Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1575 – 1584

Stimuli-responsive liposomes for the delivery of nucleic acid therapeutics Fatemeh Movahedi, MS a , Rebecca G. Hu, PhD b , David L. Becker, PhD b , Chenjie Xu, PhD a,⁎ a

School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore b Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore Received 28 November 2014; accepted 11 March 2015

Abstract Nucleic acid therapeutics (NATs) are valuable tools in the modulation of gene expression in a highly specific manner. So far, NATs have been actively pursued in both pre-clinical and clinical studies to treat diseases such as cancer, infectious and inflammatory diseases. However, the clinical application of NATs remains a considerable challenge owing to their limited cellular uptake, low biological stability, off-target effect, and unfavorable pharmacokinetics. One concept to address these issues is to deliver NATs within stimuli-responsive liposomes, which release their contents of NATs upon encountering environmental changes such as temperature, pH, and ion strength. In this case, before reaching the targeted tissue/organ, NATs are protected from degradation by enzymes and immune system. Once at the area of interest, localized and targeted delivery can be achieved with minimal influence to other parts of the body. Here, we discuss the latest developments and existing challenges in this field. © 2015 Elsevier Inc. All rights reserved. Key words: Nanomedicine; Nanoparticle; Stimuli-responsive liposome; Controlled drug delivery; Nucleic acid therapeutics

Nucleic acid therapeutics (NATs) are composed of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) based molecules that aim to enhance or eliminate specific gene expression and consequently the levels of the related protein. 1 NATs have attracted much attention over the last decade as they have proven to be useful tools in deciphering complex biological processes, and demonstrated efficacy in ameliorating disease conditions that are related to abnormal expression of specific

gene or protein. 2 Unfortunately, most of the work is still confined to basic research in the laboratory and few have been translated into clinical applications. 3 This delay has been attributed to some extracellular and intracellular hurdles faced by NATs such as degradation, non-specificity and systematic toxicity (Figure 1). 4 The first challenge is the survival of NATs in the blood stream where NATs are exposed to serum nucleases, serum

Abbreviations: DC-Chol, 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol; DDAB, dimethyldioctadecylammonium bromide; DLPC, dilauroyl phosphatidylcholine; DMRIE, 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide; DMG, dimyristoyl-sn-glycerol; DOAB, dimethyldioctadecylammonium bromide; DODAP, 1,2-dioleoyl-3-dimethylammonium propane; DOPE, dioleylphosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3phosphoethanolamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DOPG, 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt); DOTAP, 1,2-dioleoyl-3-trimethylammonium propane or N-[1-(2,3-dioleoyloxy)]-N,N,N-trimethylammonium propane methylsulfate; DSG, distearoyl-sn-glycerol; DPPC, dipalmitoylphosphatidylcholine; DPPA, 1,2-dipalmitoyl-sn-glycero-3-phosphate acid; DSPC, 1,2-Distearoyl-sn-glycero-3-phosphatidylcholine; DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoetanolamine-N-[amino(polyethylene glycol)]; DSPE-PEG2000-NH2, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt); DSTAP, 1,2-distearoyl-3trimethylammonium-propane; MPB-PE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (sodium salt); PE, phosphatidylethanolamine; POPE, 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine; SOPC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine; SPION, super paramagnetic iron oxide nanoparticles; TNS, 6-(p-toluidino)-2-naphthalenesulfonic acid; TMAG, N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride. The XCJ lab is partially supported by the Tier-1 Academic Research Funds by Singapore Ministry of Education (RG 64/12 to XCJ). The DLB lab is supported by the Start-up Funds by Lee Kong Chian School of Medicine, Nanyang Technological University. ⁎Corresponding author at: School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore. E-mail address: [email protected] (C. Xu). 1549-9634/© 2015 Elsevier Inc. All rights reserved.


F. Movahedi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1575–1584

Figure 1. Extracellular and intracellular barriers in the delivery of nucleic acid therapeutics. Adapted with permission from Jones et al. 5 Copyright (2013) American Chemical Society.

proteins and immune cells. Any surviving NAT needs circulate to the target tissue while avoiding filtration by the kidneys. Once at the target tissue NATs face another set of challenges imposed by the vascular endothelial barrier which is tightly controlled, a dense network of extracellular matrix that hinders the diffusion of NATs, and the lipophilic cellular membrane that prevents the uptake of large and charged molecules. Finally, even after successfully entering cells, NATs have to escape the endosomes/lysosomes to reach the cytoplasm and in some cases the nucleus. One way to overcome these hurdles is to deliver NATs with nanoparticle (NP)-based carriers or nanocarriers. 6 NPs offer several advantages including: (i) NPs with the appropriate surface coating may increase the concentration and efficacy of NATs at the target site by reducing the binding of serum proteins and minimize degradation by serum nucleases 7; (ii) NPs can be designed to achieve regulated controlled release during transportation or at the site of action 8; (iii) it is feasible to incorporate both hydrophobic and hydrophilic drugs into NPs and improve bioavailability by enhancing the solubility of hydrophobic drugs 9; (iv) NPs can be decorated with specific ligands targeting specific receptors that were over-expressed on the diseased cells. 10 Among the wide range of NP-based platforms, liposomes are one of the most studied nanocarriers. 11 Liposomes consist of a biocompatible phospholipid bilayer and they can protect amphiphilic, hydrophilic, and/or hydrophobic drugs against a

variety of threats that lead to immediate dilution and degradation. 12 They enter cells mainly through the endocytosis pathway, in which the liposomes are engulfed by the plasmid membrane and taken into the cytoplasm. 13 Once inside the endosomes/lysosomes, there is an electrostatic interaction between the lipoplex (complex of liposome and nucleic acid) and cytoplasmic-facing side of endosomal membrane. This interaction leads to the formation of neutralized pairs, which disrupt the endosome/lysosome and release NATs to cytoplasm. 14 The use of liposomes for drug delivery started shortly after their invention by Bangham and coworkers in 1965. 15,16 The first liposome-based drugs (Myocet and Doxil) were approved by USA Food and Drug Administration (FDA) for cancer treatment in 1995. 17 Since then, a wide range of therapeutics including both hydrophobic (e.g. paclitaxel) and hydrophilic (e.g. hydroxyurea) small molecules have been successfully incorporated in liposomes to improve their efficacy and/or efficiency. Today there are about 8 commercialized liposomebased products and another 53 liposomal formulations in different stages of clinical trials. 18,19 The success of liposomes in optimizing small molecule drugs has driven researchers to explore the possibility of addressing the challenges met by NATs in vivo. 20,21 An advantage of using liposomes is their biocompatibility and biodegradability since most are made with naturally occurring lipids. 22-25 In addition, the composition-dependent surface charge of liposomes can help

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to control the interaction with nucleic acids. The inclusion of lipophilic drugs in the lipid bilayers makes it possible to co-deliver drug and nucleic acid therapeutics. 26,27 Other attractive advantages of liposomes include their flexibility of coupling with site specific ligands for active targeting. 28 Table 1 lists some representative efforts of using liposomes to deliver NATs. While these pioneering studies confirm the potential of liposomes in prolonging the circulation time, enhancing the therapeutic efficacy and reducing the toxic side effects of NATs they also revealed some limitations such as low delivery efficiency and non-specific targeting. 36,37 One approach to address these challenges is to utilize trigger-responsive liposomes. 38,39 These liposomes change their structure or conformation when subjected to internal (e.g. pH, enzyme) or external (e.g. temperature, magnetic field) stimuli. Consequently, the release of cargo drugs can be precisely controlled and concentrated at the target site. 40 Furthermore, since it can be regulated to release the drug at the target site, the drug can be protected more efficiently and significantly improve the delivery efficiency. The research topic has been getting more and more attention in recent years more than 300 papers published last year, most of which included animal studies (Figure 2). In the following, we summarize the recent efforts in utilizing these ‘smart’ liposomes for the delivery of NATs.

