Send Orders for Reprints to reprints@benthamscience.ae 259 Current Drug Targets, 2018, 19, 259-270 REVIEW ARTICLE )##-EF  )##-E Stimuli-responsive Smart Liposomes in Cancer Targeting Impact Factor: 3.236 BENTHAM SCIENCE Ankit Jain1 and Sanjay K. Jain2,* 1 Institute of Pharmaceutical Research, GLA University, NH-2, Mathura-Delhi Road, Mathura (U.P.) - 281 406, India; Pharmaceutics Research Projects Laboratory, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Central University, Sagar (M.P.) - 470 003, India 2 Abstract: Background: Liposomes are vesicular carriers which possess aqueous core entrapped within the lipid bilayer. These are carriers of choice because of biocompatible and biodegradable features in addition to flexibility of surface modifications at surface and lipid compositions of lipid bilayers. ARTICLE HISTORY Received: September 29, 2015 Revised: January 15, 2016 Accepted: January 31, 2016 Current Drug Therapy DOI: 10.2174/1389450117666160208144143 Objective: Liposomes have been reported well for cancer treatment using both passive and active targeting approaches however tumor microenvironment is still the biggest hurdle for safe and effective delivery of anticancer agents. To overcome this problem, stimuli-responsive smart liposomes have emerged as promising cargoes pioneered to anomalous tumor milieu in response to pH, temperature, and enzymes etc. as internal triggers, and magnetic field, ultrasound, and redox potential as external guides for enhancement of drug delivery to tumors. Conclusion: This review focuses on all such stimuli-responsive approaches using fabrication potentiality of liposomes in combination to various ligands, linkers, and PEGylation etc. Scientists engaged in cancer targeting approaches can get benefited greatly with this knowledgeable assemblage of advances in liposomal nanovectors. Keywords: Stimuli-sensitive, liposomes, thermo-sensitive, pH-sensitive, ultrasound responsive, smart delivery. 1. INTRODUCTION Nanocarriers like liposomes, polymeric and inorganic nanoparticles, and drug/nucleic acid conjugates are found to be extraordinarily safe and effective as compared to "naked" drugs/nucleic acids [1-5]. Bioengineering of these carriers with suitable ligands improves in vivo performance by many folds in terms of blood retention, target selectivity, cellular internalization, and environmental responsiveness to external or internal stimuli such as pH, redox potential, enzyme, magnetic field, ultrasound, light and temperature [6-10]. The anomaly in tumor microenvironment offers the possibilities to fabricate nanocarriers for effective temporal and spatial localization of drug/bioactive by controlling release, internalization, and intracellular fate [11, 12]. Liposomes are regarded as the model biomembranes in the field of drug delivery with enhanced safety and efficacy. These have been explored for the site-specific drug delivery by extra- or intracellular targeting using various functionalization techniques [13, 14]. These are well documented cargoes for theranostic potential as they show better pharmacokinetics and efficacy for treatment of cancers. Since cancer possesses complex physiology of the microenvironment it demands multifaceted approach to design liposomal cargoes such as, remotecontrolled or tumor stimuli-responsive characteristics to enhance tumor extravasation, selective ligands to facilitate cellular internalization, and biocompatible guides for intracellular localization within tumor cells [15, 16]. However, the *Address correspondence to this author at the Pharmaceutics Research Projects Laboratory, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Central University, Sagar (M.P.) - 470 003, India; Tel: +917582-265457, +91-9425172184; E-mail: drskjainin@yahoo.com 1873-5592/18 $58.00+.00 therapeutic potential of liposomes is limited because of insufficient approachability to tumor site, non-selective uptake and non-effective release of drug within the tumor. Recently, nanocarriers based theranostics including polymer conjugates, vesicular and nanoparticulate systems, and carbon nanotubes are attracting scientists for desired co-delivery of diagnostic and therapeutic agents. These theranostic nanocarriers can bring both therapeutic and diagnostic potential at macro level as well as molecular level. Tethering bioligands and incorporating stimuli-responsive elements have emerged with great potential to control release at targeted site of interest [17]. Application potential of cell-penetrating peptide such as elastin-like polypeptide based therapeutics is also under exploration for the thermo-responsive and targeted delivery of therapeutic bioactives for the treatment of cancer. As these approaches can accomplish both passive and active targeting using liposomes for better intra-tumoral localization [18]. Dendritic cells (DC) are involved in antigen presentation and generation of cytotoxic T lymphocyte (CTL) response which are required for vaccination against cancer. Various ligands like mannose, CD11c/CD18, Fc receptor, DEC-205 and DC-SIGN on DC are reported for active targeting of cancer using pH sensitive liposomes [19]. Recently, liposome-nanoparticle assemblies (LNAs) are introduced to combine the potential of liposomes with engineered nanoparticles for better therapeutic outcomes. In particular, LNAs can protect the stimuli-sensitive nanoparticles from immune system and to initiate and control drug release upon triggering in response to external stimuli so called nanoparticle-controlled liposomal release. LNAs are not widely explored and researches are trying to build the structurefunction relationships of LNAs [20]. Paliwal et al. (2014) © 2018 Bentham Science Publishers 260 Current Drug Targets, 2018, Vol. 19, No. 3 Jain and Jain discussed the pH-sensitive liposomes for intracellular delivery of various therapeutic as well as diagnostic agents. These liposomes get destabilized at acidic milieu of endosomes to deliver bioactives within the intracellular region. To direct these liposomes to desired site, different ligands including specific antibodies depending upon receptors/antigens found over tumor cells [21]. This review summarizes various stimuli based triggers which have been employed to develop these “smart” liposomes for safe and efficacious delivery of cancer therapeutics. Figure 1 shows potential of stimuliresponsive smart liposomes. Figure 2 represents targeted intracellular stimuli responsive drug release in cancer cell. 2. pH-SENSITIVE LIPOSOMES pH-sensitive liposomes are found to