Delivery of therapeutics using nanocarriers for targeting cancer cells and cancer stem cells.

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Delivery of therapeutics using nanocarriers for targeting cancer cells and cancer stem cells

Development of cancer resistance, cancer relapse and metastasis are attributed to the presence of cancer stem cells (CSCs). Eradication of this subpopulation has been shown to increase life expectancy of patients. Since the discovery of CSCs a decade ago, several strategies have been devised to specifically target them but with limited success. Nanocarriers have recently been employed to deliver anti-CSC therapeutics for reducing the population of CSCs at the tumor site with great success. This review discusses the different therapeutic strategies that have been employed using nanocarriers, their advantages, success in targeting CSCs and the challenges that are to be overcome. Exploiting this new modality of cancer treatment in the coming decade may improve outcomes profoundly with promise of effective treatment response and reducing relapse and metastasis.

Sangeetha Krishnamurthy1,2, Xiyu Ke1,2 & Yi Yan Yang*,1 Institute of Bioengineering & Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore 2 NUS Graduate School for Integrative Sciences & Engineering, National University of Singapore, Singapore *Author for correspondence: Tel.: +65 6824 7106 Fax: +65 6478 9084 yyyang@ ibn.a-star.edu.sg 1

Keywords: cancer stem cells • cancer therapy • drug delivery • gene delivery • nanocarriers • protein delivery

Contrary to the belief about the therapeutic effect of stem cells, numerous studies in the past decade have proved that stem cells are also essential in the development of tumors. Termed ‘cancer stem cells’ (CSCs), these are a subpopulation of cells in the heterogeneous tumor environment, which exhibit self-renewal, show enhanced resistance and are able to create a tumor in immunodeficient mice. The role of CSCs in tumor initiation, relapse and metastasis is illustrated in Figure 1 [1] . The term CSCs has been used interchangeably with tumor-initiating cells or stem-like cells as there is no strict definition of CSCs being followed since the field is still at its infancy. CSCs were initially discovered in leukemia in the 1990s [2] . Later they were identified in almost every type of cancer including lung, gastric, prostate, pancreatic and brain cancer [3–7] . The identification of CSC subpopulation in each type of cancer was based on the expression of certain unique surface markers. For example, brain CSCs were identified based on expression of CD133 and nestin [5] ,

10.2217/NNM.14.154 © 2015 Future Medicine Ltd

gastric CSCs on expression of CD44 [8] and prostate CSCs on the basis of high expression of A2B1 and CD133 [9] . Various assays are routinely used to identify and isolate CSCs, primarily by using the above-mentioned surface markers via flow cytometry. Another method is relatively simpler, through which the population of stem cells is enriched from a cancer population by using specific features unique to stem and drug-resistant cells. One such example is the side population sorting, which has been proved to contain a concentrated population of stem cells than nonside population cells [10] . It is based on the enhanced activity of ATP-binding cassette subfamily G member 2 (ABCG2) efflux pumps in CSCs in comparison with somatic cells. A few nuclear staining dyes like Vybrant® Dye Cycle™ (Invitrogen, CA, USA) and Hoechst 33342 (Sigma-Aldrich, MO, USA) are used for staining the whole population and the cells are sorted by flow cytometry depending on their ability to efflux or retain the dye [11] . Similarly, stem cell population can be

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A

Asymmetrical division

BCSC Plasticity Cancer cell

B

Therapy resistance Primary tumor Relapse D Matastasis

C

Figure 1. Key roles of breast cancer stem cells in disease’s progression. (A) The small fraction of BCSCs within tumors is maintained by self-renewal of BCSCs and by ‘dedifferentiation’ of cancer cells. (B) BCSCs are more resistant to radiation and chemotherapy than the rest of tumor cells and can survive therapy. (C) The surviving cancer stem cells are able to repopulate treated tumors and cause relapse. (D) BCSCs can also be the source of metastatic lesions. BCSC: Breast cancer stem cell. Reproduced with permission from [1] ; copyright © 2012, Elsevier.

enriched by separation based on proteins like aldehyde dehydrogenase and TGF-ß. The self-renewal property of CSCs is evaluated using in vitro assays like colony and sphere formation assays. The tumorigenic potential of the isolated CSCs are confirmed by inoculation in immunocompromised mice [2] . Chemotherapy and radiation therapy have been commonly employed in clinic for cancer treatments. However, the current treatment modalities fail miserably in tackling CSCs as chemotherapy [12] and radiation therapy [13] have been ironically shown to increase CSC population. The main reason for this is the quiescent nature of CSCs and their enhanced chemotherapy and radiation resistance [14,15] . Several small molecular weight compounds have been found to effectively target CSCs like salinomycin [16] , lonidamine [17] , tariquidar [18] , tesmilifene [19] , biguanides (metformin and phenformin) [20] , plant-derived compounds like curcumin [21] , piperine [21,22] , epigallocatechin-3-gallate (EGCG) [22] and sulforaphane [23] . Some CSC-target-

