Nanoparticle-mediated delivery of siRNA for effective lung cancer therapy.

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Nanoparticle-mediated delivery of siRNA for effective lung cancer therapy

Lung cancer is one of the most lethal diseases worldwide, and the survival rate is less than 15% even after the treatment. Unfortunately, chemotherapeutic treatments for lung cancer are accompanied by severe side effects, lack of selectivity and multidrug resistance. In order to overcome the limitations of conventional chemotherapy, nanoparticle-mediated RNA interference drugs represent a potential new approach due to selective silencing effect of oncogenes and multidrug resistance related genes. In this review, we provide recent advancements on nanoparticle-mediated siRNA delivery strategies including lipid system, polymeric system and rigid nanoparticles for lung cancer therapies. Importantly, codelivery of siRNA with conventional anticancer drugs and recent theranostic agents that offer great potential for lung cancer therapy is covered. Keywords: codelivery system • lung cancer • nanoparticle • RNA interference • siRNA delivery system

Lung cancer is one of the most lethal diseases worldwide, with 1.6 million deaths and 1.8 million diagnoses annually according to the GLOBOCAN 2012 [1] . Although chemotherapy, radiotherapy and surgery have been evolved for treating lung cancer over last decades, survival rate is still less than 15% in the world, which is much lower than other types of cancer such as breast (86%), colon (62%) and prostate (97%) [2,3] . Lung cancer is generally categorized into two main types: small cell lung carcinoma (SCLC; approximately 80% abundance) and nonsmall cell lung carcinoma (NSCLC; approximately 20% abundance) [4,5] . Depending on the patterns of growth and spread of lung cancer types, various methods of treatment are used. Importantly, chemotherapy and radiation therapy are the main feasible treatments for SCLC [6] , these therapies are recommended after surgical removal of tumor for NSCLC because of its lower sensitivity to chemotherapy [7] . Although chemotherapy is a viable option for both types of lung cancer by far, it has severe limitations. Most of

10.2217/NNM.14.214 © 2015 Future Medicine Ltd

chemotherapeutic drugs not only act against actively dividing cells but also harm constantly dividing healthy cells in the hair follicles, digestive tracts and bone marrow. Furthermore, anticancer drugs adversely affect reticuloendothelial systems [8] . To overcome the aforementioned chemotherapeutic-associated problems, nano particle systems have been employed for lung cancer therapy because they have several advantages such as preferential accumulation of drug-loaded nanoparticles in the tumor cells due to enhanced permeability and retention (EPR) of nanoparticles [9] , and the ability of encapsulation and delivery of poorly soluble drugs [10] . Despite the advancement of nanoparticle-based drug delivery systems, challenges still remain in chemotherapy. In most cases, multidrug resistance (MDR) occurs by overexpressing drug-resistant genes to resist the anticancer drug-induced cell death and pump the drugs out of the cells. As a result, higher dose of drugs is required to kill the cancer cells creating a greater risk of more severe adverse effects.

Nanomedicine (Lond.) (2015) 10(7), 1165–1188

Young-Dong Kim‡,1, Tae-Eun Park‡,2, Bijay Singh2, Sushila Maharjan2, Yun-Jaie Choi2, Pill-Hoon Choung3, Rohidas B Arote1 & Chong-Su Cho*,2 1 Department of Molecular Genetics, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea 2 Department of Agricultural Biotechnology & Research Institute for Agriculture & Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea 3 Department of Oral & Maxillofacial Surgery & Dental Research Institute, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea *Author for correspondence: Tel.: +82 2880 4868 Fax: +82 2875 2494 chocs@ snu.ac.kr ‡ Authors contributed equally

part of

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Review Kim, Park, Singh et al. Recently, as an alternative to chemotherapy, nanoparticle-mediated siRNA therapy has attracted much attention in treating lung cancer [2] due to its ability to selectively regulate the problematic genes in a sequencedependent manner. Furthermore, combination of traditional chemotherapy and gene therapy via codelivery of anticancer drugs and MDR-related siRNA has been emerged as an innovative strategy for overcoming MDR in lung cancer. In this review, we present an overview of current progress and challenges in nanoparticle-based siRNA therapies for the treatment of lung cancer and cover different strategies developed for the delivery of anticancer drugs and/or imaging agents with siRNA for synergistic anticancer effect.

cal applications: siRNAs do not easily cross over cell membrane due to their size (13 kDa) and net negative charges; siRNAs are vulnerable to enzymatic digestion (RNase) and rapidly cleared by renal filtration and urinary excretion during blood circulation. In order to overcome these limitations, strategies for delivering siRNA have been developed and particularly nanotechnology has made significant advances in the fields of RNAi therapeutics providing several advantages such as biocompatibility, siRNA protection, ease of scale-up and modification, storage stability, and improved quality control [15] . In this section, nanoparticle systems using lipid, polymer, rigid-particle and specific ligands for lung cancer therapies will be discussed.

Nanoparticle-mediated siRNA delivery for lung cancer therapy

Biological barriers to nanoparticle-mediated siRNA delivery

Overview of nanoparticle-mediated siRNA delivery

Extracellular barriers

siRNA is a class of double-strand short RNA with 21–24 nucleotides in length which guide sequencedependent endonucleolytic cleavage of the mRNAs. Since publication by Elbashir et al. [11] describing the use of siRNAs as a tool for regulating mammalian gene function, siRNAs have been explored for drug development process and even as therapeutic agents. The RNAi-induced gene silencing begins as transduced siRNAs that are assembled into the RNA-induced silencing complex (RISC), where double strands are separated and one strand guides a sequence-specific recognition of mRNA [12] . Furthermore, the cleavage of targeted mRNA takes place by Argonaut 2, catalytic engine of RISC, between bases 10 and 11 relative to the 5’ end of the siRNA guide strand, leading to subsequent degradation of the cleaved transcript by cellular exonucleases in the P-bodies [13] . The siRNAs processed by RISC can be recycled for several rounds of mRNA cleavage mediating strong inhibition of target gene expression [14] . siRNAs have some important advantages as potential new drug against cancers. Theoretically, siRNAs can be easily designed for any gene of interest with known sequence, which give consequence to their therapeutic potential for numerous cancers caused by one or few genes. siRNAs have high sequence specificity with strict discrimination even a single nucleotide mismatch and thereby facilitates downregulation of oncogene with less off-target effect [12] . These benefits of siRNAs provide solutions to drawbacks of traditional pharmaceutical approaches such as difficulty in access to target proteins and time-consuming processes of drug discovery. Despite the therapeutic potentials of siRNA, there are some disputes that are hampering their practi-

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Nanomedicine (Lond.) (2015) 10(7)

For successful lung cancer gene therapy, nanoparticles need to overcome several extracellular and intracellular barriers to deliver siRNAs into cytoplasm within lung cancer cells. Extracellular barriers include all biological, chemical and physical barriers encountered following administration of nanoparticles to reach the target cells. The process of opsonization is one of the most important biological barriers, making it more visible to phagocytic cells [16] . In specific, lung is a major tissue of reticuloendothelial system consisting a large population of resident macrophage, which lowers the chance of reaching lung cancer cells [17] . After opsonization by serum proteins, nanoparticles are recognized as foreign materials and captured by phagocytic cells. The engulfed nanoparticles are eventually disrupted and removed from bloodstream. The opsonization rates of nanoparticles largely depend on their surface properties such as charge and hydrophobicity. In that sense, surface modification by PEG or hydroxyl group is an important strategy to increase colloidal stability of nanoparticles by blocking nonspecific interaction with serum protein [16] . Although nanoparticles are expected to accumulate at tumor site by passive targeting termed as ‘EPR effect’, lack of specificity is still challenging. Decoration of surface of nanoparticles with lung cancer targeting ligands is a useful method for directing them to target cells with less off-target effect [18] . Intracellular barriers

Intracellular barriers include nonspecific cellular uptake, poor endosomal escape and inadequate vector unpacking [19] . Most of nanoparticles cannot readily cross over plasma membrane due to their size and hence they are internalized by endocytosis. In the process of endocytosis, nanoparticles are surrounded by plasma membrane and bud off to the inside cells to form traf-

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ficking vesicles [20] . Depending on the uptake mechanisms, the vesicles tend to fuse with lysosomes or avoid it, which determine the siRNA delivery efficiency. Endocytosis pathways of gene delivery fall into three categories: clathrin-mediated endocytosis (CME), macropinocytosis and caveolae-mediated endocytosis (CvME) [21] . Among them, CME is the best-characterized major route for macromolecule uptake. The first step of CME is strong binding of ligand to specific receptor concentrated in specialized regions of the plasma membrane, called clathrin-coated pits. Ligandreceptor clusters opsonized by clathrin-coated vesicles form curvature, and pinch off from the plasma membrane. On reaching early endosomes, some of receptors are recycled to the membrane and internalized genes experience drop in pH and enzymatic degradation. Therefore, escape from endosome to avoid lysosomal fusion is an important strategy for internalized genes to be delivered to target sites. Macropinocytosis, a kind of nonspecific endocytosis, fulfills the necessity of massive fluid uptake. Macropinocytosis forms large endocytic vesicles, macropinosomes, which uptake bulk of soluble macromolecules accompanied by cell surface ruffling. The vesicles undergo classical endocytic pathway in the same manner of CME or partly avoid fusion with lysosome. Thus, macropinocytosis has advantageous aspect as an entry route of polyplexes because some parts of macropinosomes can avoid lysosomal degradation. On the contrary, Goncalves et al. described that macropinocytosis has a negative role in gene delivery because of delayed translocation of nanoparticles to cytosol [22] . In contrast to CME and macropinocytosis, the role of CvME in avoidance of lysosome was observed in viruses. Many pathogens including SV40 internalize the cells via CvME and delivered to the caveosome of neutral pH in which SV40 can safely move into endoplasmic reticulum or Golgi apparatus. The major difference of CvME with classical endocytic pathway is that CvME does not experience a drop in pH and follow the nonacidic and nondegradative routes [23] . Therefore, targeting of CvME for avoidance of lysosomal degradation became a key strategy to overcome intracellular barrier. Nonviral gene carriers for lung cancer RNAi therapy Lipid system

