Nanocarrier based approaches for targeting breast cancer stem cells

Abstract

Breast cancer stem cells (BCSCs) are heterogeneous subpopulation of tumour initiating cells within breast tumours. They are spared even after chemotherapy and responsible for tumour relapse. Targeting BCSCs is, therefore, necessary to achieve radical cure in breast cancer. Despite the availability of agents targeting BCSCs, their clinical application is limited due to their off-target effects and bioavailability issues. Nanotechnology based drug carriers (nanocarriers) offer various advantages to deliver anti-BCSCs agents specifically to their target sites by overcoming their bioavailability issues. In this review, we describe various strategies for targeting BCSCs using nanocarriers.

Graphical Abstract

Keywords:

• Breast cancer stem cells
• nanocarriers
• drug targeting
• surface biomarkers
• self-renewal

Introduction

Breast cancer is the most frequently diagnosed cancer and is the second leading cause of cancer deaths in women. Metastasis and relapse of tumour are emerging as major challenges in treatment of breast cancer [1,2]. Compelling body of evidences suggests that, small population of tumour initiating cells within tumours called breast cancer stem cells (BCSCs) are responsible for tumour relapse and metastasis [3–6]. The quiescence, expression of higher levels of drug efflux transporters and high DNA repair capacity of BCSCs makes them to resist apoptosis by chemotherapeutic agents [7,8]. The pool of BCSCs which are spared after chemotherapy will convert into tumour cells in future and results in tumour relapse [9]. In addition, BCSCs promote metastasis by induction epithelial-to-mesenchymal transition (EMT), in which, epithelial cells lose their intercellular adhesion, accompanied by gain of invasive and migratory properties, which is a pre-requisite for metastasis [10–13] (Figure 1). It is, therefore, necessary to eradicate BCSCs to achieve radical cure in breast cancer.

Figure 1. The role of BCSCs in tumour relapse and metastasis. (A) Conventional chemotherapeutic agents for treatment of breast cancer eliminate bulk tumour cells (non-BCSCs) while sparing tumour initiating, BCSCs and results in tumour relapse; (B) The transition from an epithelial state to a mesenchymal state (EMT) in BCSCs (or metastatic cells) occurs by activation of transcription factors such as Snail, Slug and Zeb and promotes dissemination. This EMT phenotype of cells enters blood circulation and survives by evading immune surveillance, then enter target organ. These cells can remain in a dormant state, lasting from months to decades or re-acquire epithelial properties by undergoing mesenchymal to epithelial transition (MET) in target organ to initiate secondary tumour growth; (C) Key signalling pathways implicated in the survival of BCSCs.

Expression of surface biomarkers (CD44, CD133, EpCAM and ALDH1), self-renewal pathways (Wnt, Notch, TGF-β, Hedgehog, Bmi-1 and STAT3), drug efflux transporters (ABCG2 and ABCB1), anti-apoptotic proteins (Bcl2, survivin etc.), autophagy, metabolism and specialized microenvironment are the important biological features of BCSCs [7,14,15] (Table 1). In recent years, a number of anti-BCSC agents have been proposed to eliminate BCSCs by altering these features (Table 2). However, these anti-BCSC agents have failed in clinical setting due to their poor water solubility, short circulation time, inconsistent stability and off-target effects [16–18]. There is a need, therefore, for suitable drug delivery systems to improve bioavailability and carry anti-BCSC agents specifically to BCSCs without off-target effects. Due to advantage of site specific delivery and ability to improve bioavailability and stability of drugs, nanocarriers have attracted attention of many researchers as carriers for targeting BCSCs: (1) surface markers, (2) self-renewal signalling pathways, (3) drug efflux transporters, (4) metabolism, (5) autophagy process, (6) anti-apoptotic proteins and (7) microenvironment [18–23] (Figure 2). In the present review, we discuss the current developments in these nanocarrier based approaches.

Figure 2. Possible therapeutic approaches for eradication of BCSCs using nanocarriers. (1) Targeting BCSC surface markers; (2) Silencing self-renewal pathways; (3) inhibiting drug efflux transporters; (4) inhibiting anti-apoptotic proteins; (5) altering metabolism; (6) targeting autophagy process; (7) disruption of vascular niche of the BCSCs to impair the specialized microenvironment housing.

Table 1. Molecular targets of BCSCs.

