Nanodrug delivery in reversing multidrug resistance in cancer cells

Different mechanisms in cancer cells become resistant to one or more chemotherapeutics is known as multidrug resistance (MDR) which hinders chemotherapy efficacy. Potential factors for MDR includes enhanced drug detoxification, decreased drug uptake, increased intracellular nucleophiles levels, enhanced repair of drug induced DNA damage, overexpression of drug transporter such as P-glycoprotein(P-gp), multidrug resistance-associated proteins (MRP1, MRP2), and breast cancer resistance protein (BCRP). Currently nanoassemblies such as polymeric/solid lipid/inorganic/metal nanoparticles, quantum dots, dendrimers, liposomes, micelles has emerged as an innovative, effective, and promising platforms for treatment of drug resistant cancer cells. Nanocarriers have potential to improve drug therapeutic index, ability for multifunctionality, divert ABC-transporter mediated drug efflux mechanism and selective targeting to tumor cells, cancer stem cells, tumor initiating cells, or cancer microenvironment. Selective nanocarrier targeting to tumor overcomes dose-limiting side effects, lack of selectivity, tissue toxicity, limited drug access to tumor tissues, high drug doses, and emergence of multiple drug resistance with conventional or combination chemotherapy. Current review highlights various nanodrug delivery systems to overcome mechanism of MDR by neutralizing, evading, or exploiting the drug efflux pumps and those independent of drug efflux pump mechanism by silencing Bcl-2 and HIF1α gene expressions by siRNA and miRNA, modulating ceramide levels and targeting NF-κB. “Theragnostics” combining a cytotoxic agent, targeting moiety, chemosensitizing agent, and diagnostic imaging aid are highlighted as effective and innovative systems for tumor localization and overcoming MDR. Physical approaches such as combination of drug with thermal/ultrasound/photodynamic therapies to overcome MDR are focused. The review focuses on newer drug delivery systems developed to overcome MDR in cancer cell.


INTRODUCTION
Cancer is a heterogeneous disease and use of multiple drugs simultaneously can result in drug resistance which is either intrinsic or acquired known as multidrug resistance (MDR). MDR renders cancer cells immune to standard treatments with many anticancer agents and is a major challenge in cancer therapy as it needs to address multiple phenotypes including MDR phenotypes. Tumor heterogeneity and tumor cell resistance to anticancer drugs thus remains key formidable challenges for effective targeting of drug delivery systems for successful chemotherapy. Drug resistance toward antineoplastic agents is a result of reduction in the effective concentration of drug in the cell prior to its interaction with the target or due to a combination of processes. The numerous mechanism of drug resistance reported includes (a) over expression of drug efflux pumps such as permeability glycoprotein (P-gp), multidrug resistance associated protein (MRP), and breast cancer resistance protein (BCRP) (b) alterations in lipid metabolism (ceramide pathway) (c) drug elimination by detoxification systems (d) drug test sequestration inside lysosomes and endosomes (e) reduced drug uptake due to altered surface receptors/carriers (f) inactivation of drugs via glutathionemediated reduction (g) over expression of target enzymes such as up-regulated thymidylate synthase (h) altered drug targets such as topoisomerase II (i) increased DNA repair capacity (j) reduced ability to undergo apoptosis (k) hypoxia up-regulated expression of MDR-linked genes such as ABC transporters, Bcl-2 family genes, glutathione, metallothionein, etc. through activation of transcription factor HIF1 (l) chromosomal abnormalities in cancer cells lead to over-expression of anti-apoptotic genes (m) altered signal transduction pathways in cancer cells governed via integrin receptors, growth factor receptors etc. leads to blockage of apoptosis and expression of MDR-linked genes those involved in DNA repair and drug-efflux pumps (Broxterman et al., 2003).
Drug resistance mechanism of antineoplastic agents ( Table 1) and mechanism of MDR in tumor cells is shown in Figure 1.

MULTIDRUG EFFLUX PUMPS
Drug efflux pumps expressed on human cancer cells majorly contribute to MDR (Sharom, 1997). These efflux pumps belong to ATP-binding cassette (ABC) family and include (a) P-gp also known as multidrug resistance protein 1 (MDR1) or cluster of differentiation 243 (CD243) a ATP-binding cassette sub-family B member 1 encoded in human by ABCB1 gene (b) Multidrug Resistance Associated Protein 1 (MRP1) a ATP-binding cassette sub-family C member 1 encoded in human by ABCC1 gene, Multidrug Resistance Associated Protein 2 (MRP2) also called as canalicular multispecific organic anion transporter 1 (cMOAT) a ATP-binding cassette sub-family C member 2 encoded in human by ABCC2 gene (c) BCRP also known as cluster of differentiation (CDw338) a member of white sub-family and ATP-binding cassette G member 2 encoded in human by ABCG2 gene (Ozben, 2006).