Hundreds of synthetic lipids have been developed for gene delivery since Fielgner et al. [24] first synthesized cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,Ntrimethylammonium chloride (DOTMA) to deliver plasmids into cells because they have many potential advantages such as ease of production, biodegradabil-


Review

ity, their ability to protect entrapped genes both from nuclease degradation and renal clearance, promotion of cellular uptake and endosomal escape [25,26] . As a consequence, several RNAi-based drugs using lipid systems also have been used for clinical trials (Table 1) . However, no liposome formulations have been used in lung cancer clinical trials. Anderson Cancer Center (TX, USA), for example, initiated a Phase I clinical trial of siRNAs encapsulated by neutral liposome, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine for suppression of oncoprotein Eph2. Besides, stable nucleic acid lipid particles (SNALP)-mediated siRNA delivery targeting PLK1 has entered the Phase I trials by Tekmira Pharmaceuticals Corporation (BC, Canada). SNALP referred to as lipidoid are advanced lipid-based delivery systems as new class of safer and efficient genedelivery vectors. To overcome the disadvantages associated with the lipid-state oil droplet of liposomes, liquid lipid was replaced by a solid lipid which forms micron colloid in aqueous surfactant solution. Although toxicities such as acute inflammation and pulmonary hypotension, tissue infiltration, decrease in white cell counts and tissue injury are reported, progress in nanotechnology will overcome the challenges and push toward clinical application. This section emphasizes the nanoparticle-based delivery of siRNA for lung cancer therapy. Amarzguioui et al. [27] delivered tissue factor (TF) siRNA in pulmonary tumor mouse by Lipofectamine 2000 because TF pathway inhibitor was reported to inhibit lung metastasis [28] . Intravenous (iv.) injection of cells transfected with TF siRNA/Lipofectamine 2000 complexes in C57BL/6 mice had a dramatic reduction in incidence of pulmonary tumors within 10–20 days, suggesting that TF siRNA might be a viable clinical strategy for the prevention of lung tumor metastasis. Zhang et al. [29] delivered HDM2 siRNA in different three lung cancer lines by polyarginine (R8) modified liposomes to overcome the limitation of poor cellular uptake for clinical use of siRNA. Polyarginine is one of the cell-penetrating peptides that can traverse cell membranes to enter cells [30] . The R8-modified liposome/HDM2 siRNA complexes demonstrated a significant stability against degradation in the blood serum and significantly reduced the proliferation of all three tested lung tumor cell lines due to the incorporation of R8 in the liposome, but they did not deliver the siRNA in vivo. Many liposomes used for siRNA delivery include cationic lipids such as DOTMA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP). The cationic lipids are composed of positively charged head group, linker group and

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Review Kim, Park, Singh et al.

Table 1. Clinical trials of lipid-based siRNA delivery. Drug

Target

Disease

Phase

Status

Company

ALN–VSP02

KSP and VEGF Solid tumors

siRNA–EphA2– DOPC

EPHA2

Atu027

ClinicalTrials.gov identifiers

I

Completed

Alnylam Pharmaceuticals NCT01158079 (MA, USA)

Advanced cancers

I

Recruiting

MD Anderson Cancer Center (TX, USA)

NCT01591356

PKN3

Solid tumors

I

Completed

Silence Therapeutics (London, UK)

NCT00938574

TKM–080301

PLK1

Cancer

I

Recruiting

Tekmira Pharmaceutical (BC, Canada)

NCT01262235

Cancer with hepatic metastases

I

Completed

National Cancer Institute NCT01437007 (MD, USA)

TKM–100201

VP24, VP35, Zaire ebola l-polymerase

Ebola virus infection

I

Recruiting

Tekmira Pharmaceutical (BC, Canada)

DCR-MYC

MYC

Solid tumors

I

Recruiting

Dicerna Pharmaceuticals, NCT02110563 Inc. (MA, USA)

NCT01518881

Data modified from [26].

hydrophobic tails. Many structures of hydrophobic domain, such as chain length, degree of unsaturation and domain asymmetry-gene activity relationships, have been reported [31,32] as the hydrophobic domain affects intracellular delivery of genes and cyto toxicity [33] . Biswas et al. [34] prepared cationic lipid with hydrophobic oxime ethers via click chemistry to decrease cytotoxicity. The results showed that lipoplexes derived from oxime ether-incorporated lipid had more gene silencing effect in H1792 lung cancer cells in the presence of serum than commonly used liposome formulation due to the low cytotoxicity by the introduced oxime ethers in the lipid. However, they did not perform in vivo experiments. Lipid/polymer system combines the liposome and the polymer-based delivery concept in which siRNA is first encapsulated into a polymer network and the siRNA encapsulated polymer is complexed with lipid because this hybrid system has advantages such as sustained release of siRNA and colloidal stability [35] . Jensen et al. [36] loaded EGFP siRNA into DOTAPmodified poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles to deliver in NSCLC cell line H1299 and spray-drying was performed to incorporate the siRNA-loaded nanoparticles into nanocomposites using mannitol as excipient for application of inhalation therapy to treat lung cancer. The results indicated that 73% of gene silencing was achieved in the presence of 10% serum where the silencing was dependent on the content of DOTAP in DOTAP/PLGA mixture. Moreover, the spray-drying process did not affect the physicochemical properties of the redispers-

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ible nanoparticles in the medium with the preservation of gene silencing in vitro, suggesting the sufficient deposition of siRNA in the lower airways of the lung. However, they did not perform in vivo experiment. Shim et al. [37] screened an optimum liposome by changing molar compositions of liposomes for delivery of MCL1-specific siRNA as an anticancer siRNA in metastasized lung cancer mouse models after intratracheal (IT) administration. Among the various combinations of liposomes, dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC)/cholesterol (CH)/1,2dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (8/5/2 in molar ratio)-based lipoplexes significantly silenced MCL1 mRNA and protein levels in lung tissue with a reduced formation of melanoma tumor nodules of mouse while there was not much difference of cellular uptake of siRNA with various liposome combinations in vitro. Garbuzenko et al. [38] compared silencing efficiency of GLO Red siRNA in vitro and in an orthotopic model of human lung cancer between IT route as a local delivery and iv. one as a systemic delivery by DOTAP liposome. The liposome showed efficient intracellular delivery of GLO Red siRNA in A549 cells. IT delivery of lipoplexes in vivo led to the higher peak concentration and much longer retention of the siRNA than iv. delivery because administration of the siRNA to the lung may enhance siRNA retention by lung cells, increase the drug concentration inside the cells and decrease the need for high drug dose [38] . Recently, Shen et al. [39] delivered transcription factor GATA2 siRNA in NSCLC cells and A549 xeno-



graft murine model with N,N-bis(2-hydroxyethyl)N-(methyl-N-(2-cholesteryloxycarbonyl aminoethyl) ammonium bromide (BHEM-Chol)/PEG-PLA for treating NSCLC harboring oncogenic KRAS mutations. The lipoplexes selectively inhibited cell proliferation and induced cell apoptosis in KRAS mutant NSCLC cells, however, this intervention had no effect on normal KRAS wild-type NSCLC cells. Further, systemic delivery of lipoplexes significantly inhibited tumor growth in the KRAS mutant A549 NSCLC xenograft murine model due to the synthetic lethal interaction between GATA2 downregulation and KRAS mutation. Fehring et al. [40] delivered CD31 siRNA modified with alternating 2’-O-methyl modification patterns on both blunt ended siRNA strands to improve stability of the siRNA to the lung endothelium by a combination of β-L-arginyl-2,3-L-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride, cholesterol and mPEG-DSPE (DACC) as a carrier. The repeated treatment with lipoplexes via iv. injection in mice reduced lung metastases and increased life span in a mouse model of lung metastasis. Examples of siRNA delivery by lipid systems for lung cancer therapy are summarized in Table 2. Polymeric system

Recently, polymeric carriers have received significant attention in siRNA delivery because polymers have versatile nature with biocompatibility and provide a scope for diverse modifications to embrace desirable properties in polymeric carriers. Consequently, polymeric carrier-mediated siRNA delivery is in developing stage of clinical trials (Table 3) . In this part, nanoparticlebased delivery of siRNA for lung cancer therapy by polymeric vectors is covered. Among polymeric carriers, polyethylenimine (PEI) has been used for various siRNA delivery applications as a golden standard because it confers high gene silencing effect due to the buffering capacity of the PEI [41] . However, growing concerns about the cytotoxicity of

