consistently dysregulated across many cancers. Examples of miRs with oncogenic activity include miR-155, mi-21, and miR-372; by contrast, miR let-7, miR-29, miR-26a, miR-31, miR-34a, miR-1, miR-206, and miR-199a-3p function as tumor suppressor genes.12−17 These dysregulated miRs are not limited to a particular tumor type, and the aberrantly expressed miRs correlate with the clinical status such as the tumor stage, drug sensitivity, and patient survival rate. Additionally, recent studies have observed a functional contribution of miRs to cellular transformation, tumorgenesis, and cancer stem cell maintenance. miRs provide critical functions downstream of classic oncogenic and tumor suppressor signaling pathways such as those controlled by KRAS, Myc, Met, mTOR, and p53. Finally, functional studies have directly documented the potent pro- and antitumorigenic activity of specific miRs both in vitro and in vivo.2,11−13 siRNA and miR As Therapeutic Agents. During recent years, RNAi technology not has only become a powerful tool for functional genomics but also represents a new and potentially powerful therapeutic approach for treating human diseases, especially cancer. For miR as potential therapy, since aberrantly expressed miRs play key roles in the development of human cancer, correcting these miRs’ dysregulation and deficiencies by either antagonizing or restoring miR function will provide a significant advance in cancer therapy. Because siRNA/shRNA and miR can play important roles in epigenetic regulation through RISC and their roles in controlling gene expression, both are valuable targets for therapeutic use in cancer.2,4,17 Furthermore, it is anticipated that the progress of therapeutic agents of RNAi and miR will develop in parallel, as most of the obstacles to RNAi-based cancer therapeutics are the same for both siRNA/shRNA and miRs. Challenges in Systemic Delivery of siRNA or miR Therapeutics. The universal presence of the RNA or miRs, combined with their efficacy and specificity, makes them very attractive as therapeutic agents for silencing (or increasing by antagomirs) aberrant gene expression in the clinical settings. However, one of the major obstacles to utilizing RNAi or miR as therapeutic agents is their targeted delivery. Importantly, the administered siRNA/miR must traverse through the biological barriers in the intact form to reach the target tissues and cell types and, following internalization, gain access to the subcellular location (cytosol) where the RNAi/miR machinery resides. This must be achieved so that silencing is maximized,

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TUMOR PLASTICITY AND MDR The International Agency for Research on Cancer (IARC) reports a staggering figure of 10 million new cases of cancer with over 6 million deaths occurring annually from the disease.24 The agency also estimates that, by the year 2030, the cancer burden will more than double to 21.4 million new cases with 13.2 million cancer related deaths globally.24 These figures foretell a gloomy story that, despite the progress made in cancer screening, detection, and treatment over past several decades, complete eradication still remains a distant goal due to several impediments including late stage diagnosis and lack of clinical procedures for eliminating cancer, inadequate strategies for dealing with cancer cell plasticity, and development of MDR in cancer.25,26 Despite persistent therapeutic interventions, an increasing body of evidence suggests that cancer cell plasticity contributes to evolution of the disease with MDR pheontype.27,28 Cellular plasticity can be defined as the ability of one cell type to attain properties of another cell type that can potentially be coupled to generate specific cell types afflicted with a disease condition.29−31 For instance, subpopulations of tumor cells (such as cancer stem cells, CSCs) play a pivotal role in tumor progression and development by producing bulk population of nontumorigenic cancer cell progeny through differentiation.32,33 Recent reports also indicate that a hypoxic micro2513



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cells lining the vessels.41,42 The aberrant vascular architecture and dysfunctional lymphatic drainage in tumors result in hypoxic areas with high interstitial fluid pressure relative to the low interstitial fluid pressure of well vascularized areas.41 Because of aberrant angiogenesis and inaccessible location, hypoxic cells are less likely to accumulate therapeutic concentrations of chemotherapeutic drugs.43 In addition to these properties that diminish chemotherapeutic efficacy, hypoxic cells have active mechanisms for inducing MDR in tumors cells, thus leading to active tumor progression.42,44−46 Transcriptional factors that respond to hypoxia are termed hypoxia-inducible (HIF) factors. These factors assist in preventing cell differentiation, support blood vessel formation, and regulate apoptosis.40 HIF factors activate enzymes that are involved in DNA repair and induce the development of resistance to DNA-targeting drugs. Hypoxic tumor cells also display reduced intracellular pH levels relative to normal cells.44 The acidic microenvironment is combined with the activation of a subset of proteases that contribute to tumor progression and metastasis.44 More importantly hypoxic tumor cells (that are deprived of oxygen) revert to anaerobic metabolism and obtain ATP through the conversion of glucose to lactic acid instead of oxidative metabolism, termed as aerobic glycolysis (or the Warburg effect).47 The application of therapies targeting tumor microenvironment using novel delivery approaches thus would be the most promising option for effective management of MDR cancers. Mechanisms of Drug Resistance in Tumors. As discussed above, multidrug resistance (MDR) is a major obstacle to the effective treatment of cancer, and MDR in cancer refers to a state of resilience against structurally and functionally unrelated chemotherapeutic agents. It can be intrinsic or acquired through exposure to chemotherapeutic agents. Drug resistance mechanisms represent adaptations to toxic insults and cellular stress, and these mechanisms are grouped into five categories: (i) induction of drug transporters, (ii) DNA repair, (iii) changes in drug metabolism, (iv) gene amplification or mutation of target proteins, and (v) changes in survival/apoptotic pathways.38 Despite distinct mechanisms involved, the MDR phenotype is usually the combination of several mechanisms such as blocked apoptosis and increased drug efflux.38 It is well documented that drug efflux transporters are generally found to be elevated in drug-resistant tumor cells. There are over 13 ATP-binding cassette (ABC) families of drug transporters that are selectively involved in the efflux of small molecule drugs.42,48 Some of these membrane bound transporters have been shown to play a specific role in efflux of cytotoxic drugs from within the cells preventing the critical concentration of drug accumulation, resulting in MDR.49 Among them, P-glycoprotein (P-gp, MDR1, ABCB1) is one of the well-characterized ABC transporters that show broad substrate specificity. P-gp mediated drug efflux is primarily considered to be one of the main contributors in the anticancer therapy of several cancers.50 Thus, P-gp is encoded by MDR1 gene and its overexpression in cancers has become a therapeutic target for overcoming MDR. Several other ABC transporters that are associated with MDR include MDR protein 1 (MRP-1, ABCC1), breast cancer resistance protein (BCRP, ABCG1), the mitoxantrone resistance protein (MXR1/BCRP, ABCG2), and the ABCB4 (MDR3).42 DNA repair mechanisms consist of a complex network of proteins able to identify DNA damage (e.g., ATM, Chk1/2, ATR, p53) and repair the damage.37 Cytochrome P450 or the

