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OncoDrugs 2013, Vol.

1 (1):3e-9e

Nanostrategies in the war against MDR in leukemia

Nanostrategies in the war against multidrug resistance in leukemia

Review Article AUTHORS: Alphy Rose-James1, TT Sreelekha1, Suraj K. George2*
AFFILIATIONS: 1Laboratory of Biopharmaceuticals, Division of Cancer Research, Regional Cancer Centre, Thiruvananthapuram-695 011, Kerala, India. 2Dept. of Hematopathology, Division of Pathology and Laboratory Medicine, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, Texas 77030, USA

* CORRESPONDING AUTHOR: Dr. Suraj Konnath George PhD. Dept. of Hematopathology. Division of Pathology and Laboratory Medicine. University of Texas MD Anderson Cancer Center. 1515 Holcombe Blvd, Houston, TX 77030, USA. Ph: 001-7137927692 Fax: 001-7137927273. Email: or

Abstract Chemotherapy is the cornerstone of treatment for haematological malignancies. Despite innovative advances in the understanding of this complex disease, multidrug resistance (MDR) is still a major culprit for the treatment failure, especially in leukemia. Conventional combinatorial therapy is a major decoy to circumvent MDR, but those are frequently thwarted by the evolving intricacies of the cancer paradigm. The advent of nanomedicine has ushered a new era with striking pharmacokinetic drug properties far enough to combat MDR. Physicochemical elements such as co-encapsulation, drug influx, ratiometric drug loading, temporal sequencing on drug release, and selective targeting moiety have been better tailored within the nanotherapeutics to overcome MDR. This review focuses on a plethora of nanostrategies and nanoplatforms which have been developed so far to battle MDR in leukemia.

Introduction Although cancer therapeutics have revolutionized over the past decade, there are several challenges including MDR which determines treatment success and quality of life in patients. As for the heterogeneous group of hematological neoplasms including acute and chronic leukemia, chemotherapy remains the most viable treatment mode. Leukemia accounts for the largest no of cases of adolescent and childhood cancer and are the main cause of cancer related deaths in adolescents. Antileukemic therapy, especially in acute myeloid leukemia (AML) is severely hindered by MDR, conferred by molecular pumps such as multidrug resistance-1 (MDR-1) permeability-glycoprotein and multidrug resistance-associated protein (MRP) and several other associated transport proteins which adds to the increased leukemia related death toll. These pumps act either alone or in synergy to expel toxic chemotherapy drugs from the tumors, making them resistant to drugs affecting remission, overall sur-

vival and clinical outcome in patients. Resistance to cancer chemotherapy may be either innate (intrinsic), as a result of inherent genotypic characteristics or acquired (extrinsic), which develops on course of chemotherapeutic exposure (Kerbel et al. 1994; Gottesman, 2002). MDR whether extrinsic or intrinsic, may be ascribed to various factors (Gao et al. 2012) which are summarized in Figure 1. Several other determinants such as errant drug activation and interaction, enhanced drug degradation, deregulated drug distribution etc. also provides impetus to developing chemoresistance (Luqmani, 2005). In contrast, drug resistance could be utilized in a good sense to protect bone marrow of patients from high-dose chemotherapy (Luqmani, 2005). Combinatorial chemotherapeutics delivering optimal and synergistic effects have proved successful in combating MDR to an extent, but have been impacted with severe complications (DeVita Jr et al. 1975). Hence, a more improved and targeted delivery of chemotherapy drugs is mandated for overcoming MDR,

non-specificity and dose-dependent side effects of chemotherapy. MDR in leukemia like any other cancer is a multifactorial effect governed by one or more MDR mechanisms and for the same reason, targeting a single mechanism might not be worth-

while. This fact emphasizes the need for novel approaches that introduce multifunctional nanoplatforms competently circumventing the MDR phenomena.