Molecular targets	Experimental results	References
Surface biomarkers
CD44	Required for cell–cell interaction, cell adhesion, and migration.	[114,115]
CD133	Inhibition of CD133 resulted in elimination of BCSCs and prevented tumour relapse.	[34,116]
EpCAM	EpCAM inhibition using SiRNA resulted in elimination of BCSCs.	[117,118]
Self-renewal pathways
Wnt/β-catenin pathway
Fz receptor	Monoclonal antibody against frizzled receptor inhibited Wnt pathway in BCSCs	[119]
β-catenin	β-catenin (CWP232228) resulted in elimination of BCSCs and non-BCSCs by inhibiting Wnt pathway.	[120]
Axin 2	Axin2 inhibition using small molecule inhibitor results in loss of β-catenin function and down regulates Wnt/β-catenin pathway.	[121,122]
Notch signalling pathway
γ- secretase	γ-secretase enzyme inhibition using MRK-003 (a γ-secretase inhibitor) silenced notch signalling in BCSCs and prevented mammosphere and colony formation.	[123]
MAML	ANTP/DN MAML, a fusion protein targets Notch nuclear co-activator MAML1 to inhibit notch signalling.	[124]
Delta-like 4 ligand (DLL4)	DLL4 inhibition by monoclonal antibodies or small molecule inhibitor resulted in anticancer activity by inhibiting notch signalling pathway.	[125–128]
Hedgehog (Hh) pathway
Smo	Smo antagonist (cyclopamine) prevented drug resistance in MCF-7/ADR cells and eliminates BCSCs.	[129]
Glioma-associated oncogene (Gli)	Gli inhibition using small molecule inhibitor effectively silenced Hh signalling pathway in breast cancer.	[130]
TGF-β	Inhibition of TGF-β signalling resulted in elimination of BCSCs.	[13]
Bmi-1	Bmi-1 silencing using SiRNA inhibited tumour growth and metastasis in breast cancer.	[110]
IL-6/JAK/STAT3 pathway
STAT3	STAT3 inhibition eliminated BCSCs and prevented tumour metastasis.	[131]
Apoptotic/Antiapoptotic proteins
PTEN/PI3/Akt axis	Inhibition of AKT inhibited mammosphere formation and resulted in complete eradication of tumour.	[132]
m-TOR	Inhibition of m-TOR with rapamycin sensitized BCSCs to radiation.	[133]
Bcl2	Bcl2 SiRNA enhanced chemosensitivity of BCSCs.	[134]
Fatty acid synthase (FAS)	Suppression of lipogenesis by modulating FAS expression resulted in apoptosis in BCSCs.	[135]
Death receptor (DR-5)	anti-DR5 antibody resulted in cytotoxicity of BCSCs.	[136]
Autophagy proteins
Beclin1	Depletion of Beclin1 resulted in inhibition of autophagy in BCSCs.	[137]
Metabolic enzymes and transporters
Hexose kinases (HK)	HK inhibition by metformin impairs glucose metabolism in BCSCs and resulted in tumour growth inhibition in breast cancer	[138,139]
Glucose transporters (GLUT)	Inhibition of GLUT1 transporter sensitized breast cancer cells to radiation.	[140]
Microenvironment
Hypoxia inducible factor1α (HIF1α)	HIF1α promotes growth of BCSCs. Inhibitors of HIF1α resulted in chemosensitization of BCSCs.	[141,142]
Carbonic anhydrase-IX (CAIX)	Inhibition of CAIX with novel small-molecule inhibitor eliminated BCSCs.	[143]
CXCR1	CXCR1 inhibition effectively eliminated BCSCs in vitro and in vivo.	[144]

Table 2. Various anti-BCSCs agents and their mechanism of action for elimination of BCSCs.

Anti-BCSC agent	Mechanism of action	References
Disulfiram	Modulator of MAPK signalling pathway	[145]
Flubendazole	Cell cycle arrest at G2/M phase and induced monopolar spindle formation through inhibition of tubulin polymerization	[146]
Metformin	Inhibition of Akt and hexose kinase	[147]
Niclosamide	Inhibition of Wnt, STAT-3, Notch and NF-κB pathways of self-renewal	[148]
Salinomycin	Inhibition of self-renewal	[149]
Simavastatin	Inhibition of mevalonate metabolism	[85]
Thioridazine	Antagonism of dopamine receptor on CSCs	[150]
Tranilast	Activation of aryl hydrocarbon receptor	[151]
Epigallocatechin gallate analogs	Activation of AMPK	[152]
Everolimus	Not established	[153]
Sulforaphine	Down regulation of Wnt-β catenin signalling	[154]
Cyclopamine	Smo Inhibitor	[53]

Targeting BCSCS using nanocarriers

Nanocarriers offer various advantages for targeting tumours by overcoming the bioavailability issued of chemotherapeutic agents [24]. They can passively accumulate at the tumour site owing to the leaky vasculature of tumour [25]. In addition, active targeting by surface modification of suitable ligands for tumour biomarkers improves the site specificity of nanocarriers [26–28]. Therefore, nanocarriers are being widely employed in recent years for tumour targeted drug delivery. With the evolution of “cancer stem cell model” in recent years several researchers have also applied nanocarriers for targeting CSCs [29]. In this section, we discuss nanocarrier based strategies for targeting BCSCs.

Targeting surface biomarkers of BCSCs using nanocarriers

BCSCs express specific biological markers on their surface. Fluorescence-activated cell sorting (FACS) uses these biomarkers to sort BCSCs [5]. Cluster of differentiation 44 (CD44), cluster of differentiation 133 (CD133), epithelial cellular adhesion molecule (EpCAM) and aldehyde dehydrogenase1 (ALDH1) are the important surface markers of BCSCs (Table 2). Recently, researchers focused on nanocarrier mediated targeting of these biomarkers, to deliver drugs specifically to BCSCs.

