Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 872 TThheerraannoossttiiccss 2014; 4(9): 872-892. doi: 10.7150/thno.9404 Review Nanoparticle-Mediated Systemic Delivery of siRNA for Treatment of Cancers and Viral Infections Mohamed Shehata Draz1,2*, Binbin Amanda Fang3,4,*, Pengfei Zhang5, Zhi Hu6, Shenda Gu6, Kevin C. Weng4, Joe W. Gray4,6, Fanqing Frank Chen1,3,4  1. Zhejiang-California International Nanosystems Institute, Zhejiang University, Hangzhou, Zhejiang 310029, China 2. Faculty of Science, Tanta University, Tanta 31527, Egypt 3. Life Sciences College, Fudan University, Shanghai 200433, China 4. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94127, USA 5. Translational Medicine Center, Changzheng Hospital, The Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, P.R. China. 6. Biomedical Engineering, OHSU Center for Spatial Systems Biomedicine, Oregon Health and Science University, Portland, OR 97239, USA * These two authors contributed equally.  Corresponding author: Fanqing Frank Chen, f_chen@lbl.gov or frank_chen@fudan.edu.cn. © Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/ licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2014.04.15; Accepted: 2014.05.27; Published: 2014.06.11 Abstract RNA interference (RNAi) is an endogenous post-transcriptional gene regulatory mechanism, where non-coding, double-stranded RNA molecules interfere with the expression of certain genes in order to silence it. Since its discovery, this phenomenon has evolved as powerful technology to diagnose and treat diseases at cellular and molecular levels. With a lot of attention, short inter-fering RNA (siRNA) therapeutics has brought a great hope for treatment of various undruggable diseases, including genetic diseases, cancer, and resistant viral infections. However, the challenge of their systemic delivery and on how they are integrated to exhibit the desired properties and functions remains a key bottleneck for realizing its full potential. Nanoparticles are currently well known to exhibit a number of unique properties that could be strategically tailored into new advanced siRNA delivery systems. This review summarizes the various nanoparticulate systems developed so far in the literature for systemic delivery of siRNA, which include silica and sili-con-based nanoparticles, metal and metal oxides nanoparticles, carbon nanotubes, graphene, dendrimers, polymers, cyclodextrins, lipids, hydrogels, and semiconductor nanocrystals. Chal-lenges and barriers to the delivery of siRNA and the role of different nanoparticles to surmount these challenges are also included in the review. Key words: Small interfering RNA; Nanoparticle; RNA interference; Delivery; Cancer; Virus. 1. Introduction In 1998, Fire and Mello et al. discovered that po-tent and specific RNA interference can be induced by double-stranded RNA (dsRNA) in Caenorhabditis ele-gans [1]. Further investigations confirmed that similar dsRNA-triggered phenomena also exist in many other species such as plants [2], Drosophila [3], and mam-malian cells [4, 5]. The past decade has witnessed an explosion of research on small regulatory RNAs that has yielded a basic understanding of many types of small RNAs in diverse eukaryotic species and the functions of key protein factors amidst the RNA si-lencing pathways. RNA silencing is recognized as a widespread mechanism of gene regulation in eukar-yotes. The key machinery of RNAi pathway is that dsRNA molecules, experimentally or naturally occur-ring, can be recognized and cleaved into 21-23 nucle-otide duplex termed small interfering RNA by Dicer homologues that have dsRNA binding domain and sRNaseIII-like enzyme activity, see Figure 1 [6, 7]. The siRNAs are incorporated into the multi-subunit ef- Ivyspring International Publisher Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 873 fector complex called RNA-induced silencing com-plex (RISC), therefore activate the helicase activity leading to cleavage of the sense strand of siRNA [8, 9]. The remaining antisense strand recognizes the hom-ologue region with base-pairing and degrading the target messenger RNA (mRNA) mediated by the Ar-gonaute (Ago) family proteins with endonuclease activity, which is the catalytic core of active RISC, resulting in inhibition of gene expression [10-12]. RNAi technology has become a routine laboratory research tool for gene functional study and is making its way as a revolutionary class of therapeutics for treatment of cancers and different viral infections. This paper is focused primarily on synthetic siRNA and its delivery using nanoparticulate systems. 2. RNAi: a potential revolutionary thera-peutics The discovery of RNAi raises the possibility to explore new approaches for many incurable and dif-ficult to treat diseases. The advantage of siRNA as therapeutics is that siRNA can target many undrug-gable genes. Other than antibody-based therapeutics that mainly targets receptors present on the cell sur-face, only a very small number of targets, mostly ki-nases, have been validated for traditional small mol-ecule drugs. In addition, it is found that diseases such as cancer, genes are often deregulated by high-level amplifications [13-15]. Such genes are particularly interesting as therapeutic targets for treatment of pa-tients that are refractory to existing therapies. How-ever, only very few of these genes, including FGFR1, IKBKB, ERBB2, etc., are considered druggable [13]. Some malignant diseases are known to be caused by multiple gene mutations, copy number change or epigenetic changes [16, 17]. Studies show that cancers are highly heterogeneous, resulting in each patient being \"unique” and requiring personalized treatment. Furthermore, cancers initially sensitive to conven-tional chemotherapeutics often adapt tolerance to targeted therapy by gene mutations and other mech-anisms [18]. A siRNA-based drug may target any mRNAs of interest, regardless of their cellular loca-tions or structures of the translated proteins. There-fore, siRNA therapeutics shows promises to meet these challenges and has emerged as new generation bio-drugs under intensive investigation. Figure 1. The circulation routine of siRNA and the biological mechanism of RNAi in vivo. siRNA is associated with nanoparticles either through chemical linkage via covalent bonds or through non-covalent interactions. Nanoparticles facilitate cellular uptake of siRNA cargo the process that commonly occurs through three main pathways (a) membrane fusion, (b) receptor-mediated endocytosis, and (c) direct endocytosis. The mechanism of internalized siRNA is controlled and initiated by the interaction with RNA-induced silencing complex (RISC). The remaining antisense strand recognizes the homologue region with base-pairing and degrading the target mRNA resulting in inhibition of gene expression. Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 874 Significant progress has been made for the de-velopment of siRNA based drugs since the discovery of RNAi machinery. Currently, several potential siRNA candidates are undergoing clinical trials summarized in Table 1, such as Bevasiranib, the first siRNA based drug in clinical trials, which targets vascular endothelial growth factor (VEGF) pathway for treatment of macular degeneration; ALN-RSV01 to treat virus respiratory diseases, and CALAA-01 to silence ribonucleotide reductase subunit 2 (RRM2) gene, which is highly overexpressed in advanced cancers. Lipid-based carriers of siRNA therapeutics can target the liver in metabolic diseases and are being assessed in clinical trials for treatment of hypercho-lesterolemia [19]. Phase Ib clinical trial of the first-in-human mutation-targeted siRNA Td101 against an inherited skin disorder is now completed. Table 1. List of siRNA-based drugs targeting different diseases were in clinical trials. Disease Target Vehicle Drug Name Company Status Cancers Solid tumor RRM2 Cyclodextrin, Transferrin, PEG CALAA-01 Calando Pharmaceuticals Terminated, Phase I Advanced solid tumors PKN3 Liposomes Atu027 Silence Therapeutics AG Completed, Phase I Pancreatic ductal adenocarcinoma Mutated KRAS oncogene LODER polymer siG12D LODER Silenseed Ltd Active, Phase I Metastatic melanoma absence of CNS metastases LMP2, LMP7, and MECL1 Transfection NCT00672542 Duke University Completed, Phase I Chronic myeloid leukemia Fusion genes SV40 SV40 vectors- carrying siRNA Hadassah Medical Organiza-tion ? Virus infections RSV RSV nucleocapsid Naked siRNA ALN-RSV01 Alnylam Pharmaceuticals Completed, Phase II HBV Pre gen./Pre-C, Pre-S1, Pre-S2/S, X Plasmid DNA NUC B1000 Nucleonics Completed, Phase I HBV conserved sequences DPC ARC-520 Arrowhead Research Corporation Recruiting, Phase II HIV HIV Tat protein, HIV TAR RNA, human CCR5 Lentivirus pHIV7-shI-TARCCR5RZ City of Hope Medical Center/Benitec Terminated, Phase 0 HCV miR-122 Naked LNA SPC3649 (LNA) Santaris Pharm Completed, Phase II EBOV EBOV polymerase L, VP24, and VP35 regions SNALP TKM-100201 Tekmira Pharmaceuticals Corporation Terminated, Phase I Other diseases Hypercholesterolemia APOB SNALP PRO-040201 Tekmira Pharmaceuticals Corporation Terminated, Phase I Pachyonychia Congenita keratin K6a Naked siRNA TD101 TransDerm, Inc Completed, Phase I Delayed graft function kidney transplant P53 Naked siRNA I5NP Quark Pharmaceuticals Active, Phase I/II Acute renal failure P53 Naked siRNA I5NP Quark Pharmaceuticals Terminated, Phase I Glaucoma; ocular hypertension ADRB2 Naked siRNA SYL040012 Sylentis, S.A. Completed, Phase I/II Dry eye syndrome TRPV1 Naked siRNA SYL1001 Sylentis, S.A. Recruiting, Phase II Wet AMD VEGF Naked siRNA Bevasiranib Opko Health, Inc. Terminated, Phase III Diabetic AMD VEGF Naked siRNA Bevasiranib Opko Health, Inc. Completed, Phase II Chronic optic nerve atrophy Caspase-2 Naked siRNA QPI-1007 Quark Pharmaceuticals Completed, Phase I AMD; CNV VEGFR Naked siRNA Sirna-027/AGN211745 Allergan Sirna Therapeutics Inc. Completed, Phase II AMD/DME RTP801 Naked siRNA PF-655 Quark Pharmaceuticals Pfizer Completed, Phase II Resource: http://clinicaltrials.gov. CNS, central nervous system; RSV, respiratory syncytial virus; HBV, hepatitis B virus; HIV, human immunodeficiency virus; HCV, hepatitis C virus; EBOV, Ebola virus; AMD, Age-Related Macular Degeneration; CNV, choroidal neovascularization; RRM2, Ribonucleotide reductase subunit 2; PKN3, protein kinase n3; KRAS oncogene, Kirsten rat sarcoma viral oncogene; LMP2, large multifunctional peptidase 2; LMP7, large multifunctional peptidase 7; MECL1, multicatalytic endopeptidase complex-like-1; HIV Tat protein, HIV-1-trans-activating protein; HIV TAR, HIV trans-activation response; CCR5, human CC chemokine receptor 5; VP24, virus protein 24; VP35, virus protein 35; APOB, apolipoprotein B; ADRB2, beta-2 adrenergic receptor; TRPV1, transient receptor potential vanilloid 1; VEGF(R), Vascular endothelial growth factor (re-ceptor); cysteine-aspartic proteases-2 (Caspase-2); PEG, polyethylene glycol; SV40,9 Simian virus 40; DPC, dynamic polyconjugate; SNALP, stable nucleic acid-lipid particle. Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 875 3. Challenges and barriers to the systemic delivery of siRNA As a therapeutic strategy, RNAi offers several advantages over small-molecule drugs, as virtually all genes are susceptible to targeting by siRNA mole-cules. This advantage is, however, compromised by the challenges of safe and effective delivery of oligo-nucleotides to diseased tissues in vivo, summarized in Table 2. On the top of these challenges is the targeting specificity and stability of the administrated siRNA. Any inadvertent silencing of nontargeted genes \"off-target effect” may lead to problems in interpreta-tion of data and potential toxicity. The design and selection of potent siRNAs should be carefully per-formed. The basic parameters for choosing siRNAs involve consideration of internal repeated sequences, secondary structure, GC content, base preference at specific positions in the sense strand, and appropriate siRNA length (19–22 bps). 2 -O-methyl ribosyl group substitution at position 2 in the guide strand could reduce silencing of most off-target transcripts with complementarity to the siRNA guide [20, 21]. In ad-dition, the stability remains major challenge to appli-cation of siRNA in vivo. The naked siRNAs face rapid degradation in the extracellular environment and are not efficiently internalized into cells. The RNA back-bone contains ribose, which has a hydroxyl group in the 2′ position of the pentose ring instead of a hydro-gen atom [22], which makes the RNA backbone very susceptible to hydrolysis by serum nucleases that cleave along the phosphodiester backbone of nucleic acids. Chemical modifications of siRNA on the sug-ar-phosphate backbone such as 2′-fluoro and 4′-thio modifications, incorporation of locked nucleic acids, phosphorothioation, methyl phosphonation can in-crease stability of dsRNA under serum-containing conditions [23]. The use of siRNA delivery vehicles is also essential for practical siRNA-mediated silencing. The proper delivery vehicles would provide protec-tion to siRNA from degradation in the serum during circulation. On the other hand, there are multiple mechanisms by which siRNA may be recognized by receptors of the innate Immune system, including both endosomal Toll-like receptors and cytoplasmic receptors [24] that can lead to systemic inflammation in vivo through inducing the production of type I in-terferons and inflammatory cytokines. This challenge of RNA-induced immunostimulation may be reduced by proper siRNA design considerations, including choices of siRNA target sequence, chemical modifica-tions to the RNA backbone, and the delivery formu-lation and method. So far, two cytoplasmic receptors that have long been known to recognize long dsRNA are protein kinase R [25] and 2′-5′-oligoadenylate synthetase [24]. A variety of siRNA backbone modi-fication chemistries have been investigated for their capacity to suppress immune activation while main-taining gene silencing activity. Making substitutions at uridine residues with 2′-fluoro, 2′-deoxy or 2′-O-methyl groups often reduces the immunostimu-latory capacity of siRNA [26, 27]. The termini (ends) of a siRNA are major determinants of immune recogni-tion. siRNA with added 3′ overhangs such as UU, can reduce immune recognition and induce more effi-ciently gene silencing in vivo [28]. Table 2. Challenges and barriers to the systemic delivery of siRNA. Challenge Solution/approach Specificity Well design, optimize algorism Stability/degradation Chemical modification, carrier Immune response Chemical modification Clearance by RES systems Encapsulation Targeting/biodistribution Receptor mediated Endosomal escaping pH responsive release Dissociation from carrier Cleavable polymers for siRNA Toxicity Reduce off-target effect, biocompat-ible and biodegradable carrier RES: reticuloendothelial system. The systemic delivery of siRNA is further ham-pered by many additional anatomical and physiolog-ical defensive barriers presented by the human body, and siRNA need to overcome before to reach its site of action. The first barrier includes the renal clearance through kidney or the entrapment in reticuloendo-thelial system (RES) that exists in the liver, spleen, lung and bone marrow. Many delivery systems larger than ~20 nm and less than ~100 nm in diameter are thought to be optimal for avoiding both renal and RES clearance and favorably improve the passive in-tra-tumoral delivery due to the unique features of leaky vasculature with capillary pore size of 100–800 nm and the absence of lymphatic drainage [29, 30]. Surface modifications using hydrophilic and flexible polyethylene glycol and other surfactant copolymers, e.g., poloxamers, polyethylene oxide, are also sug-gested to prepare stealth delivery carriers that can remain in the systemic circulation for a prolonged period of time [31, 32]. The second barrier is the en-dothelial lining and extracellular matrix barrier. For successful delivery, siRNA and its carriers must be readily to extravasate and move through the complex extracellular matrix to reach the diseased cells. The normal endothelial layer lining most of tissues allows the permeation of materials through abundant small pores of about 45 angstroms diameter and relatively scarce large pores of about 250 angstroms. This small pore system restricts the permeation of materials Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 876 larger than 4 or 5 nanometers [33, 34]. Only naked siRNA oligonucleotides or that are modified with molecular conjugates should be readily permeable, while other types of formulations may not be able to efficiently reach the underlying tissues. However, this represents an opportunity for nanocarriers to specifi-cally deliver siRNA to certain types of tumors that have fenestrated endothelia. The third barrier is bio-distribution, and it can be conquered by several ap-prochaces particularly via targeted delivery of siRNA [35-37]. After siRNA is successfully delivered into the cells, how it can be efficiently released from endosome also presents a big challenge. If siRNA remains inside the endosome for too long, it will inevitably be de-graded. Several methods aiming to enhance endoso-mal escape include conjugation with lipids or pep-tides, pH-sensitive lipoplexes, etc [38]. Figure 1 pre-sents the circulation routine of siRNA complexes and the biological mechanism of RNAi in vivo. 4. Nanoparticles in RNAi therapeutics Nanoparticulate systems have emerged in last few years as an alternative material for advanced di-agnostic and therapeutic applications in medicine. Compared to molecular medicine, nanoparticles offer many advantages that overcome a range of challenges and barriers summarized in the previous section, particularly, bioavailability and biodistribution of therapeutic agents. The first remarkable property of nanoparticles is their superior in vivo retention by decreasing enzymatic degradation and sequestration by phagocytes of the reticulo-endothelial systems. This is mostly attributed to their immunochemically inert surfaces in contact with the biological environ-ment. Increased deposition to the diseased sites via compromised vasculatures in the phenomenon called enhanced permeability and retention effect also con-tributes to their improved deposit to diseased sites and efficacy [37]. Various other properties of nano-particles, including size, shape, surface charge, den-sity, composition, and surface chemistry have been investigated [39]. The accumulated data presents in-teresting correlations among all these properties that led to a range of outcomes. Research has been focused on windows of optimal and controllable properties that guides the design and synthesis of nanoparticle formulations [40]. The properties that have been validated in chemotherapeutics are also exploited for siRNA packaging and delivery [41-46]. The effort began with stable association of siRNA molecules with the na-noparticles and their retention in circulation. Methods of conjugating siRNAs with other inert and biocom-patible molecules, such as cholesterol and long-chain fatty acids have been reported [47, 48]. Complexation, encapsulation, and non-covalent association of siRNA into several nanoconstructs are reported. Success has been limited to date and there are still numerous challenges associated with many stages along the de-livery process especially several recent reports on the toxicity and instability of some siRNA-nanoparticle complexes in vivo [49-51]. Different nanoparticle sys-tems offer various advantages and disadvantages based on their composition, physical, and chemical characteristics, thus leading to a range of effectiveness when associated with siRNA. It has been found that unique challenges are associated with siRNA as many relatively successful technologies for oligonucleotides and DNA delivery did not translate to expected re-sults for siRNA. An example is cationic lipid-gene complexes that are widely adapted for transfection and yet the release of siRNA during the intracellular pathways remains a major hurdle. Here we reviewed the main types of nanoparticle systems, and discuss their advantages, disadvantages, and the current state of development, summarized in Table 3 and Table 4. Table 3. Types of nanoparticle systems used in siRNA delivery. Nanoparticle systema Target geneb Silencing (%)c Delivery routed Ref Type Shape Size ζ potential Silica, silicon-based nanoparticles MSNPs Spherical ~ 220 nm ND Bcl-2 ~80% In vitro, A2780/AD cells [54] Spherical 130 nm 29-38 mV GFP 55-60% In vitro, HEPA-1 cells [55] Spherical 832 nm 25.4 mV Pgp 80 or 90% In vitro, KBV1 cells [56] Spherical 50 nm ND 50% In vivo, i.v. injection in mice with MCF-7 cells [57] Metal, metal oxides nanoparticles MNPs Irregular/ spherical ≤156.2 nm 26-46 mV GFP 54.8% In vitro, PC-3 cells [64] Spherical 70–150 nm 2±2 mV GFP 21.5% In vitro, SHEP cells [65] Spherical ~ 60 nm −2.6 mV GFP 49.2% In vitro, C6 glioma cells [66] Spherical 75 nm −30 mV GFP 20% In vitro, MDA-MB-435 and A549 cells [67] Irregular/ spherical 100 nm –2-40 mV Luc 30% In vitro, 4T1 cells In vivo, i.t. injection in mice [68] Irregular/ spherical 120 nm ~ 40 mV Luc ~75% In vitro, 4T1 cells [70] AuNPs Spherical 100 nm ND GFP 73.5 In vitro, PC-3 cells [75] Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 877 Nanoparticle systema Target geneb Silencing (%)c Delivery routed Ref Type Shape Size ζ potential Spherical 26.8 nm ND EGFP 72% In vitro, K1 Cells [77] Spherical ∼110 nm 30±9 mV β -gal 48% In vitro, SVR-bag4 cells [79] Spherical 18.2 nm ~12 mV GFP 57.8% In vitro, MDA-MB-435 cells [80] Rod L = 46.5 nm D = 17.3 nm ND Galectin-1 ~83% In vitro, MDM cells [81] Carbon-based materials CNTs SWNTs D = ~1–3 nm; L = ~200 nm ND CXCR4 /CD4 50–60% In vitro, human T cells and PBMCs [91] SWNT ND ND TERT 80% In vitro, HeLa cells [92] SWNTs L= 50-300 nm ND Lamin A/C 40 % In vitro, HeLa cells [93] SWNTs D = 1-1.4 nm L = 0.1-1 µm ND ERK 75% In vitro, cardiomyocytes [94] SWNT L = 50–300 nm ND cyclin A2 31% In vitro, K562 cells [95] MWNT D = 20–30 nm L = 0.5–2 µm ND siTOX 50% In vitro, A549 cells In vivo, i.t. injection in mice with Calu 6 cells [96] MWNT D = 9.5 nm; L = 1 µm −64 mV Luc 60–90% In vitro, H1299 cells [209] Graphene Sheet-like ~ 200 nm 55.5 mV Bcl-2 30-60% In vitro, HeLa cells [106] Dendrimers PAMAM Spherical 72-165 nm ND siGLO Red ND In vitro, A2780 cells [112] Spherical ~150 nm ND Bcl2 22-84% In vitro, A2780 cells [113, 