Review

PEI due to its nondegradable nature have accentuated the need for strategies to modify PEI. Importantly, cytotoxicity of PEI depends on the molecular weight and chemical structure of the PEI. ZamoraAvila et al. [42] delivered WT1 siRNA in lungs of mice with B16F10 lung metastasis by branched PEI (25 kDa) via aerosol administration to inhibit lung metastases growth. The WT1 siRNA/PEI complexes reduced the number and growth of visible metastatic tumor foci in the lungs with an activation of the intrinsic apoptotic pathway but they did not check cytotoxicity in vitro and in vivo. On the other hand, Kamlah et al. [43] delivered HIF1A and HIF2A siRNA in a Lewis lung carcinoma (LLC) cancer model mice by linear PEI via several different routes. Among the routes, iv. jugular vein injection of the complexes prolonged better survival and showed better reduced angiogenesis and tumor cell proliferation than intraperitoneal and IT administrations. Recently, Bonnet et al. [44] delivered BIRC5 and CCNB1 sticky siRNA in mammary adenocarcinoma cells and lung metastasis model mouse by linear PEI for lung tumor metastasis inhibition. The ssRNAs delivered by PEI inhibited target genes at the mRNA and protein levels, and showed inhibition of lung tumor metastasis after iv. administration. Thomas et al. [45] removed residual N-acetyl moieties from commercial linear PEI (25 kDa) to maximize the number of protonable nitrogens to deliver influenza nucleocapsid protein siRNA in mouse. The fully deacylated PEI afforded 77% suppression of luciferase gene expression in the mouse. However, specificity of deacylated PEI to mouse lung is not clear. Cho and his coworkers [46] delivered AKT1 shRNA in lung cancer cell line by degradable poly(amino ester) (PAE) synthesized by reaction of low molecular weight PEI with poly(ethylene glycol) diacrylate as shown in Figure 1 [47] . AKT1 is correlated to cancer cell migration, invasion and proliferation [48] . The PAE/AKT1 shRNA reduced cell migration, invasion and proliferation in A549 cells more than PEI/AKT1 shRNA. They also delivered AKT1 siRNA into K-rasLA1 and urethane-

Table 2. Examples of siRNA delivery by lipid systems for lung cancer therapy. Lipid systems

Targeted siRNA

Animal model

Route

Lipofectamine2000 ®

TF

Pulmonary tumor mouse

iv.

Ref. [27]

DOTAP liposome

GLO Red

Orthotopic human lung cancer mouse

IT and iv.

[31]

EDOPC/CH/DOPE

MCL1

Metastasized lung cancer mouse

IT

[38]

BHEM-Chol/PEG-PLA

GATA2

A549 xenograft murine mouse

iv.

[39]

DACC

CD31

Metastasized lung cancer mouse

iv.

[40]

BHEM-Chol: N,N-bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonyl aminoethyl)ammonium bromide; CH: Cholesterol; DACC: dioleoyl-sn-glycero-3-ethylphosphocholine; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTAP: N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium methyl sulfate; EDOPC: Dioleoyl-sn-glycero-3-ethylphosphocholine; IT: Intratracheal injection; iv.: Intravenous injection; PLA: Polylactide.



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Table 3. Clinical trials of polymer-based siRNA delivery. Delivery system

Drug

Target gene

Disease

Phase Status

Company

ClinicalTrials. gov identifiers

Cyclodextrin

CALAA–01

RRM2

Solid tumors

I

Active

Calando Pharmaceuticals (CA, USA)

NCT00689065

LODER Polymer

siG12D–LODER KRAS

Pancreatic cancer

II

Recruiting Silenseed (Jerusalem, Israel)

NCT01676259

JetPEI®

SNS01-T

Factor 5A

Multiple myeloma tumors

I/II

Recruiting Senesco Technologies Inc. (GA, USA)

NCT01435720

Dynamic PolyConjugate

ARC-520

Conserved regions of HBV transcripts

Hepatitis B

I

Recruiting Arrowhead Research NCT01872065 Corporation (CA, USA)

Dynamic PolyConjugate

ARC-520

Conserved regions of HBV transcripts

Hepatitis B

II

Recruiting Arrowhead Research NCT02065336 Corporation (CA, USA)

induced lung cancer model mice through a nose-only inhalation system by the same carrier [49] . After aerosol delivery twice weekly for 4 weeks, PAE/AKT1 siRNA complexes showed downregulation of Akt1 related signals and inhibited the growth of tumors in the lung cancer model of K-rasLA1 mice as shown in Figure 2. Cho and his coworkers [50] delivered EGFP siRNA in A549 cells by a reductable polyspermine carrier prepared by reaction of spermine and N,N-cystaminebisacrylamide as shown in Figure 3, and they compared AKT1 silencing efficiency among shRNA, siRNA and ssiRNA (siRNA with sticky overhangs) in A549 cells. The results indicated EGFP silencing by the carrier was about 1.5-times superior in A549 cells to PEI 25 kDa due to the cytosol specific degradation of the disulfide bonds. Moreover, AKT1 ssi showed better apoptosis in A549 cells over AKT1 siRNA and AKT1 shRNA by the first 2 days probably due to the more stable AKT1 ssiRNA than siRNA and shRNA. They also prepared spermine-based PAE based on glycerol propoxylate triacrylate (GPT) and spermine as shown in Figure 4 to deliver AKT1 shRNA in A549 cells and in K-rasLA1 lung cancer model mice via aerosol administration [51] . Aerosol delivery of carrier/AKT1 shRNA significantly suppressed lung tumor progression in lung cancer model mice through the Akt signaling pathway without toxicity due to the high buffering capacity of spermine and hydroxyl groups of GPT. Furthermore, they prepared another spermine-based PAE based on glycerol triacrylate (GT) and spermine as shown in Figure 5 to deliver AKT1 shRNA in A549 cells and in K-rasLA1 mouse model of nonsmall cell lung cancer via aerosol administration [52] . Similarly, the carrier/AKT1 shRNA complexes suppressed lung tumorigenesis in a K-rasLA1 lung cancer mice model by inducing apopto-

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sis through the Akt signaling pathway and cell cycle arrest. Kawata et al. [53] delivered PLK1 siRNA in human lung cancer cell line and lung cancer model mouse via iv. administration by biocompatible atelocollagen to prevent growth of liver metastases of lung cancer as overexpression of PLK1 is correlated with the wide variety of cancers [54] . The results indicated that PLK1 siRNA/atelocollagen complexes induced cell death in lung cancer cells and iv. administration of the complexes inhibited the growth of metastatic liver tumors of lung cancer in a mouse model. Chitosan, another biocompatible polymer, has been widely used as a gene carrier. However, the major limitation of chitosan is low gene silencing due to its poor buffering capacity for endosomal escape. Therefore, Cho and his coworkers grafted low molecular weight PEI with periodate-oxidized chitosan by an imine reaction to deliver AKT1 siRNA in A549 cells [55] . The carrier efficiently delivered siRNA and thereby silenced oncoprotein Akt1. Besides, silencing of the cell survival protein by the carrier significantly reduced the lung cancer survival and proliferation. Varkouhi et al. [56] delivered siRNA in H1299 human lung cancer cell line by thiolated N,N,N-trimethylated chitosan (TMC) to increase extracellular stability and to improve intracellular release of polyplexes. The thiolated TMC/siRNA complexes showed higher gene silencing (60–80%) with retaining their silencing activity in the presence of hyaluronic acid (HA) than nonthiolated TMC/siRNA ones (40%) due to better extracellular stability by thiol groups. Recently, an inhalable dry chitosan/siRNA powders in the presence of mannitol was prepared using the supercritical carbon dioxide technique to make stable siRNA powders [57] . The dry chitosan/siRNA



powders were stable in serum, were distributed in the lung epithelial surface of mouse at a higher concentration and significantly inhibited gene expression in the mouse lung metastasis model after pulmonary administration of the powder. Furthermore, Han et al. [58] prepared another biocompatible cationic bovine serum albumin (CBSA) by surface modification of BSA with ethylenediamine through an amide linkage to deliver BCL2 siRNA in lung cancer B16 cells and B16 lung metastasis model mouse. The polymer exhibited higher gene silencing in B16 cells which is comparable to Lipofectamine and inhibited the tumor growth in a B16 lung metastasis model mouse after iv. administration with lower toxicity and immunogenicity. Nguyen et al. [59] synthesized biodegradable branched polyester consisting of tertiary amine-modified polyvinyl alcohol backbone grafted to PLGA to deliver luciferase siRNA in H1299 as a human lung epithelial cell line for pulmonary gene therapy. The degradation of the polymer/siRNA was seen within 4 h in PBS with sustained release of siRNA and 80–90% knockdown of the gene was achieved with only 5 pmol antiluciferase siRNA even after nebulization. But, they did not check the efficiency of the system in vivo. Amphiphilic block copolymers are promising candidates for effective gene delivery system because they have several advantages such as nanoparticle stability, narrow size distribution with tunable sizes, protection of genes during blood circulation and bioconjugation with cell targeting molecules for specific targeting [60] . Du et al. [61] synthesized PEG-PLGA-PLL by ring opening polymerization of carbobenzyloxy-L-lysine N-carboxyanhydride by amino-terminated PEG-PLGA to deliver siRNA in human lung cancer SPC-A1-GFP cells. The polymer showed higher gene silencing with lower cytotoxicity than Lipofectamine. Yu et al. [62]

H

H N

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synthesized pH-sensitive poly(methacryloyloxyethyl phosphorylcholine/poly(diisopropanolamine ethyl methacrylate) diblock copolymer via atom transfer radical polymerization to deliver oncogene MDM2 siRNA in NSCLC cells and H2009 xenograft tumor model mice. The polymer showed MDM2 knockdown in p53 mutant NSCLC H2009 cells and inhibited tumor growth in tumor-bearing nude mouse via iv. repeated injection of the polyplexes. Recently, Mao et al. [63] used self-assembled nanoparticles made from the poly(ɛ-caprolactone (PCL)/poly(2-aminoethylene phosphate) (PAEP) block copolymer and PCL/PEG block copolymer to deliver CDK4 siRNA in KRAS mutant cell line and A549 NSCLC xenograft murine model mice. The polymer showed selectively decreased CDK4 expression in KRAS mutant cell lines and antitumor activity in a KRAS mutant model mice due to the synthetic lethal interaction between CDK4 downregulation and KRAS mutation. Recently, degradable cationic star polymers, prepared by dimethylaminoethyl methacrylate (DMAEMA) via reversible addition–fragmentation transfer polymerization and then chain extended in the presence of N,N-bis(acryloyl)cystamine and DMAEMA as crosslinkers, were used to deliver siRNA in H460 lung cancer cells and lung tumor model mouse [64] . The polymer suppressed GFP mRNA expression by 80%, 48 h post-transfection in the lung cancer cells and silenced target gene expression by 50% in lung tumor model mouse via IT administration of the polyplexes. Besides, Conti et al. delivered EGFP-siRNA in A549 lung alveolar epithelial cells by amine-terminated poly(amidoamine) dendrimer as a gene carrier to develop oral inhalation (OI) formulations for the local delivery of gene to the lung [65] due to fact that OI is one of the direct and noninvasive ways of targeting O

l NH2

+

O

O

m O

PEI linear (Mn : 423) CH2CI2 45°C, 48 h

H N

PEG diacrylate (Mn : 258, 575 and 700) O l N H

O

O m O

n

PEI-alt-PEG *Degree of polymerization 1 ~ 9.43 (Mn : 423) m ~3 (Mn : 258) ~ 10 (Mn : 575) ~ 13.5 (Mn : 700) Figure 1. Proposed reaction scheme for copolymer formation. PEI: Polyethylenimine.