Stimuli-responsive liposomes for NAT delivery Exogenous stimuli-responsive liposomes for NAT delivery Exogenous stimuli refer to stimuli that are applied externally such as light, heat, magnetic field and ultrasound. 41 Application of exogenous stimuli enables pulsatile drug release which can be defined as transient and fast release of a certain amount of drug in a short period of time. 42 The advantage being that since the stimuli are generated by machines protocols can be easily standardized to achieve regulated release. 38 In addition, stimuli can be applied to specific site to target release. 43 Although there are many types of exogenous stimuli, magnetic field and ultrasound have been the major strategies investigated due to their ability to penetrate into deeper tissue enhancing their potential in translating from bench to bedside. Magnetically responsive liposomes Magnetically responsive liposomes are liposomes that contain magnetic elements (e.g. Fe3O4 or Fe2O3 NPs). The presence of magnetic elements allows liposomes to be localized at a target site in response to an external magnetic field. Meanwhile, magnetic elements in liposomes can also act as contrast agents for magnetic resonance imaging (MRI). 44 Finally, when an alternating magnetic field is present, the local temperature around the magnetically responsive liposomes can be increased which may enhance the efficacy of NATs if the target gene is sensitive to temperature. 38 The idea of using magnetic systems to deliver nucleic acids is not new. 45 One decade ago, magnetic NPs were combined with nucleic acids and cationic molecules to form transfection complexes – termed magnetofection – to enhance transfection efficiency. 46,47 Here the time to reach maximum transfection


efficiency was significantly shorter with magnetically responsive liposomes. Hirao et al developed magnetic cationic liposomes composed of PE and DC-Chol and demonstrated their efficiency by transfecting plasmid DNA containing a luciferase reporter gene into the human osteosarcoma cells (Saos-2). 48 Under a magnetic field, maximum luciferase activity was achieved within 30-minutes of magnetic induction which was 3.5-fold faster than that without magnetic induction. The application of this delivery system was further explored by measuring the apoptosis rates after transfection of the p53 gene into Saos-2, which increased from 2.4% to 18.9% upon magnetic induction. Such a gene delivery system is expected to be applicable under in vivo environment and may provide several clinical advantages. Wang et al examined the magnetic complex of Lipofectamine 2000 and combiMAG (aqueous dispersion of SPIONs coated with a monolayer of polyethyleneimines) and plasmid DNA for magnetofection of plasmids expressing Green fluorescent protein (GFP) and short hairpin RNAs targeting type 1 insulin-like growth factor receptor (IGF-1R) in both cancer cells and tumor-bearing mice. SPION-mediated transfection was considerably enhanced in presence of magnetic field. In comparison with lipofection, liposomal magnetofection resulted in a 3-fold transgene expression as well as 30% higher transfection efficiency. TEM images indicate that the mechanism of the cellular uptake of transfected genes and SPIONs is similar to other transfection methods; i.e., which is a clathrin-dependent endocytosis process. 49 The key point about this system is application of commercially available SPION and Lipofectamine 2000 under the influence of a static external magnetic field which is quite straightforward to set up and offers a novel method for gene therapy of lung cancer. Namiki et al developed a novel cationic magnetic liposome (LipoMag) by encapsulating hydrophobic magnetic NPs with phospholipid. 50 The therapeutic nucleic acids were coated on the surface of LipoMag – consists of an oleic acid-coated magnetic nanocrystal core and a cationic lipid shell – through the electrostatic interaction between the positively charged groups of cationic lipid molecules and the negatively charged groups of nucleic acids. By screening the transfection efficiency of luciferase gene plasmid, the authors selected the (O,O′-ditetradecanoyl-N-(α-trimethlammonioacetyl) diethanolamine chloride) and DOPE (molar ratio: 1:0.04) as the preferred composition (D6DOM). Later, D6DOM was able to deliver glyceraldehyde phosphate dehydrogenase (GAPDH)targeting siRNA into various cancer cells in vitro to knock down the expression of GAPDH under a magnetic field. Finally, with this platform, the authors knocked down the epidermal growth factor receptor (EGFR) mRNA in tumor vessels, which suppressed the growth of tumors in mice. In summary, LipoMag system demonstrated more efficient gene silencing for 9 of 13 cell lines compared with commercially available delivery systems as well as better anti-tumor effect for mice bearing tumors without any adverse side effects. Clearly, the system has a high potential of gene delivery especially for tumor vessel-targeted RNA interference. Liposomal magnetofection under a dynamic gradient magnetic field might have even higher transfection efficiencies. Vainauska et al used liposomes composed of Lipofectamine 2000 and Fe3O4 NPs. Under a dynamic gradient magnetic field, they achieved less toxicity and 21% and 42% higher transfection


F. Movahedi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1575–1584

Table 1 Liposomes for the delivery of nucleic acid therapeutics. Vector

Nucleic acids

DMRIE/DOPE liposome

Human histocompatibility (HLA) B7 gene




The plasmid-liposome complex was evaluated in mice and pigs for safety, toxicity and efficacy in inducing an antitumor response DOTAP cationic liposome Cystic fibrosis transmembrane DOTAP liposomes with CFTR gene were conductance regulator delivered to the nasal epithelium of cystic (CFTR) gene fibrosis patients


Egg phosphatidylcholine, cholesterol, and DSPE-PEG


DDAB/DOPE liposome

DOPC liposome

Liposomes consisting of polycationic lipids and lipid-helper DOPE

Liposomes containing DOTAP, cholesterol, DOPE, polyethylene glycol (PEG) 2000-C16Ceramide