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ing drugs have been tested in clinical trials. For example, tariquidar was tested in a couple of studies as an adjuvant to sensitize P-glycoprotein (P-gp) rich drugresistant tumors and chemotherapy-resistant advanced breast carcinoma in combination with vonirelbine, doxorubicin or taxanes. The results from both studies showed that it had potential to increase drug exposure in drug-resistant tumors, but the study on chemotherapy-resistant breast carcinoma had to be stopped due to the concern of adverse effects caused by tariquidar [24,25] . Similarly, metformin is being investigated as an adjuvant in several ongoing clinical trials on patients with CSC-enriched breast cancer either alone as a neoadjuvant or in combination with anticancer drugs like letrozole. Preliminary results indicate that the drug improves the DNA damage recognition and repair along with regulation of cancer cell metabolism [26,27] . However, wide clinical applications of these drugs are hampered by their nonspecific toxicity to healthy tissues, short half-life and lack of water solubility [28–30] . For example, phenformin, an anti-diabetic drug has been withdrawn by US FDA due to incidences of fatal systemic toxicities [31] . Curcumin has very poor absorption and rapid metabolism despite the route of administration [28] . Therefore, it is critical to deliver these drugs effectively in vivo to increase water solubility, reduce toxicity, prolong blood circulation and enhance efficacy. Nanoparticles especially those that are made from biodegradable and biocompatible polymers have been studied as carriers to deliver a wide range of therapeutic molecules [32] including hydrophobic/hydrophilic drugs [33–35] , proteins and peptides [36] , imaging probes [37] , nucleic acids [38] , and antibodies [39] . They have also been explored to deliver multiple drugs simultaneously [40] . The nanoparticles are able to protect the cargo from the harsh biological conditions until they reach the intended site [41] , enhancing its pharmacokinetics and pharmacodynamics significantly [42] . PEGylated nanoparticles have been shown to evade opsonization by the reticulo-endothelial systems and hence prolong nanoparticle circulation time and reduce accumulation in healthy tissues compared with non-PEGylated nanoparticles [43] . Stimuli-responsive nanoparticulate systems, which respond to external stimuli like temperature [44] , pH [45–48] and light [49] , have been developed to deliver drugs in a controlled manner. Due to their nanosize, the nanoparticulate delivery systems are able to accumulate in the tumor preferentially due to the enhanced permeability and retention (EPR) effect [50,51] and more specific targeting can also be achieved with the use of targeting moieties like folate [52] , antibodies [53] and aptamers [51,54] . Hence with all these advantages, nanocarriers can be an excel-

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Delivery of therapeutics using nanocarriers

lent candidate for delivering therapeutics to CSCs. With efficient delivery of CSC-specific therapeutics to the tumor, relapse and metastasis can be avoided, as illustrated in Figure 2. Drug, protein and gene delivery using nanoparticles have been reported to target CSCs. In the following sections, these approaches will be reviewed, and the strategies to improve anti-CSC efficacy as well as the potential of nanomedicine in targeting CSCs will be discussed. Drug delivery Conventional cancer chemotherapeutics have been found to be ineffective in treating CSCs since CSCs exist in a state of quiescence and have abundant expression of ABC transporters and enhanced DNA repair capabilities [55,56] . Hence a CSC-targeted approach is needed to eradicate a tumor completely. As mentioned previously, a number of other classes of chemotherapeutics have been reported to be effective in CSC targeting. For example, the antipsychotic drug thioridazine is a dopamine antagonist and has an effect on CSCs which express dopamine receptors [57] , and the anthelmintic drug niclosamide targets breast cancer stem cells in vitro and in vivo [58] . The antidiabetic drugs metformin and phenformin were also found to target CSC by modulating the miRNA expression and subsequently modulating the signaling factors [21,59] . Antibiotics like salinomycin [16] , sulforaphane [23] and even natural compounds like EGCG [22] , curcumin [60] , piperine [21] and genistein [61] have been very effective in reducing CSC load. In addition, proteins [62] and genes [63] were also reported to overcome CSCs. Various nanoparticulate systems have been proposed to deliver these drugs in single or combination formulations to achieve synergistic effects. A list of drugs that have been used against CSCs is listed in Table 1. Single-drug delivery systems Several types of nanoparticles such as polymeric nanoparticles, liposomes, fullerenes, carbon nanotubes, silica and metallic nanoparticles have been used for drug delivery. However polymeric nanoparticles/ micelles and liposomes are the most preferred carriers due to several advantages that they provide, including biodegradability, biocompatibility, prolonged blood circulation, easy installation of versatile functionalities that interact with drugs for effective loading. This trend is also evident from several polymeric nanoparticle and liposome systems, which are being used clinically [64] or in clinical trials [65] for drug delivery when compared with other types of nanoparticles like metal nanoparticles. Curcumin is a very promising plant derivative, which has been found to be effective in targeting can-


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cer cells and CSCs [21] . However, its in vivo efficacy is restricted due to low bioavailability [30] . Wang et al. reported polymeric micelles self-assembled from stearic acid-g-chitosan oligosaccharide (CSO-SA), which provided high loading capacity for curcumin. Curcumin was loaded into CSO-SA micelles by sonication followed by membrane dialysis. A loading level of 29 wt% was achieved, and the curcumin-loaded micelles were tested against colorectal cancer cells. In addition to increased cytotoxicity compared with free curcumin, the curcumin-loaded micelles also significantly inhibited CD24 +/CD44 + CSC subpopulation. The reduction of CSCs was monitored by flow cytometry and also using spheroid assay in vitro. They further tested these micelles in an orthotopic colorectal tumor model in nude mice, and the curcumin-loaded micelles effectively suppressed tumor growth. In addition, there was more than 50% reduction of CSCs in the tumor [60] . In another study reported by the same group, CSOSA micelles were used to deliver a conventional chemotherapeutic drug (oxaliplatin) [66] . Experimental results showed that the delivery of oxaliplatin-loaded micelles raised the intracellular concentration of oxaliplatin significantly and reduced the IC50 of the drug by fourfold as compared with the free drug. In addition, the drug-loaded micelles reduced CD133 +/ CD24 + CSC subpopulation both in vitro and in vivo. The efficacy of oxaliplatin-loaded micelles in targeting CSCs was attributed to the ability of these micelles to bypass the ATP-binding cassette transporters overex-

Gene delivery

Protein delivery

Metastasis

Cancer stem cells

Drug delivery

Relapse

Figure 2. Illustration of various approaches to target cancer stem cells using nanocarriers. Various cancer stem cell-specific drugs, proteins and genes can be delivered using nanocarriers that can lead to reduced incidence of tumor metastasis and relapse.