Figure 1: Schematic of the various extrinsic or intrinsic factors involved in MDR. Chemotherapeutic drugs may be ineffective and develop resistance because of impairment in any of the drug-effector mechanisms like detoxification, efflux, uptake, drug-target modulation, activation, metabolism etc. Components acting on apoptotic machinery and DNA repair which interferes with cell death also contribute to drug resistance

Therapeutic nanoparticles have emerged as tumor-targeted drug delivery systems enhancing potency of chemotherapeutic drugs without causing life-threatening side effects. By virtue of their unique physicochemical characteristics, nanotherapeutics offer myriads of escape mechanisms for circumventing MDR based on drug efflux pump activity on the tumor cell surface, such as that exerted by MDR-1 (Ayers and Nasti, 2012). The overall merit of deploying nanoparticles for cancer treatment is the drastic reduction in the half inhibitory concentration for most of the chemotherapy agents; accomplished by the marked intracellular accumulation and pharmacodynamics of drugs. Nanoconjugates optimize therapeutic doses of the conventional cytotoxic agents, reversing MDR while providing ample treatment response in patients undergoing chemotherapy. Various nanoconstructs have been developed which has revolutionized cancer-targeted therapy reversing MDR (Gao et al. 2012). Some of the areas in which these nanoconstructs have been potentially useful are depicted in Figure 2. There are several nanoplatforms constructed either naturally or synthetically based on the chemical nature of drugs and pharmacological drug loading and

unloading capabilities (Shapira et al. 2011). Solid lipid based nanoparticles are used for pH dependent drug release exploiting the acidic pH of MDR cells, while polymeric nanoparticles possess the unique property of versatile acid responsive drug release kinetics (Li et al. 2012; Kang et al. 2010). Hydrogels are easily synthesized to enable stable platforms for protein conjugation and complement targeted delivery of drugs to resistant cells, such as conjugating with anti-Pgp antibody (Stastny et al. 2002). Magnetic nanoparticles enhance the passive mechanisms of extravasation and internalization in the tumor microenvironment which can be exploited as a theranostic mediator (Klostergaard and Seeney, 2012). Micelle based nanoparticles are capable of solubilizing various insoluble drugs whereas gold nanoparticles have size or shape dependent optoelectronic characteristics (Gupta et al. 2006; Mukherjee et al. 2007). Quantum dots or semiconductor nanocrystals retain intrinsic fluorescence properties which can be used to track tumor-targeting in real time (Wu et al. 2003; Michalet et al. 2005). The present review discuss on various nanostrategies that have advanced hitherto to combat MDR in leukemia.









Figure 2: Schematic of various mechanisms by which nanoconstructs overcome MDR. Nanoparticles owing to their small size can specifically enhance optimal and targeted drug delivery. These nanoconstructs can be easily internalized and solubilized along with the drug moiety at the tumor sites. Various aspects such as delayed clearance, protection of labile drugs, stimulus-dependent release also aid in combating MDR. Photolabile nanoconstructs are utilized for theranostic purposes such as photothermal therapy.

Nanoparticles in combination therapy MDR can be surpassed by therapeutic synergism attained by combining two or more drugs administered as combination therapy. However, conventional combination strategies are strangled by varying pharmacokinetic profiles of different drugs, which may result in varying rates of drug uptake and inadequate dosages at the target site. Nanoparticles carrying multiple drugs can overcome these inadequacies, ensuring spatial release of ratiometric drug doses aiming at multiple targets including metastasized sites in cancer (Lee and Nan, 2012). There are several nanotherapeutic approaches utilized, but majority included liposomes for the MDR reversal in leukemia. Following are a few successful nanoplatforms employed in combination therapy. Liposomal drug delivery system is an eloquent multifunctional design that overcomes the drug efflux mechanism of MDR tumors (Immordino et al. 2006). Co-encapsulated liposomal nanocarriers are engulfed by non-specific endocytosis which enables crossing the cellular membrane in an invisible form, preventing the drugs from being expelled by efflux pumps. Due to these specifics, the stealth liposomes have been widely studied for its potential in reversing MDR (Allen et al. 1992; Wang et al. 2005). Liposomes are one of the first nanoparticle platforms to have entered the clinical trials (Zhang et al. 2008). Liposome based combination therapy with chemosensitizers against MDR protein has been studied by several authors. In vitro cytotoxicity studies with liposomes coencapsulated with an anticancer drug, doxorubicin and MDR modulator, verapamil demonstrate reduced cardiotoxicity and reversal of MDR in doxorubicin resistant prostate cancer cell lines (Wang et al. 2005). Liposomal conjugates with cytarabrine and daunorubicin (CPX-351) have been used in clinical trials for AML due to its beneficial specificity and stability of cytotoxic agents (Feldman et al. 2012). Liposomal encapsulation of