environment in metastatic tumors facilitates phenotype switching, allowing melanoma cells to participate in blood capillary formation.34 Such phenotype alteration makes the management of the disease highly challenging. Cellular plasticity arising from epithelial to mesenchymal transition (EMT) in cancers has also been associated with tumor progression and resistance to drug therapies.27 EMT has been found to be a major contributor to cancer metastasis, through the acquisition of highly invasive and migratory potential of tumor cells.35,36 Indeed EMT has been implicated to causes tumor cells to develop resistance against a number of targeted therapies.28 Reports indicate that an EMT phenotype has been identified in several EGFR mutant non-small cell lung (NSCL) cancer patients who have progressed while on EGFRdirected Erlotinib therapy due to loss of an epithelial tumor marker called E-cadherin.28 MDR continues to be a major challenge in the treatment of several virulent cancers. MDR cancer cells show a broad range of resistance against functionally dissimilar and structurally unrelated chemotherapeutic agents. Historically, MDR has been linked to elevated expression of drug efflux transporters (such as p-glycoprotein), changes in drug kinetics, or amplification of drug targets.37,38 However, development of drug resistance in patients treated with novel targeted molecular therapies has provided far better understanding of the complexities involved in MDR cancers.37 Recent studies clearly indicate that heterogeneity in tumor cell population is a major driver of cancer drug resistance.37 Successful therapy thus heavily impinges on novel treatments that can take account of tumor heterogeneity comprising genetic variation, the microenvironment, and tumor cell plasticity.37 Tumor Microenvironment and MDR. The outcomes of several studies have demonstrated that the tumor microenvironment strongly influences cellular phenotypes, including susceptibilities to insults by a range of mechanisms. Tumor microenvironment consists of cells (tumor associated macrophages or fibroblasts), extracellular matrix, signaling molecules and mechanical cues that can act in paracrine manner to influence tumor initiation and support tumor growth and invasion, protect the tumor from host immunity, promote drug resistance, and provide niches for dormant metastases to often thrive and proliferate. Recent studies have identified that microenvironment can alter the response of tumor cells to chemotherapy and targeted therapies through production of secreted factors which might drive tumor growth and foster MDR.39 During early stages of the tumor development, cancer cells may rely on a tumor-supportive microenvironment while at the later stages (i.e., the metastatic setting) tumors could be biologically characterized by the so-called self-sufficiency of cancer cells. Such acquired self-sufficiency to survive in a microenvironment-independent manner could result in insensitivity to molecular targeted therapeutics acting by depriving cancer cells of paracrine acting stimuli. Such conditions trigger morbidity of cancer cells to targeted drug treatments in patients. Hypoxia has long been considered as another major feature of the tumor microenvironment and a potential contributor to the MDR and enhanced tumorigenicity of CSCs.40 Hypoxia is a condition in which tumor tissue is deprived of oxygen supply. Hypoxic regions are created (most commonly in the core of necrotic tumors) when aberrant angiogenesis occurs in tumor tissues or blood vessels are closed or impaired due to compression, tumor cell invasion, and discontinuity of epithelial 2514



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tumor interstitium.57,62,64,65 This phenomenon was coined the “enhanced permeability and retention” (EPR) effect, first discovered by Matsumura and Maeda more than three decades ago65 (Figure 3). In this regard, it is also important to note that

glucuronyl transferases are the drug metabolizing enzymes responsible for the biotransformation of many chemotherapeutic drugs, and their activity lowers the intracellular drug levels. Gene amplification of receptors, such as EGF receptors, can often compromise the efficacy of therapies. In addition to these, the relative activities of proapoptotic and antiapoptotic pathways contribute to the sensitivity of tumor cells to cytotoxic drugs. Taken together, these overlapping mechanisms are the main biochemical determinants of tumor cell sensitivity to cytotoxic drugs.37 Drug resistance to the broad range of chemothrapeutic drugs is demonstrative of the existence of a highly dynamic environment of the tumor tissue that makes their management highly challenging. Recent studies looking at tumor microenvironment also suggests that tumoral heterogeneity is a major driver of drug resistance.51,52 In another context, according to the CSC model, drug resistance is mostly caused by the intrinsic or acquired resistance mechanisms of accumulating CSCs.40 Indeed, several drug efflux transporters have been identified in CSCs, including P-gp, multidrug resistance-associated proteins (MRP), and breast cancer resistance protein (BCRP).53 Many studies suggest that tumors are enriched with CSCs at the completion of primary therapy, and the surviving CSCs contribute to drug resistance and ultimately cause disease recurrence.54 In one such study, a high survival of CSCs following etoposide treatment was found to be associated with expression of MRP1 and the activity of an apoptosis inhibitor β-livin in glioblastoma tumors.55 High BCRP levels were linked to increased CD133 expression and Akt signaling in drug resistant hepatocellular carcinoma (HCC).56 AKt signaling was able to alter the subcellular localization of BCRP transporters in HCC, thus determining drug efflux in CSCs.56 In the same study AKt signal inhibition by P13K inhibitors not only suppressed cancer cell proliferation but also increased the sensitivity of drug resistant cells.56 Therefore, the therapeutic strategies directed toward CSCs might improve cancer therapy, in particular for those cancers that are refractory to conventional chemotherapeutics aimed predominantly on “bulk” tumor populations.

