Role of integrated cancer nanomedicine in overcoming drug resistance

    Role of Integrated Cancer Nanomedicine in Overcoming Drug Resistance Arun K. Iyer, Amit Singh, Srinivas Ganta, Mansoor M. Amiji PII: DOI: Reference:

S0169-409X(13)00166-X doi: 10.1016/j.addr.2013.07.012 ADR 12487

To appear in:

Advanced Drug Delivery Reviews

Accepted date:

15 July 2013

Please cite this article as: Arun K. Iyer, Amit Singh, Srinivas Ganta, Mansoor M. Amiji, Role of Integrated Cancer Nanomedicine in Overcoming Drug Resistance, Advanced Drug Delivery Reviews (2013), doi: 10.1016/j.addr.2013.07.012

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ACCEPTED MANUSCRIPT Running Title: Integrated nanosystems to overcome drug resistance

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Drug Resistance

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Role of Integrated Cancer Nanomedicine in Overcoming

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Arun K. Iyer1, Amit Singh1, Srinivas Ganta2 & Mansoor M. Amiji1,* Department of Pharmaceutical Sciences, School of Pharmacy, Bouvé College of Health

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Sciences, Northeastern University, Boston, MA 02115 & 2Nemucore Medical Innovations, Inc.,

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Worcester, MA 01608

*All correspondence should be address to: Tel. 617-373-3137 Fax. 617-373-8886 Email: [email protected]

ACCEPTED MANUSCRIPT Abstract Cancer remains a major killer of mankind. Failure of conventional chemotherapy has

to the complexity and diversity of this deadly disease.

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resulted in recurrence and development of virulent multi drug resitant (MDR) phenotypes adding Apart from displaying classical

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physiological abnormalities and aberrant blood flow behavior, MDR cancers exhibits several

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distinctive features such as higher apoptotic threshold, aerobic glycolysis, regions of hypoxia,

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and elevated activity of drug-efflux transporters. MDR transporters play a pivotal role in protecting the cancer stem cells (CSCs) from chemotherapy. It is speculated that CSCs are

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instrumental in reviving tumors after the chemo and radiotherapy. In this regard, multifunctional nanoparticles that can integrate various key components such as drugs, genes, imaging agents

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and targeting ligands using unique delivery platforms would be more efficient in treating MDR

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cancers. This review presents some of the important principles involved in development of MDR

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and novel methods of treating cancers using multifunctional-targeted nanoparticles. Illustrative examples of nanoparticles engineered for drug/gene combination delivery and stimuli responsive nanoparticle systems for cancer therapy are also discussed.

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AAc: Acrylic acid AML: Acute myeloid leukemia BCP: Block copolymer BK: Bradykinin CD: Cyclodextrin Chol: Cholesterol CSC: Cancer stem cells CTAB: Cetyltrimethylammonium bromide DDS: Drug delivery system Dex: Dexamethasone DLL4: Delta-like 4 ligand DMAEMA: N,N-dimethylaminoethyl methacrylate DOPG: Dioleoylphosphatidylglycerol DOX: Doxorubicin DPPC: Dipalmitoylphosphatidylcholine DSPC: 1,2- distearoyl-sn-glycero 3-phosphocholine ECM: Extracellular matrix EGFR: Epidermal growth factor receptor EMT: Epithelial-mesenchymal transition EOEOVE: Poly[2-(2-ethoxy)ethoxyethyl vinyl ether EpCAM: Epithelial cell adhesion molecule EPR: Enhanced permeability and retention GILT: Gamma-interferon-inducible lysosomal thiol reductase HDAC: Histone deacetylase HFMF: High-frequency magnetic field HIFU: High intensity focused ultrasound HPMA: Poly(N-(2-hydroxypropyl)methacrylamide) IARC: International agency for research on cancer IPNs: Interpenetrating networks LCST: Lower critical solution temperature LTSL : Low thermosensitive liposomes MA: Maleic anhydride (MA) MAAc: Methacrylic acid MDR: Multi-drug Resistance MMPs: Matrix metalloproteinase MPS: Mononuclear phagocytic system MRI: Magnetic resonance imaging MSNP: Mesoporous silica nanoparticles MSNs: Mesoporous silica nanoparticles NGF: Nerve growth factor NIR: Infra-red NO: Nitric oxide

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NSCLC: non-small cell lung cancer NTSL: Non-thermosensitive liposomes P-gp: P-glycoprotein PAA: Poly(amidoamine) PAM: Polyacrylamide PbAE: Poly(β-amino ester) PCL: Poly(caprolactone) PDDA: Poly(dimethyldiallylammonium chloride) PDEAAm: Poly(N,N-diethylacrylamide) PDMAEMA: Poly[2-(dimethylamino)ethyl methacrylate] PEG: Poly(ethylene glycol) PEI: Polyethyleneimine PEO: Poly(ethylene oxide) PGA: Poly(glutamic acid) PGs: Prostaglandins PHEA: α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide PLGA: Poly(D,L-lactide-co-glycolide) PNIPAAm: Poly(N-isopropyl acrylamide) PSMA: Prostate-specific membrane antigen PTMC: Poly(trimethylene carbonate PVCL: Poly(N-vinlycaprolactam) RGD: Arginine-glycine-aspartic acid RNAi: RNA interference SCLC: Small cell lung cancer shRNA: short hairpin RNA siRNA: small interfering RNA SP: Side population SPIONs: Superparamagnetic iron oxide nanoparticles TF: Tegafur TTSL: Thermosensitive liposomes UCNPs: Upconverting nanoparticles UCST: Upper critical solution temperature US: Ultrasound VEGF: Vascular endothelial growth factor VPF: Vascular permeability factor

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ACCEPTED MANUSCRIPT 1. Introduction – Tumor Plasticity According to the report from the International Agency for Research on Cancer (IARC), there

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are over 10 million new cases of cancer each year and over 6 million annual deaths from the disease [1]. IARC estimates that by 2030, the cancer burden will increase to 21.4 million new

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cases and 13.2 million cancer related deaths globally [1]. Despite the remarkable progress in the

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prevention, detection, and treatment of cancer over the last five decades, adequate therapy

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remains elusive due to late stage diagnosis and lack of clinical procedures for eliminating disseminated cancer, inadequate strategies for addressing multi-drug resistance (MDR) and

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cancer cell plasticity [2, 3]. There is increasing body of evidence that suggest cancer cell plasticity contributes to the persistence of the disease in spite of therapeutic interventions [4, 5].

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Cellular plasticity is the ability of one cell type to attain properties of another cell type that can

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potentially be coupled to regenerate specific cell types affected by disease condition [6-8]. For

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example, subpopulations of tumor cells such as cancer stem cells (CSC) facilitate an active contribution to tumor development and progression by producing bulk population of nontumorigenic cancer cell progeny through differentiation (Figure 1) [9, 10]. Recent studies suggest that hypoxic microenvironment in metastases facilitate phenotype switch allowing melanoma cells to participate in blood capillary formation [11]. <<<>>> Cellular plasticity in the form of epithelial-mesenchymal transition (EMT) in epithelial cancers has been associated with tumor progression and resistance to anti-cancer therapies [4]. EMT has been implicated as a major factor driving metastasis, through the acquisition of enhanced migratory and invasive potential of tumor cells [12, 13]. By undergoing this process the tumor cells attain resistance to a number of targeted therapies [5]. An EMT phenotype has been identified in a number of epidermal growth factor receptor (EGFR)-mutant non-small cell

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ACCEPTED MANUSCRIPT lung (NSCL) cancer patients who have progressed while on EGFR-directed Erlotinib therapy due to a loss of E-cadherin (an epithelial tumor marker) [5].

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Drug resistance continues to be a major challenge in the cancer therapy, and MDR cancer cells show a broad range of resistance against functionally and structurally unrelated

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chemotherapeutic agents. Historically, MDR has been linked to elevated expression of drug

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efflux transporters, changes in drug kinetics or amplification of drug targets [14, 15]. However, the development of drug resistance in patients treated with novel targeted molecular therapies

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has provided greater understanding of the complexities involved in cancer drug resistance [14].

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Recent studies suggests that tumoral heterogeneity is a major driver of cancer drug resistance [14]. To achieve successful cancer therapy, the current therapeutic interventions have to be

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modified to include methods that estimate tumor heterogeneity comprising of genetic variation,

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the microenvironment, and cell plasticity in tumors [14]. The goal of this review is to present a

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perspective on therapeutic strategies employing multifunctional nano delivery systems to overcome MDR by addressing tumor heterogeneity. 1.1 Cancer Stem Cells and MDR The CSC hypothesis suggests that tumors consist of a small population of cells, which are also accurately called ‘tumor-initiating cells’ that have been considered to have tumorigenic potential. CSCs have an ability to undergo self-renewal by cell division generating diverse cells that form the tumor (Figure 1) [9, 16, 17]. It has been shown that CSCs can initiate new tumors following transplantation into animal models, while the majority of other cancer cells cannot [18], although aberrant CSCs share common properties with normal tissue stem cells such as self-renewal and differentiation capacity. CSC hypothesis provides functional heterogeneity that is commonly observed in solid tumors [17]. According to this model, solid tumors are shown to be hierarchically arranged and CSCs present at the apex of the hierarchy have tumorigenic ability [17, 19]. The CSC model also provides an attractive cellular mechanism to account for the 5

ACCEPTED MANUSCRIPT therapeutic refractoriness and dormant behavior displayed by many of solid tumors [9, 16, 17]. Solid tumors account for the major cancer burden, and epithelial cancers such as lung, breast,

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colon, prostate and ovary constituting nearly 80% of all cancers, leading to a major therapeutic challenge in cancer therapy [17].

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Existence of CSCs was first identified in acute myeloid leukemia (AML) in which a rare

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subset of cell population comprising 0.01-1% of the total population could induce leukemia when transplanted into immune deficient mice [20]. In general, the reported fractions of CSCs in

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tumor cell population vary from 0.1 to 30% depending on the type and the advancement of the

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tumor [21]. A number of cell surface markers have proven valuable for the isolation of subsets enriched for CSCs, shown in Table 1. CSC hypothesis has been shown consistent with data from

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distinct cancer types such as breast cancer [22], colorectal cancer [23], chronic and acute

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myeloid leukemia [24], pancreatic cancer [25], mesenchymal neoplasms [26] and head and neck

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squamous cell carcinomas [27].