doxorubicin, verapamil and transferrin receptor increased cytotoxicity in doxorubicin resistant K562 cells, which presents a promising strategy for decreasing side effects of verapamil and reversing MDR (Wu et al. 2007). Liposomal conjugates with daunorubicin and 6-mercaptopurine induced synergistic and dose dependent cytotoxicity in highly resistant leukemic cell lines, Jurkat and Hut78 than their corresponding single drug liposomes (Agrawal et al. 2005). Kobayashi et al introduced a novel approach by developing anti-MDR1 hammerhead ribozymes delivered using cationic liposomes or retroviral vectors to reverse MDR in P-gp overproducing leukemic cells (Kobayashi et al. 2001). A triple targeted strategy using transferrin receptor targeted liposomes co-encapsulating anti-Bcr/Abl siRNA and imatinib, specifically reversed MDR in Philadelphiapositive chronic myeloid leukemia patients. This conjugation enhanced the potency of imatinib mesylate by four-fold, while the presensitized cells yielded eight-fold increase (Mendonca et al. 2010). A stealth liposomal conjugate of vincristine and quinacrine was found to combat MDR successfully in mice bearing the MDR K562 human chronic myelogenous leukemia (Liang et al. 2008). A similar stealth liposomal formulation of topotecan and amlodipine reversed MDR in HL-60 cells (Li et al. 2006). All these evidences indicate their characteristic ability to conjugate with hydrophilic and hydrophobic drugs, superior biodistribution and bioabsorption properties, low toxicity and sitespecific targeted delivery, which has considerably improved antitumor drug utility. Biocompatible and biodegradable polymeric nanoparticles like PLGA, PCL, PEG etc. have higher stability, tunable physiochemical properties and controllable drug profiles. They are self-assembled nanoparticles consisting of amphiphilic diblock copolymers and hence, drug encapsulation may be achieved by mixing the drug in the nanoparticle preparation mixture. Multiple drugs may be directly co-encapsulated into the hydrophobic polymeric core or by incorporating an addi-