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A

CON

SCR

siAkt1

Akt1

carbon nanotubes (CNTs) and gold nanoparticles (GNPs) to deliver siRNA for lung cancer therapy will be explained. IONPs

Akt2

Akt3

GAPDH B

CON

SCR

siAkt1

Akt1

IONPs have been used as drug delivery system and contrast agent for MRI because selective delivery of therapeutic agents to target sites can be obtained by external permanent magnetic field. Wang et al. [68] prepared magnetic lipoplexes composed of lipid, IGF1R shRNA and super paramagnetic IONPs for lung cancer therapy. The IGF1R specific-shRNA transfected by liposomal magnetofection inhibited IGF1R expression by 85.1±3% in A549 cells while inhibition by lipofection was 56.1±6%. Further, in vivo delivery efficiency of the plasmids expressing GFP and shRNAs targeting IGF1R into the lung tumor-bearing mice was significantly higher in the liposomal magnetofection group than in the lipofection group.

Akt2

CNTs Akt3

Actin

Figure 2. Aerosol delivery of poly(ester amine)/AKT1 siRNA-inhibited Akt1 activity in lungs of K-rasLA1 mice. (A) Reverse transcriptase-polymerase chain reaction analysis of AKT1, AKT2 and AKT3 mRNA expression in the lungs of K-ras LA1 mice. (B) Western blot analysis of the Akt protein family. Lysates from the lungs of K-ras LA1 mice treated with aerosol-delivered AKT1 siRNA were analyzed for Akt1–3 protein expression. Results indicated a selective suppression of the Akt1 level only. CON: Control; SCR: Scrambled siRNA.

the lungs [66] . The siRNA formulated as dendriplexes in pressurized metered-dose inhalers as an OI device efficiently targeted A549 cells and silenced genes even after exposure of siRNA to the propellant environment, suggesting the possibility of OI formulation for the local administration of siRNA in vivo. Examples of siRNA delivery by polymeric systems for lung cancer therapy are summarized in Table 4. Rigid nanoparticles

Rigid nanoparticles such as inorganic and carbon materials have been developed as siRNA carriers because they exhibit unique tunable properties such as large surface area to volume ratios, facile surface modification, controllable size and additional function [67] . In this section, iron oxide nanoparticles (IONPs),

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CNTs have been utilized as drug carriers because they can penetrate mammalian cells without apparent toxicity. Podesta et al. [69] prepared amino-functionalized multiwalled CNTs to deliver proprietary toxic siRNA in human lung carcinoma (Calu6) cells and human lung tumor-bearing mice. The delivered cationic CNTs/TOXsiRNA complexes induced significant cytotoxic activities in lung carcinoma cells and significantly suppressed tumor volume in the human lung tumor-bearing mice via IT administration. They also reported that precisely tailored number of amino functions (dendron generations) on the CNTs can enhance cellular internalization and gene silencing in the A549 cells [70] . Similarly, Zhang et al. [71] delivered TERT siRNA in LLC tumor cells and lung tumorbearing mice by cationic single-walled CNTs. The CNTs suppressed TERT expression with a production of growth arrest in tumor cell lines and reduced tumor growth in lung-bearing mice via subcutaneous administration. GNPs

GNPs are promising gene vectors because they can be easily tailored to a desirable size and shape, they have unique surface plasmon resonance and they can be easily modified with thiolated molecules [67] . However, only one PLL-conjugated GNP has been reported to deliver siRNA in human lung cancer H1299 with a near-infrared (NIR) laser irradiation for the light-triggered endosomal release of siRNA [72] . Light-triggered delivery of siRNA by the PLL-conjugated GNPs downregulated about 49% of the targeted GFP expression with lower cytotoxicity.



Furthermore, they delivered a combination of three different siRNAs composed of MDM2, MYC and VEGF in lung metastasis model mice by PEG-AN-conjugated LPD nanoparticles [76] . The targeted nanoparticles formulation significantly prolonged the mean survival time of the mice by 30% as compared with the untreated controls and significantly reduced the lung metastasis (∼70–80%) at a relatively low dose (0.45 mg/kg) without local and systemic immunotoxicity. Huang and his coworkers coated calcium phosphate (CP)/liposome (CPL) nanoparticles with DSPE-PEGAN to specifically deliver siRNA in human H460 lung cancer cells and in vivo [77] . The targeted CPL nanoparticles could release more cargo to the cytoplasm than the targeted LPD nanoparticles, leading to a significant (∼40-fold in vitro and ∼4-fold in vivo) improvement in siRNA delivery. They also delivered a mixture of MDM2, MYC and VEGF siRNA in lung tumor-bearing mice by the targeted CPL [78] . The treatment with a mixture siRNA by the targeted CPL nanoparticles significantly reduced lung metastases (∼70–80%) at a relatively low dose (0.36 mg/kg) via a single iv. injection. Furthermore, they delivered a mixture of HDM2, MYC and VEGF siRNA in A549 cells and lung tumorbearing mice [79] . The siRNA codelivered by the targeted CPL effectively and simultaneously knocked

Specific ligand-coupled nanoparticle system

One of the strategies to overcome the extracellular barriers in nonviral gene delivery is receptor-mediated endocytosis for specific enhancement of cellular uptake. In this section, anisamide (AN), HA, antibody, folic acid (FA) and Arg-Gly-Asp (RGD) as specific ligands for delivery of siRNA to the lung will be explained. Anisamide

It is well documented that AN is a targeting ligand for the sigma receptor overexpressed in human lung cells [73] . Huang and his coworkers coated liposome/protamine/DNA (LPD) nanoparticles with DSPE-PEG-AN to specifically deliver surviving siRNA into lung cancer cells [74] . The LPD nanoparticles coated with PEGylated lipid tethered to a targeting ligand showed higher surviving protein downregulation and antitumor activity in the lung cancer cells than nontargeted nanoparticles. They also coated liposome/protamine/HA (LPH) nanoparticles with DSPE-PEG-AN to specifically deliver luciferase siRNA into lung cancer cells and in the tumor-bearing mouse [75] . The targeted LPH nanoparticles silenced 80% of luciferase activity in the metastatic B16F10 tumor in the mouse lung after a single iv. injection.

O S

N H

H N

S

O

CAM + H N

2HN

Review

N H

NH2

Spermine Michael addition reaction

H N 4

H N

2 4

4

4

1 4

1

4 N H

4 2

O

5 N H

3

6 N H

S 5

3

H N

5 S

6

O

2 n

Reductable polyspermine

10

9

8

7

6

5

4

3

2 1 0.693 3.525 4.263

5 4 3 1.037 1.141 3.189 8.945 0.586 1.097

1.000 0.176

6

2

1

ppm

Figure 3. Synthetic scheme of reductable polyspermine. CAM: N,N′-Cystaminebisacrylamide.



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Review Kim, Park, Singh et al. down HDM2, MYC and VEGF expression in A549 cells and repeated iv. injections of mice with mixture siRNA by the targeted CPL impaired NSCLC growth in vivo for both A549 and H460 tumors. Hyaluronic acid

It has already been reported that the HA recognizes CD44 receptors as an integral membrane glycoprotein highly overexpressed in majority of NSCLC [80] . HA is also known biomarker for NSCLC tumors [81] . Ganesh et al. [82] introduced HA as a specific ligand to the lung cancer cells into lipid (or PEI) to specifically deliver siRNA in A549 cells and lung tumor-bearing mice. The HA-conjugated carriers transfected siRNA into lung cancer cells overexpressing CD44 receptors and targeted specific knockdown by delivering SSB/ PLK1 siRNA in solid lung tumors as well as in metastatic tumors due to the receptor-mediated mechanism. Similarly, Taetz et al. [83] conjugated HA with DOTAT/DOPE liposomes for the targeted delivery of antitelomerase siRNA in CD44-expressing lung cancer cells. The HA-conjugated liposomes showed higher gene silencing of telomerase enzyme in A549 cells than unmodified ones. Furthermore, Wang et al. [84] introduced HA into liposome/protamine nanoparticles to deliver CD47 siRNA in lung metastasis mice. The HA-conjugated nanoparticles efficiently inhibited lung metastasis to about 27% of the untreated control without hematopoietic toxicity via iv. administration.