CD44 targeted nanocarriers

Biopolymers like hyaluronic acid and chitosan were being employed as ligands for active targeting of CD44 biomarker [30]. In a recent study, Dong Wang et al. have developed hyaluronan (HA) modified mesoporous silica nanoparticles (MSNs) loaded with 8-hydroxyquinoline supported by lipid bilayers containing docetaxel to eliminate BCSCs. In this study, HA modification resulted in enhanced uptake of nanoparticles in BCSCs via CD44 receptor. In the MCF-7 xenograft mice, this combination therapy enhanced antitumour efficacy and reduced off-target effects (Figure 3) [31]. In another study, Goodarzi et al. have developed a macromolecular conjugate of docetaxel with HA to selectively target CD44 + BCSCs The developed bioconjugates are effective in eliminating BCSCs along with non-BCSCs [32]. Ruo-Jing Li et al. have reported that, stealth liposomes of All-trans retinoic acid (ATRA) were able to decrease the expression of CD44 in the BCSCs in vitro, and prevented tumour relapse in breast cancer [9]. Wei Rao et al. have recently developed a doxorubicin-loaded polymeric nanoparticle surface modified with chitosan to specifically target the CD44 surface receptors of BCSCs [33] (Table 3).

Figure 3. The preparation procedure of HA modified mesoporous silica nanoparticles (MSN). MSN modified with an amine-containing silane were first loaded with docetaxel or 8-hydroxy quinoline. Liposomes were prepared with different components consisting of DOPC, DOPE, cholesterol and 18:0 PEG-2000 PE (60:5:30:5 mass ratio). HA was conjugated to the liposomes by an EDC-based method. Finally, hyaluronan modified mesoporous silica nanoparticle-supported lipid bilayers (HA-MSS) was constructed by fusing HA-modified liposomes to drug loaded MSN [31] (Reproduced with permission. Copy Right, Elsevier 2013).

Table 3. Targeting BCSC surface markers using nanocarriers.

Surface marker	Therapeutic agent	Nanocarrier	References
CD44	CD44 antibody Paclitaxel	Poly[(d,l-lactide-co-glycolide)-co-PEG](PLGA-co-PEG) micelles	[155]
CD133	CD133 antibody Paclitaxel	CD133 antibody conjugated polymeric nanoparticles	[34]
EpCAM	EpCAM aptamer Doxorubicin	Carbon nanotubes functionalized with EpCAM aptamer and piperazine–polyethylenimine derivative	[36]
	EpCAM SiRNA	Polymeric nanocomplex fabricated with EpCAM aptamer and EpCAM SiRNA	[37]

CD133 targeted nanocarriers

Swaminathan et al. have recently reported that, encapsulation of paclitaxel in CD133 antibody conjugated polymeric nanoparticles specifically targeted CD133⁺ BCSCs population and improved therapeutic efficacy compared to free paclitaxel. In addition of the CD133 targeted nanocarriers prevented tumour-recurrence in xenograft mice model [34] (Table 3).

EpCAM targeted nanocarriers

In order to specifically delivery SiRNA to EpCAM + BCSCs, Mohammadi et al. have devised, single-walled carbon nanotubes (SWCNTs) functionalized with EpCAM aptamer and piperazine–polyethylenimine derivative [35]. In another study, Alibolandi et al. have developed EpCAM aptamer conjugated polyethylene glycol–poly lactic acid-co-glycolic acid (PEG-PLGA) nanopolymersomes for targeted delivery of Doxorubicin to human breast cancer cells. They reported that, DOX loaded aptamer conjugated nanopolymersomes exhibited efficient cell uptake and internalization, and showed a significant cytotoxicity toward EpCAM-positive breast cancer cells [36]. Recently, Subramanian et al. have developed polyethyleneimine (PEI) nanocomplex fabricated with EpCAM aptamer and EpCAM SiRNA to target EpCAM + cells. This nanocomplex exhibited greater efficiency in elimination of EpCAM + cells [37] (Table 3).

Nanocarrier mediated silencing of self-renewal pathways in BCSCs

Self-renewal is one of the key mechanisms required for the survival of BCSCs. Wnt/β-catenin, Notch, Hedgehog (Hh), Transformin growth factor-β (TGF-β), signal transducer and activator of transcription factor 3 (STAT3) and B-cell-specific moloney-murine leukaemia virus integration site 1 (Bmi-1) are the key signalling pathways implicated in self-renewal and survival of BCSCs [38] (Table 4).

Table 4. Targeting self-renewal pathways of BCSCs using nanocarriers.