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Table 1. List of small molecular drugs used for targeting cancer stem cells. CSC origin

Drug

Delivery carrier

Head and neck squamous cell carcinoma

All-trans retinoic acid (ATRA)

N/A

Ref. [97]

Colon cancer

Indomethacin

N/A

Pancreatic cancer, glioblastoma, medulloblastoma, colorectal cancer

Curcumin

Nanocurc™ (SignPath Pharma, Inc., PA, USA) polymeric micelle

Breast cancer

Quinacrine

Liposomes

[102]

Breast cancer

ATRA

Liposomes

[72]

Breast cancer

Salinomycin

Polymeric micelle

[69]

Breast cancer

Thioridazine

Polymeric micelle

[70]

Breast cancer, head and neck cancer

Epigallocatechin gallate (EGCG)

N/A

[22,103]

Breast cancer

Metformin

N/A

[59]

Glioblastoma multiforme

Cyclopamine

N/A

[104]

Pancreatic cancer

Cyclopamine and rapamycin

N/A

[105]

Glioblastoma

Temozolomide

N/A

[105]

Leukemia

Sabutoclax

N/A

[106]

Melanoma

Phenformin

N/A

[107]

Breast cancer

Sulforaphane

N/A

[23]

Breast cancer

Piperine

N/A

[21]

Prostate cancer

Genistein

N/A

[61]

Breast cancer

Ginsenoside F2

N/A

[108]

[98] [60,99–101]

CSC: Cancer stem cell; N/A: Not applicable (i.e., no delivery carrier used).

pressed in CSCs, leading to effective delivery of the drug into the cells. Nanoparticles have also been shown in several other reports to successfully deliver drugs to multidrug-resistant cells, evading the drug-efflux pumps. This is due to the difference in mode of entry of free drugs (mainly by diffusion) and nanoparticles (by endocytosis). Dual-drug delivery systems It has been shown that nanoparticle-based systems loaded with single CSC-targeting drugs are effective in reducing the CSC population and bringing about tumor remission. However various CSC-targeting drugs by themselves are often not very effective in reducing the bulk cancer cells. This is due to the fact that CSC-targeting drugs are not highly cytotoxic as compared with conventional chemotherapeutic drugs. Therefore, studies have been reported to explore combination therapy by using a conventional chemotherapeutic drug and a CSC-targeting drug for eliminating both cancer cells and CSCs. Lu’s group reported the use of liposomes encapsulating various drug combinations for targeting breast cancer cells and CSCs. Liposomes were prepared from

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egg phosphatitylcholine (EPC), cholesterol and polyethylene glycol-distearoyl phosphosphatidyl ethanolamine (PEG2000-DSPE) and termed as ‘stealth liposomes’. In the first study, vinorelbine, a semisynthetic vinca alkaloid that inhibits cell cycle and a sesquiterpene lactone, parthenolide, which has been found to target CSCs by multiple pathways, were loaded into liposomes individually [67] . The combination of vinorelbine-loaded and parthenolide-loaded liposomes was tested in both MCF-7 and MDA-MB-231 human breast cancer cell lines. The vinorelbine-loaded liposomes inhibited normal cancer cells significantly while having a minimal effect on CSCs, and parthenolideloaded liposomes effectively killed the CSCs. In an MCF-7 xenograft mouse model, complete inhibition of tumor growth was observed in the group administered with the combination of liposomes. In the second study, the drugs chosen were daunorubicin, an anthracycline antibiotic which acts via multiple pathways to kill cancer cells including inhibition of DNA synthesis and tamoxifen, a modulator of estrogen receptor, which was hypothesized to target CSCs due to its ability to upregulate p53 expression, and reverse P-gp as well as breast cancer resistance protein [68] . Daunoru-



bicin and tamoxifen were loaded into stealth liposomes separately, and tested on human breast cancer cell line, MCF-7 and also on MCF-7 xenograft mice. Daunorubicin had very little effect on CSCs in vitro, whereas the combination of daunorubicin and tamoxifen significantly increased the inhibitory effects on both tumor cells and CSCs. The in vivo cotreatment of daunorubicin- and tamoxifen-loaded liposomes resulted in better tumor inhibition than individual treatments and even caused gradual shrinking of the tumor. Thus, compared with the free drugs, the liposomal formulation was significantly more effective and less toxic both in vitro and in vivo. Zhang’s group also reported the co-delivery of paclitaxel and salinomycin using polymeric micelles [69] . Salinomycin was found to effectively reduce CSC population hundred-folds when compared with paclitaxel. A diblock copolymer of poly(ethylene glycol) and poly(ε-caprolactone) (PEG-b-PCL) was used to form micelles and the drugs were loaded into the micelles individually. The paclitaxel-loaded micelles were further decorated with octreotide for targeting somatostatin receptors overexpressed in cancer cells. The micelles had an average size of 25–30 nm, which was considerably smaller than the aforementioned liposomes [68] , thereby increasing their ability to accumulate in tumors via the EPR effect. The co-delivery of paclitaxel-loaded and salinomycin-loaded micelles killed MCF-7 cells in vitro and their effect was similar to the free drug combination. However, in vivo studies on MCF-7 mouse xenografts, the micellar formulation showed a significantly higher anti-tumor activity than the free drugs. This could be attributed to the enhanced accumulation of drug-loaded micelles in the tumor and sustained drug release. Recently, we reported the co-delivery of doxorubicin, an anticancer drug that kills a wide range of cancer cells and thioridazine, an anti-psychotic drug that targets CSCs, using nanosized biodegradable functional polymeric micelles [70] . The micelles were formed by self-assembly of a mixture of a diblock copolymer of PEG and urea-functionalized polycarbonate (PEG-bPUC) and a diblock copolymer of PEG and acid-functionalized polycarbonate (PEG-b-PAC) as shown in Figure 3A . The mixed micelles were stable in the blood stream due to strong hydrogen bond formed between acid and urea in the micellar core, and preferably accumulated in tumor tissues after intravenous injection [71] . These micelles were around 80 nm in size and showed excellent loading capacity for both doxorubicin and thioridazine. The micelles were tested in BT-474 and MCF-7 human breast cancer cell lines and a BT-474 xenograft mouse model. The combination therapy had a significantly better effect against both cancer cells