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Recent studies have suggested that CSCs are involved in metastasis [13, 17, 28]. Metastasis is the major cause of deaths in cancer patients, and metastatic potential depends on several factors that determine overall tumor growth, survival, angiogenesis and invasion [17]. In epithelial cancers, EMT plays an important role in the metastasis, which involves disruption of epithelial cell homoeostasis and the acquisition of a migratory mesenchymal phenotype [13]. A recent study demonstrates that there may be a direct link between the EMT and acquisition of stem cell properties [28]. Cells undergoing an EMT could possibly be the precursors to metastatic tumor cells, or even metastatic CSCs. CSCs may also have a role in the creation of a particular niche for metastasis. These observations have important implications in cancer therapy, as the new therapeutic strategies that target metastatic CSC can have profound effect on tumor progression and patient survival. 6

ACCEPTED MANUSCRIPT CSCs involvement in drug resistance, metastasis and relapse of cancer can significantly affect cancer therapy [18]. Current approaches involving radio and chemotherapies eliminate the

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bulk of tumor cells, but often are not able to kill the CSCs, because they are protected by specific resistance mechanisms (Figure 1). According to the CSC model, drug resistance is closely

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associated with many intrinsic or acquired mechanisms of accumulating CSCs, such as

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quiescence, specific morphology, DNA repair ability and elevation of drug efflux transporters, anti-apoptotic proteins and detoxifying enzymes [18]. The tumor microenvironment and hypoxic

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stability provide additional protection against cancer therapy for CSCs [18]. The surviving CSCs

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give rise to new tumors and metastases, resulting in cancer recurrence [18]. The relapsed tumors become more malignant, fast spreading and resistant to radio and chemotherapy re-challenge,

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making the prognosis for cancer patients dismal [18]. These observations provide an explanation

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improving cancer therapy.

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for many treatment failures in recurrence and refractory tumors, and offer newer perspectives for

1.2 Tumor Microenvironment and MDR Numerous studies have recognized that components of tumor microenvironment strongly influence cellular phenotypes, including susceptibilities to toxic insults by a range of mechanisms. Tumor microenvironment consists of cells (cancer associated fibroblasts or macrophages), extracellular matrix (ECM), signaling molecules and mechanical cues that can act in paracrine manner to influence tumor initiation, support tumor growth and invasion, protect the tumor from host immunity, foster drug resistance, and provide niches for dormant metastases to thrive. Recent studies have identified microenvironment can alter the response of tumor cells to chemotherapy and targeted therapies through production of secreted factors which might drive tumor growth and MDR [29]. During early stages of tumor development, cancer cells may rely on a tumor-supportive microenvironment while later stages (i.e., the metastatic setting) could be biologically characterized by the self-sufficiency of cancer cells. Such acquired self-sufficiency 7

ACCEPTED MANUSCRIPT of cancer cells to survive in a microenvironment independent manner could result in insensitivity to molecular targeted agents acting by depriving cancer cells of paracrine acting stimuli.

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Hypoxia has long been considered as a major feature of the tumor microenvironment and a potential contributor to the MDR and enhanced tumorigenicity of CSCs [18]. It is a condition in

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which tumor tissue is deprived of oxygen supply. Hypoxic regions are created when aberrant

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angiogenesis or blood vessels are closed or impaired due to compression, tumor cell invasion and discontinuity of epithelial cells lining the vessels [30, 31]. The aberrant vascular architecture and

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dysfunctional lymphatic drainage in tumors results in hypoxic areas with high interstitial fluid

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pressure relative to the low interstitial fluid pressure of well vascularized areas (Figure 2) [30]. Because of aberrant angiogenesis and inaccessible location, hypoxic cells are less likely to

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accumulate therapeutic concentrations of chemotherapeutics [32]. In addition to these properties

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that diminish chemotherapeutic efficacy, hypoxic cells have active mechanisms for inducing

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MDR in tumors cells, thus leading to tumor progression [31, 33-35]. Transcriptional factors that respond to hypoxia are termed hypoxia-inducible (HIF) factors. These factors assist in preventing cell differentiation, support blood vessel formation and regulate apoptosis [18]. HIF factors activate enzymes that are involved in DNA repair and induce the development of resistance to DNA-targeting drugs. Hypoxic tumor cells also display reduced intracellular pH levels relative to normal cells [33]. The acidic microenvironment is combined with the activation of a subset of proteases that contribute to metastasis [33]. Tumor cells revert to anaerobic metabolism under hypoxic condition to obtain ATP through the conversion of glucose to lactic acid (the Warburg effect) instead of regular oxidative metabolism (aerobic glycolysis) [36]. The dynamic nature of tumors involving overlapping biochemical mechanisms and tumor heterogeneity exacerbates the treatment of cancers. The application of therapies targeting tumor microenvironment using novel delivery approaches thus would be the most promising option for effective cancer therapy. 8

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<<<>>>

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1.3 Mechanisms of Drug Resistance in Tumors Multidrug resistance (MDR) is a major obstacle to the effective treatment of cancer, and

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MDR in cancer refers to a state of resilience against functionally and structurally unrelated

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chemotherapeutic agents. It can be intrinsic or acquired through exposure to chemotherapeutic agents. Drug resistance mechanisms represent adaptations to toxic insults and cellular stress, and

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these mechanisms are grouped into five categories; 1) induction of drug transporters, 2) DNA

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repair, 3) changes in drug metabolism, 4) gene amplification or mutation of target proteins and 5) changes in survival/apoptotic pathways [15]. Despite of distinct mechanisms, the MDR

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phenotype is usually the cumulative effect of a combination of MDR mechanisms such as

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blocked apoptosis and increased drug efflux [15].

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Drug efflux transporters are generally found to be elevated in drug-resistant tumor cells which selectively involved in the efflux of small molecule drugs [31, 37, 38]. Some of these ATP binding Cassette (ABC) families of drug transporters have been shown to play a specific role in pumping out cytotoxic drugs from cells preventing the critical concentration of drug accumulation within the cells, resulting in MDR [39]. Among them, P-glycoprotein (P-gp) is one of the well characterized ABC transporters that show broad substrate specificity, which is currently considered to be one of the main hindrance in the anticancer therapy of several cancers [40]. P-gp is encoded by the MDR1 gene and its overexpression in cancers has become a therapeutic target for overcoming MDR. Several other ABC transporters that are associated with MDR include multi-drug resistance protein 1 (MRP-1, ABCC1), breast cancer resistance protein (BCRP, ABCG1), the mitoxantrone resistance protein (MXR1/BCRP, ABCG2) and the ABCB4 (MDR3) [31].

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ACCEPTED MANUSCRIPT DNA repair mechanisms consist of a complex network of proteins able to identify DNA damage (eg. ATM, ATR, Chk1/2, BRCA1 or p53) and repair the damage [14]. Cytochrome

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P450 or the glucuronyl transferases are the drug metabolizing enzymes responsible for the biotransformation of many anti-cancer drugs and their activity lowers the intracellular drug

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levels. Gene amplification of receptors, such as EGF receptors, can often compromise the

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efficacy of therapies. In addition to these, the relative activities of pro-apoptotic and antiapoptotic pathways contribute to the sensitivity of a tumor cell to cytotoxic drugs. These

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overlapping mechanisms are the main biochemical determinants of tumor cell sensitivity to

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cytotoxic drugs [14]. Drug resistance to the broad range of anti-cancer drugs is indicative of the dynamic nature of tumor tissue. Recent studies looking at tumor microenvironment suggested

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that tumoral heterogeneity is a driver of drug resistance [41, 42].

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According to the CSC model, drug resistance is mostly caused by the intrinsic or acquired

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resistance mechanisms of accumulating CSCs (Figure 1) [18]. In this regard, several drug efflux transporters have been identified in CSCs, including P-gp, multidrug resistance-associated proteins (MRP) and breast cancer resistance protein (BCRP) [43]. Many studies suggest that tumors are enriched with CSCs at the completion of primary therapy, and the surviving CSCs contribute to drug resistance and ultimately cause disease recurrence [44]. In one such study, a high survival of CSCs following etoposide treatment was found to be associated with expression of MRP1 and the activity of an apoptosis inhibitor β-livin in glioblastoma tumors [45]. High BCRP levels were associated with increased Akt signaling in drug resistant hepatocellular carcinoma (HCC) [46]. AKt signaling was able to alter the subcellular localization of BCRP transporters in HCC, thus determining drug efflux in CSCs [46]. In the same study AKt signal inhibition by P13K inhibitors, not only suppressed cancer cell proliferation, but also increased the sensitivity of drug resistant cells [46].

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ACCEPTED MANUSCRIPT Therefore, the therapeutic strategies directed towards CSCs might improve cancer therapy, in particular for those cancers that are refractory to conventional chemotherapeutics aimed

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predominantly on “bulk” tumor populations.

2. Integrated Multifunctional Nanoparticles

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2.1 Nanoparticles Based Delivery

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Chemotherapy conventionally involves the use of synthetic molecules and natural

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products that in general are low molecular weight anticancer compounds. Such agents inherently suffer from sub-optimal utilization due to rapid clearance and short blood circulation half-lives.

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Thus, their concentration in the tumors cannot be maintained within the therapeutic window for longer durations. Also, anticancer drugs, face systemic delivery challenges because they

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generally lack selectivity to tumor tissues and cells. When systemically administered via the

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intravenous (i.v.) route, small molecule anticancer drugs indiscriminately diffuse across blood

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vessels of normal and tumor tissues alike, causing undesired side effects [47]. Recent therapeutic interventions such as the ones based on RNA interference (RNAi) mechanism hold great promise in terms of targeted action [48, 49]. However, gene therapy using naked plasmids, DNA and RNA also suffer from similar drawbacks as the small molecule drug counterparts. Indeed, naked genes and siRNAs are more prone to rapid degradation by enzymes and nucleases present in the blood that make their systemic delivery even more daunting [50]. Therefore, there is a need for developing targeted delivery systems that can not only protect the labile drug/gene payload from degradation but also ferry them selectively to the sites of interest. Incidentally, encapsulation of small molecule drugs and gene therapeutics into nanoparticles can offer several advantages [51, 52]. Apart from protecting the labile payload from degradation in the blood stream, nanoparticles in turn protect the body from exposure to the cytotoxic drugs. Moreover the optimal molecular weight and size of polymeric drugs and nanoparticles can facilitate the transport of drugs and gene therapeutics encapsulated in them selectively to solid tumor tissues based on a phenomenon 11

ACCEPTED MANUSCRIPT called the enhanced permeability and retention (EPR) effect, discovered by Matsumura and Maeda more than three decades ago (Figure 3) [53].