Enhanced transfection efficiencies in vitro with a magnitude of two to seven folds. No quantification for in vivo results. Transgene DNA was detected in seven of the eight treated patients up to 28 days after treatment. Levels of gene transfer and functional correction were comparable to those reported using adenoviral vectors, but for longer and uncompromised by adverse effects. BCL2 mRNA as suppressors of Examine the suppression of the pump An additional activation of caspase pump and non-pump resistance and non-pump resistance of tumor cells dependent apoptotic pathway and in vitro and in vivo after systemic enhanced apoptosis induction by the administration of liposomes anticancer drug, doxorubicin. This enhancement led to the dramatic increase in the toxicity in vitro and tumor suppression activity in vivo of liposomal doxorubicin. HPV 16 E7 mRNA Proliferation of CaSki cells was studied Use of liposomes significantly antisense oligonucleotides by the MTT assay after exposure to increased the cellular uptake plasmid-liposome complex of oligonucleotides siRNA targeting the Liposomes encapsulating siRNA were Three weeks of treatment with oncoprotein EphA2 tested in an orthotopic mouse model of plasmid-liposome complex reduced ovarian cancer tumor growth when compared with a non-silencing siRNA. When the plasmid-liposome complex was combined with paclitaxel, tumor growth was dramatically reduced compared with treatment with paclitaxel and a non-silencing siRNA. pEGFP-C2 DNA and Plasmid-liposome complex was In comparison with the control and RNA-EGFP for tuning the introduced into dendritic cells (DCs) in 1.9-2.3-fold decrease of the lung transfection efficiency. progenitors and immature DCs of bone metastases in comparison with the RNA-B16 for suppression of marrow origin in vitro. The use of these group of animals injected with melanoma metastases. DCs induced the suppression of B16 Lipofectamine 2000/RNA melanoma metastases in vivo. B16-loaded DCs siLamin A/C siRNA Liposomes containing siRNA were highly Following systemic delivery of effective at reducing Lamin A/C mRNA liposomes, 45 ± 2% of epithelial expression in not only endothelial cells but murine lung cells receive siRNA also epithelial cells of the lung following delivery upon intravenous (IV) IV delivery. liposomal administration

efficiency in PC3 cells compared with liposomal magnetofection and lipofection respectively. 51 Also, Baryshev et al reported application of a newly developed device called “DynaFECTOR” for magnetofection under a dynamic magnetic field. 52 In contrast to a static field, application of dynamic gradient magnetic field prevents non-uniform distribution of aggregate complexes on the surface of HepG2 cells due to enhanced contact with the cellular membrane which stimulated endocytosis. 52 In addition to utilizing magnetic attraction to localize NATs at target sites, oscillating magnetic field can also be applied to induce hyperthermia treatment to enhance certain gene expressions. Mild hyperthermia has been shown to improve the effect of chemotherapy and immunotherapy treatment with TNF-α and IL2. 53,54 Such an effect was further examined in the U87MG glioblastoma cell line. Bouhon et al encapsulated both the interferon-β (IFN-β) gene and magnetic NPs within cationic






liposomes of DOPE, DLPC, and TMAG. The IFN-β gene was driven by a heat inducible promoter called the glucocorticoidinducible mouse mammary tumor virus (MMTV) 55 since in some studies, it was shown that hyperthermia can enhance the effect of chemotherapy. 56,57 Bouhon et al showed that less IFN-β expression was detected without hyperthermia and only a drop of 45-55% viability rate. By applying a high frequency magnetic field to induce mild hyperthermia a significantly higher amount of IFN-β was found in the cells and the viability rate was reduced to only ~ 10%. Thus, the combination of IFN-β cytokine gene expression and mild hyperthermia can have a synergic cell killing effect due to increasing the sensitivity of transfected cells. In the light of the above discussion, it is clear that magnetically responsive liposomes can be introduced as efficient systems for NAT delivery while they can allow the tracking of the drugs through MRI. Their biggest advantage is their response

F. Movahedi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1575–1584

Figure 2. Publication analysis in the past five years for stimuli-responsive liposomes in the delivery of nucleic acid therapeutics (search key words: liposome, stimuli-sensitive, nucleic acid, gene, oligonucleotide, and animal; search engine: Google Scholar).

to the magnetic field that has no limitation to penetrate into the deep tissue. 38 However, all existing magnetically responsive liposomes are composing of magnetic materials, whose biocompatibility and biodegradability need to be studied carefully. In addition, due to the big density of magnetic materials, the drug loading capacity of these liposomes is usually low. Finally, if these magnetically responsive liposomes are used for delivering drugs into the deep tissue/organ such as heart, there is a need to develop a technology to precisely position the magnetic field in a non-invasive way. Acoustically responsive liposomes Acoustically responsive liposomes are developed for diagnostic imaging and ultrasound mediated gene or drug delivery. 58 They were reported for the first time in 1996 by Alkan et al. 59 Later, Huang et al worked extensively on improving synthesis methods. 60-62 Acoustically responsive liposomes are usually synthesized by entrapping a small amount of echo-contrast gas within liposomes, which can function as a novel delivery tool for antisense oligonucleotide or plasmid DNA. 63 When ultrasound is applied, gas bubbles oscillate and generate stress on the lipid membrane. Liposomes rupture to release their encapsulated contents when the stress exceeds elastic limitation of liposome membrane. In addition to the controlled release, the disruption of bubble liposomes can cause cavitation on the neighboring cells/ tissue, which further facilitates the permeation of therapeutic agents. 64,65 The key determining factor for drug release from acoustically responsive liposomes is the ultrasound frequency. In general, both high and low frequency ultrasound can be used; although for accurate targeted delivery, high frequency ultrasound is preferred. 66 However, low frequency ultrasound can penetrate deeper into the tissue. 67 A recent concept is using dual frequency ultrasound 68 which can be investigated as a potential approach for NAT delivery. Un et al developed an ultrasound-responsive mannose-modified gene carriers, Mannose-PEG2000 bubble lipoplexes, by enclosing perfluoropropane gas into mannose-conjugated PEG2000-DSPE-modified cationic liposomes (DSTAP: DSPC: Man-PEG2000-DSPE)/plasmid complexes. 69 In vitro, the application of ultrasound enabled 500-fold higher gene expression


(luciferase) than without ultrasound in mouse cultured macrophages, abundantly expressing mannose receptors. It was believed that this enhanced gene expression was due to the direct entrance of bubble lipoplexes through the temporarily perforated cell membrane without mediating endocytosis and subsequently improved cellular association of plasmid from the lipoplexes. In vivo, similar results were observed. The level of gene expression with Man-PEG2000 bubble lipoplexes and ultrasound exposure was 500-800-fold higher than with Man-PEG2000 lipoplexes alone. It was also higher than with Bare-PEG2000 bubble lipoplexes and ultrasound exposure or the conventional sonoporation method using naked plasmid and bubble liposomes in the liver and spleen, which were the target organs of mannose-modified carriers. With the proven efficacy, the group demonstrated the anti-tumor effects against E.G7-OVA cells in mice immunized with Man-PEG2000 bubble lipoplexes constructed with pCMV-OVA (ovalbumin-expressing plasmid DNA) and exposed to ultrasound. Significantly higher anti-tumor effects against E.G7-OVA cells were observed. The major superiority of this system lies behind its contribution to overcome poor introducing efficiency of carriers into cytoplasm which is the main obstacle of NAT delivery by non-viral vectors. Recently, Koebis et al reported effective in vivo delivery of an antisense oligonucleotide by perfluoropropane bubble liposomes composed of DPPC and DSPE-PEG for the treatment of myotonic dystrophy type 1 (DM1). 70 The characteristic feature of the pathology of DM1 is the aberrant regulation of dozens of alternative splicing events including the abnormal splicing of the chloride channel 1 (CLCN1) gene. A promising treatment for DM1 is to correct the abnormally regulated splicing with antisense oligonucleotides such as phosphorodiamidate morpholino oligonucleotide (PMO) with great advantage of remarkable innocuity. To facilitate the penetration of PMO deeper into tissues and improving the efficiency, the researchers constructed bubble liposomes with DPPC and DSPE-PEG2000 (molar ratio of 94:6) and perfluoropropane gas. With this platform, they delivered antisense PMOs targeting exon 7A of the CLCN1 gene into skeletal muscles of HSA LR mice and reduced the inclusion of exon 7A. With some modification of the liposome composition (DSPC, cationic lipid DSDAP, and DSPEPEG2000 [molar ratio of 64:30:6]), the same team delivered micro RNA-126 (miR-126) into a hindlimb ischemia model. 71 The miR-126 promotes angiogenesis via the inhibition of negative regulators of VEGF signaling. They demonstrated that the liposomes-miR-126 complex reached the ischemic site after intravascular injection and delivered miR-126 following exposures to ultrasound. Subsequently, miR-126 caused the induction of angiogenic factors and improved blood flow. Enhancing the efficiency of PMO delivery using Bubble liposomes and ultrasound is a great achievement since it offers a promising safe treatment with high pharmaceutical potentials while its application have been limited due to insufficient cellular uptake. Apart from delivering NATs, Yin et al examined the co-delivery of siRNA and chemotherapeutic drug paclitaxel (PTX) by ultrasound-responsive liposomes composed of DPPC, cationic DSPE and DPPA to fight against drug resistance in chemotherapy. 72 The ultrasound-responsive liposomes were