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and CSCs in vitro than individual drug formulations. In particular, in the animal model, the combination therapy suppressed tumor growth more effectively than individual drug formulations (Figure 3B) . The combination therapy delivered by the micelles did not induce significant weight loss during the whole course of the study as shown in Figure 3C . Finally, the percentage of CSCs in the resected tumor was analyzed using flow cytometry. The treatment with thioridazine showed the highest reduction in the percentage of CSCs, and CSC population in the tumors treated with the combination therapy was much lower than the individual drug formulation-treated groups (Figure 3D) . A similar strategy was employed in other studies. For example, Li and coworkers used all-trans retinoic acid (ATRA) to induce differentiation of CSCs and make them susceptible to the traditional chemotherapeutic drug, vinorelbine as shown in Figure 4A [72] . ATRA and vinorelbine were individually loaded into nanosized liposomes synthesized from EPC, cholesterol and PEG2000-DSPE. The drug-loaded liposomes were tested on MCF-7 breast cancer cells and also sorted CSCs in comparison with the free drugs as shown in Figure 4B–E. Both free ATRA and ATRAloaded liposomes inhibited the growth of CSCs (i.e., side population [SP] cells) more effectively than that of noncancer stem cells (i.e., nonside population [NSP] cells) (Figure 4B & C) . In contrast, vinorelbine-loaded liposomes were more sensitive to NSP than SP cells (Figure 4D) . Combination of ATRA- and vinorelbine-loaded liposomes had a greater inhibitory effect against SP cells than individual liposome formulations (Figure 4E) . The combination therapy was tested in a relapse tumor model by xenografting the sorted MCF-7 CSCs into the nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice. Both ATRA and ATRA-loaded liposomes delayed the formation of tumors in mice compared with the control group without any treatment (15 days vs 11 days). ATRA-loaded liposomes performed better than free ATRA in reducing tumor size, and the combination of ATRA- and vinorelbine-loaded liposomes were the most effective in suppressing tumor growth. In another study, quinacrine was used in combination with daunorubicin, where quinacrine (an antiprotozoal drug) essentially acted as a resistancemodulating agent, sensitizing CSCs to daunorubicin [73] . Both drugs were individually and simultaneously loaded into liposomes, which were made of PEG2000DSPE, EPC and cholesterol by using an ammonium sulfate gradient loading method. The liposomes were further modified with dequalinium for mitochondrial targeting of CSCs. The liposomes were about 98–100 nm in size with loading efficiencies of >80%. The


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O O

O

(2) Pd/C, H2 O

O

O

O

O O

O

H

O y

n

O

H N

H N

PEG-b-PUC

O

OH

OH OH O

O

O

O

O

x

HO

O

H N

H N

H

O

O

m

H3C

O

O

H3C

PEG, DBU/TU

O

O

O

PEG-b-PAC

O O

O

(1) PEG, PUC

OH O

H

O

N CH3 H

Thioridazine (THZ) H3C

N

S

S

OH

Doxorubicin (DOX)

NH2

DOX-MM

THZ-MM TH

Target cancer cells ll

Target cancer stem cells s

Cancer cell Cancer stem cell

148

e tumor volume (%)

Figure 3. Effect of co-delivery of doxorubicin and thiroridazine using polymeric mixed micelles on breast cancer cells and cancer stem cells (see facing page). (A) Schematic showing the synthesis of polymer and preparation of micelles; (B) effect of various formulations in nude mice bearing BT-474 xenografts; (C) changes of mouse body weight during the treatments; and (D) percentage of cancer stem cells (CD44+/CD24-) in cells obtained from tumors at the end of the treatments. a: p < 0.01 vs control; b: p < 0.05 vs THZ; c: p < 0.05 vs DOX; d: p < 0.01 vs DOX; e: p < 0.01 vs THZ; f: p < 0.01 vs DOX-MM or THZ-MM; 300 g: p < 0.05 vs DOX + THZ; h: p < 0.05 vs DOX-MM; i: p < 0.01 vs DOX-MM; j: p < 0.01 vs DOXControl MM + THZ-MM 280 THZ CSC: Cancer stem cell; DBU:1,8-diazabicyclo[5,4,0]undec-7-ene; DOX: Doxorubicin; MM: Mixed micelles; 260 THZ-MM PAC: Acid-functionalized polycarbonate; PUC: Urea-functionalized polycarbonate; THZ: Thioridazine; TU: Thiourea. DOX Reproduced with240 permission from [70] ; copyright © 2013, Elsevier. DOX-MM 220 DOX+THZ 200 DOX-MM+THZ-MM 180 Nanomedicine (Lond.) 160 (2015) 10(1) 140 120


a


300

Control THZ THZ-MM DOX DOX-MM DOX+THZ DOX-MM+THZ-MM

280 260 240 Relative tumor volume (%)

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220 200 180 160

a

140 120

a, b a a a, c, e

100 80 60

a, f, g

40 20 0

0

1

120

3

4

5

6

7 8 9 Time (days)

10

11

12

13

14

15

16

Control THZ THZ-MM DOX DOX-MM DOX+THZ DOX-MM+THZ-MM

115

Relative body weight (%)

2

110 105 100

a, h, j

95

a, i, j

90 85

Percentage of CSCs from tumor (%)

80

0

1

2

3

4

5

6

7 8 9 Time (days)

10

11

12

13

14

15

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a

8

1 Control 2 DOX 3 THZ 4 DOX+THZ 5 DOX-MM 6 THZ-MM 7 DOX-MM+THZ-MM

6

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h

d a

2

a, e

0 1


2

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4 Groups

5

6

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A Drug releases from the liposomes Endocytosis of the liposomes Drug accesses the cytoplasm as a result of being destroyed by lysosomes or passive diffusion

All-trans retinoic acid stealth liposomes prevent the relapse of breast cancer by differentiating cancer stem cells and arresting cell cycle, and kill the cancer cells by a co-therapy with vinorelbine stealth liposomes Microtubules