be potential cargoes for chemotherapy of malignant tumors because of effective targeting and accumulation of anti-cancer agents in tumors and flexibility of long-circulating and fusogenic property [22]. pH-sensitive liposomes in combination of cellpenetrating proteins and peptides, and targeting moiety can be targeted to intracellular targets, nuclei, mitochondria and lysosomes [23]. In 1980, Yatvin et al. found that the release of drugs can be controlled in pH- sensitive liposomes using variation in pH in calf, horse, or human serum at 37°C. Such liposomes were targeted to primary or metastatic tumors because of lower pH in the tumor areas as compared to physiological pH and other body parts [24]. Wang and Huang (1987) developed pH-sensitive immunoliposomes composed of a plasmid bearing the E. coli chloramphenicol acetyltransferase (CAT) gene controlled by a mammalian cAMP-regulated promoter. These liposomes were functionalized with H-2Kk antibody and DOPE was used as a pH Cationic charge Hydrophilic C drug sensitive lipid. After intra-peritoneal injection of entrapped or free DNA into BALB/c mice (nude) bearing H-2Kkpositive RDM-4 lymphoma cells, there were high CAT activity and about 20% uptake of these liposomes by the RDM4 cells because of antibody specificity and pH-sensitivity [25]. pH-sensitive liposomes undergo acid-triggered destabilization. DOPE pH-sensitive liposomes are reported as first generation liposomes that lose their fusogenicity in serum (10%). Sudimack et al. (2002) developed cholesteryl hemisuccinate (CHEMS) pH-sensitive liposomes loaded calcein that showed appreciable serum stability and rapid destabilization in acidic milieu. It was found that there was no membrane fusion upon liposomal destabilization in response to acidic pH as indicated in octadecylrhodamine B chloride (R(18)) lipid-mixing assay. These liposomes when loaded with cytosine-β-D-arabinofuranoside (araC) and coupled with folate showed enhanced cytotoxicity (17-fold) in KB human oral cancer cells as compared to Folate receptor (FR)-targeted non-pH-sensitive liposomes [26]. pH-sensitive liposomes can manifest triggered release of drugs at low pH. Shi et al. (2002) reported pH-sensitive liposomes of dimethyldioctadecylammonium bromide (DDAB) and CHEMS that could undergo abrupt release of drug with irreversible aggregation at acidic conditions. These liposomes were compared to DOPE pH-sensitive liposomes and found to have better retention of pH-sensitivity in blood circulation. After coupling with folate, targeted pH-sensitive liposomes showed enhanced cytosolic dislodgment of calcein and increased cytotoxicity with cytosine-β-D-arabinofuranoside in KB cells in comparison to FR-targeted non-pH-sensitive liposomes. Upon combining polylysine-condensed plasmid Dispersal of liposomal content in response to stimulus at tumor site Thermo-sensitive component or others pH-sensitive component Lipophilic drug PEG Stimulus Ligand coupled PEG Ligand coupled cleavable PEG Transitional stage in response to stimulus (pH, temperature, redox potential, ultrasound, enzyme, and magnetic field etc. Low mol. weight ligands (e.g. folate) or peptides or protein ligands (e.g. transferrin) or antibodies or antibody fragments or radiolabel or fluorescent marker Fig. (1). Potential of stimuli-responsive smart liposomes. Stimuli-responsive Smart Liposomes in Cancer Targeting Current Drug Targets, 2018, Vol. 19, No. 3 261 STIMULI-RESPONSIVE SMART LIPOSOMES BYSTANDER EFFECT RECEPTOR ENDO-LYSOSOME ENDOSOME STIMULI-RESPONSIVE INTRACELLULAR DRUG RELEASE NUCLEUS MITOCHONDRIA Fig. (2). Targeted intracellular stimuli responsive drug release in cancer cell. DNA to these targeted liposomes, there was increased FRspecific delivery of a luciferase reporter gene into KB cells [27]. Gene therapy is found to be an emerging therapeutic approach for a number of diseases including AIDS or cancer. Nucleic acid based drugs are required to be delivered to cytosol using an efficient cargo which can be viral and nonviral (synthetic) type. Viral vectors pose a serious issue of safety which is being overcome with alternative non-viral vectors based on cationic lipids or polymers. However, there are number of difficulties such as blood stability, RES uptake, toxicity, and targeting potential. Fabrication with PEG along with functionalization with selective ligand renders increased blood retention, RES evasion, and enhanced accumulation in tumors. Neutral and anionic lipids have also been reported for nucleic acid delivery [28]. The positive charge of cationic-lipid/DNA complexes (lipoplexes) renders highly susceptible to interact with the biological milieu, leading to aggregation and destabilization, and rapid clearance from the blood circulation [29]. Lee et al. (2009) reported modularly clickable polymer-caged nanobins (PCNs) which were developed using liposome templates cross-linked with alkyne-functionalized diamine linker. Using click ligation, this cross linker helped coupling azide-modified targeting ligands. These PCNs were pH-sensitive and were found to trigger doxorubicin (Dox) release in slight acidic conditions. After conjugating folate ligand, these liposomes (f-PCNDox)) showed fifty-fold higher potency as compared to nontargeted liposomes in FR-positive cancer cells i.e. KB and OvCa432 [30]. In another study, hydrazine-functionalized PEG-PE was used to prepare pH-cleavable liposomes conjugated with monoclonal antinucleosome antibody 2C5 and antimyosin antibody 2G4, and glycoproteins concanavalin A (Con-A) and avidin. It was found that almost 80% of the hydrazone bond cleaved at pH 5 (0.5 - 1 h). Antibody coupled liposomes showed enhanced cytotoxicity and uptake in HeLa and MCF-7 cells because of targeting ability of antibody and low pH triggered cleavage of hydrozone bond allowed an exposure of the antibody to target cells for significant cellular association [31]. If pH-triggering doesn’t affect stability of liposomes in vivo then better cytotoxicity of Dox loaded liposomes can be obtained after getting internalized by clathrin-mediated endocytosis. To overcome in vivo instability of pH-sensitive liposomes, Mamasheva et al. (2011) developed pH-triggered gel-phase liposomes for the delivery of Dox. These liposomes form "leaky" interfacial borders in bilayer upon getting triggered with decreasing pH. FRtargeted liposomes were found to follow the caveolae mediated endocytosis (less acidic) in KB cancer cells. Developed liposomes showed increased Dox uptake per cancer cell thereby more killing effect (IC50 = 0.032 mM, 6 h) as compared to non-pH-triggered vesicles (IC50 = 0.194 mM) because of higher exposure of drug to the cancer cells plausibly via rapid and bulk release of drug from the developed liposomes [32]. pH-responsive CPP modified liposomes were targeted using hyaluronic acid (HA) for tumor treatment by Jiang et al. (2012). Based on tumor cellular uptake capacity (pH 6.4 or 7.4), they screened a number of synthetic tumor pH-triggered CPPs which were rich in arginines and histidines. R6H4 (RRRRRRHHHH) was found to be the optimal in pH-response. R6H4 was coupled to liposomes (R6H4-L) and HA was used to mask the positive charge of R6H4-L to obtain HA-modified R6H4-L (HA-R6H4-L). 