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prepared in two steps. First, PTX-loaded liposomes were synthesized using the traditional thin-film hydration-sonication method with DPPC, DSPE, and DPPA. Secondly, octafluoropropane (C3F8) gas was bubbled into the liposomes through sonication and siRNA targeting antiapoptosis genes (i.e. BCL-2) was complexed with the bubble liposomes. PTX/siRNA liposomes were injected into nude mice bearing human HepG2 xenografts by tail vein injection. When low frequency ultrasound was imposed at the tumor site, effective tumor-penetrating co-delivery of siRNA and PTX was achieved. Subsequently, the PTX treatment-inducible anti-apoptosis in HepG2 cells was effectively suppressed. Due to the synergistic anti-cancer effect of the two therapeutic agents, tumor growth was completely inhibited using low-dose of PTX in animal study. Such a co-delivery system can overcome the BCL-2 mediated drug resistance and enable low dose but effective PTX treatment. In a nutshell, acoustically responsive liposomes provide a promising non-ionizing radiative method for delivering NATs. It is possible to regulate the penetration depth by tuning the frequency and exposure time of the ultrasound, 38 which is their main advantage over magnetically-responsive liposomes. However, it should be kept in mind that ultrasound has difficulty penetrating bone. 73 Therefore, the acoustically responsive liposomes should not be used for tissue/organ related with bone. Additionally, for the gas-based liposomes, the stability during the long-term storage should be considered. Endogenous stimuli responsive liposomes for the NAT delivery Endogenous stimuli arise from the differential microenvironment between normal tissues and diseased areas, such as higher redox potential, reduced intercellular/intracellular pH and increased level of certain enzymes. In comparison to the exogenous stimuli, endogenous stimuli exist inherently and thus do not require external equipment to elicit stimulation. This is beneficial for the application in clinical settings. In addition, one of the key challenges in NAT delivery is the limited cytoplasmic delivery due to degradation in certain pH environments and/or enzymes in the endosomallysosomal trafficking pathway (Figure 1). 74 The pH/enzyme responsive nanocarriers can recognize the unique microenvironment in the endosome/lysosome, and destabilize the membrane to facilitate the escape of therapeutics across the endosomal barrier.38 Redox-responsive liposomes A major difference of redox potential exists between the reducing intracellular space and oxidizing extracellular space or between normal and tumor tissues. For example, intracellular concentration of glutathione (i.e. redox potential) in cancer cells is significantly higher (100-fold) than normal extracellular level of glutathione. Therefore, liposomes responding to this drastic potential difference can be utilized for the construction of stimuli-sensitive liposomes. 75,76 This type of liposome is usually made of standard phospholipids and a small portion of lipid with both hydrophilic and hydrophobic parts which are linked through disulfide bonds. Their structure is maintained under normal condition by disulfide bonds. Upon entering the intracellular space, the disulfide bonds will be perturbed, which destabilizes the liposomes and releases the encapsulated cargo. 77

As early as 1999, researchers had already shown that the disulfide-containing cationic liposomes were more efficient at transfecting GFP plasmid into brain cells than the non-disulfide ones due to potential function of reversibility of thiol–disulfide exchange reaction. 78 Balakirev et al prepared cationic liposomes with natural pro-vitamin and lipoic acid. 79 Under the oxidized condition, liposomes condensed plasmid DNA into homogenous spherical particles and the complex was stable enough to protect DNA from degradation. Upon reduction, spherical NPs swelled and transformed into toroids (due to thiol–disulfide exchange reaction), dissembled and released DNA. Using this redoxresponsive system, transgene expression was increased several fold due to glutathione and nicotinamide adenine dinucleotide phosphate-oxidase (NADPH)-dependent complex reduction. Recently, Wang et al reported the highly efficient delivery of siRNA with bioreducible lipid-like materials (termed “lipidoid”) integrating disulfide bonds, which are degradable in the presence of thiol-containing biomolecules (Figure 3, A). 80 These lipidoids were synthesized via Michael addition of aliphatic amines and acrylate, incorporating disulfide bonds, and could encapsulate siRNA via electrostatic interaction in the form of NPs. Using siRNA targeting GFP as a model the researchers demonstrated the successful suppression of GFP expression in cells with bioreducible lipidoids/siRNA complexes (Figure 3, B). Specifically, naked siRNA treatment has no effect on suppressing GFP expression, suggesting that naked siRNA has inefficient cellular uptake or instability in serum. The lipidoid/siGFP-treated cells, however, display varying levels of GFP expression depending on the specific lipidoid used in the lipidoid/siRNA lipoplexes. All nonbioreducible lipoplex treatments suppressed GFP expression slightly; the most efficient nonbioreducible lipidoid, 2-O14, reduced GFP expression to 80% of controls. In contrast, the bioreducible lipidoid/siRNA complexes silenced GFP expression with much higher efficiency than the nonbioreducible lipidoids that contained the same amine head and hydrophobic tail length. Finally, they were able to deliver siRNA-targeting polo-like kinase 1 (Plk-1) into cancer cells to deplete Plk-1 and inhibit tumor cell proliferation (Figure 3, C). It can be considered as a model system for integration of disulfide bonds into liposomes for facilitating NAT release in response to intracellular GSH. The presence of helper lipids is the other factor that may improve transfection efficiency of redox-sensitive liposomes. Gabriele et al showed that helper lipids with unsaturated dioleoyl chains and phosphatidylethanolamine head groups could improve transfection efficiency of DOPC/DOPE/SS14 redox-responsive liposomes considerably in U87-MG cells. 81 They reported 33.4% transfection efficiency of plasmid DNA (pEGFP-N1) (compared with 12.4% using Lipofectamine 2000®). In addition, their study demonstrated a linear correlation between transfection efficiency and intracellular glutathione level while no linear correlation between oxidative stress level and transfection efficiency has been observed. Their work has been limited to in vitro study and future in vivo studies for confirming the effect of helper lipids on transfection efficiency looks inevitable. Through our literature search, we realized that redox-responsive liposomes have not been fully explored for the delivery of NATs although this concept was proposed a decade ago. Clearly there is lots of work to be carried out in this field particularly by