tional media compartment into the nanoparticle for drug delivery or by covalent conjugation. All these approaches aid in temporal control of drug release associated with improved therapeutic efficacy, offering future promises for MDR reversal. PCL based nanoparticles with cyclosporine A and doxorubicin exhibited significant cytotoxic effects in the doxorubicin-resistant p388 leukemic cell line (Soma et al. 2000). This may be attributed to the synergistic effect achieved by combining the chemosensitizing compound cyclosporine A with an effective cytotoxic drug like doxorubicin. Although curcumin is a widely known anticancer agent, its ability to inhibit MDR was recently discovered. Nanoparticles of curcumin with doxorubicin enhanced systemic bioavailability, and the derived nanocurcumin doxorubicin conjugate (NDC) inhibited MDR causing significant cytotoxicity in various tumor models including acute leukemia and multiple myeloma. In addition to reducing cardiotoxicity of doxorubicin, NDC enhanced safety parameters while also overcoming MDR (Pramanik et al. 2012). Yet another group of nanocarriers of biomolecules are carbon nanotubes (Kam and Dai, 2005; Zhao et al. 2008). These functionalized or surface modified carbon nanotubes are cell compatible, non-toxic and are effectively endocytosed by living cells (Kam et al. 2006). There are also oxidized carbon nanotubes which are easily taken up by the much resistant leukemic K562 cells (Li et al. 2009a). Conjugating doxorubicin with anti-Pgp antibody and carbon nanotubes can effectively deliver desired therapeutic effect within the cells leading to the reversal of MDR in multidrug resistant leukemic cells (Li et al. 2009b). Pluronics are synthetic amphiphilic block copolymers, a novel class of polymeric inhibitors which sensitizes MDR tumors to apoptosis by inhibiting P-gp drug efflux systems and abolishing sequestration of chemotherapeutic drugs in acidic vesicles (Alakhov et al. 1996; Batrakova et al. 2003). Conjugating pluronics with doxorubicin significantly improves the therapeutic delivery of drug-micellar nanosystems leading to the reversal of MDR in the tumors (Valle et al. 2011). In a separate study, pluronic P85 inhibited doxorubicin-induced MDR in P388 murine lymphocytic leukemic cells in vitro and in vivo (Sharma et al. 2008). Dendrimers are highly branched polymeric globular macromolecules with an initiator core and functional end groups at the periphery. Due to their extensive branching nature, chemotherapeutic drugs could be loaded on the periphery of the polymeric dendrimer (Svenson and Tomalia, 2005). Although they are not exploited clinically, dendrimers have been proved to increase bioavailability of multiple drugs at optimal doses, which in turn can overcome MDR in a wide sense. 5-poly (propyleneimine) dendrimer with co-encapsulated methotrexate and all-trans retinoic acid can minimize premature drug leakage during circulation while conferring increased stability and pH dependent drug release (Tekade et al. 2009). The nanoconstruct could efficiently increase the cytotoxicity in resistant leukemic cell types. Niosomes or non-ionic surfactant-based multilamellar vesicles are self-assembly of hydrated surfactant monomers which serve as drug carriers where insolubility, instability and rapid degradation are problematic. The advantage of using niosomes as an alternative to liposomes in drug delivery is to enhance the metabolic stability and long-term tissue-targeted distribution. This will prevent toxicity to the extra-tumorous neighbouring microenvironment (Kerr et al. 1988). PEGylation and surface modification such as hydroxycamptothecin loaded pegylated

niosomes are utilized for a more tumor-targeted drug delivery (Hong et al. 2009).