O H2C

OCH2CH2CH O

Antibody, one of the common tools to target antigen in particular types, has been also used for siRNA delivery [85] . Chen et al. [86] developed LPH nanoparticles modified with tumor-targeting single-chain antibody fragment to deliver siRNA and miRNA into experimental lung metastases of murine B16F10 melanoma. The combined siRNAs (c-myc/MDM2/VEGF) delivered by the antibody-conjugated nanoparticles efficiently downregulated the target genes in the lung metastasis and significantly reduced the tumor load in the lung through two daily iv. injections. Moreover, an enhanced anticancer effect was found when miR-34a and siRNAs were codelivered in lung tumor-bearing mice by the antibody-targeted nanoparticles. Similarly, Mokhtarieh et al. [87] conjugated antihuman epidermal growth factor receptor antibody (anti-EGFR) to asymmetric liposome particles for specifically delivering of siRNA in NSCLC cell line. The antibodyliposome showed anti-EGFR-dependent target gene silencing in NSCLC cells due to the target-specific delivery of siRNA with a specific ligand. Recently, Nascimento et al. introduced EGFR-targeted peptide with PEGylated chitosan to deliver MAD2 siRNA in NSCLC cells for the induction of cancer cell death [88] because the complete abolition of MAD2, as an essential mitotic checkpoint component necessary for accurate chromosome segregation during mitosis, leads to cell death [89] . The EGFR-targeted chitosan nanopar-

OH O

OCH2CH2CH

H2C

Antibody

OH

CH2

CHCH2CH2O

+

H N

N H

HN 2

NH2

OH

GPT

SPE Michael EtOH Addition

O ~ SPE

OH O

OCH2CH2CH

SPE ~ CHCH2CH2O

OCH2CH2CH

~ SPE O

OH

OH

GPT-SPE Figure 4. Proposed reaction scheme of glycerol triacrylate–sperimine. GPT: Glycerol propoxylate triacrylate; SPE: Spermine.

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2

HN

N H

N H

Review

NH2

O

EtOH +

Michael addition

SPE~

O

~ SPE O

O

O O

O SPE ~

O O O

GT-SPE (PEA)

O O

GT, 254 Figure 5. Synthetic scheme of GT–SPE polyspermine. GT: Glycerol triacrylate; PEA: Poly(ester amine); SPE: Spermine.

ticles showed nearly complete depletion of MAD2 expression in A549 cells due to apoptosis whereas nontargeted system showed partial depletion. However, they did not validate the results in vivo.

showed high tumor growth inhibition and increased tumor mouse survival related to an enhanced inflammatory response in lung tumor tissue through IT administration.

Folic acid

Nanoparticle-mediated codelivery of anticancer drug & siRNA for lung cancer therapy Most commonly, anticancer drug-loaded nanoparticles delivered at tumor site kill drug-sensitive cancer cells but leaves behind a higher proportion of drug-resistant cells in the heterogeneous environment within the tumor. As a result, the survived and regenerated cancer cells acquire MDR properties [95] . Nowadays, MDR is the main cause of failure of chemotherapy in not only lung cancer but also various different types of cancers [96] . During chemotherapeutic treatment to lung cancer, diverse mechanisms of MDR are obtained in SCLC while they are inherent in NSCLC. To fight against MDR, codelivery of anticancer drugs with siRNA has emerged as a progressive and powerful strategy. As siRNAs selectively silence MDR-related genes, drugs are more easily delivered and effectively kill cancer cells (Figure 7) . Furthermore, synergistic effects of drugs and siRNAs reduce the highly required drug dose and prevent drug-induced undesirable side effects. In this section, two mechanisms that involved in MDR: pump resistance and nonpump resistance [97] will be covered and codelivery strategies for inhibiting MDR mechanisms will be discussed.

Folate receptors as a high-affinity membrane folatebinding protein are overexpressed in various human cancer cells and bind to FA with high affinity (Kd∼10 –10 ) [90] . Jiang et al. [91] introduced FA into chitosan-graft-PEI to deliver AKT1 shRNA in A549 cells and urethane-induced lung cancer model mouse for lung cancer cell targeting as shown in Figure 6. The FA-chitosan-graft-PEI/AKT1 shRNA complexes showed higher gene silencing in A549 cells than chitosan-graft-PEI/AKT1 shRNA ones and showed more suppression of lung tumorigenesis in lung cancer model mouse via aerosol administration through the Akt signaling pathway. RGD peptides

RGD peptide as a specific ligand has a high affinity for αv integrins on tumor and tumor angiogenic endothelial cells [92] . Yonenaga et al. [93] introduced RGD-grafted PEG into polycation liposome (PCLS) containing dicetylphosphate-tetraethylenepentamine (DCP-TEPA) to deliver luciferase siRNA in B16F10luc2 murine melanoma cells and B16F10-luc2 lung metastatic model mice. The RGD-PEG-PCLS/luciferase siRNA complexes showed higher gene silencing in vitro than PEG-PCL/luciferase siRNA ones and high gene knockdown efficiency against metastatic tumor-bearing mice in lung via iv. administration. Similarly, Conde et al. [94] introduced RGD into GNPs to deliver MYC siRNA in lung tumor-bearing mice. The RGD-GNPs/MYC siRNA complexes


Mechanisms of MDR Pump resistance

Pump resistance typically occurs as a result of energydependent drug efflux pump which include P-glycoprotein (P-gp; also known as ABCB1 or MDR-1


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Review Kim, Park, Singh et al. protein) [98] , MDR associated-protein 1 (MRP-1, also known as ABCC1) [99] and lung resistance protein [100] . Drug efflux pump proteins continually bind and eject the anticancer drugs out of the cells. P-gp that belongs to a large family of ATP-dependent transporters is widely expressed in our body and plays excretory and protective roles under normal circumstances. Some cancer cells express a surprising amount of p-gps which pump out xenobiotic compounds including neutral or positively charged anticancer drugs (doxorubicin [DOX], vinblastine, paclitaxel [PTX]) [101,102] . On the other hand, MRP-1, highly expressed in NSCLC [103] , preferably binds and pumps out not only anionic hydrophobic anticancer drugs such as daunorubicin, DOX and etoposide [102] but also drugs conjugated to glutathione (GSH) and glucuronate [104] . Nonpump resistance

Nonpump resistance is independent of drug efflux pump and not interrelated with drug accumulation

in cancer cells. Several nonpump resistance mechanisms are activated by nonpump resistance relatedproteins such as BCL-2, MCL-1, survivin, VEGF, etc. Nonpump resistances prevent the cancer cell death induced by anticancer drugs via cellular antiapoptotic and antioxidant defenses, damaged DNA repair, activation of detoxifying proteins and alteration of cell cycle checkpoints [96,97] . Among them, antiapoptotic defense related proteins have been widely investigated to overcome cancer resistance [105–108] . The overexpression of antiapoptotic proteins, such as BCL-2 family members and inhibitor of antiapoptotic proteins prevent normal apoptosis in the cancer cells. Human BCL-2 protein is the most famous antiapoptotic protein belonging to BCL-2 family; antiapoptotic proteins (Bcl-2, Bcl-XL, Bcl-w, MCL-1, etc.) and proapoptotic proteins (Bax, Bak, Bad, Bid, etc.) [109] . When anticancer drugs such as DOX enter the cancer cells, BCL-2 proteins become overexpressed [103] . Accordingly, formation of Bcl-2/Bcl-2 homodimer is more favored by increase of Bcl-2 protein concentra-

Table 4. Examples of siRNA delivery by polymeric systems for lung cancer therapy. Polymeric systems

Targeted siRNA

Animal model

Route

Deacylated PEI

Influenza nucleocapsid protein

Influenza virus infected mouse

iv.

Ref. [45]

Atelocollagen

PLK1

Murine liver metastasis model of lung cancer

iv.

[53]

Chitosan

Luciferase

Colon 26/Luc metastasis model of lung cancer

Pulmonary iv.

[57]

CBSA

BCL2

B16 metastasis model mouse

iv.

[58]

PEG-alt-PEI

AKT1

L-ras

LA1

lung cancer model mouse

Aerosol

[46,49]

Reductable AKT1 polyspermine based on spermine and GPT

L-ras

LA1

lung cancer model mouse

Aerosol

[50]

Reductable AKT1 polyspermine based on spermine and GT

L-rasLA1 lung cancer model mouse

Aerosol

[51]

Branched PEI

WT1

B16F10 metastasis model of lung cancer

Aerosol

[42]

Linear PEI

HIF1A and HIF2A

LLC cancer model mouse

iv., ip. and IT

[43]

Linear PEI

BIRC5 and CCNB1

Metastasis model of lung cancer

iv.

[44]

PMPC-b-PDPA

MDM2

H2009 xenograft tumor model mouse

iv.

[62]

PCL-b-PAEP and PCL/PEG

CDK4

A549 NSCLC xenograft murine model mouse

iv.

[63]

PDMAEMA

GFP

NSCLC lung carcinoma cancer model mouse

Intratumoral

[64]

CBSA: Cationic bovine serum albumin; GFP: Green fluorescent protein; GPT: Glycerol propoxylate triacrylate; GT: Glycerol triacrylate; ip.: Intraperitoneal injection; IT: Intratracheal injection; iv.: Intravenous injestion; LLC: Lewis lung carcinoma.