Nuclear All-trans retinoic acid Vinorelbine Breast cancer cell or breast cancer stem cell B

Inhibitory rate (%)

SP, sorted breast cancer stem cells NSP, sorted breast cancer cells

80 60 40 20

Inhibitory rate (%)

C

120 100

0 0

120 100

40 20

E SP, sorted breast cancer stem cells NSP, sorted breast cancer cells

80 60 40 20 0

0.0 0.1 0.5 Concentration of vinorelbine stealth liposomes (µM)

120 Inhibitory rate (%)

Inhibitory rate (%)

120

SP, sorted breast cancer stem cells NSP, sorted breast cancer cells

60

0

1 5 10 20 50 Concentration of free all-trans retinoic acid (µM)

A Apoptosis

80

D

100

N Necrosis

100

0 1 5 10 20 50 Concentration of all-trans retinoic acid stealth liposomes (µM) Vinorelbine stealth liposomes Vinorelbine stealth liposomes + 10 µM all-trans retinoic acid stealth liposomes ns

80 60 40 20 0

0.1 0.5 Concentration of vinorelbine stealth liposomes (µM)

Figure 4. Combined cytotoxic effect of ATRA-loaded and vinorelbine-loaded liposomes against cancer stem cells. (A) Schematic showing the effect of ATRA-loaded and vinorelbine-loaded liposomes; (B) effect of free ATRA; (C) effect of ATRA-loaded liposomes; (D). Effect of vinorelbine-loaded liposomes on SP and NSP; (E) effect of Vinorelbine-loaded liposomes on nonsorted cells alone and in combination with ATRA-loaded liposomes. **: p < 0.05; ***: p < 0.01; ***: p < 0.001. ns: No statistical significant difference; NSP: Nonside population; SP: Side population. Reproduced with permission from [72] ; copyright © 2011, Elsevier.

combined effect of the drugs loaded in mitochondrialtargeting liposomes resulted in the highest shrinkage of MCF-7 mamospheres in vitro. The in vivo anticancer efficacy was studied in an MCF-7 CSCs xenograft mouse model, where MCF-7 CSCs (dissociated mamospheres) were injected in the mammary fat pad.

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The mitochondrial targeting liposomes loaded with dual drugs exhibited a strong anti-tumor effect (∼79% inhibition), while individual drug-loaded liposomes without mitochondrial targeting resulted in 28 to 44% tumor inhibition in a relapsed MCF-7 xenografted mouse model.



The engineering of nanoparticles is crucial in the delivery of dual drugs. The availability of both drugs employed in predictable ratios at the tumor site is important for achieving a synergistic effect since one drug potentiates the effect of another drug. In cases where two drugs are loaded in separate nanoparticles, it is important to tune the size and surface chemistry of both nanoparticles to be similar, which will maximize the co-localization of these nanoparticles in the tumor site. To achieve this, it is suggested to use the same polymer to load two different drugs into nanoparticles. It is particularly challenging to load two drugs with different physicochemical properties into the nanoparticles that are formed from the same polymer at high loading capacity. Organocatalytic living ring-opening polymerization methodology, which was recently developed by James Hedrick and his coworkers, has demonstrated promise in synthesizing multifunctional biodegradable amphiphilic block copolymers with well-defined molecular structure and molecular weight [73,74] . These polymers have been successfully utilized to form micellar nanoparticles for drug delivery with excellent stability, high drug loading capacity and negligible toxicity [70,71,75,76] For different drug molecules, specific functional groups can be installed onto the polymers using this synthetic methodology so that drug molecules can form noncovalent interactions with the micellar core to enhance drug loading and micelle stability. Using this methodology, molecular weight of polymers can also be controlled to have narrow molecular weight distribution so that micelles with narrow size distribution can be formed, which is important to achieve ideal biodistribution [77] . Gene delivery Nucleic acids have long been preferred to target CSCs over chemotherapeutic drugs. Most chemotherapeutics target highly proliferative cells, but CSCs tend to stay in a state of quiescence. However they have a large repertoire of genes, which are over expressed to maintain stemness and tumorigenicity. Nucleic acids can effectively target these genes. Especially small interference RNAs (siRNAs) and microRNAs (miRNAs) have been extensively studied for the treatment of CSCs in various cancers. Due to the inherent limitations such as negative charge, large molecular weight and low stability of these small RNAs, a range of carriers have been developed to condense RNA molecules into nanoparticles for effective delivery. Gene knockdown often sensitizes CSCs to small molecular anticancer drugs. Therefore, in order to achieve synergistic effects, these small RNAs are frequently co-delivered with small molecule anticancer drugs. Viral and nonviral vectors can be used to deliver genes. Nonviral