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Figure 3. Enhanced intracellular siRNA delivery with bioreducible lipid-like NPs. (A) Schematic illustration of siRNA delivery with bioreducible Lipid-like NPs. (B) GFP expression of GFP-MDA-MB-231 cells treated with naked siRNA, lipidoid/siRNA nanocomplexes, and Lipofectamine 2000 (LPF 2000)/siGFP complexes. Data are presented as mean ± SD (n = 3, the two asterisks refer to statistical significance between bioreducible and nonbioreducible lipidoid facilitated siRNA delivery, P b 0.05, Student's t test). (C) Bioreducible lipidoid 1-O16B facilitated siPlk-1 delivery reduced the viability of different cancer cells. The cell viability was measured by AlamarBlue assay. Data are presented as mean ± SD (n = 3, P b 0.05). Adapted with permission from Wang et al. 80 Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

considering the fact that they are more complicated compared with exogenous-responsive systems. pH-responsive liposomes pH-responsive liposomes are stable in physiological condition (pH 7.4) but destabilize in acidic environments that are common in some pathological tissues such as tumors. 82 Such liposomes contain acidic groups which work as the stabilizer in neutral pH whereas under acidic conditions, carboxylic groups are protonated and destabilize the system. 83 One example is liposomes composed of PE and its derivatives like DOPE. Due to poor hydration of the head group of PE and its derivatives, they have high affinity to lipophilic cell membrane and facilitate cell entery. 84 Two decades ago, Ropert et al encapsulated an antisense complementary to the region of the initiation codon AUG of the Env gene mRNA of Friend retrovirus which was an inhibitor of the translation of Env protein in vitro. 85 They used DOPE, oleic acid and cholesterol in ratio 10:5:2 for pH-sensitive liposomes and DOPC, oleic acid and cholesterol in ratio 10:5:2 for non pH-sensitive liposomes. The results showed higher activity of AUG antisense encapsulated in pH-sensitive liposomes and the ability to avoid lysosomal degradation of oligomers. One decade later, Biddanda et al fabricated the pH-sensitive liposomes with PE, fusogenic at acidic pHs; cholesteryl hemisuccinate, liposome stabilizer at physiologic pH; and cholesterol (molar ratio of 7:4:2) to deliver antisense phosphorothioate oligonucleotide (TJU-2755) against tumor necrosis factor-α (TNF-α), one of the key cytokines associated with liver injury. 86 The formulation was stable for more than 4 weeks at pH 7.4, but readily released the encapsulated contents when exposed to an acidic environment below pH 6. In the

animal study, liposome-encapsulated TJU-2755 was administered (i.v.) into the rats followed by a single dose of lipopolysaccharide 48 hours later. The authors observed a 30% reduction in TNF-α produced by the liver. Two doses of antisense oligonucleotide daily inhibited TNF-α production by 50%. These results indicate that TJU-2755 encapsulated in pH-sensitive liposomes can be used to effectively reduce endotoxin-mediated production of TNF-α in macrophages in vivo and thus may be of value in attenuating or preventing macrophage-mediated liver injury. Recently, Harashima et al synthesized a new pH-sensitive cationic lipid (i.e. YSK05) that could self-assemble the multifunctional envelope-type nanodevice (MEND). 87 They formulated anti-polo-like kinase 1 (PLK1) siRNA (siPLK1) in YSK05-MEND that was topically administrated on subcutaneous tumors. The YSK05-MEND resulted in a more efficient gene silencing compared with MENDs containing conventional cationic lipids (i.e. DOTAP and DODAP). The researchers claimed that YSK05 facilitates the endosomal escape of the MEND and thereby enhances the efficacy of siRNA delivery into cytosol and gene silencing. In a subsequent study, this team encapsulated siRNA against vascular endothelial growth factor receptor 2 within YSK-MEND. 88 The as-synthesized NPs were further conjugated with cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD), a well-known ligand to αVβ3 integrin on tumor endothelial cells. In both in vitro and in vivo experiments, the RGD-YSK-MEND caused significant gene silencing in tumor endothelial cells, but not in endothelial cells in normal organs and cancer cells without severe toxicity. In summary, pH-triggered liposomes are attractive systems for NAT delivery particularly because of lower pH in microenvironment which is common in both primary and


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metastasized tumors. However, it is undeniable that regulation and tuning the system are not that simple due to complexity of the microenvironment that varies case by case. Other stimuli-responsive liposomes for NAT delivery Besides the above stimuli-responsive liposomes, there are other potential strategies deemed to be suitable for NAT delivery. One is the well-known thermo-responsive liposome, which have been successfully developed for drugs such as doxorubicin (with trade name as ThermoDox). Zhao et al reported co-delivery of doxorubicin and SATB1 shRNA by thermo-responsive liposomes composed of DPPC, DC-Chol, and DOAB. In vitro experiments showed considerable reduction in cancer cell viability and in vivo studies demonstrated significant decrease of tumor volume. 89 In order to improve NAT release, lysolipid-containing thermosensitive liposomes that contain lysolipid in DPPC bilayers could be a good option as well. 90 Alternatively, incorporation of metals in the liposome structure may improve responsiveness. For instance, Bouhon et al examined liposomes of DOPE, DLPC, and TMAG incorporated with magnetite NPs as heating material for delivering plasmid bearing the interferon (IFN)-β, which triggered high expression of IFN-β in glioma cells. 91 The number of surviving cells declined to 20% within 1 hour of magnetic heat generation and cytokine transfection. Another applicable system is photo-sensitive liposome. Dave et al presented DNA-functionalized Au-liposomes in which gold NPs could either promote leakage from liposomes by UV radiation or protect them against it, depending on DNA sequence. 92 The hybrid nanostructure includes a soft liposomal part composed of DOPC, cholesterol, DOPG and MPB-PE as well as a hard part of gold NPs since Au NPs can effectively absorb radiation energy. Although application of Au liposome hybrids is accompanied with the problem of non-specific targeting as well as irreversible electrostatic interactions, this system seemed to be both programmable and reversible. Hence, tuning such system makes it possible to promote leakage from liposome or inhibit it. Enzyme-sensitive liposomes might also be suitable for NAT delivery. Wan et al reported significant enhancement in tumor cell transduction with reduced toxicity and immunogenicity of the vector, by using enzymatically cleavable PEG-liposome composed of PEG/matrix metalloproteinase (MMP)-substrate peptide/cholesterol. 93 Another approach is to make use of the sensitivity of certain liposome formulation to small metabolites. El-hamed et al developed a liposomal system attached to DNA-functionalized hydrogel by a DNA aptamer linker. The DNA aptamer linker could bind to adenosine (AMP and ATP) and release the encapsulated compounds. 94,95 Outlook and conclusions The development of NATs has suffered from non-specific targeting, ineffective delivery, and low transfection efficiency. However, stimuli-responsive liposomes offer active delivery and make tailored NAT release possible. In this review, common stimuli such as magnetic field, ultrasound, redox potential, and pH have been discussed for NAT delivery using stimuli responsive liposomes. In spite of numerous successful in vitro studies, organ

specific delivery and in vivo efficiency are still low and there is a lack of translation into clinical studies. Additionally, some in vitro studies failed to perpetuate success in in vivo situations, which are often confounded with other intrinsic factors. 38 It is also vital for stimuli responsive systems to possess the capability of regulated assembly during delivery and disassembly for drug release. 96 The materials must be designed in a way smart enough to elicit ON-OFF response to stimuli (assembly or disassembly). However, this process is not always reproducible due to the inconsistent responses observed. Standardization and optimization remain a challenge. To overcome the problems mentioned above, a thorough understanding of the mechanism of action is required. Deciphering the physiological differences between normal tissue and target sites can also help to make improvements 75 and introducing more sensitive liposome structures will also be essential. Overall, more studies should concentrate on improving simple and reproducible stimuli-responsive liposomal nucleic acid delivery systems that are clinically acceptable. The number of research groups focusing their investigations on this field promises more advancement in the near future.