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Review

Chitosan CH2OH

CH2OH

O OH

CH2OH

O

O

O

O

OH

FC

O

OH X

K

NH2

M

NH

CH2OH

CH3

NHS/DCC

+

H2N

N N

N

N N

N

H N

H N O

H N

O O

OH X

NH

O

O

O

OH K

H N

N

N

CH2OH

O

O

OH

C O H2N

CH2OH

O

NH2

NH

O M

NH2

C O O

CH3

COOH

COOH

N2 IO4-

COOH

Folic acid CH2OH

CH2OH

O OH N

H2N N

N N

CH2OH

O

O

NH H N

O

O

X

K

H N

O

O

OH

C

M O

NH

O

O

CH3

O COOH

O

NaBH4

PEI 1800 Da

Periodate-oxidized FC

CH2OH

CH2OH

O

O

OH H2N

N N

N N

O OH

NH H N

O O

NH C O

O

O

O

X

K

H N

CH2OH

CH3

O

M NH

NH

PEI

PEI

COOH

FC-g-PEI

Figure 6. Proposed reaction scheme for synthesis of FC-g-polyethylenimine. Circled parts indicate regions where a chemical reaction occurs. DCC: Dicyclohexylcarbodiimide; FC: Folic acid; FC-g-PEI: Folic acid-graft-PEI; NHS: N-hydroxysuccinimde; PEI: Polyethylenimine.

tion on the mitochondria surface, which limits formation of apoptosome in the mitochondria external membrane resulting in eluding normal apoptotic pathway of cancer cells.


Codelivery of anticancer drug and siRNA for overcoming MDR of lung cancer

In this section, we introduce various outstanding strategies of codelivery of anticancer drugs and siRNAs which


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Nonviral nanoplatform Anticancer drug Pump or nonpump resistance-related MDR siRNA

Drug efflux Pump resistance gene knockdown

Nonpump resistance gene knockdown Drug influx

Inhibition of drug efflux pump

Inhibition of antiaptotic defenses

Increase in intracellular anticancer drug concentration

Apoptosis

Lung cancer cell death

Multidrug resistance lung cancer cell

Figure 7. Nanoparticle-mediated codelivery of anticancer drug and siRNA for overcoming multidrug resistance in lung cancer therapy. MDR: Multidrug resistance.

are categorized according to the targeted MDR associated with lung cancer therapy: combination of drug and nonpump resistance siRNA, combination of drug and siRNA specific for both pump and nonpump resistance. Examples of codelivery of anticancer drugs and siRNAs for lung cancer therapy are summarized in Table 5. Codelivery of nonpump resistance siRNA with anticancer drug

Survivin protein is one of the main barriers to effective lung cancer chemotherapy. Most of malignant

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tumors such as lung, breast, colon, stomach, pancreas and liver cancer cells overexpress survivin protein [119] , which plays crucial roles in nonpump resistance via anti apoptotic regulations inhibiting caspase activation [120,121] . Therefore, downregulation of survivin as a preferential target for lung cancer has been widely investigated [122] . To overcome MDR by survivin, codelivery of survivin silencing gene and anticancer drugs recently has emerged as a new strategy for effective chemotherapy. Shen et al. [110] designed P85-PEI/TPGS/PTX/shSur



complex nanoparticles (PTPNs) which consists of pluronic P85, PEI, D-a-tocopheryl polyethylene glycol 1000 succinate (TPGS), PTX and BIRC5 shRNA. P85-PEI was synthesized by N,N’-carbonyl diimidazole-mediated acylation and P85-PEI/TPGS/PTX mixed micelles (PTPs) was prepared by thin-film hydration method. P85 was used to inhibit glutathione s-transferase activity which is highly related to many acquired drug resistance in cancer by detoxification processes [123] and TPGS was used to increase micelle stability and drug-loading efficiency. According to the result from in vitro cell cytotoxicity test, PTPNs had 360-times lower IC50 value than free PTX in A549/T cells (PTX resistant cell line) whereas it had a similar value with free PTX against A549 cells. Furthermore, PTPNs showed not significant undesirable toxicity into the tumor (A549/T) bearing nude mice. Consequently, synergistic effect of BIRC5 shRNA and anticancer drug (PTX) improved chemotherapeutic efficacy on the MDR human lung cancer cells. In further research, they modified PTPNs for active targeting of integrin αvβ3 on lung cancer cells by loading nine-unit cyclic tumor-homing peptides contained RGD sequence (iRGD) [111] . It was shown that iRGD-P85-PEI/TPGS/PTX/BIRC5 shRNA complexes (iPTPNs) provided significant accumulation of PTX and BIRC5 shRNA in lung cancer cells (A549/T) and effectively suppressed expression of survivin in vitro. In particular, iPTPNs showed higher anticancer effect than PTPNs toward tumor tissue due to its penetrating facilitation ability by EPR effect and active targeting via iRGD. To investigate synergistic effect of codelivery of BIRC5 siRNA and anticancer drug (PTX) in the MDR lung cancer cells, Zhu et al. [112] designed multifunctional micellar nanocarrier, PEG-peptidePEI-1,2-dioleoyl-snglycero-3-phosphoethanolamine (PEG-pp-PEI-PE). In their platform, PEG shielded unexpected interaction with blood components and PEI provided endosomal/lysosomal escape of gene by proton sponge effect [124] . In addition, synthetic octapeptide (GPLGIAGQ) was conjugated to nanocarrier for targeting MMP2-triggered tumor. The PEG-ppPEI-mediated codelivery of PTX and BIRC5 siRNA significantly enhanced anticancer efficacy in vivo, significantly improving IC50 (15 nM) compared with IC50 of free PTX (96 nM). Another interesting approach is using BIRC5 and CCNB1 ssRNAs [44] , where complexes are more stable than with siRNA [125] . Similar with functions of survivin, cyclin B1 plays a crucial role in tumor cell survival participating in mitosis and leading uncontrolled cell growth in various carcinomas [44,126] . To demonstrate the combinational effects of ssRNAs and Cis-


Review

platin (CIS), Bonnet et al. injected ssRNA (survivin or cyclin B1)-CIS-PEI into very aggressive lung metastasis mouse model by iv. injection of TSA-Luc cells. The ssRNA (BIRC5)-CIS-PEI complexes showed significantly higher tumor inhibition (90%), compared with free CIS (40%) and increased mice survival rate by restoring sensitivity to chemotherapeutic drugs in xenograft model. VEGF is another molecular target as a nonpump resistance related protein which is an important MDR factor inhibiting apoptosis by upregulation of bcl-2 family as well as a prototypical angiogenic factor [127,128] promoting tumor growth and metastasis [129] . For the treatment of NSCLC with aggressive and metastatic properties, VEGF silencing genes and anticancer drugs codelivery strategy has appeared to enhance the efficacy of chemotherapy via synergistic inhibitory effects on angiogenesis and tumor growth. Ma et al. [113] codelivered pshVEGF (human VEGF targeted shRNA) and low-dose-CIS (Diammindichloridoplatin; DDP) into the tumor (A549) bearing nude mice using DOTAP/Chol-based liposomal carrier. Cotreatment of pshVEGF and DDP significantly reinforced antitumor efficacy tumor growth inhibition since pshVEGF inhibited angiogenesis by RNAimediated downregulation of VEGF expression and simultaneously DDP induced apoptosis by damaging DNA. The combination of angiogenesis therapy and chemotherapy by enhancing cellular apoptosis and reducing tumor angiogenesis. Zhang et al. codelivered VEGF siRNA and gemcitabine monophosphate (GMP) entrapped into lipid/calcium/phosphate (LCP) nanoparticle modified with AA [114] . The calcium phosphate precipitate (CaP) core of LCP facilitated encapsulation of both agents and promoted endosomal release by rapidly dissolving in acidic endosome. Moreover, AA ligand provided enhanced delivery efficacy via sigma receptor-mediated tumor targeting and high density of DSPE-PEG on the surface prevented undesirable interaction with blood component at lipid bilayer surface. Interestingly, encapsulation efficiency (EE%) of GMP-VEGF-LCP-AA was similar with only VEGF siRNA or GMP-LCP-AA implying no interference of two therapeutic agents. The treatment of VEGF siRNA plus GMP loaded in LCP-AA yielded remarkable tumor growth inhibition rate in tumor (human NSCLC cells) bearing nude mouse model, which was associated with decreased angiogenesis and increased induction of apoptosis. Codelivery of both pump & nonpump resistance siRNA with anticancer drug

Codelivery of anticancer drug with pump or nonpump resistant silencing gene enhances drug sensitiv-


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MRP1 + BCL2 ASO MRP1 + BCL2 siRNA MRP1 + BCL2 siRNA BIRC5 + BCL2 siRNA

PEGylatedliposome

Pyridylthiolterminated MSN

Liposome (DOTAP)

CD44-targeting HA

A549 cell line

A549 cell line

H69AR cell line, MCF-7/ AD cell line, HCT15 cell line

H460 cell line

A549 cell line

TSA-Luc cell line

CIS

A549 cell line, H69 cell line, A549DDP cell line, H69AR cell line

DOX or TAX A549 cell line

DOX + CIS

DOX

DOX

MRP1 + BCL2 siRNA

CIS

Liposome (DOTAP)

VEGF shRNA

Liposome (DOTAP/Chol)

CIS

A549 cell line, A549/T cell line



Used cells

Tumor (A549, A549DDP)bearing nude mice

Tumor (A549)-bearing nude mice



None

Tumor (H460)-bearing nude mice


Tumor (TSA-Luc)bearing nude mice


Tumor (A549/T)bearing nude mice

Tumor (A549/T)bearing nude mice

Animal model

420 ± 4.5 nm, -28.2 ± 5.5 mV

Cationic, LHRH peptide, 110 ± 20 nm, +60.3 mV, inhalation delivery

LHRH peptide, siRNA-S-SMSN, inhalation delivery

Neutral, local pulmonary delivery

Cationic, 100–140 nm, +20 mV

AA ligand for sigma receptor, +10.0 ± 4.0 mV, ER%: 75%

Cationic, 200 nm–300 nm

>70 nm, +34 mV

MMP2-cleavable octapeptide (GPLGIAGQ), 43 nm, +50.2 ± 1.1 mV

iRGD peptide, 141–160 nm, +30 mV, DL%: 1.88%, ER%: 94.02%

150–180 nm, >+20 mV, DL%: 1.92%, ER%: 95.86%

Characteristics

AD: Adriamycin; ASO: Antisense oligonucleotide; CIS: Cisplatin; DDP: Cisplatin; DL: Drug-loading coefficient; DOTAP: Liposomal transfection reagent; DOX: Doxorubicin; ER: Encapsulation ratio; GMP: Gemcitabine monophosphate; HA: Hyaluronic acid; LCP: Lipid/calcium/phosphate; LHRH: Luteinizing hormone-releasing hormone; MSN: Mesoporous silica nanoparticles; PE: Phosphoethanolamine; TAX: Paclitaxel; TSA: Trichostatin A; TPGS: Tocopheryl polyethylene glycol 1000 succinate.