262 Current Drug Targets, 2018, Vol. 19, No. 3 These liposomes were loaded with paclitaxel (PTX) and showed increased cytotoxicity in HepG2 cells at pH 6.4 than at pH 7.4 after HAase treatment [33]. Soininen et al. (2012) reported avidin-biotin technology with multi-modal liposomes for effective tumor targeting. Either Dox was biotinylated (B-Dox) or entrapped in biotinylated sterically stabilized pH-sensitive liposomes (BL-Dox). B-Dox and BLDox were targeted to the lentiviral vector transduced cells expressing an avidin fusion protein. There was improved cellular uptake with B-Dox in rat glioma (BT4C) cells expressing avidin fusion protein receptors but decreased cell toxicity (78-fold) because of affected nuclear localization. However, BL-Dox showed increased efficacy of Dox in number of cell lines [34]. Qiao-ling et al. (2012) developed lactosyl-norcantharidin phospholipid complex (LPC) loaded liposomes (Lac-NCTD). Carboxymethyl chitosan was adsorbed on the surface of liposomes to prepare pH-sensitive LPC liposomes (pH-LPC-lips). The pH-LPC-lips showed increased cytotoxicity against HepG2 cells and inhibition of tumor growth in mice bearing H22 liver tumors [35]. Pichon and Midoux (2013) developed mannosylated lipopolyplexes loaded with mRNA (Man-LPR) encoding a melanoma antigen to transfect DC for the anti-B16F10 melanoma vaccination in mice. Liposomes were histidylated to render them pH-sensitivity because of protonation of histidine groups that destabilized endosomes resulting in delivery of nucleic acid in the cytosol [36]. Bandekar et al. (2013) developed pHresponsive liposomes with tunable and reversible isolated lipid domains. While circulating in the blood the functional ligands remained masked and content was well retained. When they reached to the tumor interstitium ligands became exposed and separated within lipid domains initiating targeting to tumor cells with the effective internalization. Liposomes got burst to release the bioactive in the acidic endosomal environment due to creation of leaky boundaries around the phase-separated lipid domains. This approach was used for the delivery of Dox and liposomes were functionalized with anti-HER2 peptide. Anti-HER2 pH tunable liposomes showed Dox release in vitro studies in a pH dependent manner. These liposomes reduced tumor volumes by 159% in comparison to non-targeted ones in BT474 xenografts subcutaneously placed in nude mice [37]. Zhang et al. (2013) developed pH-responsive CPP modified liposomes to overcome the disadvantages associated with conventional CPP based approaches i.e. the lack of selectivity and uncontrolled in vivo penetration. They introduced AGYLLGHINLHHLAHL(Aib) HHIL-NH₂ (TH) over the surface of liposomes as a fabricated α-helical CPP that showed pH-responsiveness. These liposomes remained stable in systemic circulation due to the neutral pH while reaching to the tumor as pH lowers, histidines in TH peptide got protonated leading to transition of surface charge from negative to positive. This charge transition enhanced cell penetration resulting in increased tumor localization. Internalization was mediated by the endocytosis mechanism with clathrin coated pits and the liposomes were primarily localized in endoplasmic reticulum and Golgi apparatus. These liposomes were also used for the delivery of PTX that brought significant tumor inhibition both in vitro (below pH 6.3) and in vivo (C26 tumor-bearing mice) [38]. Bersani et al. (2014) reported acid-sensitive liposomes for targeting tumors using mPEG-DSPE and stearoyl-PEG-poly Jain and Jain (methacryloyl sulfadimethoxine) [stearoyl-PEG-polySDM] copolymer. Cloud point studies revealed that the hydrophilic/hydrophobic copolymer conversion was found to be at pH 7.0. The copolymer showed solubility above pH 7.0 but got aggregated at acidic pH. All liposomal formulations were stable at physiological pH including the presence of foetal bovine serum but their size was found to increase at pH 6.5 as also revealed from TEM studies. Cell lines studies showed no cytotoxicity up to 10 mg/mL. Moreover, microscopic studies (fluorescence, cytofluorimetry, and confocal) also confirmed higher cytotoxicity at pH 6.5 in MCF-7 cell line as compared to negligible cell interaction at pH 7.4. Gemcitabine was also encapsulated in these liposomes which showed similar physiological stability for several hours [39]. Chiang and Lo (2014) reported a tumor-extracellular matrix pH-induced targeting liposomes (ECM-targeting liposomes). With the intent to conquer the defects of liposomes, these liposomes were cross-linked by hydrogen bonds between methoxy-poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide-co-histidine)-cholesterol copolymers and biotin2-polyethylene glycol cross-linkers. ECM-targeting liposomes exhibited abrupt in vitro drug release in acidic environment. ECM-targeting capability and pH responsiveness in liposomes increased more in vivo accumulation of drug in tumor and anticancer potential [40]. Liu et al. (2014) developed a liposomal cocktail (LMDHV) to overcome MDR and drug insensitivity due to slow drug release over a period of time leading to decreased intracellular localization of drug. LMDHV was prepared using malachite green carbinol base (MG) as a pH-responsive agent and Her-2 antibody functionalized liposomes bearing Dox and verapamil (VER) to target Her-2 positive breast cancer. MG embedded in the lipid bilayer was found to destabilize the vesicles in low pH milieu resulting in an abrupt dispersal of the liposomal content. Her-2 antibody and MG imparted 6-times higher reversal efficiency in Dox resistant breast cancer. In vivo studies showed that there was significantly higher tumor inhibition in case of LMDHV as compared to free Dox owing to synergistic effects on tumor growth in xenografts [41]. For cancer gene therapy, Shi et al. (2014) reported a pH