Nanoparticles for increased therapeutic index MDR often results in suboptimal levels of drugs in cells due to increased drug efflux. Modifying drugs so as to increase the internalization and retention time might reverse MDR. Nanoparticles based drug delivery systems have been utilized to aid enhanced absorption of drugs in selected tissues by enhancing permeability and retention. They also can control the pharmacokinetic profiles of the drugs, drug tissue distribution profile and improve intracellular penetration. Polyelectrolyte microcapsules encapsulating imatinib have been reported to increase antitumor activity and higher drug retention within chronic myeloid leukemic stem cells, preventing dose escalation and early relapse (Palama et al. 2010). Tetraheptyl ammonium capped Fe3O4 magnetic nanoparticles increased the intracellular accumulation of daunorubicin thereby enhancing its cytotoxic effect in multidrug resistant leukemic cells. Magnetic nanoparticles of Fe3O4 containing daunorubicin inhibit the in vivo growth of leukemic cells in mice by effecting increased daunorubicin accumulation, without affecting the Pgp levels (Chen et al. 2008). Bioactive liposome based pH sensitive micelles co-delivering ceramide and doxorubicin can overcome MDR in leukemia by modulating the drug efflux system and inducing apotosis. The micellar system had high drug encapsulation efficiency, good stability and endosomal acid triggered release of doxorubicin (Wang et al. 2013). Lipid based delivery of drugs helps in minimizing off-target effects, increasing resistance to nuclease degradation, and avoiding immune responses such as / interferons, RNA-dependent kinase effects, and toll-like immunity which are major issues in oligofectamine based drug delivery. Yu et al developed a CD20 Ab (rituximab)-conjugated lipopolyplex nanoparticle (RITINP)- and Bcl-2-targeted antisense G3139 which could effectively suppress the immune response by selective B cell-targeted delivery and early endosomal compartmentalization of G3139encapsulated RIT-INPs, resulting in reduced NF-B activation, robust Bcl-2 down-regulation, and enhanced sensitivity to fludarabine-induced cytotoxicity (Yu, et al. 2013). Squalene, a precursor in sterol biosynthesis coupled with nucleoside analogues like gemcitabine is used for the construction of nanoassemblies. These increase the cytotoxicity in gemcitabine resistant leukemic cells both in vitro and in vivo. In addition to enhancing cytotoxicity, squalenoyl gemcitabine has a slow rate of metabolism in plasma which increases the therapeutic efficacy (Couvreur et al. 2006). DPV-576, a mixture of nanoplatinum and nanodiamond, exhibits chemosensitizing effect in daunorubicin resistant HL-60 cells. The formulation could decrease the IC50 value by four fold with increased accumulation of daunorubicin in the cells. It has been observed that DPV-576 could create holes in membranes which might be the possible mechanism by which the construct exhibits its MDR reversal properties (Ghoneum et al, 2013).

Nanoparticles in the theranostic avenue Theranostics refer not only the individualized therapies for diagnosis and treatment, but also a feedback mechanism determining multiple functionalities within a single particle for the

localization, biodistribution and release kinetics of treatment modalities in real time. The advent of nanomedicine in theranostics has drawn the horizons closer with respect to diagnosis and therapy (Liu et al. 2007). Microtubule stabilizing paclitaxel loaded magnetic nanoparticles (pac-MNPs) with lectin glycoprotein as theranostic mediator was studied to determine cytotoxic effect on Bcr-Abl positive K562 cells. The nanoconstructs could lower the IC50 values of resistant K562 cells with a higher cellular uptake as functionalized by C type lectins. Study results showed the activation of JNK pathway leading to Bcr-Abl instability and the induction of extrinsic apoptotic pathway (Singh et al. 2011). In a different study, magnetoplasmonic nanoparticles conjugated with polyethylene glycol and curcumin was used to characterize theranostic imaging and therapeutic modalities in HL-60 cells. High contrast magnetic resonance images and apoptotic rates were obtained which proved the versatility of this nanoassembly in the treatment of leukemia (Chen et al. 2010).