Codelivery of both pump and nonpump resistance siRNA with anticancer drug

BIRC5 and CCNB1 sticky siRNA

Linear PEI

TAX

GMP

BIRC5 siRNA

PEG-pp-PEI-PE nanoparticle

TAX

TAX

Drug

LCP nanoparticle VEGF siRNA

BIRC5 shRNA

BIRC5 shRNA

P85-PEI/TPGS nanoparticle

Codelivery of nonpump resistance siRNA with anticancer drug

P85-PEI/TPGS nanoparticle

Therapeutic gene

Carrier

Types

Table 5. Examples of therapeutic gene and drug codelivery for lung cancer therapy.

[118]

[117]

[116]

[115]

[103]

[114]

[113]

[44]

[112]

[111]

[110]

Ref.




ity through bypassing drug efflux pump and preventing cellular antiapoptotic defense [130–133] . Strategies targeting only one drug resistance causal factor are not enough to control all of the factors of MDR [103] because both pump and nonpump resistances are simultaneously activated by treating anticancer drug in genetically heterogeneous tumor environment. To overcome the limitation, Saad et al. [103] designed multifunctional liposome-based delivery system composed of MRP1 siRNA (drug efflux pump silencing siRNA), BCL2 (antiapoptotic protein silencing siRNA) and DOX-loaded positively charged DOTAP (1,2-dioleoyl-3-trimethylammonium–propane) liposome. Treatment of free DOX or DOX-loaded liposome on human MDR H69AR lung cancer cells simultaneously upregulated both MRP1 and BCL2 genes. On the other hand, siRNA (MRP1/BCL2)-DOX-loaded liposome significantly suppressed MRP1 and BCL2 both at mRNA and protein levels compared with other treatments. Targeting dual mechanisms associated with MDR provided more effective inhibition of MDR and induction of apoptosis than only one MDR gene targeted treatments using siRNA (MRP1)-DOX-loaded liposome, siRNA (BCL2)-DOX-loaded liposome. Similarly, Garbuzenko et al. [115] delivered DOX with MRP-1 antisense oligonucleotides (ASO) and BCL2 ASO in orthotopic murine model of human lung cancer using liposomal carrier through pulmonary route. Mixture of ASO (MRP1)-DOX-loaded liposome and ASO (MRP1)-DOX-loaded liposome (IC50 value; 6.64 ± 0.30 μM) and ASO (MRP1/BCL2)-DOX-loaded liposome (IC50 value; 5.58 ± 0.30 μM) provided remarkable high cytotoxicity in A549 cells with activation of caspase-dependent apoptosis. Interestingly, ASO (MRP-1/BCL-2)-DOX-loaded liposome has better antitumor effect than the mixture of ASO (MRP1)DOX-loaded liposome and ASO (BCL2)-DOX-loaded liposome in vitro and in vivo implying the effectiveness of two-in-one complex system [115] . To effectively downregulate MDR genes in the lung cancer cells, Ganesh et al. [118] investigated differential MDR gene expression levels of different cell lines: human nonsmall lung cancer cell line (A549), small cell lung cancer cell line (H69), resistant cell lines (A549DDP and H69AR). Four types of pump and nonpump resistant genes (MRP1, BCL2, MDR1 and BIRC5) were distinctively expressed at each cell line. After targeted cell specific siRNA sequences were designed and screened, a combination of BIRC5 and BCL2 siRNA was chosen as the best candidate for codelivery and delivered with CIS loaded in CD44targeting HA-based self-assembling nanosystems. As a result, codelivery of siRNA (BIRC5+BCL2) with CIS significantly inhibits tumor growth without side


Review

effects compared with single-agent treatment or single siRNA with CIS treatment. Taratula et al. suggested an innovative approach of simultaneous delivery of two types of anticancer drugs (DOX, CIS) and two types of siRNA (MRP1, BCL2) by using mesoporous silica nanoparticles (MSN) conjugated with luteinizing hormone-releasing hormone (LHRH) peptide as specific targeting moiety [116] . Four types of combinations of LHRHPEG-siRNA-anticancer drug-MSN were synthesized: LHRH-PEG-siRNA (BCL2)-DOX-MSN, LHRH-PEG-siRNA (MRP1)-DOX-MSN, LHRHPEG-siRNA (BCL2)-CIS-MSN and LHRH-PEGsiRNA (MRP1)-CIS-MSN. The mixture of LHRHPEG-siRNA (BCL2)-MSN and LHRH-PEG-siRNA (MRP1)-MSN provided effective downregulation of BCL2 (58%) and MRP1 (56%) mRNA levels in vitro. According to the result from in vitro MTT assay, mixture of both drug (DOX, CIS) and siRNA (BCL2, MRP1) offered highest anticancer efficacy by synergistic effects. Furthermore, based on their successful previous research, they delivered same siRNAs with TAX using liposomal nanoparticles, namely LHRHPEG-siRNA-anticancer drug-DOTAP liposomal complex [117] . The treatment of LHRH-PEG-siRNA (MRP1/BCL2)-TAX (Taxol)-DOTAP to the tumor (A549) bearing nude mice via inhalational local delivery significantly downregulated two types of MDR genes. Consequently, their codelivery system increased drug sensitivity to TAX and effectively induced cancer cell death, and strongly inhibited lung tumor growth. Nanoparticle-mediated lung cancer therapy by combination of imaging agent & siRNA In clinical trials of lung cancer gene therapy, one of the crucial problems is the incapacity to trace and detect biodistribution and gene expression in the target tissue, respectively. Although biopsy or autopsy give data about therapeutic efficiency at laboratory level, these methods are invasive and very limited to verify information for clinical usage of nanoparticle-based gene delivery systems. Therefore, nowadays many researchers make an effort to develop safe and noninvasive methods for imaging of gene therapy, and various imaging technologies have been developed and used in RNAi-mediated therapy; optical (fluorescence, nonfluorescence) imaging, nuclear imaging (positron emission tomography/ single photon emission computed tomography) and MRI can track nanoparticles or siRNAs and assess therapeutic effect of siRNA both in vitro and in vivo [134] . In this section, among various imaging techniques in gene therapy, we will focus on the recent technological advancement of the combinations of imaging agents and nonviral siRNA therapy for lung cancer.


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Review Kim, Park, Singh et al. Magnetic nanoparticles as MRI contrast agentbased siRNA delivery for treating lung cancer

Magnetic nanoparticles (MNPs) are already being used clinically as contrast agents for MRI, which is a noninvasive technique that can provide real-time highresolution soft tissue information [135] . Furthermore, unique features of MNPs as gene carriers hold dual therapeutic potential for imaging and gene delivery in lung cancer: uniform and controllable size, ease of multifunctional modification at their surface and contribution of their supermagnetism at magnetofection by response of magnetic fields [136] . To enhance monitoring of gene therapy in lung cancer, Taratula et al. [137] developed a multifunctional MNP-based siRNA delivery system. They prepared superparamagnetic iron oxide (SPIO)-polypropyleneimine generation 5 (PPI G5) dendrimers-siRNA complexs, performed PEGylation, and conjugation of LHRH peptide to the distal end of the PEG layer for integrating tumor-specific targeting moiety and increase steric stability. They delivered BCL2 siRNA and anticancer drug (CIS) in vitro and in vivo by using SPIO-PPI G5-PEG-LHRH platform. The results demonstrated satisfactory conditions as highly efficient gene and drug codelivery system, which are cellular internalization via receptor-mediated endocytosis, specific tumor-targeting the ability to prevent rapid clearance in vivo, no significant cytotoxicity in vitro as well as in vivo, and antitumor effect. Although they did not confirm efficacy of siRNA delivery via MRI imaging, their research offered outlook and potential of multifunctional MNPs to monitor the delivery process and the therapeutic effects in the applications of lung cancer siRNA therapy. Recently, Chen et al. [138] developed of a novel magnetic mesoporous silica nanoparticles (M-MSNs)-based VEGF siRNA delivery system for lung cancer treatment. Furthermore, M-MSNs were functionalized with PEIPEG copolymer for better accumulation in the tumor region, and modified bifunctional fusogenic peptide KALA to enhance internalization and endosomal escape capacity. They confirm significant target gene knockdown and inhibition of tumor growth in vivo. However, the best noticeable point in their research is that M-MSN@PEI-PEG-KALA/VEGF siRNA complexes were used to demonstrate therapeutic effects on lung cancer by using in vivo MRI tracking. In T2-weighted magnetic resonance (MR) images of the tumor, 24 h after iv. administration, magnetic resonance signal was significantly reduced in both the subdermal and orthotopic lung cancer models compared with before the injection, indicating that complexes were effectively delivered and accumulated at the tumor site. As a result, MNP system and MRI technique could simultaneously