Signalling pathway	Therapeutic agent	Nanocarrier	References
Wnt/β-catenin	Sulforaphane	Self-assembled poly(d, l-lactide-co-glycolide)/hyaluronic acid block copolymer-based nanoparticles	[41]
	Salinomycin	PLGA nanoparticles	[19]
Notch	DAPT	Glucose functionalized mesoporous silica nanoparticles	[20]
	Notch SiRNA	Multifunctional Core/Shell nanoparticles Cross-linked Polyetherimide-folic acid	[47]
TGF-β	LY364947 PLK1 SiRNA	PEG-PLGA nanoparticles	[49]
Hedgehog (Hh)	Cyclopamine Doxorubicin	PLGA nanoparticles	[52]
BMI-1	BMI-1 SiRNA	Polyethylenimine conjugated BMI 1 SiRNA loaded in nanoparticles	[156]
STAT-3	Niclosamide	Polymeric micelles	[61]

Nanocarriers for silencing Wnt/β-catenin pathway

Wnt pathway activation occurs when Wnt ligand binds the transmembrane receptor, which in turn results in activation of the low-density lipoprotein-related receptor (LRP). This leads to the suppression of glycogen synthase kinase-3β (GSK-3β) protein, and improve the stability of β-catenin. It then forms a complex with the transcription factor/lymphocyte enhancer factor and activates the expression of its target genes such as c-terminus of myc protein (c-myc) and cyclin D1, involved in self-renewal of BCSCs [39,40]. β-catenin, is an important molecular target for silencing Wnt/β-catenin pathway. Several nanocarriers have been developed in recent years for silencing Wnt pathway by inhibiting β-catenin. For instance, Jingbin et al. have recently developed self-assembled poly (d,l-lactide-co-glycolide)/hyaluronic Acid block copolymer-based nanocarriers loaded with β-catenin inhibitor, sulforaphane (SFN) to inhibit self-renewal of BCSCs. These SFN-loaded nanocarriers were effective in silencing Wnt pathway in BCSCs than free SFN due to improved drug accumulation. In vivo studies also showed that, these nanocarriers had the strongest antitumour efficacy than free drug [41]. In another study, to silence Wnt signalling Peng Zhao et al. devised a self-assembled nanoparticle system with immune tolerant elastin-like polypeptide (iTEP) and salinomycin, a Wnt/β-catenin signalling pathway inhibitor for elimination of BCSCs. Salinomycin was encapsulated into amphiphilic iTEP − Salinomycin conjugates. The nanoconjugates of salinomycin significantly reduced the tumour growth in 4T1-orthotopic breast cancer model [42,43].

Nanocarriers for silencing notch signalling pathway

Because of its key role in self-renewal of BCSCs, Notch signalling has attracted research attention to prevent tumour relapse and metastasis [44,45]. Binding of ligands results in cleavage of Notch receptor by γ-secretase enzyme to release Notch intracellular domain (NICD). NICD then translocates into nucleus and then activates Notch target genes [46]. γ-Secretase, therefore, is an important target to prevent proteolysis of Notch receptors and to suppress the Notch activity in BCSCs. In a recent study, it was demonstrated that, glucose functionalized MSNPs carrying γ-secretase inhibitor, specifically inhibited notch signalling in BCSCs and non-BCSCs to inhibit their self-renewal capacity and to prevent tumour recurrence [20]. Recently, Yang et al. developed Multifunctional Core/Shell nanoparticles Cross-linked Polyetherimide-folic acid to deliver Notch-1 SiRNA to breast cancer cells [47]. Nanocarriers treated MDA-MB-231 cells exhibited significantly decreased expression of Notch-1.

Nanocarriers for silencing transforming growth factor-β (TGF-β) pathway

TGF-β signalling exerts tumour suppressor effects in normal cells and early carcinomas. However, in advanced tumours, the protective and cytostatic effects of TGF-β will be lost (as a result of mutations) and promotes tumour progression, invasion and metastasis. TGF-β signalling, therefore, has dual role as both tumour suppressor and promoter [48]. Recently, Zuo et al. have reported that inhibition of TGF-β signalling pathway using small molecule inhibitor, LY364947 promoted the penetration and improved the efficacy of nanocarriers with siRNA against oncogene, polo-like kinase 1 (Plk1) [49]. In another study by Liu et al., gold-metallofullerenol based nanomaterial effectively eliminated BCSCs by inhibiting TGF-β signalling [50].

Nanocarriers for silencing hedgehog (Hh) signalling pathway

In Hh pathway, the activation of Smoothened (Smo), a seven-pass transmembrane receptor is necessary for signalling process. Binding of Hh ligand to Smo activates the glioma associated oncogene (Gli) family of transcription factors (Gli1/2/3) to carry out the further downstream signalling required for self-renewal of BCSCs [51]. Smo and Gli family of transcription factors are the important targets for inhibition of Hh pathway. Nanocarriers are being widely employed to deliver Smo inhibitors and silence Hh pathway.