114] Spherical 120-180 nm ND Bcl2 70-50% Polymers Chitosan Spherical 200 nm ND POSTN, FAK, PLXDC1 51% In vitro, SKOV3ip1, HeyA8, A2780, A2780ip2 and MOEC cells In vivo, i.v injection in mice [123] ND ND ND Src/Fgr 81.8% In vitro, SKOV3ip1 and HeyA8 cells In vivo, i.v. injection in mice [124] ND 500 nm 51 mV HPV16 E7 ~31% In vitro, CaSki cells [125] Spherical 400–500 nm 32-45 mV HPV16 E6 ~58% In vitro, SiHa cells [126] Dextran Spherical 100-300 nm −15.9 mV Luc 60% In vitro, PC-3 cells [148] Polycations ND 7-40 nm ND Luc Up to 90% In vitro, Neuro 2A cells [136] VEGF GFP ∼66% ∼80% In vitro, HepG2 cells [143] Spherical 100-300 nm 1.54 mV RFP 80% In vitro, B16F10 cells [144] ND 100 nm ND GFP 76±14% In vitro, primary human glioblastoma cells [145] ND 200 nm −2.7 mV VEGF ND In vitro, PC-3, KB, HeLa, A2780, and A549 cells [146] Spherical 314 ±15 nm −6.0 mV RFP ND In vitro, B16F10 cells [147] ND 1-5μM ND VEGF 60% In vitro, A2780 cells [149] Micelles ND 75-85 nm 3.3 mV PLK-1 ND In vitro, OSRC-2 cells [49] Star-shaped 56±3 nm ND EGFP 74±1.5% In vitro, HeLa cells In vivo, i.v. injection in mice [138] Cyclodextrin ND ~70 nm ND RRM2 77% In clinical trial, i.v. injection, advanced solid tumors [153] ND ~60–100 nm 5-10 mV RRM2 50% In vitro, Neuro2A-Luc cells [155] ND ~70 nm ND RRM2 ND In vivo, i.v. injection in monkeys [157] ND ~80 nm 10 mV Luc 50% In vitro, In vivo, Neuro2A-Luc cells [158] Lipid-based nanoparticles Liposomes Spherical 184 nm 42.9 mV siTOX 50% In vitro, A549 cells In vivo, i.t. injection in mice with Calu 6 cells [96] Spherical 190 nm 37.8 mV Luc/ GFP 70% In vitro, HT1080 cells In vivo, i.v. injection in mice [160] ND 80-100 nm ND HBV 40-50% In vitro, Huh7 liver-derived cells In vivo, i.v. injection in mice [172] ND ND ND EphA2 50% 95% In vivo, i.p. injection in mice In vitro, HeyA8 or SKOV3ip1 cells [176] ND ND ND IL-8 32-48% In vitro, HeyA8 and SKOV3ip1 cells In vivo, i.p. injection in mice [177] Spherical 85–90 nm ND HCV IRES 90% In vivo, i.v. injection in mice [178] ND 81–85 nm ND ZEBOV Lpol., VP24, VP35 66% In vivo, bolus intravenous infusion, monkeys [179] ND ND ND MARV VP24, VP35, VP40, NP, Lpol 60-100% In vitro, HepG2 cells In vivo, i.v. injection in mice [180] Hydrogels Spherical 7–8 µm 20-30mV EGFP ND In vitro, HUH7 cells [182] Spherical Rȥ ∼54 nm 0.3 mV EGFR ~35% In vitro, Hey, BG-1 cells [183] ND ~100 nm ND EGFR ND In vitro, Hey cells [184] ND ND ND GFP 80% In vitro, HEK293 cells [189] Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 878 Nanoparticle systema Target geneb Silencing (%)c Delivery routed Ref Type Shape Size ζ potential ND ND ND IL-10 80% In vitro, APCs [190] ND ND ND TG2 72-92% In vivo, s.c. injection in mice In vitro, A375SM and MDA-MB231 cells [191] Irregular 200-500 nm ND GFP 66±8.2% In vitro, HCT-116 cells [192] ND 6 mm ND mTOR 72% In vitro, NIH 3T3 cells [193] Spherical 111±15 nm 36.7mV GFP ND In vitro, MDA-MB-435 cell [194] ND 100-400 nm ND GFP/VEGF 53% In vitro, Hela cells and PC-3 cells [195] Semiconductor nanocrystals Ellipsoid ND ND EGFP 29% In vitro, HeLa cells [199] Spherical 16±1 nm ~21 mV Her-2 65% In vitro, SK-BR-3 cells [203] Spherical 17 nm 8.5 mV Human CYPB 98.19 In vitro, MDA-MB-231 cells [204] Spherical 200 nm ND VEGF 29.7±3% In vitro, PC-3 cells [205] aValues are estimated for nanoparticle delivery system (NP and siRNA) by different techniques, including TEM, SEM, AFM, and DLS for shape and size, while zeta potential for surface charge. ND, not determined; MSNPs, mesoporous silica nanoparticle; MNP, magnetic nanoparticles; MNCs, magnetic nanoclusters; AuNPs, gold nanoparticles; CNTs, carbon nanotubes; PAMAM, polyamidoamine; SWNT, single-walled nanotubes; MWNT, multi-walled nanotubes; G4, generation 4; L, length; D, diameter; PEI, polyethylenimine; PLL, poly-L-lysine; Rz, z-average radii; PEG, poly(ethylene glycol); PPD, PEG-peptide-dioleoylphosphatidyl ethanolamine; bBcl-2, B-cell lymphoma 2; Pgp, P-glycoprotein; GFP, green fluorescent protein; Luc, luciferase; EGFP, enhanced green fluorescent protein; β –gal, β –galactosidase; ERK, extracellular regulated kinase; CXCR4, chemokine receptor type 4; CD4, cluster of differentiation 4; TERT, telomerase reverse transcriptase; VEGF, vascular endothelial growth factor; RFP, red fluorescent protein; periostin; FAK, focal adhesion kinase; PLXDC1, plexin domain containing 1; Src, Sex combs reduced; F gr, gardner-Rasheed feline sarcoma viral oncogene homolog; HPV16 E7, human papilloma virus 16 E7 gene; PLK-1, polo-like kinase 1; RRM2, ribonucleotide reductase M2; EphA2, EPH receptor A2; HBV, hepatitis B virus; HCV IRES, hepatitis C virus internal ribosome entry site; ZEBOV, Zaire ebolavirus; Lpol, L polymerase; VP, viral protein; MARV, Marburg virus; IL-8, interleukin 8; IL-10, interleukin 10; TG2, tissue transglutaminase; mTOR; mammalian target of rapamycin; Her-2, human epidermal growth factor receptor 2; Human CYPB, human cyclophilin B. c from its control value d SVR bag4 is an endothelial cell line; A2780, A2780/AD, A2780ip2, SKOV3ip1, HeyA8 and BG-1 are human ovarian cancer cell lines; KBV1 is human epidermoid carcinoma; SK-BR-3, MCF-7, MDA-MB-435 and MDA-MB231 are human breast cancer cell lines; HEPA-1 is mouse liver cancer cell line; HeLa, CaSki and SiHa cells are human cervical cancer cell line; SHEP is human neuroblastoma cell line; A549, Calu 6 and H1299 are lung cancer cell lines; 4T1 is murine breast cancer cell line; PC-3, human prostate car-cinoma cell line; K1 is a Chinese hamster ovary cell line; MDM is human monocyte-derived macrophage cells; PBMCs are peripheral blood mononucleated cells; K562 is chronic myelogenous leukemia cell line; HUH7 and HepG2 are human liver cancer cell lines; B16F10 is murine melanoma cell line; KB is human oral cavity epidermal cancer cell line; MOEC is mouse endothelial cell line; Neuro2A is mouse neural crest-derived cell line; HT1080, human fibrosarcoma cell line; HCT-116 is colonic epithelial cell line; HEK293, human embryonic kidney 293 cell line; APCs are antigen-presenting cells; A375SM is metastatic human melanoma cell line; NIH 3T3 is mouse embryonic fibroblast cells; i.v., intravenous; i.t., intratumorally; i.p., intraperitoneal; s.c., subcutaneous. Table 4. Advantages and disadvantages of different nanoparticles to siRNA delivery. Nanoparticle Advantages Disadvantages Ref MSNPs Large surface area Stability Biocompatibility Tuned biodegrability Controllable porosity, allows multifunctional and sequential delivery Surface reactivity, and easy functionalization In vivo toxicity [52, 53, 58-63] MNPs Large surface area Small in size, allows longer circulation and improved tissue penetration High magnetization for remotely-controlled and fast delivery Controlled clustering Potential for multimodal applications (e.g. targeting, diagnostic, and therapy) Poor colloidal stability Limited biocompatibility and Cytotoxicity Non-biodegradability [64, 71-73, 83] AuNPs Large surface area Easy synthesis, modification and bioconjugation Rational stability and biocompatibility Potential for multimodal applications (e.g. targeting, diagnostic, and therapy) High cost of large scale production Stickiness and limited in vivo stability Non-biodegradability [74, 82, 83] CNTs Large surface area Ultra-high functionalization and loading capacities High penetration capacity to biological barriers Ultimate electrical and thermal conductivities and mechanical strength Encapsulation and storage functions in delivery of molecules Difficulty in production and handling Non-biodegradability Unresolved toxic properties [84-86, 108] Graphene Large surface area Facile synthesis Colloidal stability Easy surface functionalization Good electrical and mechanical properties High cost and difficultly of massive production Non-biodegradability Increased biosafety concerns [100, 102, 107] Dendrimers Very precise size and shape controllability Water solubility and biocompatibility Elicit negligible immune response Easy electrostatic interaction with nucleic acid and protection from nuclease activity Non-specific cytotoxicity Limited release of the associated bio-actives Rapid clearance [109, 110, 115] Polymers Easy and cheap production Fine tenability of structure and properties Simplicity for loading and complexation with nucleic acid by electrostatic interaction It can be tailored for a wide range of molecular weights Natural polymers are nontoxic, biocompatible, and biodegradable. Limited stability Synthetic polymers cause cellular necrosis and apoptosis [23, 50, 117, 125, 144] Cyclodextrin Low toxicity Act as molecular containers that can help to enhance biological properties of loaded molecules Lack of immune stimulation High cost production Concerns regarding their safety and limited solubility [150, 151, 159] Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 879 In vivo stability due to the absence of enzyme degradation in humans Liposomes Biocompatibility Rapid cellular uptake The flexibility of synthesis, modification and formulation Targeting and controlled release Easy conjugation and functionalization with components such as targeting, contrast agents, probes, and fluorophores. High production cost Limited instability and leakage of loaded materials Low solubility Rapid clearance [160, 161, 175] Hydrogels Tenable synthesis and physicochemical properties Selective surface-functionalization High degree of porosity and high loading capacity Controlled and sustained release into the target tissues Biocompatibility and biodegradability High cost production Instability [181, 185, 188] Quantum dots Size and structure-based tunable emission High molar extinction coefficient High photo and chemical stability Potential for synergistic diagnostic and therapeutic applications Potential toxicity Particle aggregation, degradation and removal [199, 200, 208] MSNPs, mesoporous silica nanoparticles; MNPs, magnetic nanoparticles; MNCs, magnetic nanoclusters; AuNPs, gold nanoparticles; CNTs, carbon nanotubes. Figure 2. Structure of mesoporous silica nanoparticles applied for delivery of siRNA. Phosphonate-modified mesoporous silica nanoparticles (MSNPs) are further modified using electrostatic attachment of a polyethylenimine (PEI) polymer, which was used for subsequent covalent attachment of poly(ethylene glycol) (PEG) or electro-static-based loading of siRNA [56, 57]. In addition, 3-isocyanatopropyltriethoxysilane (ICP) is utilized for the preparation of generation 2 (G2) amine-terminated polyamidoamine (PAMAM) dendrimers-modified MSNPs. The terminal amino groups of PAMAM are covalently reacted with ICP functional groups on the MSN surface. These dendrimer-modified MSNPs can efficiently complex with siRNAs through electrostatic interaction [54]. 