References 1. Videira M, Arranja A, Rafael D, Gaspar R. Preclinical development of siRNA therapeutics: towards the match between fundamental science and engineered systems. Nanomedicine 2014;10:689-702. 2. Pushpendra S, Arvind P, Anil B. Nucleic acids as therapeutics. In: Erdmann VA, Barciszewski J, editors. From Nucleic Acids Sequences to Molecular Medicine. Berlin Heidelberg: Springer; 2012. p. 19-45. 3. Lehner R, Wang X, Marsch S, Hunziker P. Intelligent nanomaterials for medicine: carrier platforms and targeting strategies in the context of clinical application. Nanomedicine 2013;9:742-57. 4. Mannell H, Pircher J, Fochler F, Stampnik Y, Rathel T, Gleich B, et al. Site directed vascular gene delivery in vivo by ultrasonic destruction of magnetic nanoparticle coated microbubbles. Nanomedicine 2012;8:1309-18. 5. Jones CH, Chen C-K, Ravikrishnan A, Rane S, Pfeifer BA. Overcoming nonviral gene delivery barriers: perspective and future. Mol Pharm 2013;10:4082-98. 6. Nayerossadat N, Maedeh T, Ali PA. Viral and nonviral delivery systems for gene delivery. Adv Biomed Res 2012;1:27. 7. Phillips MA, Gran ML, Peppas NA. Targeted nanodelivery of drugs and diagnostics. Nano Today 2010;5:143-59. 8. De Jong WH, Borm PJ. Drug delivery and nanoparticles: applications and hazards. Int J Nanomedicine 2008;3:133-49. 9. Gelperina S, Kisich K, Iseman MD, Heifets L. The potential advantages of nanoparticle drug delivery systems in chemotherapy of tuberculosis. Am J Respir Crit Care Med 2005;172:1487-90. 10. Gao H, Yang Z, Zhang S, Cao S, Shen S, Pang Z, et al. Ligand modified nanoparticles increases cell uptake, alters endocytosis and elevates glioma distribution and internalization. Sci Rep 2013;3. 11. Lee Y, Kataoka K. Delivery of Nucleic Acid Drugs. Advances in Polymer Science 2012;249:95-134. 12. Kulkarni PR, Yadav JD, Vaidya KA. Liposomes: a novel drug delivery system. Int J Curr Pharm Res 2011;3:10-8. 13. Ziello JE, Huang Y, Jovin IS. Cellular endocytosis and gene delivery. Mol Med 2010;16:222-9. 14. Yuan X, Naguib S, Wu Z. Recent advances of siRNA delivery by nanoparticles. Expert Opin Drug Deliv 2011;8:521-36. 15. Lindner LH, Hossann M. Factors affecting drug release from liposomes. Curr Opin Drug Discov Devel 2010;13:111-23.

F. Movahedi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1575–1584 16. Bangham AD, Standish MM, Watkins JC. Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol 1965;13:238-52. 17. Pinheiro M, Lucio M, Lima JL, Reis S. Liposomes as drug delivery systems for the treatment of TB. Nanomedicine (Lond) 2011;6:1413-28. 18. Swami A, Shi J, Gadde S, Votruba A, Kolishetti N, Farokhzad O. Nanoparticles for targeted and temporally controlled drug delivery. In: Svenson S, Prud'homme RK, editors. Multifunctional Nanoparticles for Drug Delivery Applications. Springer US; 2012. p. 9-29. 19. Etheridge ML, Campbell SA, Erdman AG, Haynes CL, Wolf SM, McCullough J. The big picture on nanomedicine: the state of investigational and approved nanomedicine products. Nanomedicine 2013;9:1-14. 20. Jarver P, Coursindel T, Andaloussi SEL, Godfrey C, Wood MJA, Gait MJ. Peptide-mediated cell and in vivo delivery of antisense oligonucleotides and siRNA. Mol Ther Nucleic Acids 2012;1:e27. 21. Kanasty R, Dorkin JR, Vegas A, Anderson D. Delivery materials for siRNA therapeutics. Nat Mater 2013;12:967-77. 22. Oh Y, Park T. siRNA delivery systems for cancer treatment. Adv Drug Deliv Rev 2009;61:850-62. 23. Portillo J, Kamar N, Melibary S, Quevedo E, Bergese S. Safety of liposome extended-release bupivacaine for postoperative pain control. Front Pharmacol 2014;5:90. 24. Rafiyath SM, Rasul M, Lee B, Wei G, Lamba G, Liu D. Comparison of safety and toxicity of liposomal doxorubicin vs. conventional anthracyclines: a meta-analysis. Exp Hematol Oncol 2012;1:10. 25. Alhajlan M, Alhariri M, Omri A. Efficacy and safety of liposomal clarithromycin and its effect on Pseudomonas aeruginosa virulence factors. Antimicrob Agents Chemother 2013;57:2694-704. 26. Wang H, Zhao P, Su W, Wang S, Liao Z, Niu R, et al. PLGA/polymeric liposome for targeted drug and gene co-delivery. Biomaterials 2010;31:8741-8. 27. Shim G, Han SE, Yu YH, Lee S, Lee HY, Kim K, et al. Trilysinoyl oleylamide-based cationic liposomes for systemic co-delivery of siRNA and an anticancer drug. J Control Release 2011;155:60-6. 28. Costa PM, Cardoso AL, Mendonca LS, Serani A, Custodia C, Conceicao M, et al. Tumor-targeted chlorotoxin-coupled nanoparticles for nucleic acid delivery to glioblastoma cells: a promising system for glioblastoma treatment. Mol Ther Nucleic Acids 2013;2:e100. 29. San H, Yang ZY, Pompili VJ, Jaffe ML, Plautz GE, Xu L, et al. Safety and short-term toxicity of a novel cationic lipid formulation for human gene therapy. Hum Gene Ther 1993;4:781-8. 30. Porteous DJ, Dorin JR, McLachlan G, Davidson-Smith H, Davidson H, Stevenson BJ, et al. Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther 1997;4:210-8. 31. Pakunlu RI, Wang Y, Saad M, Khandare JJ, Starovoytov V, Minko T. In vitro and in vivo intracellular liposomal delivery of antisense oligonucleotides and anticancer drug. J Control Release 2006;114:153-62. 32. Lappalainen K, Urtti A, Jääskeläinen I, Syrjänen K, Syrjänen S. Cationic liposomes mediated delivery of antisense oligonucleotides targeted to HPV 16 E7 mRNA in CaSki cells. Antivir Res 1994;23:119-30. 33. Landen CN, Chavez-Reyes A, Bucana C, Schmandt R, Deavers MT, Lopez-Berestein G, et al. Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res 2005;65:6910-8. 34. Markov OO, Mironova NL, Maslov MA, Petukhov IA, Morozova NG, Vlassov VV, et al. Novel cationic liposomes provide highly efficient delivery of DNA and RNA into dendritic cell progenitors and their immature offsets. J Control Release 2012;160:200-10. 35. McCaskill J, Singhania R, Burgess M, Allavena R, Wu S, Blumenthal A, et al. Efficient biodistribution and gene silencing in the lung epithelium via intravenous liposomal delivery of siRNA. Mol Ther Nucleic Acids 2013;2:e96. 36. Saul JM, Annapragada A, Natarajan JV, Bellamkonda RV. Controlled targeting of liposomal doxorubicin via the folate receptor in vitro. J Control Release 2003;92:49-67.