Hu et al. have developed amphipathic and redox-responsive polymer particles for co-delivery of cyclopamine (Smo inhibitor) and Doxorubicin to eradicate BCSCs and non-BCSCs [52]. The polymeric nanocarrier system composed of a hydrophobic PLGA head and a hydrophilic HA segment linked by a bioreducible disulphide bond. This nanocarrier system diminished the number and size of tumour spheres and also achieved active targeting of BCSCs. In addition, they enhanced tumour regression and prolonged survival compared to monotherapy using orthotopic mammary fat pad tumour growth model. In another study, it was reported that, cyclopamine inhibited Hh signalling in BCSCs and enhanced antitumour activity when given in combination with ¹⁷⁷Lu-mediated radiotherapy. The authors demonstrated that combined chemoradiation therapy is an effective strategy for elimination of both non-BCSCs and BCSCs in vitro and in vivo [53].

Nanocarriers for silencing IL-6/JAK/STAT3 signalling pathway

IL-6/JAK/STAT3 is the canonical STAT3 activation signalling pathway, reported to be deregulated in cancer. Since the receptor of IL-6 does not contain a kinase catalytic domain, it induces STAT3 phosphorylation by activating members of the JAK family [54–58]. It was demonstrated that inhibition of STAT3 pathway suppressed growth of xenograft tumours. In addition, it has been reported that IL-6/JAK1/STAT3 signalling pathway plays an important role in activation of EMT [59,60]. Misra et al. have demonstrated STAT3 modulation using mixed polymeric micelles of niclosamide (an inhibitor of STAT3 phosphorylation), to inhibit BCSCs and non-BCSCs. They have evaluated the efficacy of developed micelles in MDA-MB231 breast cancer cells. The niclosamide micelles were reported to eliminate CD44 + BCSCs and also improved the cytotoxicity of niclosamide in non-BCSCs [61].

Nanocarriers for induction of apoptosis in BCSCs

Deregulation of apoptosis and anti-apoptotic (survival) signalling pathways is a characteristic of cancer and a critical determinant of efficacy of chemotherapy [62]. The B-cell lymphoma2 (Bcl2), FLICE like inhibitory protein (cFLIP), nuclear factor-κ-B (NFκB), phosphatase and tensin homolog (PTEN), mammalian target of rapamycin(m-TOR), death receptors (DR)-4/5 proteins are the well-characterized regulators for induction apoptosis in BCSCs [21,24,63]. Nanocarrier mediated induction of apoptosis by downregulation of anti-apoptotic proteins or upregulation of apoptotic proteins specifically in BCSCs using nanocarriers is an attractive strategy. Chen et al. have proposed an inorganic nanoparticle system containing Doxorubicin and Bcl2 SiRNA to improve the efficacy of chemotherapy by eliminating drug resistant cancer cells [64]. In another recent study by Zhou et al., it was reported that, dual responsive mesoporous nanoparticles (MSNs) of Doxorubicin and Bcl2 SiRNA improved the efficacy of chemotherapy. The MSNs were prepared by modification of polyethylenimine–polylysine copolymers (PEI-PLL) via the disulphide bonds. To achieve active targeting folate (FA)-linked polyethylene glycol (FA-PEG) was conjugated to PEI-PLL. The FA-conjugated MSNs (MSNs-PPPFA) showed significantly enhanced intracellular uptake in folate receptor overexpressed MDA-MB-231 breast cancer cells than non-targeted counterparts and resulted in enhanced tumour regression because of downregulation of Bcl2 protein [23]. Shi-Yong Li et al. have developed, nanoparticles (NPs) of decitabine (DAC) for chemosensitization of BCSCs to DOX therapy and to inhibit ALDH1 expression [65]. In vitro studies revealed that treatment with nanoparticles loaded with DAC (NPDAC) combined with nanoparticles loaded with doxorubicin (NPDOX) reduced the proportion of BCSCs in the mammospheres of MDA-MB-231 cells, and overcame the drug resistance. In another study, a non-ionic surfactant (niosome) based co-delivery system containing Bcl2-SiRNA and ABCG2-SiRNA in combination with Doxorubicin to down regulate the ABCG2 and simultaneously induces apoptosis in BCSCs and non-BCSCs [21]. Recently, Shen et al. have devised a nanoparticle delivery system of proteasome inhibitor Bortezomib (BTZ) to induce apoptosis by disrupting protein homeostasis in BCSCs. BTZ nanoparticles (NPBTZ) prolonged circulation half-life of BTZ, improved cell uptake and enhanced tumour regression in both BCSCs and non-BCSCs [66]. In another study, it was reported that, the combination of vinorelbine stealthy liposomes and parthenolide stealthy liposomes resulted in simultaneous eradication of both BCSCs and non-BCSCs by affecting their apoptosis [67]. Zhang et al. have demonstrated that, targeting of daunorubicin and quinacrine using liposome resulted in elimination of BCSC and prevented of tumour relapse. The authors reported that, liposomes of daunorubicin and quinacrine resulted in prolonged circulatory effect and are selectively accumulated in mitochondria and induced apoptosis by activating pro-apoptotic protein Bax, decreasing of mitochondrial membrane potential (ΔΨm), releasing and activating caspase-9 and its downstream caspases-3 in BCSCs [68]. Sadhuka et al. have recently devised super paramagnetic iron oxide nanoparticles (SPIONs) to promote magnetic hyperthermia and induce reactive oxygen species (ROS) mediated apoptosis in BCSCs. Authors have demonstrated that SPION-transduced hyperthermia decreases side population cells, ADLH expression, tumour sphere formation, and in vivo xenotransplantation. In this study, the authors found ROS levels were increased upon hyperthermia in proportional to the time of the magnetic field exposure [69].