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<<<>>> As it was discussed earlier, tumor cells posses devastating invasive potentials that divide and

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multiply at exponential rates. When the tumor cells multiply and grow to be the size of ~ 1-2 mm

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in diameter, it starts to develop blood vessels around them, to nourish and feed the growing tumors. This process of angiogenesis was found to be essential for tumor growth beyond certain

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point [54]. As the tumor grows further, there is formation of more complex network of blood

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vessels that are highly disorganized, aberrant and “leaky”. Furthermore, solid tumors in general have dysfunctional lymphatic clearance [55-58]. Apart from the unique anatomical features

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described above, tumors cells secrete excessive levels of permeability mediators such as vascular

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permeability factor (VPF) or vascular endothelial growth factor (VEGF), bradykinin (BK),

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prostaglandins (PGs), matrix metalloproteinases (MMPs), nitric oxide (NO) and peroxynitrite [55, 56, 59, 60]. The anatomical and pathophysiological abnormalities coupled with overproduction of these vascular permeability mediators leads to extensive accumulation of blood plasma components, and nanoparticles into the tumor interstitum [55, 58, 59, 61]. As a consequence of the EPR-effect it is thus possible to attain very high local concentration of the nanoparticles and polymeric drugs in the tumor tissues. In general it is observed that drugs and genes encapsulated in nanoparticle delivery systems such as polymer conjugates, liposomes, polymeric nanoparticles, dendrimers, nanoemulsions and polymeric micelles (Figure 4) are all capable of exploiting this unique phenomenon to accumulate selectively in solid tumors [97101,19-23]. <<<>>>

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ACCEPTED MANUSCRIPT The EPR effect has been found to be more pronounced if the nanoparticulate delivery systems are designed to evade the mononuclear phagocytic system (MPS), thus prolonging their

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circulation half-life in the blood. In this regard, grafting of amphiphatic polymers such as poly(ethylene glycols) (PEGs) onto the surface of nanoparticles, micelles and liposomes have

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thereby rendering long plasma circulation times[62-66].

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been found to be very effective in providing “stealth characteristics” that enables MPS escape,

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Passive tumor targeting or the EPR effect is found to be effective for delivering drugs in the case of high-to-moderately vascularized solid tumors, however, delivering drugs using this

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approach to avascular, hypovascular (such as pancreatic and prostate cancers) or necrotic tumors still remains a challenge. Also, passive targeting does not apply for diseases other than solid In such cases, “actively targeted” systems using

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tumors such as melanomas or leukemia’s.

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antibodies or peptides decorated onto nanoparticle systems that can home to receptors or

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antigens overexpressed on the surface of cancer cells can be more efficacious (Figure 3) [67-71]. More importantly, even in the case of solid tumors, the delivery efficiency of passively targeted nanoparticle systems can be significantly enhanced when tumor homing ligands are made part of the nano delivery system [67, 69, 71, 72]. Along these lines, we have developed an epidermal growth factor receptor (EGFR) targeting peptide-decorated nanoparticles for active targeting of EGFR overexpressing cancers [70, 73]. The EGFR peptide coated nanoparticles were found to be more efficacious in comparison to non-targeted nanoparticles indicating the advantage of actively targeted nanoparticle systems.

In another example, arginine-glycine-aspartic acid

(RGD) tripeptide conjugated nanoparticles were developed to target the integrin receptors expressing tumor cells [74, 75]. The α5β5 or α5β3 integrin receptors are overexpressed on vascular endothelial cells of angiogenic blood vessels of proliferating tumors, and are a good target for

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ACCEPTED MANUSCRIPT active targeting [75, 76]. In another case, Werner et al., used folate-based active targeting to deliver nanoparticles to ovarian cancer that overexpress folate receptors [77].

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Nucleic acid construct such as aptamers that can selectively bind to prostate-specific membrane antigen (PSMA) on prostate cancer cells are also being explored as active targeting

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ligands for diseases such as prostate cancers [78, 79]. Active ligand based targeting thus in effect

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provides a more precise “pinpointed targeting” of the nanoparticles to the diseased cells (and

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affords binding/intracellular internalization) after the primary targeting based on the EPR effect (Figure 3). Such multifunctional smart delivery systems are currently intensely being explored

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for targeted cancer therapy including MDR tumors.

2.2 Multifunctional Nanoparticle-Based Delivery Systems

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As noted earlier, there are several advantages of developing multi-functional nanoparticle system for cell specific disease targeting. As such, the requirements for treating MDR cancers

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are far more complex, where multi-agent delivery has almost become essential [80]. In addition, multifunctional nano-carries can be constructed with low, moderate or high level of complexity based on the desired application. In general biodegradable and inert nanosystems are designed to protect the labile payload from degradation and reduce the side effects associated with the free drug on systemic administration [81, 82]. Also, most of the nanoparticles, micelles and liposomes delivered via the intravenous route are PEGylated to evade the immune system and MPS uptake as discussed before [62, 64]. The other attributes that make nanoparticles attractive include the encapsulation of poorly soluble drugs [83] and multi-agent delivery using a single nanoparticle that can work synergistically to augment therapeutic effects [84, 85]. In this regard, multi-agent nanoparticle also offers the flexibility to devise the system to have spatial and temporal control on drug/gene release based on the polymer coatings/surfaces [86, 87]. For example, based on the requirement of the delivery system the delivery vehicle or the 14

ACCEPTED MANUSCRIPT biodegradable polymer can be tailored to release the drugs concomitantly or if desired, after a time delay, or in a sustained fashion [88, 89]. However the most attractive feature of

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multifunctional nanoparticles for treating MDR cancers can be attributed to active targeting using targeting molecules such as antibodies, peptides or aptamers, that can help locate, bind or

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traffic the nanoparticles into the target tumor cells [31, 90, 91]. Recently, organelle specific

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targeting of nanoparticles has also been explored [92], that have tremendous potentials for diseases such as MDR cancers. Another area that has been intensely pursued is the so called

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“theranostic nanosystems” which can perform diagnostic and therapeutic functions

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simultaneously [93, 94]. In such cases, the nanoparticles containing the active payload such as drugs and genes are also conjugated or tagged with imaging agents (such as optical probes, radio

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ligands or contrast agents) for simultaneous disease diagnosis, imaging and therapy [94]. Such

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multifaceted nano delivery system have the advantage of performing several functions such as

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visualizing the localizations of the disease, measuring delivery efficiency of nanosystems and monitoring dose response, all performed concurrently, in real time, using a single delivery system. Nano-theranostics thus have the potentials to revolutionize diagnostic and therapeutic intervention of several diseases such as cancer [71]. The applications of some such multifunctional nanoparticles for cancer targeting are discussed in the latter sections.

3. Illustrative Examples of Small Molecule Approaches to Overcome MDR 3.1 Combination Drug Therapy The concept of using combination therapeutics to improve the clinical efficacy with acceptable clinical toxicity profile has evolved greatly over the past several years. The rationale for such approach is centered on targeting different biochemical pathways to overcome MDR in heterogeneous tumors. One-dimensional mechanism of action of single agent chemotherapy frequently activates and strengthens the alternative pathways, leading to the emergence of MDR phenotype and tumor recurrence [95]. Accumulating clinical experience, supported by animal 15

ACCEPTED MANUSCRIPT models suggests that chemotherapy is most effective when given in combination to achieve additive or synergistic effect, and deter the development of drug resistance [95-98]. The basis for

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combination cytotoxic therapy has been to co-deliver therapeutics that work by diverse molecular mechanisms, thereby increasing tumor cell killing while reducing the chance of MDR

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and minimizing overlapping toxicity [96]. Despite many in vitro cell-based screening

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methodologies that have generated lead molecules for combinatorial treatments, their clinical outcomes are often met with little enhancement in efficacy and at times causes higher toxicity

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[99, 100]. One important factor that separates in vitro success from significant clinical benefit is

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the varying pharmacokinetics profile among different drugs [95, 101]. A better strategy for more effective combination therapy is developing technologies for precise and controlled delivery of

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multiple drugs. For example, multifunctional nanoparticle delivery systems have been successful

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in reversing MDR in in vitro and in vivo tumor models by co-delivering drug combinations that

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chemosensitize and kill the tumor cells more effectively [90, 101, 102]. Tumor cells that acquired MDR will be effectively treated utilizing combination drugs, which consist of a chemotherapeutic agent with an MDR modulator. For example, P-gp is an efflux pump over expressed in liver, ovarian, pancreatic and gastrointestinal cancers can readily efflux out doxorubicin, vinblastine and taxanes [103, 104]. P-gp modulators like verapamil or curcumin can inhibit P-gp pumps and sensitize the cells to primary chemotherapeutic agent [85, 104, 105]. In one such study, we have performed nano delivery of curcumin to overcome P-gp mediated efflux of paclitaxel by MDR ovarian cancer (SKOV3TR) cells [85]. This combination therapeutics using nano delivery system demonstrated synergistic and additive effects against the drug sensitive and drug resistant SKOV3 ovarian tumor cells [85]. In addition, the same approach in animal models demonstrated increased paclitaxel oral bioavailability and enhanced therapeutic efficacy in SKOV3 tumor xenografts [105]. For the case of apoptotic pathway dependent MDR, pro-survival mutations such as the deregulation of BCL2 and NFkB allow 16

ACCEPTED MANUSCRIPT tumor cells to tolerate drug insults and considerably decrease their apoptotic response. Many therapies attempted to exploit this characteristic of MDR cancer through the co-delivery of pro-

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apoptotic modulators or inhibitors to restore the apoptotic signaling leading to tumor cell death [84, 106]. The rationale underlying this strategy is lowering the apoptotic threshold of MDR cells

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to chemotherapeutic agents. For example, we have employed C6-ceramide (a pro-apoptotic

paclitaxel in MDR ovarian cancer [84, 106].