sensitive targeted gene carrier i.e. folic acid coupled Schiff-base linked imidazole chitosan (FA-SLICS) that could self-assemble plasmid DNA (pDNA) into nano-scaled polyplexes. These were able to release the pDNA in the endosome owing pH sensitivity of Schiff-base moieties. FA-SLICS showed insignificant cytotoxicity to normal cells (CHO) but slight toxicity to HeLa and HepG2 cells [42]. Collagen I network in tumors may hinder the penetration of nanocarriers resulting in compromised antitumor potential. Losartan is an angiotensin inhibitor which has been used to decrease the level of collagen I leading to ease in the penetration of nanocarriers. Zhang et al. (2015) developed pH-sensitive cleavable liposomes (Cl-Lip) composed of PEG5K-Hydrazone-PE and DSPE-PEG2K-R8. PEG5K-hydrazone-PE got hydrolyzed upon reaching to the tumor region by EPR effect because of acidic pH of tumor microenvironment. This resulted in exposing the R8 peptide that helped internalization of liposomes into the cancer cells. The cell uptake studies in 4T1 cells showed enhanced cytotoxicity of Cl-Lip bearing paclitaxel (PTX-Cl-Lip). In vivo studies also documented deep penetration of these liposomes into the tumors with increased accumulation by 22.0% and enhanced oxygen Stimuli-responsive Smart Liposomes in Cancer Targeting Table 1. Current Drug Targets, 2018, Vol. 19, No. 3 263 Application potential of pH-sensitive liposomes in cancer treatment. pH-sensitive Liposome Drug Targeted Cancer Remarks Ref. DAPE or POPE based liposomes - - The developed liposomes (35 mol% DAPE or 35 mol% POPE) showed increased plasma stability but significant pH sensitivity below pH 6.5. [48] S5A8 mAb coupled immunoliposomes Dox 38C13 murine B-cell lymphoma These immunoliposomes showed enhanced in vitro cytotoxicity and increased blood circulation with prolongation in survival of mice as compared to non-targeted liposomes and free Dox. [49] Anti-B-cell lymphoma mAb LL2 conjugated PEG-PE liposomes Dox Neoplastic B cells The targeted pH-sensitive liposomes showed increased cellular interaction (3.8 times), cytotoxicity (1.5 times) and localization into low pH compartments as compared to plain liposomes. [50] ASGPr targeted pH-sensitive liposomes bearing AODN HCV363 (AODN) against HCV 5'NCR Transgenic cell HepG2.9706 There were dose-dependent inhibitory activities on luciferase expression with 86% inhibition at 1.0 µmol/L. [51] ASGPr targeted DOPE liposomes bearing AODN AODN Hepatocellular carcinoma (HCC) 18-gal modified liposomes showed increased hepatocyte specificity with high transfection potential. [52] Dox ER-positive MCF-7 cells Estrone anchored pH-sensitive liposomes having fusogenic potential (at pH 5.5) exhibited enhanced in vitro cytotoxicity. These also inhibited in vivo tumor growth as compared to non-pH-sensitive targeted liposomes and free Dox. [53] Calcein CT26.WT colon carcinoma Calcein release from these liposomes was pH-dependent and occurred within few minutes because of destabilization of liposomes at acidic pH and PR_b facilitated effective targeting to integrin αvβ 1 expressing CT26.WT colon carcinoma cells. [54] ara-C Human myeloid leukemia cells Dioctadecyl, N-isopropylacrylamide and methacrylic acid were incorporated PEGylated liposomes. Targeted liposomes showed improved pharmacokinetics and cytotoxicity in Balb/c and leukemic HL60bearing immunodepressed (SCID) mice. [55] Mouse breast adenocarcinoma model These pH-sensitive liposomes showed increased localization in cancer cells in vivo. [56] ER targeted pH-sensitive liposomes PR_b-targeted pH-sensitive PEGylated liposomes Anti-CD33 mAb coupled pHsensitive immunoliposomes pHLIP [pH (low) insertion peptide] based liposomes HER-2 targeted pH-sensitive histidine-lysine peptide based immunoliposomes anti-HER-2 siRNA Pancreatic cancer model These targeted immunoliposomes sensitized human tumor cells and silenced the target gene, with significant inhibition of tumor growth in vivo. [57] Thiolated transferrin coupled PEGylated pH-sensitive liposomes - Human T-leukaemia cells Developed liposomes showed selective internalization via receptordependent endocytosis in human T-leukaemia cells in vitro followed by pH-triggered cytoplasmic delivery. [58] pH-sensitive histidylated oligolysine HoKC based liposomes DNA DU145 human prostate cancer xenograft tumor model HoKC increased transfection efficiency in tumor bearing mice. [59] DAPE = diacetylenic phosphatidylethanolamine, POPE = phosphatidylethanolamine-beta-oleoyl-gamma-palmitoyl, Monoclonal antibody = mAb, PEG-PE = poly(ethylene glycol)modified phosphatidylethanolamine, ASGPr = asialoglycoprotein receptors, AODN = Antisense oligodeoxynucleotide, ER = estrogen receptor, PR_b = KSSPHSRN(SG)(5)RGDSP which is a fibronectin-mimetic peptide sequence, ara-C = 1-beta-d-arabinofuranosylcytosine, HoKC = K[K(H)KKK]5-K(H)KKC. distribution in tumor tissues in 4T1 tumor bearing mice [43]. Liposomes have taken great attention for the cancer therapy because of widely accepted passive targeting potential to the solid tumors using EPR effect. However, liposomes show lesser entrapment of hydrophilic bioactives like topotecan (TPT). TPT shows pH-sensitive ring-opening hydrolysis resulting in the formation of less active carboxylate form [44]. Active loading techniques by creating low intravesicular pH have been investigated to improve drug entrapment. Fugit and Anderson (2014) developed a mathematical model to predict TPT's permeability as a function of pH depending upon ionization state, membrane binding and ring-opening inter-conversion kinetics. This model fitting established TPT release from liposomes and can benefit others for active loading of TPT [45]. Tumor endothelial cells (TECs) are found to involve significantly in pathological manifestation in neovasculaturization. Small interfering RNA (siRNA) shows promising capability in gene silencing for cancer 264 Current Drug Targets, 2018, Vol. 19, No. 3 chemotherapy. Sakurai et al. (2014) reported siRNA loaded liposomal delivery based system (a MEND) to target TECs. This system possessed a pH-responsive cationic lipid i.e. YSK05 (YSK-MEND). cRGD is specific in binding to αVβ3 integrin overexpressed TECs. cRGD were incorporated into the YSK-MEND (RGD-MEND) for effective gene silencing. Upon i.v. administration of RGD-MEND, there was induction of RNAi-mediated gene reduction in TEC as obtained from RT-PCR reports. These MEND showed inhibition of tumor growth after three consecutive injections on alternate days [46]. Dual approach using receptor-mediated targeting and pH-sensitivity in liposomes are found to enhance in vivo efficacy by many folds. Xia et al. (2013) synthesized folatepoly(2-ethyl-2-oxazoline)-distearoyl phosphatidyl ethanolamine (F-PEOz-DSPE), to combine FR selectivity and pH sensitivity so as to increase cell uptake and rapid release of drug during endosomal cascade in cancer cells. F-PEOzDSPE liposomes bearing Dox showed selective targeting to human ovarian cancer cells (SKOV3) as compared to PEOzDSPE or DSPE liposomes [47]. mRNA-based vaccines are also studied for treating cancers. For selective and efficient delivery, synthetic or in vitro transcribed (IVT) mRNA encoding tumor antigen is found to be superior to plasmid DNA encoding tumor antigen. Table 1 summarizes application potential of pH-sensitive liposomes in cancer treatment. 