Inhibition of drug efflux transporters of BCSCs

ABCB1 or P-glycoprotein (P-gp) and ABCG2 or breast cancer resistance protein (BCRP) are important drug efflux transporters implicated in the chemoresistance in breast cancer [70,71]. Inhibitors of ABCG2, like Fumitremorgin C (FTC), Tryprostatin-A, Tariquidar, have been proposed to kill BCSCs in order to prevent chemoresistance. However, their low bioavailability and off-target effects hindered their clinical application [72–76].

Recently, it was reported that, pluronic block co-polymers have ability to overcome drug efflux and sensitize BCSCs to chemotherapy due to their ability to inhibit drug efflux transporters by inhibition of ATP synthesis in mitochondria of tumour cells [77–81]. Nanocarriers comprising pluronic block co-polymers are, therefore, being widely employed for enhancing drug accumulation and chemosensitization of BCSCs. In a recent study, Wang et al. have developed stimuli (pH and thermal) responsive nanoparticles comprising Pluronic F127, PLGA and chitosan for co-delivery of doxorubicin and irinotecan to eliminate BCSCs. Besides minimizing efflux of drug from BCSCs, the nanoparticles also affected the activity of topoisomerases-I & II and resulted in efficient destruction of the BCSCs both in vitro and in vivo [82]. Sun et al. have demonstrated an enhanced efficacy of chemotherapy by simultaneous suppression of multi drug resistance (MDR) proteins and anti-apoptotic genes using niosomes based co-delivery of ABCG2 SiRNA and Bcl2 SiRNA along with Doxorubicin chemotherapy [21]. In another report, novel cationic poly (lactide-co-glycolide) nanoformulation of MDR SiRNA reversed the MDR in vitro and enhanced the efficacy of Doxorubicin chemotherapy [83]. Recent research, therefore, suggest that, inhibition of drug efflux transporters using nanocarriers present an attractive strategy for sensitizing BCSCs to chemotherapeutic agents.

Targeting BCSCs metabolism

Inhibition of the mitochondrial metabolic function is one of the approaches for elimination of BCSCs [84,85]. Hexokinase 2 (HK2), which catalyzes the first step of glucose metabolism is the important target for altering the metabolism in BCSCs [86]. It was recently reported that, metformin (anti-diabetic drug) inhibit HK2 and thereby enhanced the effects of chemotherapy [87]. Considering the potential of metformin to kill BCSCs, Goodarzi et al. synthesized a hyaluronic (HA) acid conjugated metformin and docetaxel to kill both BCSCs and cancer cells. The synthesized polymer–drug conjugates were characterized using NMR, FTIR, DSC and GPC. The synthesized HA–drug conjugates have potential for CD44 mediated drug targeting of both BCSCs and non-BCSCs [22]. In another recent study, it was reported that, metformin loaded solid lipid nanoparticles (SLNs) have efficient cellular and mitochondrial uptake in MCF-7 breast cancer cells. In this study, metformin was successfully incorporated into SLNs by an emulsification and low-temperature solidification method. The pharmacokinetics results showed that SLNs could improve the bioavailability of metformin [88].

Figure 4. Potential nanocarrier systems for efficient targeting of BCSCs. (A) Nanocarriers serve as vehicles for therapeutic agents and increase drug accumulation in tumour tissues via EPR effect. (B) Surface modification of nanocarriers using suitable ligand can reduce off-target effects. (C) Stimuli-responsive nanoparticle systems, which are activated upon exposure to tumour microenvironment may further enhance the accumulation of drugs and also increase cellular internalisation. (D) Nanocarrier systems for co-delivery of dual drugs can improve therapeutic efficacy by simultaneous targeting of both BCSCs and non-BCSCs.

Autophagy

Autophagy plays an important role in breast cancer initiation or transformation of mammary epithelial cells, chemoresistance and metastasis [89]. It was reported that treatment with autophagy inhibitors (e.g. Chloroquine) or silencing of autophagy-associated genes affects stem cell renewal, differentiation and stress-resistant abilities and enhanced the sensitivity to chemotherapeutic agents by elimination of BCSCs [90,91]. In a recent study, Rong Sun et al. have reported that nanocarrier mediated autophagy inhibition significantly improved the efficacy of chemotherapeutic agents against BCSCs. In this study, they prepared nanocarrier system using PEG-PLA co polymers to deliver the autophagy inhibitor chloroquine (CQ) in combination with Doxorubicin and Docetaxel. The authors reported that, the developed nanocarriers significantly prolonged the circulation half-life of CQ and enhanced the accumulation of drugs in tumour tissues. Further, the combination therapy resulted in enhanced tumour regression by eliminating non-BCSCs as well as BCSCs in MDA-MB-231 orthotropic tumour mice model [92].