Figure 3. Concept of EPR effect for tumor targeted drug delivery. Adapted with permission from ref 57. Copyright 2006 Elsevier B.V.

polymeric drugs and macromolecules with molecular weight >40 kDa (which are above the renal excretion threshold) are able to circulate longer in the blood and show prolonged accumulation in the solid tumors.66−70 The EPR phenomenon was later observed for many types of polymer−drug conjugates, micelles, nanoparticles, and liposomal delivery systems.57,71−74 Also, the tumor tissues were found to have impaired lymphatic clearance due to which the accumulation and retention of nanoparticles continued to occur in the tumor as long as they could circulate in the blood. Furthermore, the EPR effect was found to be more effective if the nanoparticles could escape mononuclear phagocytic systems (MPS) and show prolonged circulation half-life in the blood. In this regard, incorporation of amphiphatic molecules such as poly(ethylene glycol) (PEG) on the surface of nanosystems was found very useful in facilitating MPS escape and rendering long plasma residence time, thus enhancing tumor accumulation.75,76 For instance, PEGmodified “stealth” liposomes encapsulated with doxorubicin could circulate for prolonged periods in the blood and exhibited improved tumor accumulation in addition to lowering the toxicities associated with the free form of doxorubicin.77 In general, nanosystems in the size range of 20−200 nm have been found effective in permeating and accumulating in the solid tumor tissue,78 although there has been some indication that the hyperpermeability of tumor vasculature with wide gap junctions can facilitate particles as large as 700 nm to accumulate effectively in solid tumors.79−81 Although EPR effect provides a “first pass” for selective accumulation of nanoparticles, micelles, and liposomal formulations into the tumor interstitium, their intracellular delivery still remains challenging.82 It is critical for a nanoparticle system to enter into the cancer cells and, more importantly, release the drug/gene cargo in the right location, for effective cell killing. Intracellular delivery to a specific location within the cells and organelles is essential for almost all anticancer drugs and genes. For instance, intracellular delivery of siRNAs and its release in the cytoplasm are key for the success of RNAi based gene silencing strategies.83 In this regard the specificity and targeting ability of nanosystems can be remarkably improved when tumor-targeting ligands are used as part of the nanodelivery systems (Figure 4).82,84 Such targeted delivery systems can selectively home to tumor cells that

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INTEGRATED MULTIFUNCTIONAL NANOPARTICLES Mechanism of Tumor Selective Delivery: Passive and Active Targeting. The development of tumors involves several anatomical and pathophysiological changes that could be utilized for tumor targeting.57 As the tumor cells multiply and grow to a size of ∼1 mm in diameter, they start to form neovasculatures.58 In order to sustain their growth, the soformed tumor nodules develop a more complex network of blood vessels around them by a process called angiogenesis.59 The endothelial cells lining the tumor blood vessels are highly disorganized and defective in architecture. Moreover, the tumor blood vessels have wide gap junctions leading to “leaky” vascular architectures.60,61 Apart from the unique anatomical features described above, tumor cells secrete elevated levels of permeability mediators such as vascular endothelial growth factor (VEGF) (also known as vascular permeability factor (VPF)), bradykinin (BK), prostaglandins (PGs), matrix metalloproteinases (MMPs), nitric oxide (NO), and peroxynitrite.57,62−65 The overproduction of permeability mediators coupled with the anatomical and pathophysiological abnormalities leads to extensive accumulation of blood plasma components, macromolecules, and nanoparticles into the 2515



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Figure 4. Passive and active tumor targeting. The schematic shows the passive and active targeting mechanisms of multifunctional image guided nanoparticles and the difference in the vasculature of normal and tumor tissues; drugs and small molecules diffuse freely in and out of the normal and tumor blood vessels due to their small size, and thus the effective drug concentration in the tumor drops rapidly with time. However, macromolecular drugs and nanoparticles can passively target tumors due to the leaky vasculature or the EPR effect, however they cannot diffuse back into the bloodstream due to their large size and impaired lymphatic clearance, leading to enhanced tumor accumulation and retention. Targeting molecules such as antibodies or peptides present on the nanoparticles can selectively bind to cell surface receptors/antigens overexpressed by tumor cells and can be taken up by receptor-mediated endocytosis (active targeting). The image guiding molecules and contrast agents conjugated/ encapsulated in the nanoparticles can be useful for targeted imaging and (noninvasive) visualization of nanoparticle accumulation/localization, as well as for mechanistic understanding of events and efficacy of drug treatment simultaneously. Reprinted with permission from ref 88. Copyright 2012 Bentham Science.

Figure 5. RGD peptide functionalized polycaprolactone−gold microparticle design for colon cancer screening. (A) Fabrication steps. (B) 3D design. (C) Scanning electron micrograph (SEM) revealing a size of ∼1.5 μm for the microparticle. Please see refs 89 and 93 for details.

overexpress specific receptors or antigens,85 thereby promoting intracellular delivery.82,83,86,87 Use of such mechanism is called “active” tumor targeting (Figure 4).78 Thus, active targeting in effect aids in more specific “secondary” targeting after “primary” targeting based on the EPR effect and, in combination, passive and active targeting provide for increased accumulation and penetration of nanoparticles at the tumor site thereby facilitating improved maintenance of high intracellular drug

concentrations. Such systems provide severalfold increased effectiveness as compared to free drug administration.78 Indeed, currently, active targeting has become a widely recognized potential route for increasing therapeutic indexes in cancer treatment. Several ligands or targeting agents can be used to surface decorate nanoparticle systems for active targeting to tumors.89−91 For example, RGD peptide coupled nanoparticles 2516



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can target integrin receptors (αvβ5 or αvβ3) overexpressed on vascular endothelial cells of angiogenic blood vessels and tumor cells.92 In a recent study we functionalized poly(epsilon caprolactone) (PCL) microparticles containing colloidal gold with RGD peptide that specifically homes to colon tumors overexpressing integrin receptors (Figure 5).89,93 These particles could increase the localization of fluorescent probes loaded in them for the diagnostic imaging and screening of colon cancers. There are several such examples in the literature where a combination of active and passive targeting has been utilized for tumor targeted drug delivery.78,82,83,87,94−97