P-GLYCOPROTEIN (P-gp)
P-gp is the first member of ABC super family and is an ATP-powered drug efflux pump membrane transporter (Fardel et al., 1996;Sharom, 1997). Over-expression of P-gp in mammalian and human cancer cells results in MDR. P-gp has two isoforms expressed in human, class I and III isoforms are drug transporters (MDR1/ABCB1) while class II isoforms export phosphatidylcholine into bile (MDR2/3/ABCB4) (Sharom, 1997). P-gp encoded by MDR1 gene is present in human tissues including liver, kidney, pancreas, small and large intestine while P-gp encoded by MDR2 gene is present at high levels only in liver (Fardel et al., 1996). Carcinoma of colon, kidney, adrenal gland, pancreas, and liver express high P-gp levels while intermediate P-gp levels are expressed in neuroblastomas, soft tissue carcinomas, hematological malignancies including CD34-positive acute myeloid leukemias, etc. with low P-gp levels expressed in malignancies of lung, esophagus, stomach, ovary, breast, melanomas, lymphomas, multiple myelomas, and acute promyelocytic leukemia but may display elevated P-gp levels after chemotherapy due to acquired drug resistance (Velingkar and Dandekar, 2010). P-gp interacts with structurally diverse substrates such as anticancer drugs, HIV protease inhibitors, analgesics, calcium channel blockers, immunosuppressive agents, cardiac glycosides, antihelminthics, antibiotics, H 2 -receptor antagonists, steroids, fluorescent dyes, linear and cyclic peptides, ionophores, peptides, lipids, small cytokines such as interleukin-2, intereukin-4, and interferon-γ, MDR chemosensitizers, and many more (Velingkar and Dandekar, 2010).

FIRST GENERATION INHIBITORS
These are non-selective, less potent with poor, and low binding affinity; requiring high doses to achieve plasma levels to reverse MDR, resulting in unacceptable patient toxicity. They are substrates for P-gp and act as competitive inhibitors thereby requiring high serum concentrations of chemosensitizers to produce adequate intracellular concentrations of cytotoxic drug due to which these inhibitors are unsuccessful in clinical trials (Dantzig et al., 2003). First generation inhibitors include Verapamil, Trifluoperazine, Cyclosporine-A, Quinidine and Reserpine, Vincristine, Yohimbine, Tamoxifen, and Toremifene. Due to unpredictable pharmacokinetic interactions of these substrates in presence of chemotherapy agents several novel chemosensitizers analogs were developed with less toxicity and greater potency.

SECOND GENERATION INHIBITORS
Structural modifications of first generation inhibitors resulted in more potent second generation P-gp modulators with better pharmacological profile, reduced toxicity, and better tolerability. They significantly inhibit metabolism and excretion of cytotoxic agents leading to unacceptable toxicity necessitating chemotherapy dose reductions. Successful treatment of refractory cancers and reversal of MDR in clinical trials have been possible by co-administration of these modulators with chemotherapy agents. Modulators include Dexverapamil, Dexniguldipine, Valspodar (PSC 833), and Biricodar citrate (VX-710).

THIRD GENERATION INHIBITORS
They have high potency and specificity for P-gp transporters over second generation agents. They do not interfere with cytochrome P450 3A4 unaffecting drug pharmacokinetics with no dose alterations in chemotherapy. They include Tariquidar-XR9576, Zosuquidar-LY335979, Laniquidar-R101933, ONT-093 (substituted diarylimidazole), Elacridar-GF120918, OC 144-093, Mitotane (NSC-38721), Annamycin, and R101933 (Ozben, 2006). Most promising Tariquidar (non-transported P-gp inhibitor) which inhibits ATPase by interaction with protein is currently in phase III trials for non-small cell lung cancer but still suspended due to unfavorable toxicity. Clinical trial studies revealed that Tariquidar, LY335979, R101933, and ONT-093 can be administered with therapeutic doses and minimal interference with pharmacokinetics of cytotoxic agents. They have shown promise in clinical trials and continued development of these agents may establish the true therapeutic potential of P-gp mediated MDR reversal. Tariquidar a third generation inhibitor with no limitations of first and second generation inhibitors, have highest specificity which specifically and potently inhibits P-gp. Inhibition of ATPase activity of P-gp suggests that the modulating effect is derived from inhibition of substrate binding, inhibition of ATP hydrolysis and or both (Fox and Bates, 2007). Clinical trials of third-generation inhibitors (Thomas and Coley, 2003) showed better tolerability of Tariquidar with no significant pharmacokinetic interaction with chemotherapy. This makes Tariquidar an ideal agent for demonstrating P-gp inhibition activity in cancer. Targeted delivery of paclitaxel and tariquidar co-encapsulated in biotin functionalized PLGA nanoparticles revealed significantly higher cytotoxicity in vitro and greater tumor growth inhibition in vivo in drug-resistant tumor mouse model compared to paclitaxel nanoparticles alone with promising results in clinical trials (Patil et al., 2009b).