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3.2 Cancer Stem Cell Targeting and Therapeutics

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modulator) with paclitaxel and observed that the combination approach improved the efficacy of

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CSCs are high profile drug targets due to their key role in tumor progression, maintenance and MDR. However, targeting CSCs is likely to be challenging, since both bulk tumor cell

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population and CSCs must be eradicated, requiring combination drugs and targeting strategies as

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shown in Figure 5. Since CSCs are molecularly distinct from bulk tumor cell population, they

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can be targeted by exploiting these molecular differences. CSCs can be identified as a small subpopulation of cancer cells characterized by specific phenotypes expressing surface markers (Table 1). The overexpression of CD44 in tumor cells was strongly linked to drug resistance [107]. CD44 binds to hyaluronic acid present in ECM and assists in attachment of CSCs to the ECM, which can contribute to proliferation and migration of malignant tumor cells [17]. Another marker, CD133, is also found to be expressed in CSCs of many cancers including brain tumors with strong resistance to chemotherapy [17]. Therefore, a number of CSC surface markers including CD44, CD133, CD24, epithelial cell adhesion molecule (EpCAM) can be used as potential targets to deliver the therapeutics to CSCs in the tumors [108]. For example, bevacizumab, a vascular EGF neutralizing monoclonal antibody, significantly inhibited CD133 + glioblastoma cells ability to initiate tumors in vivo and depleted both blood vessels and selfrenewing CD133+ cells from tumors. These data suggest that glioblastoma CSCs have potent

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ACCEPTED MANUSCRIPT angiogenesis in tumors. Therefore, anti-angiogenic therapy in combination with chemotherapy may prove effective in targeting CSCs in glioblastoma tumors [109].

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ABC drug transporters, which are ATP-dependent drug efflux pumps render protection to the CSCs from cytotoxic drugs. Thus, CSCs could be targeted by ATP-competitive agents [110].

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The ABC drug transporters efflux a broad range of amphiphilic drugs including taxanes,

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anthracyclines and vinca alkaloids from the tumor cells. The ability to efflux different compounds is currently exploited for the Hoechst dye efflux assay, a method used for CSCs

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isolation that identifies them as side population (SP). The first and second generation of ABC

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inhibitors failed to demonstrate any therapeutic benefit, however, a more potent third generation inhibitors have been developed and are undergoing clinical evaluation [111].

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In another strategy, high-throughput compound screening was employed to selectively target

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the CSCs in tumors. For example, the antibiotic salinomycin was identified, which preferentially

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killed breast CSCs by inducing the differentiation of mesenchyme-like tumors in vivo, as characterized by increased E-cadherin and reduced vimentin expression [112]. This study provided an important concept that the CSC population could be treated by facilitating differentiation to a more “epithelial-like” cell state. Histone deacetylase (HDAC) inhibition could also cause apoptotic response or chemosensitization by differentiation of mesenchymallike tumor cells and CSCs. In this regard, HDAC inhibitors such as Trichostatin A and Vorinostat have now entered clinical trials for various cancer therapies as single agents or in combination with targeted and conventional therapeutics [113]. It has been demonstrated that the combination of chemotherapy and differentiation causing agents could cure the majority of patients affected by cancers [114, 115]. For example, a randomized Phase II clinical trial showed an enhanced therapeutic efficacy in NSCL cancer patients when all-trans retinoic acid was combined with platinum containing therapy [115].

18

ACCEPTED MANUSCRIPT The aberrant activation of self-renewal pathway associated signals is thought to be the main cause of CSCs fate. The Hedgehog (Hh), Notch, and Wnt/β-catenin are the most studied self-

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renewal pathways (Figure 5) [17, 94]. Novel combination therapies including small molecular drugs and RNA inhibitors (such as siRNAs) targeting these pathways are critical in order to

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completely eradicate MDR cancer cells and CSCs. The aberrant Hh signaling pathway plays a

SC

crucial role in tumor development. The control of the self-renewal by Hh has been found to be mediated through the modulation of Bmi-1, in breast CSCs [116]. The Hh pathway inhibition by

NU

cyclopamine effectively hampered glioblastoma multiforme stem-cell clonogenicity and

MA

selectively depleted pancreatic CSCs [117, 118]. Feldmann et al., suggest that by targeting CSCs that are most likely involved in tumor initiation at metastatic locations using Hh inhibitors may

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provide a new paradigm for therapy of metastasis tumors, particularly when used in combination

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with conventional antimetabolites that reduce bulk tumor size [118]. The other pathway, Notch

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has been implicated in various tumorigenic functions covering from cell survival to motility [119], and it is one of the most intensively investigated and recognized therapeutic targets in CSC [120]. Notch also communicates with other oncogenic pathways including the NFkB pathway, rhw hypoxia sensor HIF1α, and the estrogen receptor alpha, and plays a pivotal role in tumor survival [120]. Delta-like 4 ligand (DLL4) is a key component of the Notch pathway and facilitates stem cell self-renewal and blood vessel formation. In a colon cancer study, the inhibition of the DLL4 combined with irinotecan has shown to reduce the KRAS mutant colon CSCs frequency [121]. KRAS mutations in colorectal cancers confer resistance to anti-EGFR therapies such as cetuximab and panitumumab, thus, Notch antagonists could play a potential role in the management of the large segment of patients unsuitable for anti-EGFR therapies. Finally, Wnt is another pathway that is involved in several cellular processes covering proliferation, motility and stem cell maintenance [122]. The direct evidence linking the Wnt pathway with CSCs comes from a recent observation that high Wnt activity functionally 19

ACCEPTED MANUSCRIPT designates the colon CSC population [123]. A number of Wnt pathway inhibitors has been identified including the ones identified from a high-throughput based screening that acts at

<<<>>>

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various levels of the pathway[94].

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Novel gene targeting approaches against CSC regulatory pathways based on gene silencing

SC

using siRNA were also evaluated for the treatment of aggressive cancers. For example, anti-TG2 siRNA incorporated in liposomes showed enhanced therapeutic efficacy when combined with

NU

gemcitabine in pancreatic cancer xenografts and also this combination efficiently inhibited the

MA

spread of metastases [124]. Silencing the MDR1 gene by siRNA in MDR tumors can reduce the expression of P-gp and make chemotherapy more efficient. However, as discussed before,

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siRNA therapeutics has poor stability in blood circulation and low cell accumulation efficiency

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hampering their direct therapeutic applications. Remarkable progress has been made in recent

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years in order to design nanoparticle-based drug delivery platforms such as nanoparticle or MNS for small molecular drugs and siRNA therapeutics. These systems can potentially overcome known drawbacks of many anticancer therapeutics such as low solubility, stability, poor pharmacokinetics, non-specific toxicity, while maintaining high bioavailability of drugs at the target site. The CSC surface markers (Table 1) could be exploited by nano delivery systems for selective therapy against CSCs in the tumors. The multifunctional delivery using targeting ligands (e.g., hyaluronic acid to target CD44[125]) and combination therapeutics (e.g., siRNA with small molecular drug[126]) can add another dimension to effectively combat the CSCs and non-CSC population of tumor cells. In later sections we will discuss major multifunctional nano delivery approaches to target tumor heterogeneity using recent examples of therapeutic strategies applied against drug resistance and CSCs.

4. Illustrative Examples of Drug/Nucleic Acid Combination to Overcome MDR As discussed earlier, there are several mechanisms for the development of MDR in cancers 20

ACCEPTED MANUSCRIPT such as prolonged drug exposure to cancer patients or patients with recurrent cancers (such as after surgical resection) possessing CSC like characteristics, which exhibit highly complex

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phenotypic altercations. Treating MDR thus requires multipronged approaches such as coadministration of a drug cocktails or drug/gene combination treatments using targeted

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nanoparticles [82, 101]. Some such advanced delivery strategies are being intensely pursued and

SC

have shown promising potentials in preliminary in vitro and in vivo settings. Wang et al., have developed a cationic core shell nanoparticle system using biodegradable amphiphilic copolymer

NU

for DNA/siRNA and paclitaxel co-delivery [127]. The polymer namely, poly (N-

MA

methyldietheneamine sebacate)-co-((cholesteryl oxocaronylamido ethyl) methyl bis(ethylene) ammonium bromide)sebacate formed self-assembling core-shell nanoparticles encapsulating the

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drug and gene payload. These core-shell nanoparticles demonstrated superior tumor suppression

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both in the in vitro cell cultures and in vivo animal models [127].

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Natural polymers such as dextran, chitosan, gelatin and hyaluronic acid have several advantages compared to synthetic polymers in terms of biological safety of the engineered nanoconstructs.

Along these lines, we recently designed and evaluated a novel lipid-modified

dextran-based self-assembling polymeric nanosystem that could encapsulate MDR-1 siRNA as well as anticancer drugs such as doxorubicin [128, 129]. These systems were intended to overcoming the dose-limiting side effects of conventional chemotherapeutic agents and the therapeutic failure due to MDR. A combination therapy of the MDR1 siRNA loaded nanocarriers with doxorubicin revealed pronounced increase in doxorubicin uptake in the nucleus of MDR cells demonstrating that our approach may be useful for reversing drug resistance by increasing the amount of drug accumulation in MDR cells. Also, nano delivery using non-toxic and biologically inert polymers offers a versatile new platform that can be favorably translated to clinical applications.

21

ACCEPTED MANUSCRIPT Researchers from Minko’s group have developed liposome based nano-carrier systems for the co-delivery of siRNA and doxorubicin for the treatment of MDR small cell lung cancer

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(SCLC)[130]. By combining the anticancer drug, doxorubicin with suppressors of pump and non-pump mediated cellular resistance (such as MRP-1 and BCL2 siRNAs) within liposomal

RI

delivery systems, enhanced efficacy of chemotherapy was achieved to a level that could not be

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achieved by separate treatment of chemotherapy or gene silencing alone [130].