In order to overcome the dose-limiting side effects of conventional chemotherapeutic agents and the therapeutic failure due to multidrug resistance (MDR), we designed and evaluated a novel biocompatible lipid-modified dextran-based self-assembled polymeric nanosystem that could encapsulate MDR-1 siRNA as well as anticancer drugs such as doxorubicin.100,101 Further, in order to image the transfection efficacy of siRNA-loaded nanoparticles on cell lines, we utilized an EGFP (green fluorescence protein) expressing BHK-21 cells and performed confocal fluorescence microscopy to visualize the downregulation of EGFP. It was observed that the EGFP siRNA was efficiently incorporated into cells and effectively inhibited the expression of EGFP in a dose dependent manner. Similarly, the MDR1 siRNA loaded dextran nanoparticles efficiently suppresses P-gp expression in the drug resistant osteosarcoma cell lines. A combination therapy of the MDR1 siRNA loaded nanocarriers with doxorubicin revealed pronounced increase in doxorubicin uptake in the nucleus (assessed by confocal fluorescence microscopy imaging) of multidrug resistant cells. These results demonstrate that our approach may be useful for reversing drug resistance by increasing the amount of drug accumulation in MDR cells. Thus the delivery using nontoxic and biocompatible dextran based nanosystems offers a versatile platform for incorporation of multiple payloads for simultaneous imaging and delivery of therapeutic molecules that can be translatable for human clinical applications. In another study, Huh et al. designed a siRNA delivery system containing glycol chitosan biodegradable/biocompatible polymer (GC) and PEI.102 The polymers were conjugated with 5β-cholanic acid (CA) to stabilize the nanoparticles and endow them with tumor-homing ability. The nanoparticles were formed by mixing GC−CA and PEI−CA to form selfassembled nanostructures (GC−PEI NPs), due to the strong hydrophobic interactions of 5β-cholanic acids in the polymers. The cationic charge on the GC−PEI nanoparticles was used to complex with the negatively charged red fluorescence protein (RFP) gene silencing siRNA designed to inhibit RFP expression. In vitro studies with RFP expressing B16F10 tumor cell incubated with siRNA−GC−PEI nanoparticles revealed time-dependent cellular uptake of the nanoparticles and lead to specific mRNA knock down. More importantly, the siRNA loaded nanoparticles displayed significant inhibition of RFP gene expression in RFP/B16F10-bearing mouse models, demonstrating their potentials as a promising vector for siRNA delivery.102 Moore’s group have developed a dual-purpose probe for in vivo delivery and simultaneous imaging of siRNA accumulation in tumors by using high-resolution magnetic resonance imaging (MRI) and near-infrared (NIR) in vivo optical imaging techniques.103 The siRNA was covalently conjugated to nanoparticles consisting of NIR dye conjugated magnetic nanoparticles. Furthermore, the nanoparticles were decorated with specific membrane translocation peptide that enables their intracellular delivery and cytosolic availability.103 Taken together, their study demonstrates the feasibility of tracking the tumor uptake and silencing ability of the siRNA conjugated nanoparticles in vivo.103 In another study, Kumar et al.104 used superparamagnetic iron oxide nanoparticles as a construct for delivery of siRNA to solid tumors and combined optical/MR imaging. The nanoconstruct, apart from performing its function as a vehicle for siRNA delivery, also served as a tool to study basic tumor

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ILLUSTRATIVE EXAMPLES OF NANOPARTICLE-MEDIATED SIRNA DELIVERY As discussed in prior sections, gene therapy based on RNA interference (RNAi) mechanism has been well established and has currently become a major area of research that hold great promise for management of diseases such as cancers. Although siRNAs have high efficiency and specificity, the major hurdle still remains its delivery to target tumor cells and intracellular availability after localization in the sites of interest.98 In one study, researchers from Park’s group have developed a polyelectrolyte complex micelle-based VEGF siRNA delivery system for antiangiogenic gene therapy (Table 1).99 The Table 1. Illustrative Examples of Nanosystems Used for siRNA Delivery To Cancer Cells nanodelivery system polyelectrolyte complex (PEC) micelle dextran nanoparticles glycol chitosan (GC)/ polyethylenimine (PEI) nanoparticles superparamagnetic iron oxide nanoparticles HA−PEI nanoparticles superparamagnetic iron oxide nanorods HA−PEI nanoparticles liposomal nanoparticles liposome polycation DNA nanoparticles mesoporous silica nanoparticles

siRNA/ target

cancer/cell type

ref

VEGF

prostate cancer

Kim et al.99

MDR-1 RFP

osteosarcoma melanoma

Susa et al.100,101 Huh et al.102

survivin

colorectal cancer lung cancer breast cancer

Medarova et al.103 Ganesh et al.83,105,106 Kumar et al.104

melanoma lung, ovarian, and breast cancer ovarian cancer

Jiang et al.109 Pakunlu et al.;110,111 Saad et al.113 Chen et al.112

ovarian cancer

Chen et al.114

survivin, Bcl-2 BRIC5 VEGF Bcl-2, MDR1 c-Myc/ VEGF Bcl-2

complexation of the PEG-conjugated VEGF siRNA was achieved by charge interaction with poly(ethyleimine) (PEI). This complex formed a stable core−shell nanostructure with the PEI/siRNA forming the central core and PEG chains forming the corona. Intravenous (i.v.) and intratumoral (i.t.) injection of the micelles in mice demonstrated significant inhibition of VEGF expression in the tumor tissue and suppressed tumor growth without detectable toxicities.99 Furthermore, to confirm the feasibility of siRNA-based gene therapy and imaging in vivo, optical imaging was undertaken using a Cy5.5 labeled siRNA. The Cy5.5 labeled siRNA containing polyelectrolyte complex micelles predominantly accumulated in the tumor, affirming the feasibility of this approach.99 2517



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Figure 6. Synthesis of HA-based functional macrostructures. A series of functional hyaluronic acid (HA) based derivatives were synthesized using a simple and versatile EDC/NHS conjugation chemistry as shown: 1, hyaluronic acid of 20 kDa molecular weight; 2, HA conjugated to monofunctional fatty amines with the general formula CH3(CH2)nNH2 (where n = 3, 4, 5, ...); 3, HA conjugated to poly(ethylene glycol) (PEG) of 2000 Da molecular weight; 4, HA conjugated to bifunctional fatty amines with the general formula NH2(CH2)nNH2 (where n = 4, 5, ...); 5, HA conjugated to thiol-containing derivative; and 6, HA conjugated to polyamines such as poly(ethyleneimine) (PEI) of 10 kDa molecular weight. Reproduced with permission from ref 83. Copyright 2013, Elsevier Ltd.