entity and extensively studied for its pharmacokinetic and pharmacodynamic characteristics in the tumor microenvironment rather than generalizing their behavior based on their physico-chemical aspects. Nanoparticle research has been spreading at immense pace as they reduce the systemic toxicity and adverse side effects of the parent drugs which are attached to it. Until recently, they were believed to pose no serious threats to the health of the patients, but now it is realized that the carrier systems themselves may impose risks to the patients. The reasoning behind the toxicity of nanoparticles causing reproductive and developmental toxicities is still unclear. But they are suspected to translocate from the respiratory tract to the circulation, pass the blood-brainbarrier, and affect the brain as demonstrated in several animal studies (Elder et al, 2006; Shimizu et al, 2009; Ma et al, 2010). Other in vitro studies with carbon nanotubes have been shown to accumulate reactive oxygen species, lipid peroxidation, oxidative stress, and mitochondrial dysfunction (Shvedova et al, 2003). Fullerenes, a C60 closed cage structure, exhibit ecotoxicity that may have an impact on microbial communities. They were found to induce lipid peroxidation in brain and glutathione depletion in gills of large-mouth bass. Several hypotheses have also explained fullerenes reaching the brain via circulation or axonal translocation exhibiting direct redox activity in the brain tissue, or production of reactive fullerene metabolites via cytochrome P450 (Oberdrster, 2004). Though very little published data emphasize on the toxicity of dendrimers and gold nanoparticles, the so far limited clinical experience makes it impossible to designate them either as safe or toxic. To date, the present literature is unsatisfactory and early for a proper risk assessment regarding nanoparticles in humans. But not far from the reality, at least a comprehensive understanding on the toxic aspects of the nanoparticulate drugs is ethically mandated before these are tested in patients. Conclusions Overcoming MDR to improve treatment efficacy of chemotherapy has been a subject of interest in the recent years. Novel nanotherapeutics aiming to reverse MDR in leukemia has not been successful clinically due to the formidable P-gp/MDR1 expression. Although several investigative studies utilized Nano approaches for overcoming MDR, it is rather surprising that there are no clinical trials on overcoming MDR in leukemia so far, and only a single clinical study where the dosage safety of atomic nano-generators employing actinium-225Ac-HuM195 is being tested in AML patients. In this scenario, the current landscape of the nanomedicine is poised at a critical stage lacking the vigorous commitment to benefit the bedside patients. Nonetheless, with further advances in nanotechnology and thorough understanding of the nanomechanisms relating MDR in cancer cells, let us hope that the future nanoplatforms will prove effective in tumor-targeted therapies for multidrug resistant phenotypes. However, the ultimate goal should be to bring safer and potent nanoweapons that could effectively fetter MDR in patients.

Multimodal strategies involving nanoparticles Thermal, ultrasound, magnetic and photodynamic therapies can also be employed in conjunction with nanoconstructs to augment the efficacy of MDR reversal mechanisms. Multifunctional magnetic Fe3O4 nanoparticles coupled with daunorubicin and 5-bromotetrandrine with hyperthermia treatment efficiently reversed MDR phenotype in leukemia. This is achieved by downregulating P-gp and upregulating core apoptotic proteins (Ren et al. 2012). Pluronic P105 micelles conjugated with doxorubicin and ultrasound treatment increased the efficacy of MDR reversal in the leukemic resistant phenotypes (Batrakova et al. 2010). By combining the highly reactive titanium dioxide nanoparticles with daunorubicin, MDR in leukemic K562 cell line could be reversed. This reversal of MDR is accomplished by the nanocarrier titanium dioxide which increases the intracellular concentration of the target drug, daunorubicin. In addition, UV irradiation could further enhance reversal of MDR of the already photoexcited nanoconjugate titanium dioxide (TiO2) in conjunction with daunorubicin (Song et al. 2006). Thus photodynamic therapy employing TiO2 and UV irradiation with various targeted drugs could be exploited for effectively overcoming multidrug resistance in various subtypes of leukemia and other forms of cancer.

Risk assessment and challenges Nanotechnology is not exclusively a positive concept, and like any other emerging field it is not without risk, and there are no proven toxicity screening methods to evaluate them. Research is still in its infancy to determine how these may impact safety, health and environment of common man. Further, these challenges need to be addressed and viewed in a greater detail. Nanoparticles easily enter the tumor vasculature via the EPR effect owing to their small size, but the same property hinders its entry to the scarcely vascularized regions (Ayers and Nasti, 2012). Hence, a better understanding of various novel concepts like selective pressure in the tumor microenvironment, vasculature leakiness, pH variation in the intracellular and extracellular niche, transport dynamics and active delivery mechanisms of the nanoconstructs would definitely contribute towards improving the application of nanotherapeutics in cancer treatment. In addition, each of these nanomaterials should be considered as a new

Conflict of Interests The authors declare no conflicts of interest.

Received: 08/07/2013 Revised: 12/03/2013 Accepted: 12/9/2013 Published: 12/14/2013

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