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Premature release or “burst effect” of drugs can be major concern that needs to be overcome to arrive at safe and effective nano delivery systems. In this regard, inorganic nanoparticles have

MA

been found to very useful as robust carrier systems. In one such study Chen et al., evaluated the co-delivery of doxorubicin and Bcl-2 siRNAs using mesoporous silica nanoparticles to MDR

D

cancers [131]. This study demonstrated minimal premature release of the drug, and significant

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enhancement in efficacy in MDR cancer cells due to the effective silencing of Bcl-2 mRNA and

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significant suppression of the non-pump mediated resistance. By this strategy, remarkable enhancement in the anticancer action of doxorubicin was achieved [131]. In another recent study, Meng et al., used multifunctional mesoporous silica nanoparticles (MSNP) to overcome doxorubicin resistance in MDR human breast cancer xenograft models [132]. siRNA that downregulates P-gp drug efflux pump was co-delivered with doxorubicin using mesoporous silica nanoparticles. After the establishment of MDR tumors in mice, it was demonstrated that polyethyleneimine/polyethylene

glycol

(PEI-PEG)

functionalized

mesoporous

silica

nanoparticles could efficiently deliver both doxorubicin and MDR-1-siRNA to the tumor site. The study further confirmed that the co-delivery resulted in synergistic inhibition of tumor growth in mice models and had better outcome than when treated with the free doxorubicin or the inorganic nanoparticles loaded either with siRNA or doxorubicin alone [132]. The use of biodegradable synthetic polymers such as the ones based on poly(D,L-lactide-co22

ACCEPTED MANUSCRIPT glycolide) (PLGA) are FDA approved for human use and hence could fast track the development of nano platforms for clinical development. In one study Patil et al., have used biotin tagged

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PLGA based biodegradable nanoparticles for the targeted co-delivery of paclitaxel, and MDR1 siRNA [133]. An in vitro screening of the targeted nanoparticles co-loaded with paclitaxel and

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MDR1 silencing siRNA demonstrated much higher cytotoxicity than identical PLGA

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nanoparticles loaded with paclitaxel alone. The report suggests that the enhanced therapeutic efficacy and cell killing of dual-agent loaded targeted PLGA nanoparticles could be correlated

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with MDR1 gene knock down that encodes for the P-gp, causing paclitaxel efflux. Furthermore,

MA

a detailed in vivo studies using MDR tumor bearing mice supported the in vitro results. Interestingly, it was observed that the tumor growth was significantly suppressed following

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treatment with dual siRNA and drug loaded in biotin tagged PLGA nanoparticles, suggesting that

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the combination treatment yielded synergistic effect [133].

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Dendrimers are a class of hyperbranched polymers that have immense potentials for combination treatment using drugs and genes [134-140]. In one example, a new class poly(Llysine) dendrimer of generation 3 called “nanoglobules” was developed by Kaneshiro and Lu for the co-delivery of siRNA and doxorubicin to glioblastoma cells [141]. Furthermore, in order to target these dendrimer to glioblastoma tumors, they were decorated with RGD-peptide, that homes to v3-integrins receptors overexpressed on the tested U87 glioblastoma cells. The targeted dendrimers showed marked gene silencing activity and tumor growth inhibition due to their efficient internalization in U87 glioblastoma cells demonstrating their utility for co-delivery [141]. Nakamura et al., recently investigated the antitumor effect of co-treatment of PEG-coated bcl2 siRNA-lipoplexes and a prodrug of 5 fluorouracil called Tegafur (TF), in mouse bearing colorectal cancers [142]. It was demonstrated that a single treatment with either siBcl-2-lipoplex 23

ACCEPTED MANUSCRIPT or TF resulted in a moderate inhibition of tumor growth whereas the co-treatment of Bcl-2 siRNA-lipoplexes with TF, induced remarkable tumor growth suppression, resulting in a tumor

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weight reduction of as much as 62%. More importantly, there were no signs of acute toxicity or

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body weight loss in mice indicating the safety of the co-treatment regimen [142].

While the strategies based on siRNAs has been well established, its utility can sometimes be

SC

suboptimal in treating diseases such as cancers due to its transient effect per dose [143]. In this

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regard, optimized short hairpin RNA (shRNA) constructs allow for high potency and sustainable long-term expression of therapeutic RNAs in targeted cells [144]. In one such interesting shRNA

MA

mediated strategy, researchers from Li’s group have attempted to reverse MDR breast cancers by silencing two different genes, by packaging two kinds of short hairpin RNAs (shRNA’s) using

polyester

polymer

TE

D

one polymer delivery platform. For this purpose Yin et al., first synthesized a bioreducible namely,

poly[bis(2-hydroxylethyl)-disulfide-diacrylate-β-

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tetraethylenepentamine] (PAP) using Michael addition reaction. It was demonstrated that codelivery of MDR1 gene silencing shRNA and survivin silencing shRNA using a single nano delivery vector resulted in synergistic effect on reversing MDR tumors in vitro and in vivo [145]. Taken together these studies demonstrate the plethora of strategies and the potentials of polymeric nanoparticles based drug/gene combination therapy for treating MDR cancers.

5. Illustrative Examples of Stimuli Responsive Delivery Systems The physicochemical properties of nanoparticles can be customized to develop stimuliresponsive systems that exploit the biological milieu of the disease target to improve the delivery efficiency and achieve the desired therapeutic efficacy. Stimuli-responsive systems have unique capability to elicit a large “response” to a small change in the chemical or physical conditions. Such systems are often also termed as “environmentally-sensitive”, “smart” or “intelligent” materials and such polymeric, polymer-lipid hybrid and polymer-inorganic hybrids have been 24

ACCEPTED MANUSCRIPT extensively used for designing stimuli-responsive delivery vehicles for biomedical application. These materials can be further classified into externally regulated and self-regulated delivery

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systems depending on the source of requisite stimuli being external or internal respectively, for initiating the payload release.

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5.1 Self regulated DDS

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Self-regulated DDS (often also known as closed-loop systems) respond to a change in the biological signal from within the body (e.g. pH, redox potential) to modulate the drug release

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profile. Such systems rely on differences in the biological milieu at the target site of actions to

MA

automatically switch the drug release on or off. The triggered release of the payload is therefore controlled entirely by the physiological condition of the diseased site and cannot be altered by an

D

external stimulus.

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5.1.1 pH-responsive systems

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As discussed earlier, the tumor microenvironment differs significantly from the surrounding normal tissues. For instance, the low pH environment presented by tumors has been exploited for designing smart materials for controlled drug delivery to tumor sites[146]. pH-responsive materials such as ionisable polymers with pKa value in the range of 3-10 thus have been actively researched to facilitate controlled site-specific delivery. Weak bases such as amines and weak acids such as carboxylic and phosphoric acids show a pH dependent ionization characteristics that alters the conformational and solubility profile of polymers as well as swelling/de-swelling properties of crosslinked hydrogels resulting in controlled payload delivery. In this regard, monomers of carboxylic acids such as acrylic acid (AAc), methacrylic acid (MAAc), maleic anhydride (MA), N,N-dimethylaminoethyl methacrylate (DMAEMA) are most commonly used while phosphate containing acids like 2-(methacryloyloxy)ethyl dihydrogen phosphate have also been reported as pH-responsive materials [147]. Poly(amidoamine) (PAA or PAMAM), an amphoteric polymer showing excellent 25

ACCEPTED MANUSCRIPT protonation/deprotonation based size variation resulting in endosomolytic properties for delivery of gelonin [148, 149]. These studies demonstrate that PAA undergoes a pH-driven coil

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expansion/rearrangement where the amphoteric backbone expands at lower pH and slowly collapses at neutral pH resulting in release of the payload. Such PAA-based pH responsive

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dendrimers have been therefore been explored as stimuli-responsive drug delivery vehicles in

SC

cancer therapy. Duncan and his group have created a library of PAA-derived delivery systems, studied physicochemical property dependent biological characteristics of these derivatives and

NU

their preliminary screening revealed several promising candidates that show low material

MA

induced toxicity compared to other polycationic vectors such as poly (ethyleneimine) and polyL-lysine [150-155]. They further studied the biodistribution of two PAA derivatives (ISA4 and

D

ISA22) to demonstrate that while ISA4 rapidly accumulated primarily in liver, ISA22 continued

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to accumulate in sub-cutaneous B16F10 melanoma tumors even after 5h of administration [150].

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PAA derivatives have therefore been explored for pH-dependent delivery of biomacromolecules [156] as well as small molecules [157] and prove to be a promising candidate for designing smart nano-delivery vectors for cancer therapy. Poly(β-amino ester) are another class of polymers that show excellent pH sensitivity. The solubility of this biodegradable polymer strongly depends on the solution pH such that the unprotonated polymer is insoluble in aqueous media at physiological pH but rapidly solubilizes at pH below 6.5 [158]. This unique property renders it as material of choice for designing pH responsive vehicle to achieve delivery in the intracellular conditions. In an extensive study, poly(ethylene oxide) modified poly(β-amino ester) (PEO-PbAE) was explored as pH-responsive material for in vitro delivery of paclitaxel [159], in vivo biodistribution [160] and efficacy/safety analysis [161]. In vitro analysis of paclitaxel loaded pH sensitive particles demonstrated higher accumulation and better cytotoxicity in MDA-MB-231 human breast cancer cells compared to drug in PCL particles or in solution. Tumor accumulation studies in SKOV-3 human ovarian 26

ACCEPTED MANUSCRIPT cancer xenograft model showed 5.2-fold and 2.2 fold higher concentration by PEO-PbAE and PEO-PCL particles respectively compared to drug in solution 1 h post-administration. Efficacy

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and safety studies at 20 mg/kg dose of paclitaxel in SKOV-3 xenograft model further revealed that drug loaded in PEO-PbAE particles showed enhanced tumor regression compared to the

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other controls while none of the formulations showed any signs of toxicity during the period of

poly(ethylene

glycol)-b

-poly-(allyl

ethylene

SC

study. In a recent report, Du et al., synthesized a dual pH responsive monomethoxyl phosphate)-doxorubicin-3-dimethylmaleic

NU

anhydride (PPC-Hyd-DOX-DA) that could effectively delivery the payload at acidic pH in a

MA

time dependent fashion (Figure 6). This formulation was shown to successfully curb the sphere forming ability of SK-3rd, a stem cancer cell line. Sphere forming property is a measure of in

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vitro self-renewal ability of these cells, which is an important yardstick for their “stemness”

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[162].