Figure 7. Whole body optical imaging of indocyanine green loaded HA−PEI/PEG nanoparticles in A549/A549DDP lung cancer tumor bearing mice. Mice were injected with ICG/HA−PEI/PEG NPs and imaged at different time points using the IVIS live imaging system as shown. The uptake of the nanoparticles in the tumor with progression of time can be clearly seen. Reproduced with permission from ref 105. Copyright 2013 Elsevier Ltd.

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biology and therapy.104 The nanoconstruct consisted of magnetic nanoparticles (for MR imaging), a Cy3 fluoropore (for optical imaging), a targeting peptide (EPPT) that could home to a specific antigen (uMUC-1) overexpressed by majority of human breast cancer cells, and a synthetic siRNA that downregulates the BIRC5 antiapoptotic gene in breast cancer cells.104 The nanoconstruct demonstrated specific tumor uptake and significant downregulation of BIRC5 gene, in animal tumor models. More importantly, the tumor uptake could be visualized both by MRI and NIR optical imaging.104 In a recent study, we engineered a series of CD44 targeting hyaluronic acid (HA) based self-assembling nanosystems for targeted delivery of siRNA. The HA polymer was functionalized with lipids of varying carbon chain lengths and nitrogen content, as well as polyamines for assessing siRNA encapsulation (Figure 6). We identified several HA derivatives that could stably encapsulate/complex siRNAs and form selfassembled nanosystems, as determined by gel retardation assays and dynamic light scattering (DLS). Many HA derivatives could transfect siRNAs into cancer cells overexpressing CD44 receptors. Interestingly, blocking the CD44 receptors on the cells using free excess soluble HA prior to incubation of cy3labeled-siRNA loaded HA nanoassemblies resulted in >90% inhibition of the receptor mediated uptake, confirming target specificity. In addition, SSB/PLK1 siRNA encapsulated in HA− PEI/PEG nanosystems demonstrated dose dependent and target specific gene knockdown in both sensitive and resistant A549 lung cancer cells overexpressing CD44 receptors (>90%). More importantly, these siRNA encapsulated nanosystems demonstrated tumor selective uptake and target specific gene knock down in vivo in solid tumors (Figure 7) as well as in metastatic tumors. The HA based nanosystems thus portend to be promising siRNA delivery vectors for systemic targeting of CD44 overexpressing cancers including tumor initiating stem cells and metastatic lesions.83,105,106 In a similar study, researchers from Korea demonstrated targeted delivery of the HA nanoparticles to CD44 expressing cells.107 Researchers in this study conjugated 5β-cholanic acid to HA using the EDC chemistry. It was demonstrated that, by varying the degree of substitution of the hydrophobic moiety, the size of the self-assembled particles could be controlled well. Another group from Korea demonstrated how the percentage of chemical modification of HA changed the distribution of those HA particles in mice.108 According to their real time imaging study using quantum dots (QDots), the HA−QDots with 35 mol % HA modification maintaining enough binding sites for HA receptors mainly accumulated in liver, kidney, and tumors (tissues with higher CD44 expression levels) while those with 68 mol % HA modification lost much of the HA characteristics and evenly distributed to the tissues in body. Jiang et al. also developed and investigated the effect of HA modification on the receptor-mediated endocytosis by labeling HA derivatives with QDots.109 Based on their real time imaging study of their HA−QDot conjugates, they have reported that the HA−QDots with a degree of modification less than 25 mol % appeared to be more efficiently taken up by CD44 expressing cells by receptor mediated endocytosis. siRNA/Drug Combination Delivery. Drug−gene combination delivery has promising potential for the treatment of refractory diseases such as MDR cancers. For instance, the pump mediated resistance can be suppressed by delivering specific siRNAs using nanoparticles that can dowregulated resistance causing genes thereby sensitizing the cancer cells to

drug treatment. Since one of the goals of our laboratory was to reverse the drug resistance in cisplatin resistant tumors, we engineered HA based systems to effectively deliver siRNA and cisplatin to ultimately reverse the resistance. To do this, we initially picked the resistant tumors that overexpress CD44 and identified the resistant genes that are expressed in those cells. After careful designing and screening of multiple siRNA sequences to target those resistant genes in the resistant cells, we picked the most efficacious sequences.106 We also modified those siRNAs to minimize the off-target effects coming from the unmodified sequences. Using our systems described before, we managed to first downregulate the resistant genes such as survivin and bcl2 using our HA−PEI/HA−PEG system carrying the corresponding siRNAs and sensitized the resistant tumors to cisplatin. Then we delivered cisplatin using our selected HA−ODA system to enhance the cell killing of the already sensitized cells. By delivering siRNA and a chemotherapeutic drug separately in 2 different systems, we demonstrated a synergistic effect in killing.106 The tumor growth inhibition was significantly improved in the tumors that had combination treatment (∼60%) when compared to the tumors that had single agent treatment (∼30%). Some reports suggest that antisense oligonucleotides targeted to pump and nonpump resistance causing genes have to be simultaneously delivered with the drug in order to achieve significant drug efficacy.110,111 In this regard, the codelivery of siRNA with the anticancer drug of interest using a single nanoconstruct can be challenging due to the differences in the property of siRNAs and the anticancer drug, and the way in which they form, by self-assembly or loading into the corresponding nanosystem. Also, the timing of siRNA and drug delivery has to be synchronized to achieve the necessary “combination” effect, which can be affected by several factors such as dose of the siRNA/drug and differences in the internalization and release mechanism of the payload. In this regard, it may be worthwhile to develop individual nanosytems tailored to encapsulate drugs and genes separately and then codeliver them. In one such study, Leaf Huang and colleagues developed two different but novel nanoparticle formulations such as cationic liposome polycation−DNA (LPD) and anionic liposome polycation−DNA (LPD-II) for systemic delivery of doxorubicin and therapeutic siRNA to MDR tumors to overcome drug resistance for cancer therapy.112 Authors in this study demonstrated for the first time the codelivery of siRNA and chemotherapeutic agent with two different multifunctional delivery systems. Minko’s group from Rutgers University managed to codeliver siRNA and doxorubicin together using a cationic liposome system and demonstrated synergistic effect in resistant cells.113 In addition, researchers from the same group also developed a codelivery system based on mesoporous silica nanoparticles (MSNs) to deliver doxorubicin and Bcl-2-targeted siRNA simultaneously to human ovarian cancer cells for enhanced chemotherapy efficacy.114 These results demonstrate the capability of codelivery strategies to effectively manage MDR cancers.