4.1 Silica and silicon-based nanoparticles Silica and silicon-based delivery systems have emerged in drug delivery applications due to their controllability in nanopore formation and surface modifications. This enabled a multi-component and multi-functional bio-delivery systems that can possi-bly perform a sequence of functions at various stages of the delivery route [52, 53]. Several spherical meso-porous silica nanoparticles (MSNPs) with core size range from 50 to 200 nm and modified with polyeth-yleneimine (PEI) or PEI- polyethylene glycol (PEG) or polyamidoamine (PAMAM) are applied for siRNA, see Figure 2 for more details on particle structure and surface modifications. This surface layer of cationic polymer or quaternized dendrimers was added to permit the electrostatic loading of the siRNA, while the presence of protective polymer of PEG was showed to improve the cellular uptake and delivery of MSNPs-siRNA conjugates. For example, PAMAM dendrimer-modified MSNPs carrying both doxorubi-cin (Dox) and B-cell lymphoma 2 (Bcl-2) gene silenc-ing siRNA has been demonstrated to enhance the cy-totoxicity of Dox by 64-fold due to the suppression of membrane pumps that mediate drug resistance [54]. Cationic PEI-MSNPs were particularly efficient for transducing mouse hepatoma HEPA-1 cells with a siRNA construct that was capable of knocking down green fluorescent protein (GFP) expression with minimal or no cytotoxicity [55]. Recently, Meng et al. have demonstrated that encapsulation of Dox by mesoporous silica nanoparticles could enhance and restore Dox sensitivity in drug resistant cancer cell lines by codelivery of a siRNA that knockdown P-glycoprotein drug exporter [56, 57]. It is worth not-ing that the application of silica nanoparticles are ad-ditionally promoted by its intrinsic biocompatibility [55, 58], biodegradability [59], and its efficient bio-elimination in vivo [60, 61]. However, several obser-vations indicating disadvantageous metabolic chang-es and toxicity especially in vivo have pronouncedly been recorded. These observations were mainly cor-related to the particle surface functionalization and the presence of active silanol groups [62, 63]. Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 880 Figure 3. Common surface modifications of metal and metal oxides-based nanoparticles reported in siRNA delivery. (a) Magnetic nanoparticles (MNPs). Branched PEI polymer is utilized for preparation of magnetite nanoparticle clusters (MNCs) [64, 68]. PEI is directly reacted with MNPs to form stable nanocomplexes or indirectly anchored through the covalent binging to 3,4-dihydroxy-L-phenylalanine (DOPA). N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) is applied to activate bovine serum albumin (BSA)-MnMEIO nanoparticles. The SPDP-activated MnMEIO nanoparticles are then treated with thiolated poly(ethylene glycol) (PEG) functionalized with a cyclic Arg-Gly-Asp (RGD) peptide, or thiolated siRNA at the distal end (red circle) [67]. MNPs can be coated with two different polymers of poly(oligoethylene glycol) methyl ether acrylate (P(OEG-A)) and poly(dimethylaminoethyl acrylate) (P(DMAEA)). P(DMAEA) forms an internal layer with a slight positive charge for electrostatic immobilization of siRNA and P(OEG-A) forms an outer antifouling shell for long circulation in vivo [65]. (b) Gold nanoparticles (AuNPs). Amine functionalized AuNPs are directly prepared by surface modification with cystamine hydrocholoride (CA) that carries amine-derived positive charge [75]. AuNPs modification with a positive charged polymer layer of PEI [77] or triethylenetetramine (TETA) [79] are reported for the simple electrostatic conjugation and delivery of siRNA. These modifications are commonly accomplished with Au-thiol non-covalent binding through thiol-terminated linkers such as 11-mercaptoundecanoic acid [75, 79]. 4.2 Metal and metal oxides nanoparticles Different kinds of metal nanoparticles have so far been tested for gene delivery purpose. Among them, magnetic nanoparticles (MNPs) have received increasing attentions owing to their unprecedented capabilities to achieve remotely-controlled transfec-tion by applying an external magnetic field. This process of magnetic-based transfection, referred as magnetofection, is one of the most powerful tools recently reported to enhance gene transfection effi-ciency. It reduces transfection time via magnetic field and force cellular transport processes [64]. Generally, magnetic nanoparticle intracellular siRNA delivery systems comprise surface polymer layers in order to enhance the siRNA-particle complex formation and their total cellular uptake (Figure 3a). Iron oxide na-noparticles modified with two different polymers, i.e. poly(oligoethylene glycol) methyl ether acrylate and poly(dimethylaminoethyl acrylate) were efficiently applied in siRNA transfection into human neuro-blastoma SHEP cells both in the presence and in the absence of a magnetic field [65]. This potential mag-netic nanoparticle-based transfection has further been expanded into several multifunctional systems suita-ble for concurrent targeting, diagnostic and therapeu-tic applications. A nanovector for siRNA comprised of iron oxide magnetic nanoparticle core coated with PEI, siRNA, and chlorotoxin and possesses dual tar-geting specificity and dual therapeutic effects has also been described for targeted cancer imaging and therapy [66]. Lee et al. developed a can-cer-cell-targeted gene silencing system prepared from a magnetic-nanoparticle platform on which a fluo-rescent dye, siRNA, and a Arg-Gly-Asp (RGD)-peptide targeting moiety were attached [67]. These multifunctional magnetic nanovectors exhibit-ed significant targeting, intracellular uptake and gene silencing effects in cancer cells. On the other hand, magnetic nanocrystal clusters (PMNCs)-cross-linked with PEI were developed to magnetically trigger in-tracellular delivery of siRNA [64, 68, 69]. Such new nanocomposite carriers are highly controlled cluster-ing structures with multi-nanoparticles cores sur-rounded by a shell of alkylated PEI [64, 68, 70]. The clustering approach in these composites greatly im-proves their magnetic responsiveness compared to individual nanoparticles, which provide more en-hanced, site-specific and retained release of siRNA [64]. In addition, the presence of PEI shells, with fa-vorable transfection properties of PEI, enables rapid and improved conjugation and transduction efficien-cy of siRNA molecules. PEI is highly cationic helping condensed electrostatic conjugation of anionic siRNA forming compact polyelectrolyte complexes with siRNA. Also, its role in nucleic acid protection and rapid endocytosis enhances the total delivery effi-ciency of these systems [71-73]. Park et al. reported Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 881 that the gene silencing effect of siRNA mediated clustering magnetic nanocrystals was considerably higher than that of siRNA complexed with single magnetic nanoparticles [64]. Recently, Chen group validated both magnetic iron oxide [68] and magne-sium oxide [70] nanoparticles-based cluster systems for the delivery of siRNA at in vitro and in vivo levels. Their initial results for luciferase siRNA delivery to luciferase transfected 4T1 murine breast cancer (fLuc-4T1) cells, and to a fLuc-4T1 xenograft model concluded the efficiency of the systems to cause sig-nificant knockdown of luciferase and demonstrated efficient protection of the loaded siRNA [68, 70]. Other classes of metal nanoparticles for deliver-ing siRNA have been proposed, including the use of gold nanoparticles (AuNPs). Due to their easy syn-thesis, amenable modification and bioconjugation, and rational stability and biocompatibility, AuNPs have recently been considered as excellent siRNA delivery systems [74]. Thus far, the strategies devel-oped to deliver siRNA by AuNPs can be classified into two categories: 1) siRNA conjugated directly to the AuNP surface via a gold–thiol bond or electro-static interaction, and 2) siRNA adhered to the AuNP surface modified with polymer layers [75-77]. As shown in Figure 3b, both strategies incorporate PEG, PEI or other passivating molecules to assist in stabili-zation and/or promoting endosomal escape into the cytoplasm [75, 77, 78]. The polyvalent siRNA/AuNPs conjugates showed a greater half-life and prolonged gene knockdown compared to free RNA duplexes [75-77, 79, 80]. Positively charged AuNPs electrostat-ically complexed with negatively charged siR-NA–PEG conjugates were described by Lee et al. to successfully suppress the expression of GFP within the human prostate carcinoma cells [75]. PEI-capped AuNPs/siRNA that target endogenous cell-cycle ki-nase, an oncogene polo-like kinase 1 (PLK1), dis-played a significant gene expression knockdown in MDA-MB-435s cells, whereas it was not obvious when the cells are treated with PLK1 siRNA using PEI as the carrier [80]. Elbakry et al. proposed lay-er-by-layer (LbL) assembly of the oppositely charged siRNA and PEI on the surface of AuNPs. The result-ant LbL-coated nanoparticles were able to reduce the cellular enhanced green fluorescent protein (EGFP) production to about 28% in CHO-K1 cells [77]. Also, dendronized PEI AuNPs suppressed β-gal expression by ∼50% with minimal toxicity [79]. Recently, Reyn-olds et al. utilized gold nanorods-galectin-1 siRNA complexes to inhibit the gene expression of galectin-1. The prepared siRNA complexes significantly attenu-ated the expression of methamphetamine potentiates galectin-1 in human monocyte derived macrophages and significantly reduced the effects of metham-phetamine on human immunodeficiency virus type 1 (HIV-1) infection [81]. These applications are gener-ally demonstrating the potentiality of metal nanopar-ticles for siRNA delivery. However, determining the cytotoxicity and stability of this class of nanoparticle is a recent challenge and more extensive in vivo stud-ies are needed to address several concerns about bio-compatibility, biodistribution and tissue accumula-tion [82, 83]. 4.3 Carbon-based materials Allotropic nanostructures of carbon nanotubes and graphene nanosheets are hosting enormous in-terests as shuttle nanovehicles for drug and siRNA delivery applications. Their extremely large surface area, with every atom exposed on its surface, allows for ultra-high functionalization and loading capaci-ties. In addition to excellent material properties such as ultimate electrical and thermal conductivities and mechanical strength, enlarge and intensify their im-portance for a wide range of biomedical applications [84-87]. Carbon nanotubes (CNTs) are 1-dimentional hollow carbon nanostructures with a typical diameter of 1–2 nm and length ranges from 50 nm up to 1 cm. This tubular morphology renders them to act as cel-lular scale nano-needles that are easily and efficiently taken up by cells [88-90]. In the same time, they per-mit encapsulation of molecules and provide material storage functions as well as protection and controlled release of loaded molecules that are becoming in-creasingly important in modern delivery applications. Both forms of single-walled (SW) and multi-walled (MW) carbon nanotubes were modified and conju-gated with siRNA to be employed against HIV infec-tion and various cancers. Ammoni-um-functionalization of CNTs and surface modifica-tion with cationic polymers such as PEI and poly(diallyldimethylammonium)chloride (PDDA) are commonly applied for simple electrostatic loading of siRNA to their surface (Figure 4). SWCNs grafted with siRNA showed effective silencing activity by 50–60% to both cluster of differentiation 4 (CD4) re-ceptor and chemokine receptor type 4 (CXCR4) co-receptor of T cells, which are required for HIV en-try into human and infection [91]. Zhang et al. pro-posed SWCNTs as an efficient vector for siRNA to suppress murine telomerase reverse transcriptase expression in murine tumor cells on both in vitro and in vivo levels [92]. This approach also has been suc-cessfully used in the control of key signaling regula-tors in cancer cells, including suppressors of cytokine signaling 1, cyclin A, and extracellular sig-nal-regulated kinase harnessing the potentiality of SWCNTs to deliver siRNA for cancer therapy [93-95]. Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 882 In a recent study, 10–30% silencing activity of MWCNs-siRNA conjugates have been reported in human lung cancer cell line H12991, and efficient suppression and prolonged survival of an animal model were previously reported in another study by Podesta et al. [96]. Graphene is a newly discovered two-dimensional carbon nanosheet structure [97-99]. It was described to have additional remarkable prop-erties of facile synthesis, high water dispersibility, good colloidal stability, easily tunable surface func-tionalization, and good biocompatibility that high-light its promise as a novel nano-carrier for safe and efficient gene transfection [100-106]. Recently, its oxi-dized form of graphene oxide (GO) were used in a pioneering study by Zhang et al. after PEI modifica-tion step for sequential delivery of Bcl-2-targeted siRNA and the anticancer drug Dox (Figure 4). The results demonstrated an excellent and effective deliv-ery of siRNA and chemical drugs using GO that sig-nificantly enhance the chemotherapy efficacy [106]. The major disadvantage of carbon based nanomateri-als to siRNA is their non- biodegradability in vivo that can cause a range of adverse health effects. This point was highlighted in several studies and there is need for more future investigations [107, 108]. 4.4 Dendrimers Dendrimers are spherical and symmetrically branched molecules in which branches radiating from a central core molecule and terminating at functional chemical groups depending on the building blocks, such as charged amine in PAMAM dendrimers (Fig-ure 5a,b). Dendrimers have a very precise size and shape defined by the number of generations through con-trolled chemical synthesis [109, 110]. Dendrimers are water soluble, biocompatible, elicit negligible immune re-sponse, and have been suc-cessfully used in DNA and oligonucleotides complexa-tion and delivery [111, 112]. Presumably, nucleic acids were stably incorporated into amine-terminated dendrimers via the interaction of negative charges on the phosphate on the backbone with the high positive charges of the amino groups, thus protected from nuclease activity. Minko group has previously designed and synthesized a surface-acetylated polyamidoamine generation four dendrimer (QPAMAMNHAc) in which surface amine groups are modified with acetyl group and internal tertiary nitrogen are quaternized (Figure 5c), as a nanocarrier for the targeted intracellular delivery of Bcl-2 siRNA in A2780 human ovarian cancer cells [112-114]. This type of dendrimer has low cytotoxicity and the internal cationic charges for complexation with siRNA to enhance siRNA protection, while the lesser degree of quaternization offers free tertiary amines for potential proton sponge effect [112, 114]. The same group later was able to achieve a targeting siRNA delivery by the addition of LHRH peptide (Figure 5c) that further improved internalization of siRNA by cancer cells and significantly enhanced its intracellular activity leading to a substantial silencing of a targeted Bcl-2 gene [114]. They further improved this design by the addition of PEG and poly-L-lysine (PLL) to form a triblock PAMAM-PEG-PLL nanocar-rier, see Figure 5c. In this design, PEG acts as a linker connecting PLL and PAMAM dendrimers and ren-ders nuclease stability for more protection of siRNA, while PLL enhances the electrostatic interaction with siRNA and also acts as penetration enhancer. This Figure 4. Carbon-based nanoparticles for delivery of siRNA. Applied carbon nanostructures include multiwalled- and singlewalled carbon nanotubes (CNTs) and single-atom-thick sheets of graphene oxide (GO). Ammoni-um-functionalized CNTs are prepared through non-covalent binding with cetylpyridinium chloride [209], tert-butyl-n-(6-aminohexyl)carbamate [92], and 1, 6 hexanediamine [95]. Polymer coated-CNTs and GO are prepared through the addition of different positively charged polymers to their surfaces, including polyethylenimine (PEI) [106, 209] and poly(diallyldimethylammonium)chloride (PDDA) [94]. Both ammonium-functionalized CNTs and polymer-coated CNSs are conjugated to siRNA by electrostatic interaction. CNTs can also be modified with non-covalent adsorption of phospholipid molecules carrying poly(ethylene glycol) (PL-PEG) chains with terminal amine or maleimide groups. This amine or maleimide terminal on the PL-PEG (red circle) permits the incorporation of thiolated siRNA by disulfide bond formation [91, 93]. Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 883 triblock carrier showed excellent stability and effi-ciency to suppress the target Bcl-2 gene [113]. Despite of these applications that generally introduce PAMAM dendrimers as highly efficient delivery vectors with many possible modifications, further addressing of several limitations, including non-specific cytotoxicity, release kinetics of the asso-ciated bio-actives and rapid clearance issues is re-quired to initiate in vivo applications [109, 115]. 4.5 Polymers (polycations, micelles) Polymers offer a great promise because their unit-by-unit construction allows for fine-tuning of the properties required for efficient transfection and re-lease of nucleic acids. Their role as carriers for siRNA delivery was extensively reviewed [23, 116, 117], and currently there is a large number of natural and syn-thetic polymers is already investigated. Natural polymers, such as cationic polypeptides [118-121], chitosan (CH) [122-126], poly(lactic acid-co-glycolic acid) [127-129] and atelocollagen [130-133] have been used due to their nontoxic, biocompatible, and bio-degradable properties. Among available natural polymers, chitosan, which is a linear polymer com-posed of β-(1–4)-2-amino-2-deoxy-D-glucopyranose units (Figure 6a), has been most commonly developed for in vitro and in vivo delivery of siRNA [122]. siRNA electrostatically incorporated into chitosan nanopar-ticles (siRNA/CH-NP) successfully delivered siRNAs against POSTN, FAK and Src family in animal model of ovarian cancer [123, 124]. Malhotra et al. described a novel method for preparing PEGylated chitosan na-noparticles by chemically modifying C6 position of its repeating units with PEG and the amine groups at the C2 position of the chitosan were protected using phthalic anhydride. This conjugated nanoparticle de-livered siRNA with no toxicity in neural cells. Also, several chitosan-based siRNA formulations were proposed for silencing the expression of human pap-illoma virus (HPV) E6/E7 oncogenes in human cer-vical cells. The electrostatic complexation of chitosan with HPV16 E7 siRNA efficiently helped to deliver siRNA into CaSki cervical carcinoma cells and was observed to induce apoptosis of cells [125]. A PEI-introduced chitosan-shell/ poly methyl methac-rylate-core nanoparticle was also able to efficiently suppress the expression of HVP oncogene in the SiHa human cervical cancer cells [126]. On the other hand, synthetic polymers applied to siRNA delivery include branched or linear PEI, PLL (two of the most common synthetic polycations), PEG, dimethylaminoethyl methacrylate, polyfluorene and cyclodextrin-based polycations (CDP). Among those synthetic polymers, PEIs, is a class of extensively used cationic polymers, with a wide range of molecular weights and many protonable amino groups, leading to a high cationic charge density at physiological pH, see Figure 6b. However, synthetic polymers like PEI and PLL can trigger cell death through necrosis and apoptosis [134] causing toxicity in living cells. This toxicity is also associated with higher polymer mo-lecular weights and increasing branching. The effect can be reduced by using block copolymers containing PEG and a polycation block [135]. Figure 5. Composition of the PAMAM delivery systems of siRNA. (a) Generation 4 PAMAM-OH dendrimer. (b) Internally quaternized PAMAM to form QPAMAM-OH dendrimer with inner cationic charges. PAMAM are frequently quaternized by methyl iodide (ICH3) and the terminal surface become very positive allowing the efficient electrostatic binding/loading of negatively charged backbone of siRNA. (c) QPAMAM with different surface modifications, including the addition of acetyl group by direct reaction with acetic anhydride (Ac2O) [112], poly(ethylene glycol) (PEG) and poly-L-lysine (PLL) [113], LHRH peptide [114]. This addition of polymer structures such as PEG and PLL is reported to enhance the surface positive charge and circulation of PAMAM nanoparticles. While, the conjugation to LHRH peptides confers targeting ability in PAMAM based delivery applications. Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 884 Figure 6. Common polymer based siRNA delivery systems. (a) Chitosan systems. The RGD modified chitosan nanoparticles (RGD-CH-NP) are reported for targeting siRNA delivery. siRNA/RGD-CH-NPs are prepared based on ionic gelation of anionic tripolyphosphate and siRNA [123, 124]. (b) Polycations prepared from polyethylenimine (PEI) or poly-L-lysine (PLL). Their highly cationic nature facilitate strong electrostatic interactions with the negatively of siRNA. PEGylation of polycations with PEG modified with heterobifunctional N-succinimidyl 3-(2-pyridyldithio)propionate allow subsequent coupling of thiolated siRNA via the N-terminal cysteine [136]. (c) Micelle-based nanoparticles. Micelles are prepared from the triblock copolymer poly(ethylene glycol)-poly(n-butyl acrylate)-poly(2-(dimethylamino)ethyl methacrylate) (PEGPnBA-PDMAEMA). The pres-ence of amine groups on PDMAEMA allows the electrostatic complexation with siRNA [138]. (d) Cyclodextrin containing polymer (CDP). A commonly reported targeted CDP system is prepared through the addition of adamantane-polyethylene glycol-transferrin (AD-PEG-Tf) [153, 155, 157, 158]. The advantage of polymer-based delivery lies in the unparalleled simplicity for formulation of siRNA and polymer complexes. Cationic polymers with high positive charge usually self-assemble into uniform micellar, polycationic nanoparticle complexes with negatively charged siRNA upon simple mixing (Fig-ure 6a-c), which is driven by electrostatic interactions [116, 136-138]. However, despite this advantage, the electrostatically formed polymer-siRNA complexes are often loose and unstable in vivo. This is due to the low charge density of short double stranded siRNA and the subsequent inability to form robust particles with cationic polymer carriers. Recently, several ap-proaches have been proposed to improve the stability of the formed complexes through the use of pol-ymerized siRNA with a high charge density and chemical crosslinking of the complexes [50, 139, 140]. Following these approaches, Mok et al. utilized the multimerized siRNA that possesses increased charge densities to produce more stable and compact poly-electrolyte complexes with