TUMOR MICROENVIRONMENT AND MDR
Tumors are core-shell structures with hypoxic core surrounded by tissues and proliferative cells. Tumor microenvironment is made of complex tissues containing extracellular matrix, activated fibroblasts, immune cells, pericytes, adipocytes, epithelial cells, glial cells, vascular and lymphatic endothelial cells, and numerous proteins (van Kempen et al., 2003;Weber and Kuo, 2012). The proliferative cells are highly vascularized, unorganized and discontinuous resulting in enhanced permeability and retention (EPR) effect widely exploited for passive targeting. The major factors contributing to tumor progression and metastasis, enhanced drug resistance, poor prognosis, and response to therapies includes cell mobility, survival potential, capacity to degrade extracellular tissue matrix, and ability to adjust in new tissue environment (Otranto et al., 2012;Singh and Kaur, in press). All solid tumor microenvironment possess the following characteristics (Milane et al., 2011) (Table 2) (a) leaky and unorganized tumor vasculature (b) hypoxia region (c) up-regulation of oncogenes (d) DNA repair mechanisms (e) down regulation of tumor suppressors and cell cycle regulation (f) increased growth factor receptors (g) low nutrients. Tumor microenvironment significantly contributes to drug resistance by reducing drug accessibility to tumor cells and reduces the oxygen radicals generated by antitumor drugs (Otranto et al., 2012;Singh and Kaur, in press). Hypoxia and acidity with low nutrient levels remains the two key factors characterizing tumor microenvironment (Schornack and Gillies, 2003;Wouters et al., 2003). Tumor hypoxia is low oxygen regions with partial oxygen pressure (pO 2 ) levels below 10 mm-Hg where normal tissues range from 24 to 66 mm-Hg (Rofstad, 2000). Hypoxia microenvironment is characterized by low pH (acidic cell environment) and can be associated with activation of proteases that contributes to metastasis, low glucose levels, high interstitial fluid pressure due to leaky vasculature, impaired lymphatic drainage, and high levels of P-gp (Tomida and Tsuruo, 2002). Hypoxia Inducible Factor (HIF) (Harris, 2002) is another mechanism that induces MDR and metastasis by up-regulating target genes by binding to hypoxia-response element (HRE) in the target. HIF-1 is a transcription factor activated in hypoxia. While tumor acidic pH results in poor tumor perfusion due to abnormal vascularization, hypoxia, and metabolic abnormalities are associated with cell growth and increased capacity for transmembrane pH regulation (Simon et al., 1994). Both pO 2 and pH are important determinants of tumor growth, metabolism, and response to variety of therapies (Fukumura and Jain, 2007). Acidic extracellular pH restricts uptake of weak base drugs such as Adriamycin, Doxorubicin, and Mitoxantrone. Both hypoxia and acidic pH contributes to growth and tumor metastasis (Harris, 2002). Hypoxia upregulates various angiogenic growth factors including Vascular Endothelial Growth Factor (VEGF), Angiopoietin (Ang) 2, Platelet Derived Growth Factor (PDGF), Placenta Growth Factor (PGF), Transforming Growth Factor α (TGFα), Interleukin (IL)-8, and Hepatocyte Growth Factor (HGF) of which Hypoxia Inducible Factor 1α (HIF1α) is considered the master regulator of oxygen homeostasis.

MODIFICATION OF CHEMOTHERAPY REGIMENS
Chemotherapy regimen includes "induction regimen" and "maintenance regimen" refers to initial disease treatment and ongoing chemotherapy to reduce chances of cancer recurrence or prevent growth of an existing cancer, respectively. Combination chemotherapy utilizes synergistic effect of multiple antineoplastic drugs acting through different mechanisms, but due to their different dose-limiting adverse effects they are given together in chemotherapy regimens. Chemotherapy regimen needs to balance efficacy and toxicity through proper dosing schedule. Dose-dense regimens have more toxic effects than standard regimen causing treatment delays and toxicity with few survival improvements and early treatment discontinuation. A dosedense approach is more effective than standard approach, as it hampers formation of blood vessels that feed tumors and tumor shrinkage following treatment promoting tumor dormancy by maintaining tumor size and preventing outgrowth. Chemotherapy regimens are identified by acronyms, identifying the drug combination agents. E.g., (i) Breast cancer: AC (Adriamycin, Cyclophosphamide), CAF (Cyclophosphamide, Adriamycin, Flurouracil), EC (Epirubicin, Cyclophosphamide), FEC (Flurouracil, Epirubicin, Cyclophosphamide); (ii) Colorectal cancer: FL (Fluorouracil, Leucovorin), FOLFOX (Fluorouracil, Leucovorin, Oxaliplatin), FOLFIRI (Fluorouracil, Leucovorin, Irinotecan). Chemotherapy regimen is based on the assumption that the mutations conferring drug resistance will not convey resistance to all the agents in the regimen and high-dose chemotherapy regimens could be given to cancer patients. Such approach assumes that despite resistance to standard doses of anticancer drugs, a dose-response relationship exists for tumors and high doses of chemotherapy might overcome the resistance.