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ILLUSTRATIVE EXAMPLES OF NANOPARTICLE-MEDIATED MICRO-RNA DELIVERY miR and Antagomir Delivery in Cancer. When oncogenic miRs are upregulated, they block the tumor suppressor genes and lead to tumor formation. On the other hand, when there is downregulation or loss of miRNAs with

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in proliferation of lung cancer cells, especially in cells with K-ras mutations. Given this information, this group also demonstrated significant tumor growth inhibition/antitumor efficacy in a genetically engineered K-ras mutated oncogenic mouse model, a model that resembles very closely actual human lung cancer.119 Liposomal delivery of miR-7-expressing plasmid has been shown to overcome EGFR inhibitor resistance in lung cancer cells. The miR-7 expression level of the transfected cells was approximately 30-fold higher. Injection of the miR-7-expressing plasmid revealed marked tumor regression in a mouse xenograft model.120 The data demonstrate the potential of therapeutic applications of miR-7-expressing plasmids against EGFR oncogene-addicted drug resistant lung cancers by liposomal delivery. A cationic lipoplex (LP) based nanosystem can capably delivered miR-29b both in vitro and in vivo in lung cancer. LPs containing miR-29b can efficiently deliver miR-29b to NSCLC cells and inhibited cell growth and clonogenicity, as well as reduced the expression of miR-29b targets CDK6, DNMT3B, and MCL1. In addition, the IC50 for cisplatin in the miR-29b-treated cells was effectively reduced. In the xenograft murine model, LPs efficiently accumulated at tumor sites. Systemic delivery of LP-miR-29b increased the tumor miR-29b expression by approximately 5-fold, downregulated the tumor mRNA expression of CDK6, DNMT3B, and MCL1, and significantly inhibited tumor growth as compared with LP-miRNC (negative control).121 These findings demonstrate that cationic LPs represent an efficient delivery system that holds great potential in the development of miR-based therapeutics for cancer treatment. In another study, a liposome−polycation−hyaluronic acid based nanoparticle has been shown to effectively deliver miR-34a in a syngeneic model of B16F10 lung metastases.122 A dicetyl phosphate−tetraethylenepentamine based polycation liposome (TEPA-PCL) is able to deliver miR-92a, one of the miRs regulating angiogenesis.123 Other liposome-based systems for delivery of miR-34a or Let-7 to inhibit tumor growth have also been reported.119 EGFR overexpression is oftentimes found in the majority of the NSCLC samples. Although the EGFR-TKI (inhibitors) has a striking effect in NSCLC with longer survival rate, the efficacy is attributed to the development of EGFR-TKI acquired resistance due to additional mutations. Thus a method to suppress the restored EGFR pathway is needed to overcome the resistance. Kiura and his group developed an approach to deliver miRNA (miRNA7) which targets multiple sites in the 3′ UTR of EGFR mRNA to suppress the EGFR expression.120 Compared to the conventional targeting therapies such as antibodies and inhibitors, the miRNA strategy seems to be independent of unexpected conformational changes due to secondary mutations. In this study, the authors used a cationic liposome system to deliver miRNA 7 expressing plasmid and demonstrated significant inhibition of cell growth in number of different lung cancer cells. Delivery of miRNA 7 also resulted in marked tumor regression and EGFR suppression in mouse xenograft lung models, which ultimately overcomes the EGFRtyrosine kinase inhibitor resistance in lung cancer cells/ tumors.120 Another group has developed transferrin targeted, protamine containing liposomes to deliver miRNA. miR29b is a member of miR29 family, which downregulates the cellular expression of antiapoptotic Mcl-1 proteins. The formulation (designated as Tf−LPmR) has protamine to increase the delivery efficiency and transferrin ligand for receptor mediated internalization in leukemia cells (K562) that overexpress

suppressor functions, they may increase the translation of oncogenes and hence formation of tumors.12115116 One of the strategies to target overexpressed miRNA in cancer is the use of oligonucleotides or antagomirs to block the expression. Antagomirs are a novel class of chemically modified stable oligonucleotides consisting of 20−24 bases that are used to block the functions of endogenous miRNAs. The other one involves the use of miRNA mimics to substitute for the loss of expression of a tumor suppressor miRNA. The key challenge in achieving effective miRNA therapeutics in those cases is the development of an efficient delivery system, which can specifically deliver antagomirs or miRNA mimics to target cells in living animals. To overcome these delivery hurdles, nonviral and viral strategies have been explored. The nonviral strategies include encapsulation of chemically modified oligonucleotides in liposomes or polymers.12 Liposomes are composed of a phospholipid bilayer with an enclosed aqueous compartment. During complexation, they interact with oligonucleotides to form complexes that are stabilized by electrostatic interactions. These liposomes protect the oligonucleotides from degradation by nucleases and increase the circulation half-life. Let-7 is one of the best studied miRNAs found to be altered in the majority of human lung cancers.13117 Bader and his group identified this tumor suppressor miRNA in the majority of NSCLC cells and patient samples and used the concept to reintroduce this miRNA mimic to cells/tumors to reactivate cellular pathways to drive a therapeutic response. Studies have shown that the reduced let-7 was significantly associated with shortened postoperative survival and overexpression resulted in the inhibition of lung cancer cell growth.118 Using a neutral lipid delivery system, Trang et al. efficiently delivered the miRNA mimic to cells and demonstrated significant growth inhibition and proliferation (Table 2).118 It was demonstrated using the same delivery system that lung tumor growth could be inhibited in s.c. NSCLC tumors (H460) that lacked the expression of let-7.118 In addition, it was also demonstrated that let-7g works against the oncogene K-ras that has a crucial role Table 2. Illustrative Examples of Nanosystems Used for miRNA Delivery to Cancer Cells nanodelivery system