3. THERMO-RESPONSIVE LIPOSOMES Shibata et al. (1992) reported thermo-sensitive DPPC (Dipalmitoylphosphatidylcholine, Tc = 41°C) liposomes bearing ADR (TS-Lip-ADR) which were functionalized with mAb against human alpha-fetoprotein (AFP). They found temperature dependent drug release and increased cytotoxicity at tumor site under controlled hyperthermia (HT) conditions [60]. Chelvi and Ralhan (1995) developed thermosensitive DPPC liposomes bearing decarbazine which were found to have enhanced in vivo efficacy in combination with HT in murine fibrosarcomas [61]. Kim et al. (2014) exploited mild HT-mediated approach for tumor-specific delivery of Dox using DPPC (thermo-responsive lipid)-based liposomes with surface anchored elastin-like polypeptide (ELP) as a thermo-responsive peptide and cyclic arginineglycine-aspartic acid (cRGD) for targeting αvβ3 integrin overexpressed in angiogenic vasculature. These liposomes showed 8 and 10-times higher cellular uptake of Dox into αvβ3 integrin-overexpressing U87MG and HUVEC cells, respectively, as compared to non-targeted liposomes, owing to cRGD targeting and thermo-responsive release. Confocal microscopy showed nuclear localization of Dox and cytosolic abundance of the liposomes under the mild HT (42 °C). There was 5-times higher accumulation and anticancer activity of these liposomes in tumor bearing mice in comparison to the non-targeted liposomes [62]. Dicheva et al. (2014) reported multifunctional targeted thermo-responsive liposomes for effective delivery of Dox. Dox loaded cationic thermo-responsive liposomes (Dox-CTLs) showed increased uptake and cytotoxicity in different cancer cell lines and endothelial cells in comparison to non-targeted TLs and it was also confirmed as fluorescent Dox nanobursts in live cell confocal microscopy. After applying heat treatment, DoxCTLs in high resolution intravital microscopy showed 3times higher Dox release into the tumors causing noticeable Jain and Jain tumor vessel damage than TLs in vivo [63]. Multifunctional thermo-sensitive polymer-modified liposomes (MTPLs) bearing anticancer agent, MRI agent, and fluorescent dye were reported as a "theranostic" nanocarrier. In vivo MTPLs showed increased accumulation in tumor cells with enhanced MR signal after mild heating (above 41°C, 15 minutes) because of triggered release of drug and contrast agent from the MTPLs. Such approaches using MTPLs can be quite useful for drug delivery with effective monitoring at the site of interest [64]. Zhu et al. (2014) developed Dox-lysine/singlewalled carbon nanotube-thermo-sensitive liposomes that showed rapid release of drug at 42 °C. These liposomes were found to exhibit increased cell uptake with higher anti-tumor activity upon near-infrared laser irradiation at 808 nm on the human hepatic carcinoma cell line (SMMC-7721) and sarcoma 180-bearing mice as compared to free Dox [65]. To bring a rapid burst release of drug in response to an external stimulus, ultrasound (low intensity) is found to be a promising approach. For this purpose, drug loaded liposomes having ultrasound responsive perfluorocarbon gas microbubble has been designed to accomplish a secondary control over spatial and temporal localization of drug at site of interest like tumors [66]. Mild HT (40-45°C) is continuously explored for theranostic applications. Magnetic resonance guided high intensity focused ultrasound (MR-HIFU) is a non-invasive technique to heat solid tumors with an effective image guidance. For such applications, low temperaturesensitive liposomes (LTSLs) are found to release drug above 40°C in solid tumors. Partanen et al. (2012) investigated LTSLs in combination to MR-HIFU in the rabbits bearing VX2 tumors using a binary algorithm for real-time mild HT feedback control (target=40-41°C). Temperature was achieved in between 40.4°C and 41.3°C after controlled exposure of ultrasound in VX2 tumors along with good 3D spatial offset both in vitro (0.1-3.2 mm) and in vivo (0.6-4.8 mm). This combinatorial approach resulted in enhanced drug delivery upon a controlled heating of the tumor [67]. The selective drug delivery upon HT using TSLs has emerged greatly and ultrasound with better control is becoming a promising technique to heat a specific area like microvessels of tumor leading to abrupt drug release. For example, ThermoDox® (Celsion) showed increased drug release and uptake under HT condition (~41°C) in tumors. Recent technique named magnetic resonance-guided focused ultrasound (MRgFUS) is combined with TSL to render localized drug delivery to tumor along with measurement of drug release. Such real-time monitoring techniques can be promising tool to control drug delivery to enhance efficacy [68]. Tumorassociated macrophages (TAMs) are significantly involved in malignant ascite (MA) progression because of presence of a tumor-promoting M2 phenotype in TAMs. Kono et al. (2014) reported ultrasound (US)-responsive and mannosemodified liposomes/NF-κB decoy complexes for transferring a nuclear factor-κB (NF-κB) decoy into TAMs in a mouse model bearing Ehrlich ascites carcinoma. These lipoplexes upon intra-peritoneal administration followed by US exposure showed effective transfer of the NF-κB decoy into TAMs resulting in the reduction of Th2 cytokine interleukin (IL)-10 and vascular endothelial growth factor while Th1 cytokines (IL-12, tumor necrosis factor-α, and IL-6) levels were found to be elevated. These manifestations led to fall in Stimuli-responsive Smart Liposomes in Cancer Targeting volume and number of Ehrlich ascites, and an increase in the life span of mouse [69]. Drug resistance being a major problem with PTX in the treatment of HCC can be overcome using siRNA targeted to anti-apoptosis genes like BCL-2. Yin et