less cytotoxic cationic car-riers than naked siRNA [139]. While, Naito et al. uti-lized phenylboronate with its capability to form re-versible covalent esters with 1,2- or 1,3-cis-diols exist on the ribose ring of nucleic acids to assist stabilizing siRNA-polyion complex micelles for intracellular controlled release of siRNAs [49]. Currently, the di-sulfide-based crosslinking is particularly interesting approach widely reported to enhance the stability and delivery efficiency of many polymeric carriers. Based on the redox potential gradient existing between ex-tracellular and intracellular environments, this strat-egy was further tailored to provide a capability for selective and controlled release of siRNA in the cyto-solic space of target cell [141, 142]. Several studies have reported the utilization of bioreducible PEI and PEG-based carriers for siRNA. Chung et al. indicated that the reducible dimerized siRNAs showed far en-hanced complexation behaviors with cationic poly-mers of PEI or PEG as compared to monomeric siRNA at the same ratio. The reducible siRNA dimeric con-jugates showed greatly enhanced cellular uptake and gene silencing effects in vitro. These results were mainly explained to be due to the higher charge den-sity and promoted chain flexibility of the dimerized siRNAs that provide more compact and stable siRNA complexes [143]. Also, a bioreducible PEI complexed with VEGF-siRNA was successful to reduce the level of VEGF protein in HepG2 human liver carcinoma cells in vitro and to inhibit HepG2 tumor growth in a xenograft mouse model. An important recent ap-proach involved the combined use of bioreducible PEI Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 885 with a polymerized siRNA (poly-siRNA) was sug-gested for more enhanced delivery and gene silenc-ing. The poly-siRNA/PEI complexes exhibited a su-perior intracellular uptake by murine melanoma cells accompanied with red fluorescent protein (RFP) gene silencing efficiency of about 80%, compared to un-treated cells [144]. Furthermore, several other poly-mers, including poly(β-amino ester)s [145], arginine conjugated poly(cystaminebisacrylamide-diamino-hexane) [146], hyaluronic acid-graft-poly(dime-thylaminoethyl ethacrylate) [147], and dextran [148] were investigated as bioreducible siRNA carrier for efficient gene silencing in various human cancer cell lines, see Table 3. The overall, results suggest their potentiality for the tumor-targeted delivery of siRNA. Most recently, this bioreducible polymer-based strat-egy for siRNA-carrier complex stabilization was combined with different delivery technologies such as microbubbles, and ultrasound to construct a multi-modal delivery system, which successfully synergizes the advantages of each delivery approach to signifi-cantly enhance the siRNA uptake and activity in vitro and in vivo [149]. 4.6 Cyclodextrin Cyclodextrins (CDs) are a group of cyclic (α-1,4)-linked oligosaccharides of α-d-glucopyranose containing a relatively hydrophobic central cavity and a hydrophilic outer surface. CDs form water-soluble inclusion complexes with many poorly soluble drugs. Thus, CDs are widely used in pharmaceutical formu-lations, and as such, their long-term biocompatibilities are defined in humans [150, 151]. Because of the low toxicity, the lack of immune stimulation, and the ab-sence of enzyme degradation in humans of cyclodex-trins, they were chosen as one of the building blocks for the cyclodextrin-containing polymer component. Cyclodextrin-containing polycation nanoparticles can self-assemble with siRNA to form colloidal particles (Figure 6d). The first targeted delivery of siRNA in a human was accomplished with cyclodextrin nano-particle formulation denoted as CALAA-01 consisting of CDP, which is the main feature of the delivery system, a PEG steric stabilization agent, siRNA against RRM2 gene and human transferring to trans-ferrin receptors for targeted delivery [122, 124, 152-157]. Once approved for use, the four component formulation is self-assembled into 70 nm nanoparti-cles in the pharmacy and administered intravenously to patients with advanced solid tumors [152-158]. Despite of this wide reputation of cyclodextrins in drug delivery applications, their large-scale commer-cial is limited by their high cost and concerns regard-ing their safety [159]. 4.7 Lipid-based nanoparticles Liposomes are among the most advanced nano-particle drug delivery systems successfully applied in the clinic [160-163]. Several liposomal formulations have been approved by FDA and many more with enhanced features such as stabilized chemotherapeu-tics encapsulation and tumor-specific targeting ver-sions are currently in development or in clinical trials [164-166]. Liposomes are controlled self-assembly of amphiphilic lipids that form a spherical unilamellar lipid bilayer enclosing an aqueous interior for en-capsulation of drug or functional agents (Figure 7). Hydrophobic drugs like paclitaxel can be embedded in the lipid bilayers. Lipid nanoconstructs have also been successfully used in gene delivery. For example, cationic lipids and oligonucleotides complexes have shown good in vivo properties as those with stealth liposomes [167]. The flexibility of liposome function-alization allows control over multiple aspects of the formulation. For example, the charge balance can be tuned by the selection of lipid components, as many cationic lipids suitable for nucleotide complexation are available [168]. Figure 7a shows the structure of the most common lipid types reported in siRNA de-livery. The composition of lipid bilayers can be chosen to present specific transition temperatures that de-termines their mechanical rigidity and fluidity in spe-cific temperature range. This has allowed for post-insertion of targeting component, such as human epidermal growth factor receptor 2 (HER2)-targeted single-chain variable fragment (scFv)-PEG-1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) to form ad hoc immunoliposomes [169]. Conjugation of functionalized lipids makes liposomes a derivatiz-able carrier for functional components, such as tar-geting, contrast agents, probes, and fluorophores, see Figure 7b that represents schematic structure of lipo-some-based nanoparticle and possible modifications with different functional moieties. PEGylation, i.e., incorporation of PEG chains, is a commonly adapted strategy for prolonging in vivo circulation. The PEG chains incorporated in Doxil and most liposomal formulations is around 4-8 mol%. Although the ad-vantages of PEGylation is apparent especially in im-proving the tumor deposition, the potential down side includes increased difficulty in cell uptake and release of siRNA once in the cellular environment due to inhibition of intracellular trafficking, which is re-ferred to as the PEG dilemma [170]. Hatakeyama et al. thus developed a tumor-specific cleavable PEG-lipid to overcome this problem [160]. A PEG-peptide- 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine that is cleaved in a matrix metalloproteinase-rich environ-ment was incorporated into a lipid-based multifunc-tional envelop-type nano device. Similarly, Yingyuad Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 886 et al. prepared putative enzyme-triggered PEGylated siRNA-nanoparticles through using PEG2000- peptidyl lipids with peptidyl moieties sensitive to tumor-localized elastase or matrix metalloprotein-ase-2 digestion [171]. Another type of triggered siR-NA-nanoparticle system was described by Carmon et al. to specifically knockdown hepatitis B virus (HBV) replication in cell culture and in murine hydrody-namic injection models in vivo. The described system is generated by the condensation of siRNA with cati-onic liposomes to form core particles subsequently post-coupled with PEG. In this system, the step of PEG coupling was included to facilitate acidic pH-triggered release of siRNA from endosomes in the target infected cells to suppress HBV [172]. The charged lipids have been initially used widely for siRNA packaging, simulating the success in DNA formulation [173, 174], one of the main chal-lenges has been the concern over toxicity of cationic components and lipids [175]. Therefore, efforts have been made in the development of neutral lipids for the stable association and delivery of siRNA. siRNA encapsulated in neutral liposomes for RNA interfer-ence reported by Sood et al. is among the more suc-cessful examples thus far. They proved conclusively that the neutral liposome 1,2-dioleoylsn-glycero-3- phosphatidylcholine-encapsulated siRNA targeting the oncoprotein EphA2 and proangiogenic cytokine interleukin 8 (IL-8) were highly effective in reducing in vivo target gene expression in an orthotopic mouse model of ovarian cancer [176, 177]. In addition, sev-eral studies reported the application of neutral lipids for siRNA delivery against viruses. A multicompo-nent lipid nanoparticles (LNPs) system comprising a mixture of titratable amino lipids (DLin-MP-DMA), PEG lipids (PEG-CDMA), and neutral lipids (choles-terol and dipalmitoylphosphatidylcholine) was de-scribed for fully encapsulating short synthetic hairpin RNAs (sshRNAs) that target the internal ribosome entry site of the hepatitis C virus (HCV). This system showed the efficacy of sshRNA against the HCV ge-nome in reducing HCV infection in vivo [178]. Stable nucleic acid-lipid particles (SNALPs) were also suc-cessfully utilized by Geisbert et al. to deliver antiviral siRNA constructs to challenge viral infections of Zaire Ebola virus (ZEBOV) and Marburg virus (MARV). A combination of modified siRNAs targeting the ZEBOV L polymerase (Lpol), viral protein (VP) 24 (VP24), and VP35 formulated in SNALPs were able to protect the tested non-human primate model from lethal ZEBOV infection [179]. For MARV, the treat-ment with different siRNA targeting MARV VP24, VP35, VP40, NP, and Lpol genes formulated in SNALPs resulted in 60%–100% survival of guinea pigs infected with MARV [180]. Figure 7. Lipid-based nanoparticles for delivery of siRNA. (a) Common lipid types and structures reported in siRNA delivery. Cationic lipids include dioleoylphosphatidyl ethanolamine (DOPE), oleic acid (OA) [173], dimethyldioctadecylammonium bromide (DDAB), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1,2-dioleoyl-3-(trimethyammonium) propane (DOTAP) [167]. Neutral lipids include 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)[176, 177] and choles-terol [173]. (b) A schematic showing lipid-based nanoparticle, e.g., liposome. siRNA molecules are encapsulated in the aqueous interior of liposome. Liposome nanoparticles can additionally carry surface protective polymer layer (e.g. PEG) and cellular receptor-specific moieties such as homing peptides and antibodies for enhanced targeting delivery. Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 887 4.8 Hydrogels Hydrogels can be defined as three-dimensional networks of responsive polymer materials that are hydrophilic in nature and able to retain large amounts of water or biological fluids. They may exhibit dif-ferent geometries like macroscopic, microscopic or nano-gels [181, 182]. Both macro- and micro-hydrogel particles were previously prepared and applied for siRNA delivery [182-184]. Nanogels are of current intensive investigations for its nanobased enhanced biomedical application [181, 182]. Due to their char-acteristic biocompatibility and biodegradability, hy-drogels offer excellent opportunities for development of a wide range of controlled siRNA delivery strate-gies [181, 185-187]. Of particular interest in delivery applications, hydrogels that possess a high degree of porosity, permitting a high loading capacity of siR-NA, and can also be selectively surface-functionalized to enable specific targeting applications. Moreover, hydrogel delivery could control and tailor siRNA re-lease time into the target tissues by adjusting the physicochemical properties of gel matrices [182]. This advantage offer great chances for sustained delivery, which has been extensively reported to be necessary for efficient gene silencing by siRNA, and considered as a major disadvantage of the other nanoparticulate siRNA delivery systems that rapidly dispersed in tissues [188]. Naked siRNA have been encapsulated and de-livered by multiple hydrogel systems, including al-ginate [189], collagen [189], dextran [182, 190], poly-acrylamide [183, 184], chitosan [191], hyaluronic acid (HA) [192], PEG [193], PEI-catechol [194], and PEI/pluronic [195], see Table 3 and Figure 8a,b. Al-ginate and collagen were formulated by Krebs et al. into hydrogel macro-particle systems that were then successfully applied for localized and sustained de-livery of siRNA [196]. Dextran hydrogels loaded with siRNA were adapted to target EGFP in HUH7 human hepatoma cells [182]. Also, Singh et al. presented an in-situ crosslinkable, injectable formulation containing dendritic cell (DC)-chemo-attractants and dual-mode DNA–siRNA loaded dextran microparticles to attract immature DCs and simultaneously deliver, to the migrated cells, immunomodulatory siRNA and plasmid DNA antigens [190]. Peptide-labeled core/shell hydrogel nanoparticles of poly(N-isopro-pylmethacrylamide) with a high loading capacity for siRNA have been developed and effectively targeted to ovarian carcinomas by receptor-peptide binding (Figure 8a). The encapsulated siRNA is transported into the cell interior, where it is available for epider-mal growth factor receptor (EGFR) gene knockdown [183]. This approach was similarly applied by the same group as effective strategy for the sensitization of ovarian cancer cells to taxane chemotherapy [184]. Chitosan hydrogel (CH-HG) is another type of par-ticular importance for siRNA applications due to its low toxicity and irritation effects. In another study, CH-HG loaded with either tissue transglutaminase (TG2) siRNA/CH-HG or docetaxel plus TG2 siR-NA/CH-HG were effectively delivered in mice bear-ing A375SM and MDA-MB231 tumors showing en-hance localized therapeutic efficacy without risk for systemic side effects [191]. Hyaluronic acid is a natu-rally occurring nonsulfated glycosaminoglycan poly-saccharide, and known to be involved in many bio-logical functions [197]. Hydrogel nanosystems fabri-cated from HA and physically entrapped GFP siRNA were readily taken up by HA receptor positive HCT-116 cells, and showed a significant extent of GFP gene silencing in both serum and non-serum condi-tions [192]. In a trial by Takahashi et al. to mitigate the soft tissue implant foreign body response by sup-pressing fibrotic responses around implants; siR-NA/branched-PEI complexes released from PEG-hydrogel were applied to inhibit the expression of mammalian target of rapamycin (mTOR) in fibro-blasts. This trial resulted in mTOR knock-down and subsequent 70% inhibition in fibroblast proliferation [193]. Similarly, PEI conjugated with catechol groups [194] or crosslinked pluronic acid [195] was adopted by several groups to produce nanogel systems, which were used as a new class of siRNA delivery carriers. The thermally responsive pluronic/PEI system is most interesting. It exhibits a thermally reversible swelling/deswelling volume expansion behavior was synthesized and used for siRNA delivery (Figure 8b). This thermal responsive behavior interestingly al-lowed for efficient cellular uptake and effective en-dosome breaking, and a subsequent enhanced deliv-ery for the electrostatically loaded siRNA-PEG on its surface. Recently, Hong et al. conducted the first at-tempt to prepare biologically active siRNA-based mi-crohydrogels using sense/antisense complementary hybridization between single-stranded linear and Y-shaped trimeric siRNAs. These siRNA microhy-drogels interestingly exhibited super cellular uptake efficiency and gene silencing activity [198]. 4.9 Semiconductor nanocrystals The superior and unique physical and photonic characteristics of semiconductor quantum dots (Qdots), including size and structure tunable emis-sion, high molar extinction coefficient, and high photo and chemical stability propelled them in a plethora of imaging applications [199-202]. Building efficient de-livery systems generally involves the synergistic use of diagnostic and therapeutic tools. In this context, Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 888 Qdots attracted the interest by being a new generation of nanoparticle carriers that allows real-time fluores-cent, multiplexed imaging and efficient delivery of therapeutic agents in living cells [203-207]. Using a PEGylated Qdot core as a scaffold for siRNA and tumor-homing peptides (F3), targeted Qdots conju-gates were prepared for siRNA delivery. Delivery of these F3/siRNA-Qdots to EGFP gene-transfected HeLa cells and release from their endosomal entrap-ment led to significant knockdown of EGFP signal [206]. Also, Qdots modified with different polymers (e.g., amphipols and proton-sponge) were specifically designed for real-time imaging and delivery of siR-NA. The polymeric-Qdots systems addressed longstanding barriers in siRNA delivery such as cel-lular penetration, endosomal release, carrier unpack-ing, and intracellular transport and showed enhanced silencing activities [203, 204]. Through a FRET based assay, Lee et al. recently utilized cationic Qdots in complex with siRNA for studying intracellular traf-ficking, unpacking, and gene silencing proving the advances brought by using the photonic properties of Qdots in exploring siRNA delivery processes [205]. Despite of these reports on the apparent applications of Qdots in siRNA delivery, potential problems may arise in their in vivo applications. Among these prob-lems, the toxicity of Qdots is a major issue. Their sur-face coating can be cytotoxic such as mercaptoacetic acid [200, 208]. Also if this coating are compromised, the metallic core made of cadmium and selenium can be highly toxic [208]. Other issues, including particle aggregation, degradation and removal within the body are still largely unknown [200]. Figure 8. Hydrogel based siRNA delivery systems. (a) Different core/shell hydrogel systems, including alginate [189], dextran [182], acrylamide [183, 184], chitosan [189, 191], poly(lactide-co-glycolide) (PLGA) [190], hyaluronic acid [192], PEG [193], poly(ethylenimine) (PEI)-catechol [194] are reported for siRNA delivery. These systems are physically encapsulating, where siRNAs are incorporated in the hydrogel network based on electrostatic interaction or non-covalent binding. (b) Thermally responsive plu-ronic/PEI system [195]. 5. Concluding remarks siRNA therapeutics have great potential to im-pact the future of medicine and, it is recently ac-claimed as a revolutionary class of drug molecules. However, there are still many challenges and barriers to achieve their full potential. Systemic delivery is one of the most important challenges. Nanoparti-cles-mediated delivery is a rapidly emerging and po-tentially powerful technology. Different nanoparticle systems offer various advantages and disadvantages based on their composition, physical, and chemical characteristics, thus leading to a range of effectiveness when associated with siRNA. Silica nanoparticles, due to their controllable porosity, allow multi-functional sequential delivery. The metal nanoparticle cores provide multiple functions, including targeted con-centration to cells or tissues, multiplexed imaging and real-time tracking. Carbon nanostructures with every atom exposed on its surface hold ultra-high surface modification and loading capacities. The flexibility of liposome functionalization allows control over multi-ple aspects of the formulation. On other, hydrogels possess a high degree of porosity and biodegradabil-ity that permit a high loading capacity and excellent opportunities for development of different controlled delivery strategies. Polymers are rather unique in their unit-by-unit construction that allows for fi- Theranostics 2014, Vol. 4, Issue 9 http://www.thno.org 889 ne-tuning of the properties required for efficient transfection and release. Here, most of the reported studies were successful in implying these unique ad-vantages of nanoparticles to overcome the known siRNA delivery barriers and to enhance the efficiency of siRNA delivery. However, the overall success has been limited to date by toxicity observations and ad-ditional challenges associated with many stages along the delivery process. 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FirsovViewEnzyme-triggered PEGylated siRNA-nanoparticles for controlled release of siRNAArticleFull-text availableJan 2014 Mathieu Mevel Peerada Yingyuad Carla PrataAndrew D MilleA key goal of our recent research efforts has been to develop novel ‘triggerable nanoparticle’ systems with real potential utility in vivo. These are designed to be stable from the point of administration until a target site of interest is reached, then triggered for the controlled release of therapeutic agent payload(s) at the target site by changes in local endogenous conditions or through the application of some exogenous stimulus. Here we describe investigations into the use of enzymes to trigger RNAi-mediated therapy through a process of enzyme- assisted nanoparticle triggerability. Our approach is to use PEG2000-peptidyl lipids with peptidyl moieties sen- sitive to tumour-localized elastase or matrix metalloproteinase-2 digestion, and from these prepare putative enzyme-triggered PEGylated siRNA-nanoparticles. Our results provide initial proof of concept in vitro. From these data, we propose that this concept should be applicable for functional delivery of therapeutic nucleic acids to tumour cells in vivo, although the mechanism for enzyme-assisted nanoparticle triggerability remains to be fully characterized.ViewShow abstractNon-viral gene delivery systemsArticleApr 2002CURR OPIN BIOTECHMark E. DavisNon-viral gene delivery systems have the potential to create viable pharmaceuticals from nucleic acids. These DNA delivery systems contain lipids and/or cationic polymers. In order for these systems to be developed into commercial products, several barriers must be overcome. These include obstacles in manufacturing, formulation and stability. In vivo, problems of extracellular non-specific interactions and intracellular trafficking to the nucleus are also encountered. Recent progress has been made in overcoming these issues.ViewShow abstractHydrogels in biology and medicineArticleJan 2006ADV MATERNicholas A. PeppasJ. Zachary Hilt