miRNA/target

cancer/cell type

neutral lipid emulsion

miR-34a, Let7

lung cancer

liposomal nanoparticles

miR-7 expressing plasmid miR-29b

lung cancer

polycation liposomes

miR-92a/ antiangiogenesis

liposome-polycationhyaluronic acid PAMAM dendrimer

miR-34a

angiogenic endothelial cells melanoma lung metastasis glioma

cationic lipoplexes

as-miR-21 miR-29b

lung cancer

transferrin-conjugated anionic lipopolyplex PLGA nanoparticles

acute myeloid leukemia lymphoma

miR-155

gold nanoparticles

miR-3, miR-1323

polylysine nanoparticle

miR-10b

ovarian cancer, neuroblastoma breast cancer

functionalized graphene oxide

miR-21

breast cancer

ref Trang et al.118,119 Rai et al.120 Wu et al.121 Ando et al.123 Chen et al.122 Ren et al.127 Huang et al.128 Babar et al.129 Ghosh et al.133 Jin et al.130 Zhi et al.131 2520



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Figure 8. Cellular uptake of fluorescently labeled A546-let7a encapsulated with dextran nanoparticles. U-2OS cells were transfected with 100 nM A546-let7a (red) encapsulated with dextran nanoparticles (A, B, C) or 100 nM A546-let7a mixed with Lipofectamine RNAiMax (D, E, F). The cells were washed with PBS and visualized by florescence microscopy at 2 h, 4 h, and 24 h after transfection.

this study also demonstrated tumor regression with the delivery of antagomirs. Their study results indicated that the antagomir delivery not only is efficient in crossing cell membrane but also maintained the threshold of intracellular antagomir levels by its slow release profile for prolonged therapeutic effect. A highly branched dendritic polymer including polyamidoamine (PAMAM) has attracted interest as nucleic acid delivery vectors.126 The presence of primary amine groups on their branched surface binds nucleic acids and compacts it into polyplexes, promoting cellular uptake in a wide variety of cultured cells. The primary amines on the surface also make it possible to conjugate suitable ligands such as transferrin for tumor or brain delivery. Overexpression of miR21 plays a key role in the majority of cancers. Downregulation of miR-21 therefore leads to repression of cell growth, increased cellular apoptosis, and cell cycle arrest, which can theoretically enhance the chemotherapeutic effect in cancer. Zhou et al.127 used PAMAM dendrimer as a carrier to codeliver anti-miR21 and 5fluorouracil to achieve delivery of miRNA to human glioblastoma cells and enhance the cytotoxicity of 5-FU. With the help of positively charged primary amine groups, the miRNA gets encapsulated easily. In addition, because of the presence of well-defined cavities and open architecture in PAMAM, guest molecules such as 5-FU could be encapsulated into the macromolecular interior through hydrophobic interactions.127 With this system, the authors demonstrated significant improvement in cytotoxicity of 5-FU and dramatic increase in apoptotic percentage of U251 cells. Authors from this study also showed that the codelivery approach brought down the migration ability of tumor cells, suggesting that this strategy may have an important clinical potential in the treatment of miR21 overexpressing glioblastoma. There are several nanocarriers that are currently pursued for clinical use in cancer therapy. More recently, biodegradable polymer nanoparticles have also been used as carriers for target specific delivery of miR to treat different types of cancer. A novel transferrin-conjugated nanoparticle delivery system for synthetic miR-29b (Tf−NP-miR-29b) has been established in acute myeloid leukemia (AML) cells. Tf−NP-miR-29b treatment of AML cells resulted in more than 200-fold increased

transferrin receptors. It was demonstrated that this Tfr targeted system containing miR29b liposome formulation resulted in enhanced biological effects on the suppression of target gene’s expression (efficient inhibition of Mcl-1 expression at the mRNA and protein levels) compared with the nontargeted or non protamine containing liposomes.120 Leaf Huang’s group demonstrated efficient miRNA delivery using a tumor targeted delivery system into experimental lung metastasis of murine B16F10 melanoma. This study reported a liposome−polycation−hyaluronic acid nanoparticle system modified with a tumor targeting single chain antibody fragment (scFv) for systemic delivery.122 Delivery of miR34a using this system demonstrated apoptosis, inhibition of survivin expression, and downregulation of MAPK pathway in B16F10 cells. Systemic delivery of miR34a using this system significantly downregulated survivin expression in the metastatic tumor and reduced tumor load in the lung. Apart from using liposome systems, a number of groups also worked on polymer delivery system for the same purpose. One such example was using PLGA based nanoparticles. PLGA was extensively studied for its ability to deliver different therapeutic agents. This polymer-based nanoparticle was shown to escape from the endosomal compartment to the cytoplasmic compartment and release its contents over longer periods of time. These features rendered PLGA particles as potential tool for oligonucleotide/miRNA delivery efficiently. A group of investigators from China has modified the biodegradable PLGA nanoparticles with a polyplexed PEI coating, in which the PLGA acts as the core and cationic PEI as the shell with an aim of encapsulating negatively charged nucleic acids. With the help of the cationic shell, they efficiently encapsulated miR-26a, a cell cycle suppressor, and demonstrated efficient delivery to human hepatocellular carcinoma cells such as HepG2 cells.124 This significantly increased the expression levels of miR-26a and inhibited the cell cycle progression by induction of G1 phase arrest in transfected HepG2 cells. These PLGA nanoparticles also showed a better safety profile compared to PEIs and liposomes. In another instance, Sekar et al.125 encapsulated chemically modified antagomirs in PLGA nanoparticles and showed efficient internalization and sustained release into cells. Authors from 2521



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Expression of miR-21 was inhibited with concomitant increase of the Au nanoparticle fluorescence that can be used to assess the silencing effect.134 These data showed that a single nanostructure can be used to target all RNA regulatory pathways while allowing for direct assessment of effective silencing and cell localization via a quantifiable fluorescence signal, making cancer nanotheranostics highly promising.