INACTIVATION OF MDR-ASSOCIATED GENES BY TARGETING SPECIFIC mRNA FOR DEGRADATION
Strategies to overcome multi drug resistance by silencing the expression of gene encoding P-gp efflux transporter, i.e., MDR-1 or Survivin through RNA interference (RNAi) or small interfering RNA (siRNA) has been explored. Transient RNAi mediated silencing can be achieved by siRNA or stable RNAi-mediated gene silencing through short hairpin RNA (shRNA) transfection. The siRNAs assembles into endoribonuclease inside the cells containing complexes known as RNA-Induced Silencing Complexes (RISCs) which guides the RISCs to complementary RNA molecules, cleaving and destroying the target RNA. Antisense oligonucleotides and catalytic RNAs have been successful in inhibiting P-gp, MRP, and BCRP expression and sensitized drug-resistant cells (Nadali et al., 2007;Ren et al., 2008). In vitro and in vivo studies with biotin-functionalized nanoparticles coencapsulating paclitaxel and P-gp targeted siRNA partially overcame tumor drug resistance (Patil et al., 2010). Two groups, Nieth et al. (2003) (Milane et al., 2011).

MONOCLONAL ANTIBODIES FOR P-gp
Monoclonal antibodies (MAbs) have potential for targeting P-gp and kill MDR tumor cells. Anti-P-gp MAbs such as MRK-16 and MRK-17 along with chemosensitizers reverses P-gp mediated MDR and conjugated MAbs such as bispecific antibody, immunotoxin and radioisotope conjugates enhance anti-tumor activity. Combination of MRK-16 with Cyclosporin-A or PSC-833 reversed Doxorubicin resistance in K562/ADM cells and inhibited tumor growth in athymic mice bearing HCT-15/ADM2-2 xenografts. MRK-16 increased Cyclosporine-A accumulation in MDR cells but not affected intracellular PSC-833 accumulation in MDR cells, instead Cyclosporin-A and PSC-833 increased MRK-16 binding to P-gp revealing a synergistic MDR reversal activity. MAbs with other anti-P-gp MAbs such as UIC2, 4E3, and series of HYB antibodies have potential to inhibit drug transport (Tomida and Tsuruo, 2002).

DEVELOPMENT OF NEW ANTICANCER DRUGS THAT ARE NOT SUBSTRATES OF P-gp
Drug analogs such as Taxane analogs DJ-927 (Phase I), BMS-184476 (Phase I), RPR 109881A (Phase II), Ortataxel (Phase II), Trabectedin-ET-743 (Phase II and III) are not recognized by P-gp transporter and are evaluated in clinical trials for their broad spectrum activity in sensitive and resistant tumor cell lines to overcome MDR (Dong and Mumper, 2010). DJ-927 was more potent and cytotoxic than paclitaxel and docetaxel when compared in vitro and in vivo in various P-gp expressing tumor cell lines with high intracellular accumulation in P-gp positive cells. The expression of P-gp levels or P-gp modulators did not affect the tumoricidal efficacy of DJ-927. Phase I study of DJ-927 in combination with capecitabine was acceptable with no pharmacokinetic drug interactions in patients with advanced solid tumor malignancies and is recommended for further clinical studies. Preclinical studies showed that BMS-184476 was more potent than paclitaxel against taxane sensitive and resistant tumors. The P-gp over-expressing human colon cancer cell line (HCT-116/MDR) was 62-fold more resistant to paclitaxel and 15-fold resistant to BMS-184476. Also the human ovarian cancer cells with acquired taxane resistance expressed 9-fold resistance to BMS-184467 and 32-fold to paclitaxel. Studies of BMS-184476 against human tumor xenografts with both acquired and primary taxane resistance models revealed superiority of BMS-184476 (Yared and Tkaczuk, 2012).

INHIBITORS OF ABC TRANSPORTERS TO REVERSE MDR
Inhibition of ABC transporters should reverse MDR by increasing intracellular drug concentrations in tumor cells and restore drug sensitivity. These inhibitors transport themselves and then act as competitive antagonists while others are not transported but affect transporter function (Dong and Mumper, 2010). Preclinical trials of first and second generation ABC transport inhibitors were not successful. They failed in clinical trials due to their non-specificity, high concentrations to inhibit activity, undesirable drug interactions due to co-administration of inhibitors and anticancer drugs (e.g., verapamil and doxorubicin), substrates of cytochrome P-450 and increased toxicity of anticancer drugs. Clinical trials of third generation inhibitors with LY335979 (Zosuquidar), GF120918 (Elacridar), R101933 and XR9576 (Tariquidar) are ongoing. Tariquidar in phase I studies revealed high potency in in vitro and in vivo studies. LY335979 prolonged survival by reducing tumor growth in mice with drug resistant tumors, GF120918 enhanced topotecan bioavailability in mice by sensitizing human MDR sarcoma MES-Dx5 cells. Although phase I and II clinical trials of third generation inhibitors are promising but are limited to unpredictable pharmacokinetic drug interactions, simultaneous involvement of several drug transporters and variability in drug transporter expression levels among individuals restricts restoration of drug sensitivity of such modulators in clinic (Wu et al., 2008).

NANOTECHNOLOGY BASED APPROACHES TO OVERCOME MDR
Nanocarriers to overcome MDR are extensively discussed in section "Nanocarriers as potential drug delivery systems in cancer therapy." Nanocarriers have been developed encapsulating anticancer drugs as P-gp substrates and/or with P-gp substrates.