al. (2014) prepared US-responsive nanobubbles bearing both PTX and siRNA (PTX-NBs/siRNA) for the treatment of HCC by combining polymeric micelles and liposomes with synergistic effects. Co-delivery of siRNA and PTX with the exposure of US after i.v. administration in the nude mice bearing human HepG2 xerografts decreased PTX inducible anti-apoptosis in HepG2 cells and tumor growth [70]. Recently, nanocarriers are explored for mechanical and thermal responsiveness by means of US. Ninomiya et al. (2014) developed US-responsive targeted liposomes fabricated with single stranded DNA aptamers [targeted to platelet-derived growth factor receptors (PDGFRs)] for breast cancer chemotherapy. Poly(NIPMAMco-NIPAM) was used as a thermo-sensitive polymer (TSP). TSP-modified liposomes (TSP liposomes) showed increased calcein release in response to US (1 MHz/30 s/0.5 W/cm2) and at HT conditions (42°C/5 min) because of elevation in local temperature upon cavitation bubble collapse caused by ultrasound rather than increase in temperature of the medium. PDGFR aptamers modified liposomes (APT liposomes) showed selective binding in MDA-MB-231 cells as compared to binding affinity to primary human mammary epithelial cells (HMECs). Dox loaded APT/TSP liposomes with the use of US irradiation exhibited enhanced cell viability in MDA-MB-231 at 24 h than plain drug and other control liposomes [71]. Lin et al. (2014) developed nanoemulsion based liposomes (eLiposomes). DPPC liposomes were loaded with perfluoropentane nanodroplets and Dox in the aqueous interior. Dox loaded eLiposomes (eLipoDox) showed controlled and triggered in vitro release of Dox in the cell cytosol in response to US (low intensity i.e. 20-kHz, 1.0-MHz and 3.0-MHz) against HeLa cells as found in the confocal images [72]. Ahmed et al. (2015) reviewed US dependent release of drugs in the tumor area to evade harmful effects to normal cells. For such applications, liposomes and micelles are found to be emerging in this field both in vitro and in vivo [73]. 4. LIGHT-RESPONSIVE LIPOSOMES Photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) was loaded into liposomes which were functionalized with polyclonal sheep anti-mouse-Ig antibody. When these liposomes were exposed to red light, the AlSPc got activated to generate singlet oxygen and the antibody resulting in killing DW-BCL cells (an Epstein Barr virus immortalized B-cell line) [85]. Tunable peptides (or like materials) can be conjugated to liposomes for increasing selectivity to the tumors. For instance, Xie et al. (2015) developed liposomes that could utilize the synergistic cell surface interactions between photo-sensitive peptides (PSPs) and targeting ligands. Lysine residues on the cell-penetrating peptides (CPPs) possessed the cationic charges that were masked temporarily by the photolabile-protective groups (PG) resulting in formation of a PSP. This PSP increased selective uptake into tumor cells after unmasking the PG upon an exposure to near-infrared (NIR) light. In contrast, the asparagine-glycine-arginine (NGR) peptide moieties that Current Drug Targets, 2018, Vol. 19, No. 3 265 selectively bind to CD13-positive tumors were attached to the liposomes to facilitate their active accumulation in the tumor tissues. The dual-modified liposomes (PSP/NGR-L) were prepared by emulsification method, and the concentrations of DSPE-PEG2000-psCPP and DSPE-PEG5000-NGR in the liposomes were chosen to be 4% and 1% (molar ratio), respectively. Cellular uptake results demonstrated an enhanced cancer cell recognition and specific uptake potential of the PSP/NGR-L. Furthermore, the PSP/NGR-L demonstrated a stronger antitumor efficacy in the HT-1080 tumor model in nude mice with the aid of NIR illumination [86]. Small interfering RNA (siRNA) is continuously getting attention for the targeted delivery to tumor cells. Recently, liposomes (PSP/NGR-L) were fabricated with photosensitive (UV) cell-penetrating peptides (PSP) and NGR peptide that enhanced the targeting specificity PSP/NGR-L in HT-1080 cells. It was suggested that the developed liposomes could escape endosomal degradation and increase cell apoptosis upon exposure to UV light [87]. Table 2 summarizes application potential of thermo-sensitive liposomes in cancer treatment. 5. REDOX-RESPONSIVE LIPOSOMES The application potential of poly(ethylene glycol) (PEG) for increasing blood circulation time and cell-penetrating peptides (CPPs) for enhancing cellular uptake have been combined in recent studies. Fu et al. (2015) developed TAT functionalized PTX loaded liposomes with cleavable PEG via a redox-responsive disulfide linker (PTX-C-TAT-LP) which remained stable in physiological conditions with increased blood circulation owing to shielding effect of PEG. Upon reaching to tumor site, this cleavable bond was broken in response to exogenous reducing agent (Glutathione, GSH) resulting in removal PEG shield to expose TAT that facilitated the cellular internalization of PTX loaded liposomes. This led to increased tumor localization both in vitro and in vivo (B16F1-bearing mice) with enhanced tumor inhibition (69.4%). Moreover, these liposomes were not found to show toxicity to the vital organs such as liver, kidney and heart [88]. Antibody-mediated targeting therapy with a great success in the treatment of cancers is also being explored because of better selectivity and efficacy. Li et al. (2015) reported a human epidermal growth factor receptor-2 (HER2) antibody-coupled NIR light-sensitive liposomes loaded with Dox. Targeted liposomal systems in combination to NIR exposure showed selective binding and increased toxicity against HER2-positive tumor cells and a doubletumor model in nude mice bearing both HER2-positive and negative tumors with more than 2-times enhanced accumulation in the tumors and 92.7% tumor inhibition efficiency as compared to non-targeted liposomes [89]. To win over chemotherapy associated MDR in cancer, Tang et al. (2014) developed reduction-sensitive liposomes (CL-R8-LP) composed of reduction-sensitive cleavable PEG and octaarginine (R8) that helped escaping lyososomes leading to increased cellular uptake of Dox and VER (P-gp inhibitor) in the tumor cells. Ex vivo studies in MCF-7/ADR cells or tumor spheroids revealed that there was increased cytotoxicity and apoptosis in case of CL-R8-LP (Dox + VER) as compared with Dox and control liposomes. Increased cell uptake was accounted to endocytosis mediated by clathrin and macropi- 266 Current Drug Targets, 2018, Vol. 19, No. 3 nocytosis with subsequent