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<<<>>>

Lipid conjugates have similarly being used as acid-labile zwitterionic groups to develop fusogenic liposomes to facilitate uptake and delivery. 1,5-hexadecyl N-glutamyl-L-glutamate and 1,5-hexadecyl N,N-diglutamyl-lysyl-L-glutamate were used as pH-labile components to deliver doxorubicin in vitro and in vivo as liposomal formulation [163]. In vitro analysis in HeLa cells confirmed 5-fold higher co-localization of doxorubicin in nucleus when delivered by pHresponsive liposomes compared to regular liposomes where the doxorubicin was largely sequestered in endosomes. In vivo studies in human breast cancer xenograft (MDA-MB-231 cells) further ascertain improved efficacy from pH-responsive liposomes when compared to the free doxorubicin as well as pH-non-responsive liposomes. Hybrid systems, having two or more independent delivery components, have also been actively researched to harvest the beneficial delivery aspects of both the materials. In one such study, an acid-labile polymer-caged liposomal hybrid system was designed by coating cholesterol-terminated poly(acrylic acid) (Chol-PAA) on 27

ACCEPTED MANUSCRIPT to

preformed

liposomes

containing

dipalmitoylphosphatidylcholine

(DPPC),

dioleoylphosphatidylglycerol (DOPG) and cholesterol, which showed a pH-dependent release

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profile of the encapsulated drug [164]. Mesoporous silica nanoparticles (MSNs) have been similarly used as a drug reservoir for

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delivery applications in tandem with surface modification with acid-labile materials as

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gatekeepers to achieve a controlled release. MSNs as drug reservoir serves several advantages including biocompatibility, high surface area and drug loading efficiency, immunity to

NU

hydrolysis and enzymatic degradation and physical protection of the loaded therapeutic moiety

MA

against any in vivo degradation due to narrow channels of the particles. Silica rods (SBA-15) modified with carboxylic acid and coated with poly(dimethyldiallylammonium chloride)

D

(PDDA) are versatile system with high drug loading capability (35.4 wt %) where the

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polyelectrolyte layer closes the channels in the silica nanorods loaded with the antibiotic

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vancomycin. Change of pH to acidic range leads to protonation of -COO- groups to the nonionized –COOH state leading to detachment of the polyelectrolyte layer and subsequent release of the payload [165]. In other reports, MSNs were grafted with poly(ethyleneimine)-cyclodextrin (PEI-CD) polypseudorotaxanes

[166], lysozyme [167] and cucurbit[6]uril (CB[6]) [168]

respectively to design pH-responsive nano-valves effecting a controlled release of the loaded therapeutic agents. Meng et al. studied drug delivery capability of MSNs modified with β-cyclodextrin, making them responsive to endosomal acidification. These particles when loaded with Hoechst 33342 dye or doxorubicin drug, showed efficient payload release at acidic pH in a time dependent fashion in solution as well as in human differentiated myeloid (THP1) and squamous carcinoma (KB-31) cells [169]. In yet another recent report, pH responsive MSNs could successfully deliver doxorubicin along with cetyltrimethylammonium bromide (CTAB) as chemosensitizer to human breast cancer cell line MCF-7 and MCF-7/ADR (drug resistant cell line). The MSNs show a 28

ACCEPTED MANUSCRIPT sustained drug release over a time period of up to 14 days at pH 4 in solution and resulted improvement in cell killing efficiency of the drug, especially in resistant cells by a synergistic

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cell cycle arrest/apoptosis-inducing effect. Further in vivo analysis in MCF-7/ADR tumor bearing mice confirmed that the drug loaded MSNs could successfully localize in the tumor and

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deliver the drug by pH activated trigger while the particles localized in liver, spleen or lung did

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not show any drug release, confirming a stimuli-specific payload delivery [170]. 5.1.2 Redox responsive systems

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The presence of oxidizing extracellular and reducing intracellular environment within a

MA

tumor is a well-documented fact, which leads to a high redox potential difference and opens upon a unique opportunity to use redox-responsive delivery systems for therapeutic applications

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[171]. Redox-responsive materials tend to disassemble when internalized within a cell in

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response to the reducing environment primarily due to 2 to 3-fold higher level of glutathione

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(GSH) tripeptide (2-10 mM) in the cytosol as well as nucleus, thereby releasing the drug [172]. The nano delivery vehicles are initially localized within the endosomal compartment of the cell, where an enzyme called gamma-interferon-inducible lysosomal thiol reductase (GILT) regulates the redox potential in the presence of cysteine [173]. Reduction sensitive materials have therefore been looked upon as an attractive choice for designing nanocarriers for delivery of drug, gene and imaging agents. The continued efforts in the past decade have led to development of polymer, protein, lipid and inorganic-based redox responsive delivery systems that largely rely on reduction-sensitive disulfide linkage. Cavallaro et al., designed α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) backbone based thiopolyplexes cross-linked by disulfide linkage for gene delivery application [174]. In a similar work, crosslinked gelatin particles were studied as redox-sensitive vehicles for DNA delivery [175, 176]. The biodistribution studies further reveal that PEG-modified thiolated gelatin particles demonstrated long circulating/enhanced residence properties and improved 29

ACCEPTED MANUSCRIPT tumor-selective accumulation due to reducing environment [177]. In yet another study, intravenous administration of plasmid DNA encoding the soluble form of extracellular domain of

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vascular endothelial growth factor receptor-1 (VEGF-R1 or sFlT-1) formulated in PEG-modified thiolated gelatin in orthotopic MDA-MB-435 breast adenocarcinoma xenograft model showed

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efficient suppression of tumor growth and angiogenesis [178].

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Redox-sensitive liposomal delivery vehicles can be prepared by conjugating a small lipid molecule to a standard phospholipid such that the hydrophilic domain of one is conjugated to the

NU

hydrophobic domain of other through a disulfide linkage. Liposomes prepared using such lipid

MA

conjugates maintain their structure in a normal environment but undergo disintegration when exposed to a reducing environment due to the cleavage of the disulfide bridging [179].

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5.2 Externally regulated DDS

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Externally regulated DDS (also known as open loop systems) are indifferent to the biological

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makeup of the site of action and their drug release characteristics depend on an external stimulus. Such systems provide much precisely controlled delivery/release profile depending on the strength or duration of the external stimuli and therefore have been actively pursued for disease targeting and therapeutics. For such cases, heat, light, ultrasound, magnetic and electrical energy have been commonly used to regulate the delivery of the drug at the site of interest. 5.2.1 Thermo-responsive systems Thermo-responsive materials include systems that undergo a phase change above a certain critical temperature, which can be lower critical solution temperature (LCST) or upper critical solution temperature (UCST). LCST and UCST respectively represent the temperature above or below which the material makes a clear homogenous solution in aqueous phase. Poly(N-isopropyl acrylamide) or PNIPAAm, the most extensively studied smart polymer as thermo-responsive material, changes from hydrophilic to hydrophobic at 32 °C in water and at slightly lower temperature in physiological pH, thereby extruding out its aqueous swelling 30

ACCEPTED MANUSCRIPT solution. The LCST strongly depends on side chain [180, 181], molecular weight and architecture of the polymer [182] as well as presence of additives such as salts, surfactants or co-

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solvents [183]. The LCST of PNIPAAm could be therefore modulated to lower and higher temperature by copolymerizing it with hydrophobic or hydrophilic monomers respectively [184-

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186]. Poly(N,N-diethylacrylamide) (PDEAAm) with LCST range of 25-32 °C [187], poly(N-

SC

vinlycaprolactam) (PVCL) with an LCST between 25-35 °C [188], poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) with an LCST around 50 °C [189] and poly(ethylene glycol) (PEG)

NU

with LCST around 85 °C are some other examples of thermo-responsive polymers that have

MA

been used for controlled drug delivery applications.

The thermo-responsive polymers for delivery application have been classified into hydrogels,

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interpenetrating networks, micelles, polymersomes, films and particles. Hydrogels are three-

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dimensional polymeric particles where the architecture could be supported by physical

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interaction (physical gels) or by chemical cross-linking (cross-linked gels). Interpenetrating networks (IPNs), on the other hand, are network of two different chemically linked polymeric conjugates that interact by physical entanglement and provide independent unique properties to the hydrogel. Wang et al., synthesized IPNs of polyacrylic acid (PAA) and polyacrylamide (PAM) grafted with β-cyclodextrin (UCST ~ 35 °C) to achieve a controlled release of ibuprofen [190]. Block copolymers with distinct hydrophobic and hydrophilic domains are known to form self-assembled micellar architecture with hydrophobic core that has been used as reservoirs for delivery applications of lipophilic drugs [191, 192]. Polymersomes on the other hand are selfassembled structures derived from block copolymer such that they have a hydrophilic core as well as shell with an intermediate hydrophobic layer and thus have been used for the delivery of water-soluble therapeutic payloads [193]. A comprehensive review on thermo-responsive polymeric delivery system has been published recently and is recommended for detailed overview of the field [194]. 31

ACCEPTED MANUSCRIPT Lipids similarly show phase change upon thermal stimulus and thus have been explored to develop temperature-sensitive delivery vehicles. Dipalmitoylphosphatidylcholine (DPPC) is one

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such lipid with a transition temperature of 41.5 °C, which is just above the body temperature and is ideal for such applications [195]. The transition temperature and drug release profile of the

RI

liposomes can be further tailored by changing the composition of the constituent lipids. Mixing

SC

lysolipid 1-palmitoyl 2-hydroxy-sn-glycero 3-phosphocholine (MPPC) with DPPC leads to enhanced release of payload at 39-40 °C compared to much slower release from DPPC liposome

NU

at 40-45 °C and DPPC-1,2- distearoyl-sn-glycero 3-phosphocholine (DSPC) liposomes at 43-45

MA

°C [196]. Kong et al., prepared non-thermosensitive liposomes (NTSL), traditional thermosensitive liposomes (TTSL) and low thermosensitive liposomes (LTSL) for a systematic

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in vivo drug accumulation and efficacy study in FaDu human squamous cell carcinoma xenograft

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model [197]. All three liposomal formulation loaded with doxorubicin showed little therapeutic

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benefit under normothermic conditions (34 °C) however with induction of hyperthermia at 42 °C, LTSL formulation showed highest doxorubicin accumulation and most potent antitumor effect compared with other formulations. Thus, hyperthermia-triggered drug release from liposomes accounted for the largest differential therapeutic effect and demonstrated the importance of rapid drug release from the drug carriers at the tumor site for most efficacious treatment [197], that could be extended for MDR tumors. In yet another report, thermoresponsive liposomes were loaded with fluorescein and DOX and in vivo imaging in FaDu tumor model as well as eNOS-GFP transgenic mice (B16BL6 melanoma cancer) revealed that induction of hyperthermia (41 °C) leads to enhanced uniform drug release in a time depended manner [198]. Thermosensitive liposomes can also be prepared by incorporating LCST polymeric materials into the lipid membrane forming lipid-polymeric hybrid stimuli responsive material. Paasonen et al., synthesized poly(N-(2-hydroxypropyl)methacrylamide) (HPMA)-mono/dilactate polymer conjugated with cholesterol that serves as anchor in the lipid bilayer. The liposomes released 32

ACCEPTED MANUSCRIPT their payload above the LCST (42 °C) where the polymer coating becomes hydrophobic resulting into destabilization of the lipid bilayer [199]. Kono et al., have similarly used PNIPAM

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on liposomal surface to develop temperature-responsive liposomes [200]. In a recent report, doxrubicin-loaded poly[2-(2-ethoxy)ethoxyethyl vinyl ether (EOEOVE)] coated liposomes

RI

demonstrated an efficient drug release at 40 °C and enhanced anti-tumor activity in mouse colon

SC

carcinoma 26 cells xenograft model in BALB/c mice [201]. In an extended study, researchers from the same group prepared EOEOVE coated multifunctional liposome with temperature-

NU

sensitive therapeutic and magnetic resonance imaging (MRI)-guided imaging modality [202]. A

MA

comprehensive review on thermosensitive liposomes highlights the strengths and benefits of such stimuli-responsive delivery systems in biomedical applications [203].