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vectors are attractive as they have low immunogenicity and large gene-loading capacity, and are easy to manufacture and install functionalities for targeting [50,78] . In this section, nonviral gene delivery systems for the delivery of RNAs and those systems on the co-delivery of siRNA and anticancer drugs for targeting CSCs are reviewed. Pan’s group discovered that miRNA-107 expression in head and neck squamous cell carcinoma (HNSCC) was downregulated and functioned as a tumor suppressor gene, which indicated that miR-107 might be used as an anticancer therapeutic [63] . In a follow-up study, they used cationic lipid nanoparticles to deliver pre-miRNA-107 (nanoparticles [NP]/premiRNA-107) to HNSCC cells in vitro and in vivo [79] . The cationic lipid nanoparticles were prepared using dimethyldioctadecyl ammonium bromide (DDAB), cholesterol and α-tocopheryl polyethylene glycol 1000 succinate (TPGS), and the encapsulation efficiency of pre-miRNA-107 was 98.9 ± 1.5%. In vitro studies showed that the transfection efficiency of NP/premiR-107 in HNSCC cells was 80,000-fold higher than free pre-miR-107, and the NP/pre-miRNA-107 significantly decreased the levels of miR-107 targets such as protein kinase Cɛ (PKCɛ), cyclin-dependent kinase 6 (CDK6) and hypoxia-inducible factor 1-β (HIF1-β). More importantly, it was found that NP/pre-miR-107 could inhibit the population of cancer-initiating cells and downregulate the expression of the core embryonic stem cell transcription factors both in vitro and in vivo. In preclinical studies, NP/pre-miRNA-107 showed a strong anti-tumor activity and prolonged survival in an HNSCC mouse model. The miRNA-34a was reported to be an important regulator, inhibiting both CSCs differentiation and metastasis by directly repressing the CSC marker CD44 [80] . Shi et al. reported the use of solid lipid nanoparticles (SLNs), which were made of glyceryl monostearate, soy phosphatidylcholine, cholesterol, polyoxyethylene 50 stearate and DDAB to condense miRNA for enhanced cellular uptake, for the delivery of miRNA-34a against lung CSCs [81] . DDABcontaining SLNs were prepared by a film-ultrasonic method, and miRNA-34a was complexed with the SLNs by incubating the SLNs with miRNA-34a at different ratios. The SLNs increased the stability of miRNA-34a in serum and enhanced its intracellulartransfection efficiency. Through downregulating the expression of CD44, the SLNs/miRNA-34a complexes induced apoptosis and inhibited migration of B16F10CD44 + cells. In vivo studies in mice bearing B16F10CD44 + tumors in lungs further showed that the SLNs/ miRNA-34a complexes accumulated in lungs due to the EPR effect, and significantly inhibited B16F10-


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A

W/O

Water phase

W/O/W

Emulsify with

Emulsify with PVA

PVA

Oil phase PEG-Pep-PCL copolymer B

1h

4h

96 h

120 h

8h

DOC

24 h

miR-200c 48 h

72 h

144 h Tumor

Heart

Lung

Kidney

Tumor volume (mm3)

C

2000

Control Empty NPs miR-200c NPs DOC DOC NPs miR-200c/DOC NPs

Stomach

Spleen

Intestine

Brain

Liver

D Control DOC DOC NPs

1000

miR-200c NPs 0

miR-200c/DOC NPs 0 2 4 6 8 10 12 14 16 18 20 22 Time (day)

Figure 5. In vivo biodistribution and anti-tumor efficacy of docetaxel-loaded nanoparticles/miR200c complexes. (A) Schematic showing the preparation of DOC-loaded nanoparticles (NPs)/miR200c complexes; (B) real-time in vivo imaging of NIR-797-loaded nanoparticles administered to mice bearing BGC-823 xenografts through the tail vein; (C) anti-tumor efficacy of DOC-loaded NPs/miR200c complexes in a BGC-823 xenograft mouse model in comparison with other formulations; and (D) tumor photographs of different groups. DOC: Docetaxel; miR-200c: ; NP: Nanoparticle; PEG-Pep-PCL: Copolymer of polyethylene glycol, gelatinase cleavable peptide and poly (ε-caprolactone); PCL: Poly (ε-caprolactone); PVA: Polyvinyl alcohol; W/O: Water-in-oil; W/O/W: water-in-oil-in-water. Reproduced with permission from [89] ; copyright © 2013, Elsevier.

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CD44 + tumor development and tumorigenicity with no observed toxicity. The miRNA145 was reported as a tumor-suppressive miRNA that was associated with tumor growth and metastasis in several types of cancer [82,83] . In addition, miR145 modulates embryonic stem cell (ESC) differentiation and simultaneously regulates multiple stemness genes, including Klf4, Oct4 and Sox2 [84] . Yang et al. and Chiou et al. reported the use of cationic graft polymer polyurethane-short branched PEI (PUPEI) for the delivery of miRNA145 to inhibit CSCs in glioblastoma multiforme (GBM) [85] and lung adenocarcinoma (LAC) [86] , respectively. PU-PEI was mixed with miR145 to form nanosized polymer/miRNA complexes. In the first study, GBM CSCs were sorted out as CD133 positive (CD133 +) cells. In vitro studies indicated that PU-PEI/miR145 complexes significantly inhibited the tumorigenic and CSC-like abilities of CD133 + GBM cells by targeting Oct4 and Sox2, and facilitated the differentiation of these cells into CD133non-CSCs. In addition, PU-PEI/miR145 complexes suppressed the expression of drug-resistant and antiapoptotic genes of CSCs, and sensitized these cells to the treatment of radiation and the chemotherapy drug temozolomide. In vivo studies demonstrated that PU-PEI/miR145 complexes significantly suppressed tumorigenesis with stemness, and synergistically improved the survival rate when used in combination with radiotherapy and temozolomide in orthotopic CD133 + GBM-transplanted immunocompromised mice. In another study using PU-PEI/miR145 complexes for the treatment of LAC, LAC-CSCs were sorted out by side population analysis or identification of CD133 markers, and it was found that LAC-CSCs exhibited low miR145 and high Oct4/Sox2/Fascin1 expression, CSC-like properties and chemoradioresistance. After treating with PU-PEI/miR145 complexes, the LAC CSCs showed reduced CSC-like activity and chemoradioresistance. In vivo studies showed that the systemic delivery of PU-PEI/miR145 complexes inhibited tumor growth and metastasis, sensitized tumors to chemoradiotherapies and prolonged the survival time of tumor-bearing mice in an in vivo orthotropic and metastatic LAC-transplanted xenografts in mice. In addition to miRNAs, siRNAs have also been used to target and sensitize CSCs to chemotherapy. For instance, Liu et al. designed and prepared a synthetic gene carrier composed of a cationic oligomer (PEI1200), a hydrophilic polymer (PEG) and a biodegradable lipid-based cross-linking moiety [87] . MDR1targeting siRNA was complexed with PEI-Lipid1:16 nanoparticles, and this complex effectively reduced the expression of MDR1 in human colon CSCs (CD133-enriched cells), and sensitized these cells to