37. Lestini BJ, Sagnella SM, Xu Z, Shive MS, Richter NJ, Jayaseharan J, et al. Surface modification of liposomes for selective cell targeting in cardiovascular drug delivery. J Control Release 2002;78:235-47. 38. Mura S, Nicolas J, Couvreur P. Stimuli-responsive nanocarriers for drug delivery. Nat Mater 2013;12:991-1003. 39. Yatvin MB, Weinstein JN, Dennis WH, Blumenthal R. Design of liposomes for enhanced local release of drugs by hyperthermia. Science 1978;202:1290-3. 40. Zhu L, Torchilin VP. Stimulus-responsive nanopreparations for tumor targeting. Integr Biol (Camb) 2013;5:96-107. 41. Lu Y, Sun W, Gu Z. Stimuli-responsive nanomaterials for therapeutic protein delivery. J Control Release 2014;194:1-19. 42. Kikuchi A, Okano T. Pulsatile drug release control using hydrogels. Adv Drug Deliv Rev 2002;54:53-77. 43. Nakayama M, Okano T, Miyazaki T, Kohori F, Sakai K, Yokoyama M. Molecular design of biodegradable polymeric micelles for temperatureresponsive drug release. J Control Release 2006;115:46-56. 44. Yang HW, Hua M-Y, Liu HL, Huang CY, Tsai RY, Lu YJ, et al. Selfprotecting core-shell magnetic nanoparticles for targeted, traceable, long half-life delivery of BCNU to gliomas. Biomaterials 2011;32:6523-32. 45. Mykhaylyk O, Sánchez-Antequera Y, Vlaskou D, Hammerschmid E, Anton M, Zelphati O, et al. Liposomal magnetofection. In: Weissig V, editor. Liposomes. Humana Press; 2010. p. 487-525. 46. Scherer F, Anton M, Schillinger U, Henke J, Bergemann C, Kruger A, et al. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Ther 2002;9:102-9. 47. Xenariou S, Griesenbach U, Ferrari S, Dean P, Scheule RK, Cheng SH, et al. Using magnetic forces to enhance non-viral gene transfer to airway epithelium in vivo. Gene Ther 2006;13:1545-52. 48. Hirao K, Sugita T, Kubo T, Igarashi K, Tanimoto K, Murakami T, et al. Targeted gene delivery to human osteosarcoma cells with magnetic cationic liposomes under a magnetic field. Int J Oncol 2003;22:1065-71. 49. Wang C, Ding C, Kong M, Dong A, Qian J, Jiang D, et al. Tumortargeting magnetic lipoplex delivery of short hairpin RNA suppresses IGF-1R overexpression of lung adenocarcinoma A549 cells in vitro and in vivo. Biochem Biophys Res Commun 2011;410:537-42. 50. Namiki Y, Namiki T, Yoshida H, Ishii Y, Tsubota A, Koido S, et al. A novel magnetic crystal-lipid nanostructure for magnetically guided in vivo gene delivery. Nat Nanotechnol 2009;4:598-606. 51. Vainauska D, Kozireva S, Karpovs A, Čistjakovs M, Bariševs M. A novel approach for nucleic acid delivery into cancer cells. Medicina (Kaunas) 2012;48:324-9. 52. Baryshev M, Vainauska D, Kozireva S, Karpovs A. New device for enhancement of liposomal magnetofection efficiency of cancer cells. World Acad Sci Eng Technol 2011;2011:306-9. 53. Lee YJ, Hou Z-Z, Curetty L, Cho JM, Corry PM. Mechanism of synergistic effects of cytokine and hyperthermia on cytotoxicity in HT-29 and MCF-7 cells: expression of MnSOD gene. J Therm Biol 1993;18:269-74. 54. Fleischmann Jr WR, Fleischmann CM. Enhancement of MuIFN-gamma antitumor effects by hyperthermia: sequence dependence and time dependence of hyperthermia. J Biol Regul Homeost Agents 1994;8:101-7. 55. Bouhon IA, Shinkai M, Honda H, Mizuno M, Wakabayashi T, Yoshida J, et al. Synergism between mild hyperthermia and interferon-β gene expression. Cancer Lett 1999;139:153-8. 56. Issels RD. Hyperthermia adds to chemotherapy. Eur J Cancer 2008;44:2546-54. 57. Mohamed F, Marchettini P, Stuart OA, Urano M, Sugarbaker P. Thermal enhancement of new chemotherapeutic agents at moderate hyperthermia. Ann Surg Oncol 2003;10:463-8. 58. Rapoport N. Phase-shift, stimuli-responsive perfluorocarbon nanodroplets for drug delivery to cancer. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2012;4:492-510. 59. Alkan-Onyuksel H, Demos SM, Lanza GM, Vonesh MJ, Klegerman ME, Kane BJ, et al. Development of inherently echogenic liposomes as an ultrasonic contrast agent. J Pharm Sci 1996;85:486-90.