INHIBITION OF MDR USING PEPTIDES
Synthetic P-gp peptides derived from fragments of extracellular loops of murine P-gp coupled with polyethylene glycol and loaded in Doxorubicin liposomes have shown MDR reversal with 83% increase in survival time of mice inoculated with P388R cells. Antitumor effect of peptide-conjugated Doxorubicin in human erythroleukemic (K562/ADR) resistant cells showed dosedependent inhibition of cell growth against K562/ADR cells as compared with Doxorubicin alone (Dong and Mumper, 2010).

NANOCARRIERS AS POTENTIAL DRUG DELIVERY SYSTEMS IN CANCER THERAPY
Nanovehicles such as polymeric nanoparticles, solid lipid nanoparticles, magnetic nanoparticles, dendrimers, liposomes, micelles, quantum dots, etc. are extensively explored for cancer diagnosis, treatment, imaging, and as ideal vectors to overcome drug resistance by diverting ABC-transporter mediated drug efflux mechanisms. The major classes of nanocarriers utilized for chemotherapeutic drug delivery are listed in Table 3 (Ayers and Nasti, 2012).

POLYMERIC NANOPARTICLES
Polymeric nanoparticles have emerged as a versatile nanotechnology platform for controlled, sustained and targeted delivery of anticancer agents including small molecular weight drugs and macromolecules such as genes and proteins Sahay et al., 2010;Tang et al., 2010). A significant reduction in tumor size and increased animal survival rate in rat xenograft glioma model with indomethacin loaded nanocapsules was observed by Bernardi et al. (2009). PLGA loaded cystatin nanoparticles and PLGA loaded cytokeratin specific monoclonal antibody nanoparticles neutralized excessive proteolysis preventing metastatic and invasive potential of breast tumor cells (Kos et al., 2009). Paclitaxel loaded PLA immunonanoparticles covalently coupled with humanized monoclonal antibodies (antiHER2) actively targeted tumor cells over expressing HER2 receptors (Cirstoiu-Hapca et al., 2009). Folic acid receptors over-expressed on human cancer cells (Antony, 1996;Wang et al., 2010) are studied in tumor models including mouse M109 carcinoma, KB human epidermal carcinoma cell line and mouse J6456 lymphoma (Alberto et al., 2004). Paclitaxel loaded PLA-PEG-ligand conjugated nanoparticles functionalized with biotin and folic acid enhanced drug accumulation in MCF-7 tumor xenograft model (Patil et al., 2009b). Lee et al. found that folic acid conjugated chitosan nanoparticles showed higher transfection activity than unmodified chitosan nanoparticles . Wang et al. (2010) observed 35% reduction in tumor growth, inhibition of P-gp and mdr1 gene levels in KB-A-1 cells implanted in Balb/c-nu/nu mice targeted by folic acid conjugated antisense oligodeoxynucleotides-hydroxypropyl-chitosan nanoparticles compared to bare antisense oligodeoxynucleotides to overcome tumor drug resistance. Folate functionalized PLGA nanoparticles loaded with anti-cancer drug nutlin-3a and chemosensitizer Curcumin enhanced therapeutic potential of nutlin-3a by modulating MDR of Y79 retinoblastoma cell through Curcumin and enhanced the anticancer activity of nutlin-3a in drug resistance Y79 cells. Dual drug loaded nanoparticles revealed better therapeutic efficacy with enhanced expression or down regulation of proapoptotic/antiapoptotic proteins and down-regulation of Bcl2 and NF-κB protein. Study demonstrated the role of Curcumin as MDR modulator to enhance the therapeutic potential of nutlin-3a for targeting MDR cancer (Das and Sahoo, 2012). Silencing P-gp expression by RNAi with reduction-sensitive linear cationic click polymer nanoparticles (RCPNs) loaded with plasmid iMDR1-pDNA for gene delivery revealed higher transfection efficiency and lower cytotoxicity than PEI/DNA nanoparticles against human breast cancer MCF-7 cells and drugresistant MCF-7/ADR cells . Vincristine sulfate loaded nanoassemblies enhanced cytotoxicity by 36.5-fold and cellular accumulation by 12.6-fold in MCF-7 and P-gp over expressing MCF-7/ADR cells compared to vincristine sulfate solution and overcome MDR by clathrin and caveolae mediated endocytosis pathways (Zhang et al., 2011b). Co-delivery of Paclitaxel and survivin shRNA nanoparticles lowered IC 50 by 360-fold in Paclitaxel resistant lung cancer cells against A549/T cells compared to free Paclitaxel and enhanced efficacy with Paclitaxel induced apoptosis and cell arrest in G2/M phase. Nanoparticles facilitated drug accumulation in tumor cells and down-regulated of survivin shRNA into nuclei of lung cancer cells lowering the apoptosis threshold of drug resistant cells and renders chemotherapeutic agents more effective to overcome MDR (Shen et al., 2012). Docetaxel loaded poly(ε-caprolactone)/Pluronic F68 nanoparticles increased drug uptake and enhanced cytotoxicity in docetaxel-resistance human breast cancer cell line and MCF-7 TAX30 compared to poly caprolactone nanoparticles indicating its potential to overcome MDR (Mei et al., 2009). Lipid/particle assemblies (LNPs) loaded with Doxorubicin in DMAB-modified PLGA nanoparticles coated with DPPC lipid shell significantly increased accumulation and improved nucleus targeting in MCF-7 cells and P-gp over expressing resistant MCF-7/ADR cells relative to free drug and reversed the transporter-mediated drug resistance in human breast cancer. Cytotoxicity (IC 50 ) of Doxorubicin loaded-LNPs was 30-fold lower than free Doxorubicin in MCF-7/ADR, indicating intracellular retention of Doxorubicin and bypassing drug resistance (Li et al., 2012a). Co-delivery of MDR1 siRNA via lipid-modified dextran-based polymeric nanoparticles with Doxorubicin increased intracellular drug concentration in MDR cell nucleus and efficiently suppressed P-gp expression in drug resistant osteosarcoma cell lines (KHOS R2 and U-2OS R2 ) (Susa et al., 2010). Pramanik et al. developed composite nanoparticles of Doxorubicin with Curcumin a potent MDR inhibitor to overcome Doxorubicin resistance in multiple in-vivo models such as multiple myeloma, acute leukemia, prostate and ovarian cancers. Composite nanoparticles revealed no cardiac toxicity or bone marrow suppression compared to free Doxorubicin (Pramanik et al., 2012). P-glycoprotein mediated efflux can be effectively circumvented by co-administration of P-gp inhibitor/s and anticancer drug/s in nanoparticles which evades P-gp recognition at cell membrane and delivers drug in the cell cytoplasm or nucleus thereby sustaining delivery of the drug inside the cell. Chavanpatil et al. encapsulated paclitaxel a P-gp substrate and verapamil a P-gp inhibitor in PLGA nanoparticles to circumvent P-gp-mediated drug efflux in MDR tumor cells (Chavanpatil et al., 2006). Doxorubicin loaded aerosol OT (AOT)-alginate nanoparticles enhanced the cellular delivery and therapeutic efficacy of P-gp substrates in P-gp over expressing cells (Chavanpatil et al., 2007). Novel polymer-lipid hybrid nanoparticle loaded with doxorubicin and chemosensitizer (GG918) evaluated in human MDR breast cancer cell line (MDA435/LCC6/MDR1) demonstrated nuclear drug localization and anticancer activity toward MDR cells, while co-administration of the single-agents loaded nanoparticles resulted in high cellular internalization but were ineffective (Wong et al., 2006). Encapsulation of paclitaxel with P-gp modulator tariquidar in poly (D, L-lactide-co-glycolide) nanoparticles functionalized with biotin revealed higher in-vitro cytotoxicity and increased intracellular accumulation compared to paclitaxel nanoparticles alone in drug-resistant tumor cells to overcome tumor drug resistance through biotin receptormediated endocytosis (Patil et al., 2009a).