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<<<>>>

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Induction of hyperthermia to facilitate delivery from thermosensitive delivery systems is an

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important aspect that has been extensively researched to achieve a controlled desired local temperature without causing damage to the tissues. Release of payload from material having LCST above body temperature has been achieved by external heating using circular resistive electric heating coils [198, 204], temperature controlled microwave ring radiator [205], ultrasound energy induced hyperthermia [206] or by incorporating superparamagnetic materials for magnetic field induced hyperthermia [207]. The depth of heat penetration for in vivo applications depends on several factors including energy source (electromagnetic radiation, ultrasound energy etc.), type of applicator (capacitive, radiative, inductive etc) frequency, power intensity, tissue composition and time of application [208]. A recent report suggests that local hyperthermia in the tumor also improves the delivery efficiency by altering the vasculation permeability, perfusion and interstitial fluid flow [209] (Figure 7). Application of thermosensitive delivery systems therefore holds tremendous promise in curbing cancer growth due to improved particle localization in tumor combined with enhanced drug delivery efficiency. 33

ACCEPTED MANUSCRIPT 5.2.2 Light-responsive systems Light as external stimuli is a popular choice since it can be readily tuned and focused on the

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surface and its spatial and temporal exposure can be easily controlled. Light sensitive materials have therefore been extensively used for various applications including self-healing systems

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[210], specific shape transformation [211] and “gated” membranes for controlled ions or gas

SC

flow [212]. Block copolymer (BCP) micelles, hydrogels and liposomes are the most commonly used nanocarriers to design light-sensitive DDS. Such systems usually incorporate a

NU

chromophore that can undergo light-assisted isomerization, cleavage, ionization or dimerization

MA

to destabilize the structural integrity of the delivery vehicle, thereby facilitating the payload release. Photosensitization of the chromophore may lead to change in hydrophobic-hydrophilic

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balance [213], reversible cross-linking [214], cleavage of block junctions [215] and main chain

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integration [216]. Near Infra-red (NIR) wavelength is preferred over high energy UV-visible for

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in vivo photosensitization since it has a deeper penetration and the component of biological issues have low absorption in that region of light. Azobenzene, o-nitrobenzene, pyrene and coumarin are the most commonly used groups to design photosensitive DDS though several other derivatives have also been explored. BCPs

composed

of

hydrophilic

poly(ethylene

oxide)

(PEO)

and

hydrophobic

polymethacrylate with pyrene on the side chain (PPy) form light-responsive micelles, which upon UV irradiation undergo solvolysis, rendering PPy hydrophilic and subsequent disintegration of the micelles [217]. Prolonged UV exposure however can be detrimental to the cells; two-photon absorption [218] and recently upconverting nanoparticles (UCNPs) have therefore been looked upon as promising alternatives. Upconversion is a process where material can be efficiently excited with a low excitation density of light, where sequential absorption of two or more photons lead to emission at wavelengths shorter than the excitation wavelength [219]. Upconverting nanoparticles absorb light in NIR wavelength and emit higher energy 34

ACCEPTED MANUSCRIPT photon in UV, visible or NIR to effect photosensitization process. Yan et al., loaded PEOpolymethacrylate bearing photosensitive o-nitrobenzyl group (PNBMA) micelles with

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NaYF4:TmYb core-shell UCNPs as light-responsive DDS [220]. Upon irradiation with 980 nm NIR light, the UCNPs emit UV and visible wavelength photon resulting in cleavage of o-

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nitrobenzyl group and causing disruption of the micellar structure. Nanoparticles made of such

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smart materials offer unique possibility of designing integrated multifunctional therapeutic and diagnostic (theranostic) DDS that could be extended for MDR cancers. Photoresponsive

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liposomal delivery vehicle are similarly designed using lipids with photolabile functional

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moieties. Yavlovich et al., studied the light wavelength dependent release profile of doxorubicin loaded in liposomes composed of DPPC, photopolymerizable DC8,9PC and DSPE-PEG2000.

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Cytotoxicity studies in lymphoblastoid (Raji) and human breast adenocarcinoma (MCF7) cells

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demonstrated a 2-3 fold improved cell death on exposure to these liposomes followed by

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exposure to light (514 nm) for 5 min [221]. A review by Alvarez-Lorenzo et al., provides an indepth outlook on photosensitive intelligent delivery vehicles [222]. 5.2.3 Ultrasound-responsive systems Ultrasound (US) is an oscillating pressure wave of frequency higher than 20 kHz. Application of ultrasound energy for imaging and diagnostics is a well-established area even though its therapeutic potential was realized earlier. While the diagnostic US is expected to minimize energy deposition within the tissue to reduce “biological effect”, therapeutic US should be able to maximize the energy deposition to generate effect. Similar to light, US energy can be easily focused over a small volume leading to very high intensity and deeper penetration efficiency, which is important for therapeutic application. US energy used for therapeutic application is therefore popularly termed as high intensity focused ultrasound (HIFU). Initial use of therapeutic US was majorly limited to thermal ablation of tumor, recent endeavors have largely focused on improving the performance of DDS. US energy enhances the delivery 35

ACCEPTED MANUSCRIPT efficiency through three major mechanisms namely heat generation, acoustic cavitation and acoustic radiation forces. The nanoparticle design parameters for US mediated heat-induced

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delivery are similar to thermosensitive DDS discussed above though other important considerations include time of US application, nature of application (continuous or pulsed),

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frequency and power [223, 224] Dromi et al., compared the performance of NTSL and LTSL in

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vitro as well as in vivo by HIFU mediated induction of hyperthermia (42 °C for 2 min). LTSL supplemented with pulsed-HIFU exposure led to increased drug release, higher drug

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concentration in tumor, as well as an increase in antitumor activity [206]. The use of US

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mediated drug release in combination with other strategies could be highly beneficial for MDR cancers.

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Microbubble technology has been extensively studied for US-mediated drug delivery.

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Microbubbles are gas filled microspheres where the core is composed of water insoluble gas (e.g.

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perfluorocarbons, sulfur hexafluoride) surface stabilized by protein, lipid, surfactant or biocompatible polymer. This technology was initially developed for contrast enhancement in US imaging since the acoustic backscatter of microbubbles could be several orders higher to biological tissues due to high acoustic impedance mismatch. Exposure to US energy in the presence of microbubbles lead to acoustic cavitation at low frequency but could lead to complete loss of structural integrity at higher frequencies. When US exposed microbubbles are in near vicinity of cells, they can significantly alter the cell permeability (procedure popularly known as sonoporation) thereby facilitating delivery across the membrane [225]. Alternatively, microbubbles can be used as drug or gene carriers for direct delivery application by US energy mediated disruption [226, 227]. US responsive micellar polymeric particles are yet another technology that has been extensively developed by seminal contributions from Prof. Rapoport’s group over the years. The rationale of their approach involves synthesis of drug-loaded pluronic micellar nanoparticles that 36

ACCEPTED MANUSCRIPT could be localized in the tumors by passive targeting followed by controlled release by focused US pulse. Initial in vitro experiments confirmed that the micellar delivery systems could rapidly

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release the payload by a 15-30 s short HIFU pulse [223, 228, 229] resulting in higher intracellular concentration of DOX in sensitive and MDR cancer cell [230, 231]. In vivo drug

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accumulation studies in intraperitoneal (i.p.) and subcutaneous (s.c.) ovarian cancer tumors

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confirmed an enhanced drug accumulation and distribution in the tumors upon application of a 30 s US pulse [232]. Efficacy studies in i.p. ovarian carcinoma model showed a decrease in

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tumor volume from 70 % for DOX administered in solution to 36 % for same dose of drug in

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micellar formulation combined with US pulse [233]. It was further ascertained that application of US pulse resulted in a uniform distribution of the drug across the tumor volume compared to a

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non-uniform distribution in its absence. Even in s.c. tumors, micelle-based DOX delivery

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combined with localized US pulse resulted in delay in the tumor growth compared to the

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administration in the absence pulse [233]. 5.2.4 Magnetically regulated systems Materials responsive to magnetic field such as magnetic nanoparticles can be potentially incorporated into a delivery vector to design magnetically regulated DDS. Delivery vehicles incorporating magnetic responsiveness offer remarkable advantages over other types of regulated system. External magnetic field can be applied to concentrate the delivery vehicle to desired site of action as well as trigger magnetic field induce direct release or hyperthermia induced heating to release the payload. Superparamagnetic iron oxide nanoparticles (SPIONs) could additional be used as MRI contrast agent for integrated imaging and stimuli-responsive drug delivery. Hua et al., demonstrated that paclitaxel loaded magnetically responsive core shell nanoconstruct of poly[aniline-co-sodium N-(1-one-butyric acid) aniline] (SPAnNa) showed enhanced cell killing efficiency under external magnetic field (Figure 8). IC50 values of free drug, drug loaded nanoparticle and drug loaded nanoparticles under magnetic field targeting was found to be 7.1, 37

ACCEPTED MANUSCRIPT 4.2 and 1.7 µg/mL for CWR22R prostrate cancer cells and 11.1, 9.7 and 4.6 µg/mL for PC3 prostrate cancer cell line respectively [234].