F. Movahedi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1575–1584

60. Huang SL, Hamilton AJ, Nagaraj A, Tiukinhoy SD, Klegerman ME, McPherson DD, et al. Improving ultrasound reflectivity and stability of echogenic liposomal dispersions for use as targeted ultrasound contrast agents. J Pharm Sci 2001;90:1917-26. 61. Huang SL, Hamilton AJ, Pozharski E, Nagaraj A, Klegerman ME, McPherson DD, et al. Physical correlates of the ultrasonic reflectivity of lipid dispersions suitable as diagnostic contrast agents. Ultrasound Med Biol 2002;28:339-48. 62. Buchanan KD, Huang S, Kim H, Macdonald RC, McPherson DD. Echogenic liposome compositions for increased retention of ultrasound reflectivity at physiologic temperature. J Pharm Sci 2008;97:2242-9. 63. Negishi Y, Fukuyama T, Endo Y, Suzuki R, Tanaka K, Sawamura K, et al. 553. Development of echo-contrast gas entrapping liposome as gene and siRNA delivery tool. Mol Ther 2006;13:S213. 64. Ferrara KW. Driving delivery vehicles with ultrasound. Adv Drug Deliv Rev 2008;60:1097-102. 65. Rychak JJ, Klibanov AL. Nucleic acid delivery with microbubbles and ultrasound. Adv Drug Deliv Rev 2014;72:82-93. 66. Zacharakis E, Ahmed HU, Ishaq A, Scott R, Illing R, Freeman A, et al. The feasibility and safety of high-intensity focused ultrasound as salvage therapy for recurrent prostate cancer following external beam radiotherapy. Int Br J Urol 2008;102:782-92. 67. Schroeder A, Avnir Y, Weisman S, et al. Controlling liposomal drug release with low frequency ultrasound: mechanism and feasibility. Langmuir 2007;23:4019-25. 68. Schroeder A, Avnir Y, Weisman S, Najajreh Y, Gabizon A, Talmon Y, et al. Rapid skin permeabilization by the simultaneous application of dual-frequency, high-intensity ultrasound. J Control Release 2012;163:154-60. 69. Un K, Kawakami S, Suzuki R, Maruyama K, Yamashita F, Hashida M. Development of an ultrasound-responsive and mannose-modified gene carrier for DNA vaccine therapy. Biomaterials 2010;31:7813-26. 70. Koebis M, Kiyatake T, Yamaura H, Nagano K, Higashihara M, Sonoo M, et al. Ultrasound-enhanced delivery of Morpholino with Bubble liposomes ameliorates the myotonia of myotonic dystrophy model mice. Sci Rep 2013;3:2242. 71. Endo-Takahashi Y, Negishi Y, Nakamura A, Ukai S, Ooaku K, Oda Y, et al. Systemic delivery of miR-126 by miRNA-loaded Bubble liposomes for the treatment of hindlimb ischemia. Sci Rep 2014:4. 72. Yin T, Wang P, Li J, Wang Y, Zheng B, Zheng R, et al. Tumorpenetrating codelivery of siRNA and paclitaxel with ultrasoundresponsive nanobubbles hetero-assembled from polymeric micelles and liposomes. Biomaterials 2014;35:5932-43. 73. Zhao YZ, Du LN, Lu CT, Jin YG, Ge SP. Potential and problems in ultrasound-responsive drug delivery systems. Int J Nanomedicine 2013;8:1621-33. 74. El-Sayed ME, Hoffman AS, Stayton PS. Smart polymeric carriers for enhanced intracellular delivery of therapeutic macromolecules. Expert Opin Biol Ther 2005;5:23-32. 75. Ganta S, Devalapally H, Shahiwala A, Amiji M. A review of stimuliresponsive nanocarriers for drug and gene delivery. J Control Release 2008;126:187-204. 76. Saito G, Swanson JA, Lee KD. Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities. Adv Drug Deliv Rev 2003;55:199-215. 77. Deshpande PP, Biswas S, Torchilin VP. Current trends in the use of liposomes for tumor targeting. Nanomedicine (Lond) 2013;8:1509-28. 78. Ajmani PS, Tang F, Krishnaswami S, Meyer EM, Sumners C, Hughes JA. Enhanced transgene expression in rat brain cell cultures

79. 80.



83. 84.











95. 96.

with a disulfide-containing cationic lipid. Neurosci Lett 1999;277:141-4. Balakirev M, Schoehn G, Chroboczek J. Lipoic acid-derived amphiphiles for redox-controlled DNA delivery. Chem Biol 2000;7:813-9. Wang M, Alberti K, Varone A, Pouli D, Georgakoudi I, Xu Q. Enhanced intracellular siRNA delivery using bioreducible lipid-like nanoparticles. Adv Healthc Mater 2014;3:1398-403. Gabriele C, Daniele P, Laura C, Roberto C, Sandra R. Bioreducible liposomes for gene delivery: from the formulation to the mechanism of action. PLoS One 2010;5. Bersani S, Vila-Caballer M, Brazzale C, Barattin M, Salmaso S. pHsensitive stearoyl-PEG-poly(methacryloyl sulfadimethoxine) decorated liposomes for the delivery of gemcitabine to cancer cells. European Journal of Pharmaceutics and Biopharmaceutics 2014;88:670-82. Torchilin VP. Recent advances with liposomes as pharmaceutical carriers. Nature 2005;4:145-60. Liu X, Huang G. Formation strategies, mechanism of intracellular delivery and potential clinical applications of pH-sensitive liposomes. Asian J Pharm Sci 2013;8:319-28. Ropert C, Lavignon M, Dubernet C, Couvreur P, Malvy C. Oligonucleotides encapsulated in pH sensitive liposomes are efficient toward Friend retrovirus. Biochem Biophys Res Commun 1992;183:879-85. Ponnappa BC, Dey I, Tu GC, Zhou F, Aini M, Cao QN, et al. In vivo delivery of antisense oligonucleotides in pH-sensitive liposomes inhibits lipopolysaccharide-induced production of tumor necrosis factor-alpha in rats. J Pharmacol Exp Ther 2001;297:1129-36. Sato Y, Hatakeyama H, Sakurai Y, Hyodo M, Akita H, Harashima H. A pH-sensitive cationic lipid facilitates the delivery of liposomal siRNA and gene silencing activity in vitro and in vivo. J Control Release 2012;163:267-76. Sakurai Y, Hatakeyama H, Sato Y, Hyodo M, Akita H, Ohga N, et al. RNAi-mediated gene knockdown and anti-angiogenic therapy of RCCs using a cyclic RGD-modified liposomal-siRNA system. J Control Release 2014;173:110-8. Zhao P, Chenxiao W, Erhu F, Xiaoming L, Guobin W, Qiang T. Codelivery of doxorubicin and SATB1 shRNA by thermosensitive magnetic cationic liposomes for gastric cancer therapy. PLoS One 2014;9:e92924. Ta T and Porter TM. Thermosensitive liposomes for localized delivery and triggered release of chemotherapy. Journal of Controlled Release. 169: 112–125. Bouhon I, Shinkai M, Honda H, Kobayashi T. Enhancement of cytokine expression in transiently transfected cells by magnetoliposome mediated hyperthermia. Cytotechnology 1997;25:231-4. Dave N, Liu J. Protection and promotion of UV radiation-induced liposome leakage via DNA-directed assembly with gold nanoparticles. Adv Mater 2011;23:3182-6. Wan Y, Han J, Fan G, Zhang Z, Gong T, Sun X. Enzyme-responsive liposomes modified adenoviral vectors for enhanced tumor cell transduction and reduced immunogenicity. Biomaterials 2013;34:3020-30. El-Hamed F, Dave N, Liu J. Stimuli-responsive releasing of gold nanoparticles and liposomes from aptamer-functionalized hydrogels. Nanotechnology 2011;22:494011. Mo R, Jiang T, Gu Z. Enhanced anticancer efficacy by ATP-mediated liposomal drug delivery. Angew Chem 2014;126:5925-30. MacEwan SR, Callahan DJ, Chilkoti A. Stimulus-responsive macromolecules and nanoparticles for cancer drug delivery. Nanomedicine (Lond) 2010;5:793-806.

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

Nucleic acid therapeutics (NATs) are valuable tools in the modulation of gene expression in a highly specific manner. So far, NATs have been actively ...
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