SOLID LIPID NANOPARTICLES (SLNS)
In-vitro cytotoxicity in resistant P388/ADR cell line and in-vivo studies in P388/ADR leukemia mouse model revealed lowering of IC 50 value by 9-fold and greater median survival time about 20 days (3.5 mg/kg dose) with Doxorubicin SLN compared to Doxorubicin solution. While comparable cell uptake and IC 50 values were obtained with both Idarubicin SLN and free Idarubicin in P-gp over expressing P388/ADR and HCT-15 cells mouse tumor models. Study revealed the potential of Doxorubicin SLN in overcoming P-gp-mediated MDR both in-vitro in P388/ADR leukemia cells and in-vivo in murine leukemia mouse model (Ma et al., 2009). Greater accumulation of Doxorubicin SLN in MCF-7/ADR cells over expressing P-gp with enhanced apoptotic cell death and decreased cell viability compared to plain Doxorubicin revealed the potential of Doxorubicin SLNs to overcome chemoresistance in adriamycin-resistant breast cancer cell line. Decrease in the intensity of 116-kDa PARP band (DNA repair enzyme activated by DNA damage and used as apoptosis biochemical marker) in MCF-7/ADR cells treated with 3 μM either of Doxorubicin or SLN alone or Doxorubicin SLN indicated efficiency of Doxorubicin SLN to cause cell death through induction of apoptosis in Doxorubicin resistant cancer cells. Cellular uptake of Doxorubicin SLN was 17.1-fold and 21.6-fold higher than Doxorubicin alone implying potential of SLNs in diminishing P-gp mediated drug efflux (Kang et al., 2010). SLNs being easily internalized enhanced cellular uptake and cytotoxicity of Doxorubicin and Paclitaxel loaded solid lipid nanospheres in human promyelocytic leukemia cells (HL60) and human breast carcinoma cells (MCF-7) compared to free drug solutions. Paclitaxel solid lipid nanospheres were 100-fold more effective than free Paclitaxel in MCF-7 cells with low sensitivity on HL60 cells. Doxorubicin SLN enhanced cytotoxicity and sensitivity on MCF-7 cells (10-fold) and on HL60 cells (>40-fold) with IC 50 at 1 ng/ml compared to Doxorubicin solution reducing drug cell resistance. Such increased cytotoxicity of Doxorubicin nanocarriers compared to solution has been earlier reported with polymeric nanoparticles, micelles and liposomes. Enhanced intracellular accumulation and cytotoxicity of Doxorubicin loaded pluronic copolymer micelles have been reported by Kabanov and coworkers. Couvreur reported that Doxorubicin loaded polyalkylcyanoacrylate nanoparticle were more cytotoxic than Doxorubicin solution against P388 leukemia cells overcomed MDR and decreased cell viability against resistant MCF-7 cell-lines (Couvreur and Vauthier, 1991). Paclitaxel SLN enhanced cytotoxicity (100-fold) at concentration >5 ng/ml on HL60 cells and at 1 ng/ml on MCF-7 cells (Miglietta et al., 2000). Polymer-lipid hybrid nanoparticle with Doxorubicin and chemosensitizer (GG918) or their combination revealed high Doxorubicin uptake in human MDR breast cancer cell line (MDA435/LCC6/MDR1) compared to co-administration of two single-agent/s loaded hybrid nanoparticles (Wong et al., 2006). Tween®80 coated Edelfosine lipid nanoparticles revealed antiproliferative effect due to P-gp inhibitory action on C6 glioma cell lines and significantly reduced the tumor growth within 14 days post treatment in nude mice bearing C6 glioma xenograft tumor (Mendoza et al., 2011). Paclitaxel and Doxorubicin SLN exhibited higher cytotoxicity in human breast tumor drug sensitive MCF-7 and drug resistant MCF-7/ADR cells compared to Taxol and Doxorubicin solution. Paclitaxel and Doxorubicin loaded SLN revealed 31.0-and 4.3-fold reversal in drug resistance of MCF-7 cells compared to MCF-7/ADR cells respectively . Doxorubicin-mitomycin co-loaded stealth polymer-lipid hybrid nanoparticles enhanced efficacy in sensitive and MDR human mammary tumor xenografts with 3-fold increase in life span, 10-20% tumor cure rate, inhibition of tumor angiogenesis with no severe tissue toxicity compared to liposomal Doxorubicin (Prasad et al., 2013). P-glycoprotein efflux at the brain limits entry of Docetaxel for cancer treatment. Folic acid modified solid lipid nanoparticles loaded with docetaxel and ketoconazole (P-gp inhibitor) evaluated in brain endothelial cell lines for cytotoxicity and cell uptake revealed a brain permeation coefficient 44 times higher than that of Taxotere® (Venishetty et al., 2013). Docetaxel loaded hepatoma-targeted SLNs revealed high cellular uptake by hepatoma cells, better biodistribution and enhanced antitumor efficacy due to increased drug accumulation and cytotoxicity in murine model bearing hepatoma and hepatocellular carcinoma cell line BEL7402 compared to Taxotere® or non-targeted SLNs for treatment of advanced and metastatic hepatocellular carcinoma (Xu et al., 2009). A lipophilic paclitaxel derivative (2 -behenoyl-paclitaxel) (C22-PX) conjugated in lipid nanoparticle for metastatic breast cancer improved antitumor efficacy, tumor retention, better tolerability, and higher plasma levels compared to Taxol in a subcutaneous 4T1 mouse mammary carcinoma model (Ma et al., 2013).