Targeting BCSCs microenvironment using nanocarriers

BCSCs require a specialized microenvironment or niche which is regulated by various factors for their survival. The factor that regulate BCSCs microenvironment include fibroblast stimuli, immune cells, autocrine signals and extracellular matrix (ECM) components, as well as physical/chemical factors such as oxygen pressure, nutrients levels and low pH [93,94]. Growth factors and cytokines released by tumour cells and cancer-associated fibroblasts and immune cells have strong effects on the survival and metastasis of BCSCs by maintaining microenvironment [95,96]. Nanocarriers have been developed recently to disrupt these components of BCSC microenvironment. For instance, it was reported that, nanocarrier mediated inhibition of lysyl oxidase (an enzyme required for collagen cross-linking and rigidity of extracellular matrix) results in disruption of tumour microenvironment [97,98]. In another study, PLA microspheres loaded with IL-12, TNF-α and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used as anti-tumoural therapy in a murine breast cancer model. The microspheres were administered by intratumoural injection, where they initiated a major infiltration of polymorphonuclear cells and CD8 + cytotoxic T-cells that resulted in increased the number of tumour free mice at the end of the study. This therapy also resulted in specific memory T-cells that could prevent tumour relapse in a murine breast cancer model [99].

Future directions

Compelling body of evidences suggest that, there is a clear promise in application of nanocarriers for detection, targeting, and eradication of BCSCs. However, in order to improve efficacy in specific targeting of BCSCs, nanocarriers should be rationally designed and must possess long circulation time, enhanced tumour accumulation and sufficient internalization in BCSCs. In this section, we discuss several potential design strategies to improve efficacy of future nanocarriers in targeting BCSCs (Figure 4).

Enhancing tumour accumulation using long circulating nanocarriers

Modification of nanocarriers with poly ethylene glycol (PEG) or other hydrophilic polymer modifications is a widely recognized approach for prolonging circulation time, by stabilizing nanoparticles, minimising non-specific binding and reducing clearance by immune cells [100]. Prolonging circulation time of the nanocarriers may some extent mitigate the effect of their poor extravasation into tissues due to increased exposure of the therapeutics in the body [101].

Enhancing intracellular penetration usingstimuli-responsive nanocarriers

Although PEGylation improves the accumulation of nanocarriers at tumour site, it was reported that, PEG reduces the cellular uptake of nanoparticles, affects intracellular trafficking and also hinders anti-cancer efficacy [102]. Stimuli-responsive nanocarriers with cleavable PEG moiety have been proposed in recent years to overcome this limitation. Stimuli-responsive nanocarriers target cancer cells based on characteristic microenvironmental changes occur in neoplastic diseases. These stimuli include lowered interstitial pH, and changes in the levels of certain components such as matrix metalloproteinases (MMPs) [103]. For instance, environment-responsive liposomes anchored by phospholipids with cell-penetrating peptides and a pH-sensitive PEG shield have improved cellular internalization. The liposomes lost their PEG coating at low pH and were internalized in the cells via the exposed cell-penetrating peptide moieties [104]. The application of stimuli-sensitive carriers to facilitate cellular internalization in BCSCs have rarely been reported, it could be a potential area worth further exploration.

Designing surface-tailored nanocarriers to reduceoff-target effects

Increasing body of evidence suggests that EPR effect does not provide specific targeting and sufficient intracellular delivery to BCSCs, which are located in the necrotic sites or otherwise deeply embedded tumour sites [28]. The concept of active targeting approach by conjugating an appropriate ligand for specific tumour marker on the surface of nanocarriers is an attractive strategy for selective delivery and improved accumulation of drugs and also to reduce off-target effects [105,106]. However, the markers currently used to target nanocarriers to BCSCs were also reported to be expressed on normal stem cells, this further makes BCSC-specific nanocarriers less discriminative and also can result in undesired toxicities [107]. Identifying highly specific BCSC markers is a major challenge as very few markers are available that exhibit sufficient selectivity in vivo. Active targeting should, therefore, be more comprehensively defined and evaluated to avoid the aforementioned pitfalls associated with receptor-specific targeting and to achieve the ultimate goal of enhanced efficacy. In addition, targeted nanocarriers must be able to reach the surfaces of BCSCs to interact with these cells through their targeting moieties, and this is possible only when there are no interactions of nanocarriers with biological objects in the body (cell surfaces, biomacromolecules, etc.). Therefore, the surface of nanocarriers must be modified with an “inert” polymer that would have minimal interactions with the cells, and then targeting moieties should be attached at the inert layer to allow for specific binding of the targeting moieties with their receptor. PEG is widely used inert polymer to minimize non-specific interaction of nanocarriers. However, there are clear indications that even this polymer attached to nanomaterials can activate complement, and otherwise engage the body immunity [108].