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facilitate dual function in both imaging and siRNA delivery for lung cancer such as real-time imaging of the tumor site, real-time tracking of nanoparticle and detecting of efficiency of therapeutic siRNA. Optically traceable siRNA delivery for treating lung cancer by using fluorescent tag

Imaging labels such as fluorescent tags, radionuclides and other biomolecules for different imaging modalities can lead to a dramatic signal amplification [139] . The optical imaging is relied on fluorescence, however, the small number of fluorescent imaging agents available in the NIR spectrum restricts the use of low-energy lasers to interrogate specimens and in vivo applications [140] . To overcome these limitations, commercially available imaging labels were developed such as cyanine 5 dye (Cy5) and quantum dot. These may offer better optical imaging as NIR fluorescent probes, which provide an optimal tool for in vivo applications such as investigating tumor physiology, metastasis pattern and tumor response to drug treatments [141] . In lung cancer siRNA therapy, Cy5 is the most commonly used anticancer theranostics. Wei et al. [142] orally codelivered TERT siRNA and anticancer drug (PTX) by using N-((2-hydroxy-3-trimethylammonium) propyl) chitosan chloride nanoparticle (HTCCP) to suppress lung tumor in murine LLC xenograft mouse model. Through capturing in vivo real-time images (merge of NIR fluorescence and X-ray) by labeling NIR fluorescent probe (Cy5) at siRNA, they confirmed biodistribution of HTCCP/siRNA, and demonstrated the passive targeting capability of HTCCP. Furthermore, codelivery system, HTCCP:Cy5-siRNA/PTX complexes were delivered in the tumor tissue cells to compare their fully complex system with a mixture system of siRNA and PTX, and they investigated distribution of anticancer agents (siRNA, PTX) by using flow cytometry. As expected, HTCCP:siRNA/PTX complexes exhibited high colocalization of siRNA and PTX in the same cell compared with the mixture of HTCCP:siRNA+HTCCP:PTX, and colocalization powerfully triggered synergistic anticancer effects of siRNA and drug. Thus, their ‘two in one system (HTCCP:siRNA/PTX)’ significantly inhibited tumor growth and showed potential for codelivery system of siRNA and hydrophobic drug in anticancer applications. Similarly, Liu et al. [143] used Cy5-labeled siRNA to detect tumor targeting and biodistribution of their carrier/siRNA complexes. They delivered Cy-5 siRNA in xenograft tumor models of human NSCLC by using tumor-targeted and self-assembled peptide nanoparticles; cyclo(Arg–Gly–Asp–d–Phe–Lys)-8–amino– 3,6–dioxaoctanoic acid–β–maleimidopropionic acid



(RPM). In fluorescent images, intense fluorescence was detected at the tumor site for at least 24 h after injection in the RPM/ Cy-5 siRNA group. However, in case of both naked Cy-5 siRNA and nontargeted control peptide/Cy-5 siRNA groups, no fluorescence was detected in the tumors. Their targeted siRNA delivery system was highly efficient on the tumor-specific accumulation, which resulted in a high inhibition rate of tumor growth, and antiangiogenic. In the above-mentioned studies, both MRI and NIR fluorescent probe were used as an effective imaging tool, which is served better and easier at tracking therapeutic nanoparticles and siRNA localization in vitro and in vivo. By simultaneously applying imaging agents and siRNAs in nanoparticle-based gene delivery system, real-time assessment may offer to verify information for clinical usage of lung cancer siRNA therapy, and will be a promising tool for effective demonstration of therapeutic efficacy of nonviral siRNA delivery systems. Conclusion & future perspective RNAi therapeutics for cancer have gained significant momentum with recent advances of nanotechnology. Although conventional chemotherapy has been available for few decades, MDR and nonspecific toxicity still remained unsolved. As a potential new drug against lung cancer, RNAi provides solutions to such limitations by directly downregulating oncogenes or MDR causative genes. The clinical advancement of RNAi therapeutics for lung cancer relies on develop-

Review

ment of effective and practical gene delivery system. There are several approaches on nanoparticle-based nonviral gene delivery system such as lipid-, polymerbased system and rigid nanoparticles. Among them, few lipid-based systems and PEI derivatives are recently being in clinical trials offering their promising practical application in the future. For enhancement of drug effects on lung cancer cells, surface modification of nanoparticles with lung cancer targeting ligands is an important strategy using specific ligand-receptor interaction. In recent, combination of chemotherapy and gene therapy has emerged as promising strategy overcoming MDR due to strong synergistic effects on lung cancer cells. The nanoparticle-based codelivery approaches provide strong therapeutic effects which could not be achieved by using only one MDR gene targeted therapy or single therapeutic agent delivery. Blocking the MDR using RNAi, anticancer drug is not only able to bypass efflux pump but also more effectively induce apoptosis at the same time, thus anticancer drug can get more opportunities to kill cancer cells. Given the challenging obstacles and great potentials of gene therapy, we envision that ultimate RNAi therapeutics for lung cancer would essentially be accompanied by well-organized knowledge of nanotechnology, genetic engineering and traditional pharmaceutical research. Financial & competing interests disclosure This work was supported partly by the National Research Foundation of Korea Grant funded by the Korean Govern-

Executive summary Nanoparticle-mediated siRNA delivery for lung cancer therapy • Lung cancer is one of the most lethal diseases worldwide with 1.6 million deaths annually. • Chemotherapy and radiation therapy are the conventional treatments for lung diseases. • Conventional therapies are being replaced by nanoparticle-mediated therapies recently. • Nanoparticle-mediated siRNA delivery has emerged as a promising approach to target the gene of interest.

Biological barriers to nanoparticle-mediated siRNA delivery • Nanoparticles need to overcome several extracellular and intracellular barriers to deliver siRNAs into cytoplasm within lung cancer cells. • Mode of endocytosis play a pivotal role in internalization and intracellular fate the nanoparticles.

Nonviral gene carriers for siRNA delivery in lung cancer therapy • Innumerable nonviral gene carriers have been developed for siRNA delivery so far, but only a few of them have entered clinical trials.

Nanoparticle-mediated codelivery of anticancer drug & siRNA for lung cancer therapy • Conventional chemotherapeutic treatments are associated with multidrug resistance, the most common cause of failure of chemotherapy. • To overcome multidrug resistance related genes, codelivery of anticancer drugs with siRNA has demonstrated higher efficacy in cancer treatment.

Nanoparticle-mediated codelivery of imaging agent & siRNA for lung cancer therapy • In clinical trials of lung cancer gene therapy, one of the crucial problems is the incapacity to trace and detect biodistribution and gene expression in the target tissue. • Codelivery of drugs and imaging agents together could trace the therapeutic effect of drug and imaging at the same time.



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Review Kim, Park, Singh et al. ment through the Ministry of Science, ICT and Future Planning (MSIP) (2014R1A1A2007163) and partly by the Oromaxillofacial Dysfunction Research Center for the Elderly at Seoul National University in Korea (#2012000912) of the National Research Foundation (NRF) funded by the Ministry of Science. The authors have no other relevant affiliations or

financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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High-Yield Synthesis of Monomeric LMWP(CPP)-siRNA Covalent Conjugate for Effective Cytosolic Delivery of siRNA.

Mono-arginine Cholesterol-based Small Lipid Nanoparticles as a Systemic siRNA Delivery Platform for Effective Cancer Therapy.

Gold nanoclusters-assisted delivery of NGF siRNA for effective treatment of pancreatic cancer.

Highly effective inhibition of lung cancer growth and metastasis by systemic delivery of siRNA via multimodal mesoporous silica-based nanocarrier.

Liposome-based co-delivery of siRNA and docetaxel for the synergistic treatment of lung cancer.

Design and development of effective siRNA delivery vehicles.

Targeted delivery of CXCR4-siRNA by scFv for HER2(+) breast cancer therapy.

Evaluation of dendrimer type bio-reducible polymer as a siRNA delivery carrier for cancer therapy.

siRNA Delivery from an Injectable Scaffold for Wound Therapy.

Targeted Therapy for Breast Cancer Stem Cells by Liposomal Delivery of siRNA against Fibronectin EDB.

Oral delivery of shRNA and siRNA via multifunctional polymeric nanoparticles for synergistic cancer therapy.

Liposomal siRNA nanocarriers for cancer therapy.

Antibody targeting facilitates effective intratumoral siRNA nanoparticle delivery to HER2-overexpressing cancer cells.

The pH-Triggered Triblock Nanocarrier Enabled Highly Efficient siRNA Delivery for Cancer Therapy.

Tumor-targeting multifunctional nanoparticles for siRNA delivery: recent advances in cancer therapy.

Multifunctional Envelope-Type siRNA Delivery Nanoparticle Platform for Prostate Cancer Therapy.

Pulmonary Codelivery of Doxorubicin and siRNA by pH-Sensitive Nanoparticles for Therapy of Metastatic Lung Cancer.

Co-delivery of hydrophobic paclitaxel and hydrophilic AURKA specific siRNA by redox-sensitive micelles for effective treatment of breast cancer.

Delivery materials for siRNA therapeutics.

Ligand-conjugated mesoporous silica nanorattles based on enzyme targeted prodrug delivery system for effective lung cancer therapy.

Co-delivery of HIF1α siRNA and gemcitabine via biocompatible lipid-polymer hybrid nanoparticles for effective treatment of pancreatic cancer.

Therapy. Targeted delivery of packaged siRNA promotes osteogenesis.

Functional polyesters enable selective siRNA delivery to lung cancer over matched normal cells.

Targetable micelleplex hydrogel for long-term, effective, and systemic siRNA delivery.