Review

paclitaxel. In another study, O6 -methylguanine-DNA methyltransferase (MGMT)-siRNA was delivered by using a cationic lipid (LipoTrust) to sensitize GBM cells to chemotherapy [88] . This system downregulated the expression of MGMT in temozolomide-resistant glioma-initiating cells and sensitized these cells to the treatment of the conventional chemotherapy agent temozolomide both in vitro and in vivo. Recently, Liu et al. reported gelatinases-stimuli nanoparticles (NPs) synthesized from PEG-peptide-poly(ɛ-caprolactone) (PEG-Pep-PCL) for the co-delivery of miR-200c and docetaxel (DOC) as shown in Figure 5A to inhibit both cancer cells and CSCs [89] . The peptide indicated is a tumor-specific gelatinases-cleavable peptide. It can be selectively cleaved in cells with high gelatinase expression, which most CSC do and are responsible for many of their resistance characteristics. These NPs effectively delivered miR-200c into cells and achieved sustained miR-200c expression in tumor cells for 9 days, as well as decreased the level of targeted gene class III betatubulin (TUBB3). The DOC-loaded NPs/miR-200c complexes significantly enhanced anti-tumor activity of DOC in BGC-823 cells. It was also shown through in vivo bioimaging that the complexes accumulated in the tumor within 4 h apart from some accumulation in the abdomen, and stayed in the tumor up to 144 h (Figure 5B) . In addition, in vivo anti-tumor efficacy studies conducted in Balb/C mice subcutaneously injected with BGC-823 cells demonstrated that the DOC-loaded NPs/miR-200c complexes suppressed tumor growth and inhibited the tumorigenicity of gastric cancer in mice more effectively than DOC-loaded NPs and miR-200c/NPs alone (Figure 5C & D) . Although nucleic acids have demonstrated success in targeting CSCs, there are still a number of hurdles to overcome before they can be applied in clinic. There are a myriad of pathways that contribute to the stemness of CSCs [90] . Hence, it is important to choose the right genes to knockdown and to deliver genes specifically to the intended target CSCs with minimal damage to normal somatic stem cells. In addition, the use of effective and biocompatible polymers as carriers is crucial to achieve successful in vivo gene delivery. The size, size distribution and stability of polymer/gene complexes are important parameters that should be taken into consideration in the design of gene-delivery carriers as they determine the in vivo biodistribution of the gene complexes. To exploit the EPR effect in tumor tissues, it is essential to design and synthesize biocompatible polymers that can condense genes into complexes having sizes below 200 nm, narrow size distribution and good in vivo stability. Moreover, combination therapy of nucleic acids with chemotherapeutic drugs is likely to achieve greater efficacy when administered


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Review Krishnamurthy, Ke & Yang sequentially than simultaneously. This is because maximal gene knockdown using siRNA or miRNA takes about 24–72 h [91] , and it is at this point of maximal knockdown that the target cell gets sensitized to the chemotherapeutic drug. Protein delivery Several CSC-specific antibodies and tumor suppressor proteins have also been used for killing CSCs. Delivering proteins like tumor suppressors is to increase the amount of cell-regulatory proteins and restore the nonfunctional apoptotic pathway in CSCs. For example, Perlstein et al. conjugated TNF-related apoptosis inducing ligand (TRAIL) with NPs (NP-TRAIL) to inhibit both glioma cells and glioma CSCs [62] . Compared with free TRAIL, the NP-TRAIL showed stronger apoptotic activity in various human glioma cell lines and glioma CSCs. Combined treatment using NP-TRAIL and γ-radiation or bortezomib sensitized TRAIL-resistant glioma CSCs to NP-TRAIL. In vivo studies in a U251 glioma cells-derived xenograft mouse model indicated that NP-TRAIL accumulated in the tumor, thus enhancing the apoptosis of both CSCs and cancer cells, decreasing tumor volume and increasing animal survival time. Apart from using antibodies for targeting therapeutic molecules to CSCs (Table 2), therapeutic antibodies have also been exploited for specifically killing CSCs based on their stark differences in characteristics from cancer cells. Nanoparticles help to enhance the circulation half-life of these antibodies and reduce their nonspecific toxicity by accumulating in the tumor through

the EPR effect. Dou et al. used anti-ABCG2 antibodies to decorate the surface of silver nanoparticles along with vincristine for treating myeloma CSCs. ABCG-2 pump is a type of ABC transporters and plays a critical role in drug efflux from cancer cells. It is found to be overexpressed in drug-resistant CSCs. The nanoparticles showed good inhibitory effects on CD44 +/CD24CSC population isolated from SP2/0 murine myeloma cells. It also enhanced the survival significantly in Balb/C mice injected with isolated CD44 +/CD24CSC population apart from increasing the bone mineral density and ameliorating lytic bone lesions [92] . Ginestier et al. reported the successful eradication of breast CSCs in an MCF-7 mouse xenograft model by using anti-CXCR1 blocking antibody specific to IL-8 receptor CXCR-1. The treatment of CSCs with recombinant IL-8 stimulated their self-renewal, which was reversed by blocking CXCR-1 receptors. The blockade also induced massive apoptosis by FASL/FAS signaling [93] . In addition to recombinant proteins and antibodies, a number of peptides have also been used to kill CSCs (Table 3) . However, they were delivered to cells without using nanoparticles. Conclusion & future perspective Nanoparticulate systems have been shown to effectively deliver small and macromolecular drugs to target cancer and CSCs. A number of studies have shown that there is a significant increase in therapeutic outcome with the use of nanoparticles-based drug, protein or nucleic acid delivery. However, the full potential of nanomedicine has not been exploited

Table 2. List of antibodies used for targeting cancer stem cells. CSC origin

Antibody target

Ref.