Nanocarrier based nucleic acid delivery to BCSCs

SiRNA and miRNA are the two main nucleic acid therapeutic drugs, which act by suppressing the genes required for survival of cancerous cells. RNAi may also have poor specificity or off-target effects, posing potential safety concerns and sometimes decreasing the effectiveness of treatment [109]. Nanocarriers are being employed to overcome the limitations associated with nucleic acid drugs [110]. After being taken up by cells, nanocarriers carrying nucleic acids should be able to induce endosomal escape and release their load in cytosol.

Designing nanocarriers for co-delivery of drugs or nucleic acids to BCSCs

Nanocarriers can be tailored for encapsulating multiple pay loads for simultaneous targeting of different cell populations. Combination of different anticancer drugs or a drug and nucleic acid using nanocarriers is being studied in recent years for treating cancers [19,111,112]. Similarly the nanocarrier-based delivery systems can also be explored for both identification and targeting BCSCs by co-encapsulation of a diagnostic and therapeutic agents. Although loading multiple drugs inside a single nanocarrier provides advantages in terms of less toxicity and better efficacy, this may result in complex pharmacokinetic and pharmacodynamics for each drug. When designing combination therapy these factors should be evaluated to justify the efficacy or synergistic effect achieved by the use of drug combination [113]. Combinatorial nanocarriers can be designed to target multiple pathways simultaneously with a combination of inhibitors and conventional therapeutics.

Conclusion

BCSCs are the major cause of tumour relapse, metastasis and chemoresistance in breast cancer. As discussed in this review, many molecular targets like surface markers, proteins involved in self-renewal and apoptotic pathways, autophagy, metabolism and microenvironment have been identified for eradication of BCSCs. The current research is focused on developing anticancer agents against these targets to eliminate BCSCs and to achieve radical cure in breast cancer therapy. Although several agents are proposed to eliminate BCSCs, their clinical utility is limited due to their poor water solubility, instability and off-target effects. We have discussed the potential of nanocarrier based drug delivery systems to overcome the limitations associated with current anti BCSC agents. The overview of recent applications of nanocarriers for targeting BCSCs suggests that nanocarriers offer various advantages for targeting of BCSCs. However, application of nanocarriers for BCSC-targeting is in its infancy, and many issues need to be well studied in clinical setting.

Abbreviations
ABC	=	ATP-Binding Cassette
ATM	=	Ataxia Telangiectasia Mutated serine/threonine kinase
Bcl2	=	B-cell lymphoma 2
BCRP	=	Breast cancer resistance protein
BCSCs	=	Breast cancer stem cells
BIK	=	Bcl2 interacting killer
Bmi-1	=	B-cell-specific moloney murine leukaemia virus integration site 1
BMP2	=	Bonje morphogenetic protein 2
CAIX	=	Carbonic anhydrase-IX
CAT	=	Catalase
CSL	=	CBF-1/RBPJ-κ in Homo sapiens/Mus musculus respectively, Suppressor of Hairless in Drosophila melanogaster, Lag-1 in Caenorhabditis elegans
CDK	=	Cyclin dependent kinases
ChKs	=	Checkpoint kinases
c-myc	=	C-terminus of myc protein
DHh	=	Desert Hedgehog
DLL4	=	Delta-like 4 ligand
DOX	=	Doxorubicin
DRs	=	Death receptors
EMT	=	Epithelial to mesenchymal transition
EpICD	=	EpCAM intracellular domain
FAS	=	Fatty acid synthase
FTC	=	Fumitremorgin C
Gli	=	Glioma-associated oncogene
GLUT	=	glucose transporter
GM-CSF	=	granulocyte-macrophage colony-stimulating factor
GPO	=	glutathione peroxidase
GSK-3β	=	Glycogen synthase kinase 3 β
HDR	=	Homology-directed recombination
Hh	=	Hedgehog
HK	=	Hexose kinase
IHh	=	Indian Hedgehog
IL	=	Interleukin
JAK	=	Janus Kinase
LRP	=	Low-density lipoprotein-related receptor
MAML	=	Master mind like
mTOR	=	Mammalian target of rapamycin
Nanog	=	Gene named after the Tír na nÓg legend
NHEJ	=	Non-homologous end joining
NICD	=	Notch intracellular domain
Non-BCSCs	=	Non-Breast cancer stem cells or bulk tumour cells
Oct-4	=	Octamer binding transcription factor
PDZ	=	Dishevelled PDZ domain
PI3-k	=	Phosphoinositide 3-kinase
PTEN	=	Phosphatase and tensin homolog
SCs	=	Normal Stem cells
SHh	=	Sonic Hedgehog
SMADs	=	Homologs of Sma and MAD proteins
Smo	=	Smoothened
SOD	=	Superoxide dismutase
SOX	=	Sry-related HMG box
STAT	=	Signal transducers and activators of transcription
STAT3	=	Signal transducer and activator of transcription factor 3
TGF-β	=	Transforming growth factor-β
TR	=	Thio-redoxin

Disclosure statement

No potential conflict of interest was reported by the authors.