Ali KhademhosseiniViewThe rise of grapheneChapterJan 2009A. K. GeimK. S. NovoselovGraphene is a rapidly rising star on the horizon of materials science and condensed-matter physics. This strictly two-dimensional material exhibits exceptionally high crystal and electronic quality, and, despite its short history, has already revealed a cornucopia of new physics and potential applications, which are briefl y discussed here. Whereas one can be certain of the realness of applications only when commercial products appear, graphene no longer requires any further proof of its importance in terms of fundamental physics. Owing to its unusual electronic spectrum, graphene has led to the emergence of a new paradigm of relativistic condensed-matter physics, where quantum relativistic phenomena, some of which are unobservable in high-energy physics, can now be mimicked and tested in table-top experiments. More generally, graphene represents a conceptually new class of materials that are only one atom thick, and, on this basis, offers new inroads into low-dimensional physics that has never ceased to surprise and continues to provide a fertile ground for applications. © 2010 Nature Publishing Group, a division of Macmillan Publishers Limited and published by World Scientific Publishing Co. under licence. All Rights Reserved.ViewShow abstractHydrogels in controlled release formulationsArticleJan 2006 Chien-Chi LinA.T. MettersViewAdministration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNAArticleApr 2007Jeremy D HeidelZhongping Yu Joanna Liu Mark DavisThe results of administering escalating, i.v. doses of targeted nanoparticles containing a siRNA targeting the M2 subunit of ribonucleotide reductase to non-human primates are reported. The nanoparticles consist of a synthetic delivery system that uses a linear, cyclodextrin-containing polycation, transferrin (Tf) protein targeting ligand, and siRNA. When administered to cynomolgus monkeys at doses of 3 and 9 mg siRNA/kg, the nanoparticles are well tolerated. At 27 mg siRNA/kg, elevated levels of blood urea nitrogen and creatinine are observed that are indicative of kidney toxicity. Mild elevations in alanine amino transferase and aspartate transaminase at this dose level indicate that the liver is also affected to some extent. Analysis of complement factors does not reveal any changes that are clearly attributable to dosing with the nanoparticle formulation. Detection of increased IL-6 levels in all animals at 27 mg siRNA/kg and increased IFN-{gamma} in one animal indicate that this high dose level produces a mild immune response. Overall, no clinical signs of toxicity clearly attributable to treatment are observed. The multiple administrations spanning a period of 17–18 days enable assessment of antibody formation against the human Tf component of the formulation. Low titers of anti-Tf antibodies are detected, but this response is not associated with any manifestations of a hypersensitivity reaction upon readministration of the targeted nanoparticle. Taken together, the data presented show that multiple, systemic doses of targeted nanoparticles containing nonchemically modified siRNA can safely be administered to non-human primates.ViewShow abstractKnocking down barriers: Advances in siRNA deliveryArticleMay 2010NAT REV DRUG DISCOV Kathryn A WhiteheadRobert LangerDaniel G AndersonViewIntroductionChapterSep 2007Torvard Laurentpolysaccharides;hyaluronan;Karl meyer;sodium hyaluronate;hyaluronidasesViewShow abstractSpecial issue on carbon nanotubesArticleAug 2002CARBON Francois BeguinP. EhrburgerViewShow moreLinked ResearchNanoparticle-Mediated Systemic Delivery of siRNA for Treatment of Cancers and Viral InfectionsJune 2014Mohamed Shehata Draz · Binbin Amanda Fang · Pengfei Zhang · Zhi Hu · Fanqing Frank ChenDownloadAdvertisementRecommendationsDiscover moreProjectDigital Health and Nanoparticle Integrated Microfluidic Technologies Private ProfileDeveloping advanced systems for the detection and control of infectious diseases View projectArticleFull-text availableNanoparticle-Mediated Systemic Delivery of siRNA for Treatment of Cancers and Viral InfectionsJune 2014 · TheranosticsMohamed Shehata DrazBinbin Amanda Fang Pengfei Zhang[...] Fanqing Frank ChenRNA interference (RNAi) is an endogenous post-transcriptional gene regulatory mechanism, where non-coding, double-stranded RNA molecules interfere with the expression of certain genes in order to silence it. Since its discovery, this phenomenon has evolved as powerful technology to diagnose and treat diseases at cellular and molecular levels. With a lot of attention, short interfering RNA (siRNA) ... [Show full abstract] therapeutics has brought a great hope for treatment of various undruggable diseases, including genetic diseases, cancer, and resistant viral infections. However, the challenge of their systemic delivery and on how they are integrated to exhibit the desired properties and functions remains a key bottleneck for realizing its full potential. Nanoparticles are currently well known to exhibit a number of unique properties that could be strategically tailored into new advanced siRNA delivery systems. This review summarizes the various nanoparticulate systems developed so far in the literature for systemic delivery of siRNA, which include silica and silicon-based nanoparticles, metal and metal oxides nanoparticles, carbon nanotubes, graphene, dendrimers, polymers, cyclodextrins, lipids, hydrogels, and semiconductor nanocrystals. Challenges and barriers to the delivery of siRNA and the role of different nanoparticles to surmount these challenges are also included in the review.View full-textArticleAbstract LB-208: Small interfering RNA library screen to identify therapeutic transcription factor.August 2013 · Cancer Research Zhi Hu Shenda GuJuha Rantala Joe W GrayPost-genomic analyses of major transcription factor families, in both malignant and non-malignant cell types, have resulted in a broader revision of understanding the biology of transcription factor families. A number of oncogenic transcription factors, such as activator protein (AP)-1, estrogen receptor(ER) and signal transducer and activator of transcription (STAT)-3/STAT-5, are overactivated ... [Show full abstract] in human carcinomas. Up to now, three main groups of transcription factors are known to be important in human cancer. Transcription factor-directed anticancer drug development has focused on membrane or cytosolic targeting of molecules acting as ligand receptors. Recent technologies, such as small interfering RNA (siRNA), have shifted transcription factor targeting toward a more sophisticated rationale to elucidate the mechanisms that govern gene expression by analyzing the functions of proteins commonly over-expressed in transformed and malignant cells. In this study, we ve applied a cell spot microarray (CSMA) method which provides reproducible multi-parametric phenotypic readouts for large-scale gene knockdown genetic screens. A human transcription factor siRNA library including 350 transcription factor genes which were significantly overexpressed in human breast cancer was screened against 6 breast cancer cell lines including (Triple negative breast cancer, HER2 positive ER positive) SUM149, BT20, HCC1569, SKBR3, MDAMB361 and MCF7 for measuring cell proliferation using EdU incorporation, cell apoptosis with c-PARP staining. We ranked the candidate gene list by the change of the rate of EdU or level of c-PARP or the combination of both. Our data showed that the silencing of LHX1, BCL6, GABPA or SMAD1 increased the c-PARP levels in most of cell lines. The Knockdown of ZNF215, NFATC1, HOXD4 or ZNF169 reduced the levels of EdU staining. Selected siRNAs were then tested on more different breast cancer cell lines to confirm the spectrum of activity. In the near future, based on this pilot screen, a complete siRNA library of transcription factors including 1000 gene list will be tested on our panel of 50 breast cancer cell lines. Our screen will provide comprehensive information on the central role of transcription factor in breast cancer, and will lead to the identity of novel therapeutics selectively against transcription factors.Citation Format: Zhi Hu, Shenda Gu, Joe Gray, Juha Rantala. Small interfering RNA library screen to identify therapeutic transcription factor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-208. doi:10.1158/1538-7445.AM2013-LB-208Read moreArticleFull-text availableAdvanced Theranostic Application of Bio-Nanoparticle SystemsApril 2019 · Current Topics in Medicinal Chemistry Pengfei ZhangMohamed Shehata DrazView full-textArticleAbstract LB-258: Developing therapeutic siRNA against HER2 in breast cancer cellsJune 2012 · Cancer Research Zhi Hu Shenda GuJuha Rantala[...] Joe W GrayOver-expression of oncogene HER2 was found in 20-30% primary breast cancers which involved in tumor initiation and progression. Breast cancer patients with high levels of HER2 (HER2 positive) showed poor clinical outcome. Increasing evidence indicated that the majority of patients with HER2 positive did not respond well to anti-HER2 drugs Trastuzumab (Herceptin) and Lapatinib. Even those who ... [Show full abstract] showed initial response ultimately became resistant to the treatment. The molecular mechanism of resistance includes the mutation of HER2 gene and the blocking of antigen of HER2 protein. The discovery of small interfering RNA (siRNA) raises the possibility of exploring new approach for cancer treatment. The siRNA against HER2 (siHER2) can reduce HER2 protein level by degrading mRNA expression. In our study, seventy-six siRNA sequences targeting the coding region of human HER2 gene were designed and synthesized. We quantified the RNA levels of HER2 and cell viability after HER2+ breast cancer cells (BT474, SkBr3 HCC1954) were transfected with siHER2 and scramble control siRNAs. Ten siRNA sequences were selected with greater than 50% knockdown efficiency and 40% inhibition of cell viability. We also measured the dose of 50% inhibition of cell growth (GI50) of top 5 siHER2 sequences in 20 HER2+ cell lines. The GI50 data showed that most of cell lines which were resistant to Trastuzumab were still sensitive to siHER2 treatment. Therefore, our data indicated that the siRNA against HER2 can potentially be used to overcome resistance of HER2 drugs, making it an alternative cancer therapeutic for HER2 positive patients. Currently, in vivo delivery of optimized siHER2 using nanoparticle is under evaluation.Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-258. doi:1538-7445.AM2012-LB-258Read moreDataFull-text availableS1 DatasetJune 2018 Shenda Gu Worapol Ngamcherdtrakul Moataz Reda[...] Wassana YantaseeFull panel RPPA raw data.(XLSX) View full-textDiscover the world s researchJoin ResearchGate to find the people and research you need to help your work.Join for free ResearchGate iOS AppGet it from the App Store now.InstallKeep up with your stats and moreAccess scientific knowledge from anywhere orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleWelcome back! Please log in.Email · HintTip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleNo account? 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