LIPOSOMES
Liposomal anthracyclines approved by US FDA for treatment of AIDS-related Kaposi's sarcoma are pegylated liposomal doxorubicin (Doxil®/Caelyx®) and liposomal daunorubicin (DaunoXome®) which preferentially accumulates in tumor tissues via EPR effect to overcome drug resistance or accumulates within extracellular space of tumor stroma and leaks into tumor environment which provides pharmacologic advantage for liposomes over free drug to overcome drug resistance (Table 4).
Currently liposomes of Paclitaxel, Camptothecins and Vincristine are in clinical development. Liposomal strategies to enhance drug bioavailability and efficacy in drug-resistant cancer include (i) liposomes modified for controlled release (ii) ligand targeted liposomes such as immunoliposomes for intracellular drug delivery in tumor cells.
Liposomes directly interact with P-gp and inhibit P-gp through endocytosis. Liposome co-encapsulating Doxorubicin and Verapamil conjugated with human transferrin (Tf) showed greater cytotoxicity, selective targeting and reversal of P-gp mediated drug resistance in resistant leukemia K562 cells than nontargeted co-loaded liposomes. Doxorubicin liposomes increased cytotoxicity on HL60 cells and Vincristine resistant HL60 cells due to rapid internalization and drug release inside the cells (Gokhale et al., 1996). Robert Lee et al. found that uptake of folate-PEG-liposomal Doxorubicin by KB cells was 45-fold higher than non-targeted liposomal Doxorubicin (Lee and Low, 1995). Liposomes overcome drug resistance due to endothelial P-gp efflux mechanism at blood-brain and blood-tumor barriers in brain tumors where the barriers allows extravasation of long circulating liposomes and circumvent drug resistance with stabilized liposomal Doxorubicin in rat intracranial sarcoma model (Siegal et al., 1995) and rat intracranial 9L gliosarcoma model (Zhou et al., 2002).