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<<<>>> An alternative mechanism of controlled drug release from magnetically active delivery

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vehicles could be through magnetic field induced hyperthermia. Magnetic nanoparticles

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integrated thermoresponsive materials have therefore been extensively studied as drug delivery vectors [235], with potentials for future development for treating MDR cancers. Purushotham et

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al. designed PNIPAM coated SPIONs with dual modality as MRI contrast agent as well as DOX

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delivery vehicle. In vitro studies suggested that an initial rapid hyperthermia induced drug release could be achieved above LCST (14.7% drug release in initial 47 min) followed by a

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slower release profile or a slow sustained release below LCST. In vivo tumor targeting under

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external magnetic field was further demonstrated in buffalo rat model implanted with

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hepatocellular carcinoma. MRI imaging and histological analysis revealed that the drug-loaded particles could be successfully localized in tumors for a successful and efficient delivery [207]. The versatility of the magnetically regulated delivery systems cements them as an attractive multifunctional smart material and several such platforms are actively researched. Hu et al., have developed a gelatin-based ferrogel containing Fe2O3 nanoparticles as stimuli responsive system that triggers drug release on exposure to high-frequency magnetic field (HFMF) [236]. The same group further developed a silica core-iron oxide shell type nanoparticles system, which shows a burst release profile of the guest molecule loaded in the core on exposure to HFMF [237]. In a recent report, SPIO nanoparticles embedded polymersomes made of poly(trimethylene carbonate)-block-poly(L-glutamic acid) (PTMC-bPGA) copolymer loaded with DOX demonstrated 18 % greater cytotoxicity effect on exposure to HFMF due to increased drug release [238]. 5.2.5 Electrically regulated systems 38

ACCEPTED MANUSCRIPT Electrically regulated DDS can be externally controlled by application of electric field. Such systems are preferred over light, US or magnetically controlled systems, which require elaborate

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instrumental set-ups to realize the desired output. Electric field, on the other hand, can be easily induced, precisely controlled and most importantly is amenable to miniaturization on a chip.

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Advancement in understanding of conducting polymers has further augmented designing and

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development of electric field regulated delivery vehicles [239]. George et al., used a biotinylated film of polypyrrole to bind streptavidin that was further functionalized by biotinylated nerve

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growth factor (NGF). Application of 3V electric field for less than 3 min across the film resulted

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in the release of biotin bound to polypyrrole surface, thereby releasing almost 100% loaded NGF [240]. Wadhwa et al., similarly studied triggered controlled release of Dexamethasone (Dex)

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from a polypyrrole-Dex film by application of an electric field [241]. Ge et al., alternatively

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explored the possibility of making conducting nanoparticles of polypyrrole loaded with

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daunorubicin and fluorescein, which were further embedded in thermoresponsive hydrogel made of poly[(D,L-lactic acid)-co-(glycolic acid)]-b-poly(ethylene oxide)-b-poly[(D,L-lactic acid)-co(glycolic acid)] (PLGA-PEG-PLGA). The hydrogel remained solid at body temperature and introduction of an electric pulse of -1.5 V/cm every 20 min facilitated the release of the cargo. In vivo subcutaneous injection of the fluorescein loaded particles in FVB mice followed by electric pulse led to release of the fluorescent dye after every electric pulse, which could be imaged at the site of interest [242]. Though clinical significance is yet to be established, platform technologies strongly suggest that such electrically regulated nanoparticle systems can be engineered for controlled release of drugs, genes and imaging agents for simultaneous drug/gene delivery and imaging of MDR cancers.

6. Conclusions and Future Outlook Cancer as a disease has truly followed the Darwin’s principle of “survival of the fittest”. Although some forms of cancers can be repressed by aggressive chemo and radiotherapy, a 39

ACCEPTED MANUSCRIPT majority of them overcome drug insults and undergo phenotypic and genotypic alterations, possessing MDR characteristics. Despite major advancements in understanding of the disease at

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the cellular and molecular level and the underlying mechanisms involved in tumor survival and maintenance, clinicians still continue to face a daunting task of managing recurrent and

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metastatic cancers. In this regard, researchers over the past few decades have been actively

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developing alternative strategies for treating several forms of cancers such as drug cocktails and drug-gene combination therapies employing organic and inorganic nanoparticle systems. Among

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them, the most popular ones such as the polymer-drug conjugates, liposomes and micellar

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delivery systems have been intensely researched upon, and have already made it into preclinical and clinical development. Such systems utilize the anatomic and pathophysiological

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abnormalities of the tumor tissues to selectively accumulate in the tumor tissues. In the recent

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times, in an effort to maximize the utility and capability of nanoparticles based delivery systems

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researchers have engineered more sophisticated delivery systems by incorporating tumor homing ligands and integrating stimuli responsive features to multifunctional (targeted) nanosystems. These systems are capable of utilizing both passive and active tumor targeting principles and a combination of drug/gene release mechanism, to produce better therapeutic outcome. More interestingly, multifunctional nanosystems are currently being designed to incorporate diagnostic intervention and therapeutic functions built into the same nano delivery system. The so-called “theranostic” nanosystems are intended to simultaneously perform diagnostic and therapeutic functions in real time. Such precision-guided multifunctional nanosystems portend to have tremendous utility for the better management of MDR tumors.

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Surface marker expression

SC

CD44,

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CD34+CD38-

Acute myeloid leukemia

PT

Cancer

Bladder

CD47,

NU

Lin-CD44+CK5+CK20CD44+CD24-/loLin-EPCAM+

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CD133+

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CD133+ ,

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EPCAMhiCD44+Lin-(CD166+) CD44 and CD133+

medulloblastomas Head and neck

CD44+Lin-

Liver

CD90+

Melanoma

ABCB5+

Ovarian

CD44+CD117+ CD44+CD24+EPCAM+,

Pancreatic CD133+

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ACCEPTED MANUSCRIPT FIGURE LEGENDS Cancer stem cells (CSC) contributing to drug resistance and tumor recurrence. A. Malignant transformation of somatic cells through genetic mutations of oncogenes. In addition, dysregulation of microenvironmental factors can contribute to the carcinogenesis. B. CSCs are capable of driving tumorigenesis through 3 key steps: (i) their ability for long-term selfrenewal, (ii) their capacity to differentiate into bulk tumor cells without CSC characteristics, and (iii) their unlimited potential for proliferation and tumorgenesis growth, and C. CSCs are highly resistant to chemo and radiation therapy. In addition they might evade immunemediated rejection. These features can drive tumor progression, tumor recurrence, and metastasis. (Adapted with permission from Ref [10]The J Clin Invest, 120 (1) 2010, 41-50). Copyright permission obtained

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Figure 1.

Tumor microenvironment associated with multidrug resistance. Hypoxic regions (A) formed in the tumor microenvironment due to aberrant vasculature (B and C), genetic mutations in regulatory genes and altered regulation of apoptotic factors (D) can lead to cellular plasticity and aggressive MDR characteristics such as increased expression of growth factors receptors (E), over-expression of drug efflux transporters (F), reversion to anaerobic metabolism (G), decreased pH (H), and increased interstitial fluid pressure (H). (Adapted with permission from Elsevier Publication /Cancer Treat Rev. 34(7) 2008, 592–602). Copyright Permission obtained

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Figure 3. Passive and active tumor drug targeting. The schematic shows the passive and active targeting mechanisms of multifunctional nanoparticles and the difference in the vasculature of normal and tumor tissues; drugs and small molecules diffuse freely in and out of the normal and tumor blood vessels due to their small size and thus the effective drug concentration in the tumor drops rapidly with time. However, polymeric drugs, nanoparticles, liposomes and micelles can passively target tumors due to the leaky vasculature or the EPR effect. Targeting molecules such as antibodies or peptides conjugated on the surface of multifunctional nanoparticles can selectively bind to cell surface receptors/antigens overexpressed by tumor cells and can be taken up by receptor-mediated endocytosis (active targeting). The image guiding molecules and contrast agents conjugated/encapsulated in the nanoparticles can be useful for targeted imaging and (non-invasive) visualization of nanoparticle accumulation/localization, as well as for mechanistic understanding of events and efficacy of drug treatment simultaneously. Copyright Permission not required

Figure 4. Schematic illustration of nanoparticulate systems used for combinatorial drug delivery to treat multi-drug resistance in cancer; a) liposome, b) polymeric micelles, c) polymer drug conjugate, d) dendrimer, e) nanoemulsion, f) mesoporous silica nanoparticles, and g) iron oxide nanoparticle (Adapted with permission from Ref [95], Elseiver Publication B.V.). Copyright Permission obtained

Figure 5.

Schematic illustration of strategies directed at cancer stem cells (CSC) targeting that include self-renewal pathway antagonists and chemo resistance-reverting agents. Copyright Permission not required Figure 6.

(A) Cellular uptake of PPC-Hyd-DOX-DA NPs (red) at pH 6.8 or 7.4 after incubation with MDA-MB-231 cells for 1 h. DAPI (4’,6-diamidino-2-phenylindole, blue) and Alexa Fluor488 phalloidin (green) were used to stain cell nuclei and F-actin, respectively. (B) Subcellular 57

ACCEPTED MANUSCRIPT distribution of PPC-Hyd-DOX-DA NPs (red) at pH 6.8. DAPI (blue) and Lysotracker Green (green) were used to stain the cell nuclei and acidic organelles. Cells were imaged using a 60 X water-immersion objective. (The figure has been reprinted from reference [159], American Chemical Society) Copyright Permission obtained a) In vivo whole body optical imaging of liposome accumulation in s.c. human BLM melanoma tumors in mice, images at 0 and 4 h post-injection with and without local HT at 41 °C for 1 h. b) Liposome accumulation in human BLM tumors described as tumor-tobackground ratios (TBR) over time with and without local HT (mean±SD) (n=3 for HT group, n=2 for NT group (Adapted with permission from Ref [209], Elseiver Publication B.V.). Copyright Permission obtained

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Figure 7.

Fluorescence micrographs of (A) PC3 and (B) CWR22R cells after 8 h of treatment with (a) SPAnH/MNPs, (b) 2 mM, (c) 5 mM, and (d) 10 mM bound-PTX. Cells were incubated with (e) SPAnH/MNPs and (f) 10 mM bound-PTX in the presence of a magnetic field (800 Gauss) applied to the plates (red ring) (Adapted with permission from Ref [234], Elseiver Publication B.V.). Copyright Permission obtained

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Figure 8.

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