Lung cancer

CD133, CD117, ABCG2

Breast cancer

CXCR1, CD44, BCRP1, ESA

[110–112]

Melanoma

ABCB5, CD271, CD20, CD133

[113–115]

Gastric cancer

CD44, CD90, ABCB1, ABCG2, CD133

Colon cancer

CD133, ESA, CD166, CD29, CD24, CD26

Leukemia

CD34, CD123, CD96

[109]

[8,116,117] [118,119] [120–122]

Pancreatic cancer

CD44, CD133, CD24, ESA

Liver cancer

CD133, CD13, CD24, CD44, CD90, EpCAM

Brain cancer

CD133, CD15, Bcrp1

Bladder cancer

CD133, CD44v6, EMA, CD47

Ovarian cancer

Brcp1, CD133, ABCB1

Prostate cancer

CD44, Integrin α2β1, CD133, ABCG2, CD24

[134–136]

Head and neck cancer

CD44, CD133

[137–139]

Cervical cancer

Brcp1, CK17, CD44

[6,123] [124,125] [126–128] [129–131] [11,132,133]

[140,141]

CSC: Cancer stem cell.

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yet as there are a few roadblocks, which have to be overcome. In the case of multiple drug delivery, different drugs were often loaded separately into nanoparticles as loading several drugs into the same nanoparticle is challenging due to different physicochemical properties of the drugs. Thus two different nanoparticles of drugs that are synergistic may not actually reach a particular cell together, and this may reduce the effectiveness of the therapy. There is a need to develop multifunctional nanoparticles, which can load multiple drugs simultaneously with high capacity. Another persistent problem is nonspecific toxicity of drugs/proteins/genes toward healthy cells. Nanoparticulate drug-delivery systems can significantly reduce the amount of therapeutics being delivered to healthy tissues through the EPR effect. In addition, the use of stimuli-responsive nanoparticle systems, which are active only in the tumor microenvironment, may further enhance the accumulation of therapeutics at the tumor site. Peptides are an important part of the biologics used for cancer treatment, interfering with the most ‘undruggable’ intracellular targets and have many advantages over classic therapeutic proteins with smaller size, cell-penetrating ability, easier modification and lower cost of synthesis. Several classes of peptides including pro-apoptotic [94] , anti-angiogenic [95] and antiadhesion peptides [96] have been found to be effective in targeting cancer and CSCs (Table 3), but they easily undergo proteolytic degradation in vivo. This issue can be overcome with the use of nanocarriers. However, there have been no reports on peptide delivery using nanoparticles for targeting CSCs, and this could be a potential area for exploration. The next underexplored class of biologics is antibodies. Though, a few studies have reported on the use of antibodies as therapeutic/targeting entities, their potential is not completely utilized. Since CSCs have a variety of unique surface markers, adding specific targeting ligands to nanocarriers may significantly improve the therapeutic effect of the drug/gene, and also reduce unwanted side effects. CSC targeting is a tricky science and success in targeting CSCs in vitro might not always translate to success in vivo. There are obvious limitations in reaching the CSCs in vivo owing to inaccessibility of the entire tumor area and microenvironmental factors, which may be circumvented by using environmentally sensitive nanoparticles (such as pH and reduction potential, among others). Also, it is important to better understand the key characteristics of CSCs in order to target them effectively. For example, CSCs always remain a scarce population within a tumor but are able to repopulate when inoculated in a new environment, indicat-


Review

Table 3. List of peptides used for targeting cancer stem cells. CSC origin

Peptide name

Delivery carrier

Gastric cancer

Anticancer N/A bioactive peptide-3 (ACBP-3)

Ref. [142]

Breast cancer AD-01

N/A

[95]

Colorectal cancer

N/A

[143]

Ovarian and A6 peptide breast cancer

N/A

[96]

Prostrate and gastric cancers

N/A

[94]

Synthetic peptide P17 and P144

TMTP1-DKK

CSC: Cancer stem cell; N/A: Not applicable (i.e., no delivery carrier used).

ing that there is some level of organization within the apparent anarchy. Knowing the factors that dictate the fate of CSCs can greatly change the way they are targeted. CSCs have been shown to exist in nearly every type of cancers known and there are many reports correlating detrimental outcomes with higher percentages of CSCs in all cancers. Yet, efforts of CSC-targeted nanoparticle approaches have been heavily explored only for a few types of cancers like brain, breast, lung and to a very less extent on others. Generally, the field of CSCs is relatively new and there are no wellestablished protocols to distinguish and separate them. Different researchers use different surface markers and biochemical assays for identification. This should be standardized so that therapeutic outcomes of different nanoplatforms can be cross-compared for accelerating the development of effective therapeutic approaches to overcoming cancer relapse and metastasis. Finally, the toxicity and long-term effects of nanoformulations need to be studied in depth before they can be used in a clinical setting. Financial & competing interests disclosure. This work was funded by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore). Graduate Research Scholarship from the National University of Singapore to S Krishnamurthy, X Ke is gratefully acknowledged. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.


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Executive summary • The presence of cancer stem cells (CSCs) causes drug resistance, cancer relapse and metastasis. Efforts are increasingly being made to tackle CSCs for improved cancer therapy. • A number of new therapeutics including small molecules, proteins and genes have been discovered to specifically target CSCs, and polymeric nanoparticles/liposomes have been demonstrated to be promising carriers for the delivery of these compounds toward tumor tissues through the enhanced permeability and retention effect. • More efforts need to be made to develop multifunctional polymeric nanoparticles for the simultaneous delivery of multiple drugs with high loading capacity. • The nanosize, narrow size distribution and in vivo stability of nanoparticles need to be taken into consideration in the design of nanoparticles for effectively targeting the therapeutics to tumor tissues without causing significant toxicity toward healthy tissues. • Safe nanocarriers are also needed to deliver anti-CSC peptides. • Protocols for identification of CSCs should be standardized so that therapeutic outcomes of different nanoplatforms can be cross-compared for accelerating the development of effective therapeutic approaches to overcoming cancer relapse and metastasis. ovarian cancer stem cell-like cells leading to an increased tumor burden. Mol. Cancer 12(1), 24 (2013).

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