expression of miR-29b as compared with free miR-29b and was approximately twice as efficient as treatment with nontransferrin-conjugated NP-miR-29b. Tf−NP-miR-29b treatment significantly downregulated target genes DNMTs, SP1, CDK6, KIT, and FLT3, decreased AML cell growth by 30% to 50%, and impaired colony formation by approximately 50%. Mice engrafted with AML cells and then treated with Tf−NPmiR-29b had significantly longer survival compared with Tf− NP-scramble or free miR-29b. Furthermore, priming AML cells with Tf−NP-miR-29b before treatment with decitabine resulted in marked decrease in cell viability in vitro and showed improved antileukemic activity compared with decitabine alone in vivo.128 A surface modified (with the cell-penetrating peptide, penetratin) PLGA nanoparticles has been shown to successfully delivery miR-155 inhibitor (anti-miR-155) in mouse model of lymphoma. Systemic delivery of anti-miR-155 nanoparticles inhibits miR-155 expression and slows the growth of these “addicted” pre-B-cell tumors in vivo, suggesting a promising therapeutic option for lymphoma/leukemia.129 Poly(L-lysine) (PLL) nanoparticle, which showed excellent nuclei acid condensation property, has been used for delivery of miR-10b recently. miR-10b is highly expressed in metastatic breast cancer cells and positively regulates breast cancer cell migration and invasion through inhibition of HOXD10 target synthesis. The results showed in this study that PLL-RNA nanoparticles could deliver the anti-miR-10b molecules into cytoplasm of breast cancer cells in a concentration-dependent manner that displayed sustainable effectiveness.130 In a multifunctional nanocomplex, composed of polyethylenimine (PEI)/poly(sodium 4-styrenesulfonates) (PSS)/graphene oxide (GO) and termed PPG, both chemotherapy drug adriamycin and anti-miR-21 can be efficiently and simultaneously delivered and overcome multidrug resistance in breast cancer.131 In our recent study, we found that dextran-based polymeric nanoparticles can efficiently deliver miR let-7a into tumor cells (Figure 8) (unpublished results). Metal nanoparticles have also been used for miR delivery. Functionalized gold nanoparticles (AuNPs) have been used for anti-miR oligonucleotide (AMO) transfection. When the AuNPs and anti-miR-29b were delivered into HeLa cells, MCL-1 protein and mRNA levels were increased significantly. Furthermore, apoptosis induced by TNF-related apoptosisinducing ligand (TRAIL) was inhibited, proving that AMOs targeting miR-29b were effectively delivered.132 Recently, a robust method for delivering unmodified miRs into cells has been developed by using cysteamine-functionalized gold nanoparticles (AuNPs). The efficiency of this method has been validated in two different tumor models and found that the best formulation of miR(1)-AuNP(10)-S-PEG(0.5) had the highest payload (10−20-fold higher than lipofectamine, a toxic transfection reagent for miRs in vitro), lowest toxicity (98% of cell viability following treatment), efficient uptake (96% uptake rate), fastest endosomal escape, and increased half-lives (at least 5 days) impacting cell proliferation and patterns of target gene expression.133 These studies represent an essential step toward the advancement of gold nanotechnology-based miR delivery systems and evaluation of their therapeutic potentials in preclinical models. The advantage of gold nanosystems is that they not only are capable of efficient delivery of miR but also yield a quantifiable fluorescence signal directly proportional to the level of miR delivery. A gold nanosystem (gold nanobeacons) has also shown efficiency at targeting and silencing miR-21, an endogenous miR involved in cancer development.

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CONCLUSIONS The discovery of RNAi-mediated gene silence heralds a powerful new strategy for treating human diseases. Despite the tremendous efforts from academia and industry in providing a glimpse of this breakthrough discovery in molecular biology, there still remains a great challenge in its clinical translation into viable therapies. Although there are several obstacles for successful RNAi therapeutics, a major hurdle is in overcoming the delivery barriers to the targeted tissue and cells, without disrupting their effectiveness and trigging unwanted immune responses. Toward this end, we have discussed some recent developments in addressing some of the delivery issues for successful RNAi therapies in vitro and in vivo. Although each system demonstrates its own merits and shortcomings, multifunctional nanosystems have shown tremendous potentials for RNA interference therapy. Thus further advancements in gene delivery research greatly hinge on the continued progress in development of new biomaterials and formulation platforms that can pave the way forward for the next generation of mature technologies for their successful clinical translation.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 617-373-3137. Fax: 617-373-8886. E-mail: m.amiji@neu. edu. Present Address ‡

Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201. Notes

The authors declare no competing financial interest.

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REFERENCES

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Stimuli-responsive liposomes for the delivery of nucleic acid therapeutics.

Prospects for nucleic acid-based therapeutics against hepatitis C virus.

Epigallocatechin Gallate Nanodelivery Systems for Cancer Therapy.

Nucleic acid detection systems for enteroviruses.

Nucleic acid aptamers: research tools in disease diagnostics and therapeutics.

Nucleic acid based logical systems.

Novel diagnostics and therapeutics for drug-resistant tuberculosis.

Non-viral nucleic acid containing nanoparticles as cancer therapeutics.

Preclinical and Clinical Advances of GalNAc-Decorated Nucleic Acid Therapeutics.

Independent blood-brain barrier transport systems for nucleic acid precursors.

Therapeutic Potential of Epigallocatechin Gallate Nanodelivery Systems.

Nucleic acid aptamer-mediated drug delivery for targeted cancer therapy.

Natural and artificial small RNAs: a promising avenue of nucleic acid therapeutics for cancer.

Phospholipid-modified PEI-based nanocarriers for in vivo siRNA therapeutics against multidrug-resistant tumors.

Nanodelivery: An Emerging Avenue for Nutraceuticals and Drug Delivery.

Advancing polymeric delivery systems amidst a nucleic acid therapy renaissance.

Phenotypic drug profiling in droplet microfluidics for better targeting of drug-resistant tumors.

Future trends and emerging issues for nanodelivery systems in oral and oropharyngeal cancer.

Strategies to target tumors using nanodelivery systems based on biodegradable polymers, aspects of intellectual property, and market.

Drug-nucleic acid interactions: conformational flexibility at the intercalation site.

Aminomethyl spectinomycins as therapeutics for drug-resistant respiratory tract and sexually transmitted bacterial infections.

Immunological and hematological toxicities challenging clinical translation of nucleic acid-based therapeutics.

Nucleic Acid Therapeutics Using Polyplexes: A Journey of 50 Years (and Beyond).

HDL as a drug and nucleic acid delivery vehicle.