lysosomal escape. In vivo studies with these liposomes showed significant MCF-7/ADR tumor inhibition [90]. Park et al. (2010) developed reductionsensitive from an amphiphilic cucurbit[6]uril (CB[6]) derivative having disulfide bonds between hexaethylene glycol units and a CB[6] core. Folate-spermidine conjugate and fluorescein isothiocyanate-spermidine conjugate were used as the ligands for SSCB[6]VC. These functionalized liposomes enhanced cytotoxicity of Dox against cancer cells [91]. In another study, Goldenbogen et al. (2011) reported reduction-sensitive liposomes having disulfide bonds and a biotin moiety. Disulfide bond containing used compounds were tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), l-cysteine, or glutathione (GSH)). When this bond got broken it removed the hydrophilic head group resulting in release of the entrapped drug. Therefore, biotin-tagged anti-HER2 antibody coupled liposomes showed enhanced uptake of Dox in breast cancer cell line BT-474 [92]. 6. ENZYME-RESPONSIVE LIPOSOMES Liposomes are biodegradable and biocompatible drug delivery cargoes which can be feasibly modified for passive and active targeting to tumors with reduced toxicity and enhanced therapeutic efficacy. Active targeting to tumors involves recognition of specific tumor receptors via tumorselective ligands or antibodies, or by stimuli-responsive drug release using pH, temperature, redox, or enzyme-responsive ingredients in the development of liposomes. Anomalous tumor microenvironment poses the hurdles in drug delivery but also offers the ways to utilize these differential characteristics as tools for selective delivery to tumors. Such targets in tumors are tumor endothelium (neovasculature), tumor associated macrophages (TAMs), fibroblasts, and pericytes [93]. There are number of disadvantages of viral vectors such as adenovirus (Ad) for cancer gene therapy. Wan et al. (2013) introduced an enzyme-responsive liposomal system that could significantly increase cell transduction in the tumor and decrease the immunogenicity of Ad vectors. Cleavable PEG-lipids in responsive to enzyme were consisted of PEG, matrix metalloproteinase (MMP), substrate peptide, and cholesterol. These MMP-cleavable lipids were incorporated in the anionic liposomal Ad vectors (AL-Ad) using the postinsertion technique. In vitro infection assays in tumor tissues revealed higher gene expression in case of enzymeresponsive liposomes as compared to free Ad5 and the noncleavable PEG-lipid based Ad vectors [94]. Cancerassociated proteases (CAPs) are found to show overexpression in tumors and can be suitable cancer targeting tool. Protease-triggered caged liposomes were developed using urokinase plasminogen activator (uPA) specific peptide sequence and poly(acrylic acid). These liposomes prevented osmotic swelling and leakage of the entrapped materials but a uPA responsive release of liposomal contents with fast release kinetics [95]. MMP2 are upregulated in the tumor region and they can be targeted for cancer treatment. Zhu et al. (2012) developed MMP2 responsive PEGylated liposomes which were functionalized with mAb 2C5 (a tumor cell-specific antinucleosome) and TAT peptide (TATp). MMP2-sensitive bond between PEG and lipid got cleaved in the tumor area by the highly expressed extracellular MMP2 resulting in the removal of PEG chains. TATp allowed the enhanced intracellular delivery of the system after long-chain Jain and Jain PEG removal and exposure of the previously hidden surfaceattached TATp. It is shown that such a design can enhance the targetability and internalization potential of nanocarriers to cancer cells [96]. 7. MAGNETO-RESPONSIVE LIPOSOMES Magneto-responsive delivery of anticancer drug with active targeting approaches is under extensive expansion for treating solid tumors but this approach has so far limited applications in intravenous administration. For example, magnetic liposomes bearing ADR were developed and studied in osteosarcoma (Os515) inoculated in Syrian male hamsters. After administering these liposomes or drug solution (equivalent to 5 mg ADR/kg, i.v.) and providing a magnetic force (0.4 tesla), there was 4-times higher drug accumulation of ADR in case of magnetic liposomes in the tumor as compared to that of drug solution. This study explored the application of implanted permanent magnet for facilitating the drug release [97]. Data is limited in targeting potential of nanocarriers in magnetic resonance (MR) imaging. Yan et al. (2013) evaluated the tumor angiogenesis targeting potential of anti-αvβ3 antibody using magneto-liposomes. These liposomes were modified with PEG and loaded with superparamagnetic iron oxide (Fe3O4). Three-step pretargeting MR imaging involved: i.v. administration of (1) biotinylated anti-αvβ3 mAb in the mice model bearing MDA-MB-435S breast cancer, (2) subsequently giving avidin and streptavidin, and (3) then administering biotinylated magneto-liposomes or magneto-liposomes. PEG protected Fe3O4 cores from macrophage uptake. MR imaging showed enhanced signal in tumor periphery and accumulation of these functionalized liposomes in neovasculature decreased MR signal as predicted from histological examination [98]. Thermo-sensitive liposomes bearing paramagnetic lanthanide-based complexes such as Gd(III) or Ln(III) chelate can be a promising MR molecular imaging agent. These nanoprobes can be used for visualization (MR) of tumor cellular targets like the glutamine membrane transporters [99]. 8. MISCELLANEOUS APPROACHES Liposomes based therapy involve active targeting which can be ligand-mediated and stimuli-responsive, and passive targeting by virtue of EPR effect in tumors [100]. Smart liposomes could trigger release of entrapped bioactives upon various internal and external stimuli. In case of internal triggers, lysosomal low pH has been used as a demarcating tool to facilitate release of drug. Nahire et al. (2014) discussed an alternative approach for synthesizing pH sensitive lipids to impart pH sensitivity by incorporating a precursor that creates gas bubbles in situ in low pH. These gas bubbles resulted in abrupt release of liposomal content as indicated in atomic force microscopy. Moreover, these liposomes can also be used as echogenic nanodevices to permit US imaging owing to in situ generation of the gas bubbles. These liposomes were also loaded with Dox and modified with folate to target pancreatic ductal cancer. It was found that there was increased Dox accumulation (14%) in response to low pH in the lysosomes and an exposure to US (1 MHz) [101]. The M-type phospholipase A2 receptor (PLA2R1) is reported to internalize secreted phospholipase A2 (sPLA2) in normal cells by means of endocytosis. sPLA2 is found to be overexpressed in prostate cancer and it could be a mediator Stimuli-responsive Smart Liposomes in Cancer Targeting Table 2. Current Drug Targets, 2018, Vol. 19, No. 3 267 Application potential of thermo-sensitive liposomes in cancer treatment. Thermo-sensitive Liposomes Drug Targeted Cancer Remarks Ref. VB loaded thermo-sensitive liposomes Vinorelbine bitartrate (VB) Lung tumor Developed liposomes showed significant inhibition of tumor growth in lung tumor bearing mice as compared to free VB after a HT for 30 min. [74] Thermo-sensitive liposomes Calcein or DOTAphenylboronate conjugate Human BLM melanoma Bulk release of calcein or DOTA-phenylboronate conjugate was obtained at 41°C that can be used both in therapy and diagnosis applications. [75] CD13(+) cancer cells LTSLs showed rapid release of drug (above 75% in 4s) at 41.3°C. There was active uptake of LTSLs by CD13(+) cancer cells (3.6 times greater affinity) as compared to linear NGR-containing peptide coupled liposomes. [76] [77] cKNGRE coupled thermosensitive liposomes (LTSLs) Dox Folate coupled thermo-sensitive magnetic liposomes (MagFolDox) Dox KB and HeLa cells These liposomes showed more than 70% calcein release at 43°C as compared to only 5% release at 37 °C. MagFolDox showed increased cellular uptake of Dox than Caelyx, non-magnetic targeted liposomes and free Dox in vitro. HIFU triggered liposomes Dox Murine adenocarcinoma tumors HIFU (42 °C, 2 min) triggered 50% Dox release in vitro and decreased tumor growth as compared with all other liposomes and free Dox. [78] [79] ADR loaded long circulating thermo-sensitive liposomes (ALTSL) ADR H22 tumor ALTSL increased the anti-tumor effects and decreased the sideeffects of ADR. MPS in combination with ALTSL showed increased tumor growth inhibition (80.4%) with elevated activity of NK cells and transformation of T cells. Dox loaded thermo-sensitive liposomes Dox - These liposomes showed 100% release of Dox within 10-20 s at 41 °C and enhanced tumor regression as compared to free Dox. [80] ALTSL ADR H22 tumor rhIL-2 in combination with ALTSL potentiated targeting efficiency to H22 tumor-bearing mice with increased survival of mice and activity of NK cells in synergistic manner. [81] Thermo-sensitive liposomes bearing melphalan Melphalan B16F10 melanoma Liposomes showed enhanced in vivo efficacy in combination with HT in C57B1/6 mice bearing B16F10 melanoma with inhibition of tumor growth. [82] ADR loaded thermo-sensitive liposomes ADR Hepatic tumors There was high tumor/liver ADR level (25.2 µg/g of wet tissue) in rats bearing implanted hepatic tumors at 42°C. [83] Cisplatin loaded thermosensitive liposomes Cisplatin - These liposomes showed increased leakage of drug at 41°C in tumor bearing mice. [84] cKNGRE = cyclic NGR-containing peptide, Caelyx = commercially available Dox liposomes, HIFU = Pulsed-high intensity focused ultrasound, ADR = Adriamycin, MPS = monkshood polysaccharide. of metastasis. Quach et al. (2014) investigated PLA2R1 expression in primary prostate cells (PCS-440-010) and human prostate cancer cells (LNCaP, DU-145, and PC-3) for cytotoxicity because of PLA2R1 knockdown caused by free or drug loaded liposomes. Immunoblot analysis showed higher expression of PLA2R1 in prostate cancer cells than that of primary prostate cells. PLA2R1 knockdown was found to be increased in case of sPLA2 responsive liposomes (SPRL) bearing Dox leading to enhanced in vitro cytotoxicity. This revealed cell proliferation in response to PLA2R1 expression and modulation of efficacy of liposomes [102]. NF-κB regulated therapy can get better selectivity for gene expression in cancer cells and may overcome resistance caused by the same. Adil et al. (2014) reported multifunctional liposomes for αvβ1 integrin targeting and subsiding resistance by modulation of NF-κB. Under the control of an NF-κB responsive element (pNF-κB-Luc), PR_b peptide modified stealth liposomes bearing a luciferase gene was delivered and it resulted in higher gene expression in vitro in DLD-1 colorectal cancer cells in comparison to BJ-fibroblast normal cells. There was a decrease in the expression of cellular protein in a dose-dependent manner and enhanced cytotoxicity with these liposomes in vitro [103]. Prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) are reported to be potential markers for advanced stage of prostate cancer (PC). Recently, Xiang et al. (2013) developed a dual-modified liposomes that incorporated PSA-responsive and PSMA-mediated liposomes that provided an enhanced 268 Current Drug Targets, 2018, Vol. 19, No. 3 selectivity to PC. The engineered liposomes i.e. folate targeting and CPPs dual-modification increased cellular uptake in the tumor because of penetrating potential of polyarginine and receptor-mediated endocytosis. Multimodal approach using engineered liposomes loaded with siRNA showed significantly higher cytotoxicity potential in PC cells as compared to single-modified liposomes i.e. folate, CPP, PSAresponsive only and non-modified liposomes. Cellular uptake into 22Rv1 cells was found to follow multiple endocytic pathways such as clathrin-mediated endocytosis and macropinocytosis and favored endosomal escape of loaded siRNA for cytoplasmic release. These liposomes also showed enhanced accumulation and tumor growth inhibition and induced apoptosis in a 22Rv1 xenograft murine model [104]. CONCLUSION AND FUTURE SCENE Stimuli responsive liposomes with the use of various triggers such as pH, temperature, light, enzyme, and redox potential etc. are emerging tactics for the effective localization of the bioactives at tumor site with desired molecular manifestations. Safety and efficacy is still to be warranted for cancer therapy or diagnosis even after a tremendous growth in nanotechnology based approaches particularly in concern to multi-drug resistance, long-term chemotherapy, and gene therapy. siRNA based cancer targeting in recent scenario is rapidly growing field as a potent sequencespecific transcription silencer. However, there are number of hurdles in systemic delivery of siRNA delivery such as low transfection and non-specific immune response. A wide sky of tumor anomaly is still open to the scientists to engineer novel stimuli-responsive and multi-pronged liposomes to achieve safe and efficacious treatment of cancer. 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