MODIFIED LIPOSOMES TO OVERCOME DRUG RESISTANCE
New liposomal systems developed for treatment of drug-resistant cancers are listed in Tables 5, 6.

MICELLES
Micelles are efficient drug carriers with potential P-gp inhibitory action, altered drug internalization, subcellular localization and selective targeting. Seven anti-tumor drugs loaded polymeric micelles in clinical trials are Genexol®-PM, NK105, NC-6004, NC-4016, NK012, NK911 and SP1049C (Gong et al., 2012). Micelles overcome drug resistance by combination of mechanisms including EPR effect, active internalization, endosomaltriggered release and drug escape. Folate decorated pH-sensitive Doxorubicin micelles showed high drug concentration in cytosol and nucleus due to triggered release in early endosomes (∼pH 6) and high cytotoxicity in Doxorubicin resistant MCF-7 (MCF-7/DOXR) cells due to internalization via folatereceptor mediated endocytosis to overcome P-gp (Lee et al., 2005). Folate functionalized micelles co-encapsulating Paclitaxel and Verapamil in O-carboxymethylated chitosan modified with deoxycholic acid revealed greater cytotoxicity and higher cellular uptake in drug resistance MCF-7 and multi-drug-resistant MCF-7/ADR cells through synergistic effect of folate receptormediated endocytosis and Verapamil mediated efflux mechanism to overcome drug resistance in tumor cells . Wei et al. revealed that pluronics lowered the IC 50 in human lung adenocarcinoma cell lines SPC-A1 (8.7 ± 0.4 ng/ml) and A-549 (0.10 ± 0.04 μg/ml) with Paclitaxel-Pluronic P123/F127 Liposomes delivers hydrophobic drugs that are not substrates for P-gp or not effluxed by P-gp Gene therapy approaches Non-viral delivery of nucleic acid to tumor cells circumvents drug resistance, non-viral delivery of resistance genes to normal tissues gives chemoprotection   (Lu et al., 2011). Cambón et al., synthesized reverse poly(butylene oxide)-poly(ethylene oxide)-poly(butylene oxide) block copolymers with potential P-gp inhibitory action in MDR cell line. Doxorubicin loaded in these polymeric micelles enhanced cell accumulation and cytotoxicity in MDR ovarian NCIADR-RES cell line over expressing P-gp (Cambón et al., 2013). Methotrexate conjugated mixed micelles of pluronic F127 and P105 suppressed tumor growth in KBv MDR cells compared to physically entraped mixed micelles due to combined effect of tumor chemosensitization by pluronic and passive targeting by micelles . Vincristine sulfate nanocarriers improved cellular uptake, cytotoxicity in MCF-7 and P-gp over expressing MCF-7/Adr resistant cancer cells by bypass P-gp due to endocytosis mediated by clathrin and caveolae pathways (Zhang et al., 2011b). Chemosensitizing ability of pluronics suppressed Doxorubicin induced MDR in murine lymphocytic leukemia cells (P388) and in BDF1 mice bearing (P388) ascite cancer cells with Doxorubicin-Pluronic P85 micelles (Sharma et al., 2008). Pluronics modulate MDR by intracellular ATP depletion, decreased mitochondrial potential and passive targeting. IC 50 values of Paclitaxel-Pluronic P123/F127 mixed micelles revealed anti-proliferation activity against lung resistance protein over expressing human lung adenocarcinoma A-549 cells with 3-fold longer mean residence time and 31.8% reduction in tumor volume compared to Taxol after 28 days . Polyethylene glycol-polycaprolactone or Pluronic P105 micelle down-regulated the mitochondrial membrane potential and reduced ATP level to improve cytotoxicity (4 times), intracellular accumulation and overcome Doxorubicin resistance in human myelogenous leukemia (K562/ADR) cells compared to Doxorubicin solution at 12 ng/mL (Han et al., 2011).

MESOPOROUS SILICA NANOPARTICLES (MSNPS)
Mesoporous silica nanoparticles (Figure 2) have high drug loading due to high pore volume and surface area, multifunctionalization for targeted and controlled delivery, enhanced cellular uptake and delivers therapeutics at cellular levels in cancer (Mai and Meng, 2013;Mamaeva et al., 2013). Doxorubicin MSNPs surface conjugated with TAT peptide facilitated intranuclear drug localization in multidrug resistant MCF-7/ADR cancer cells and overcome MDR compared to free Doxorubicin or non-TAT peptide conjugated nanoparticles (Pan et al., 2013). Doxorubicin MSNPs lowered the IC 50 value 8-fold compared to free Doxorubicin and overcome MDR in Doxorubicin resistant and P-gp over expressing cancer cell line MCF-7/ADR by increased cell proliferation suppression effect . Chemotherapy efficacy was enhanced bypassing the efflux pump resistance in multidrug-resistance cancer cells by co-delivery of Doxorubicin and siRNAs in MSNPs (Chen et al., 2009a). Rapid internalization of siRNA loaded magnetic MSNPs coated with polyethylenimine and surface modified with fusogenic peptide (KALA) in the tumor cells resulted in knockdown of enhanced green fluorescent protein (EGFP) and VEGF and inhibited tumor growth by suppression of tumor neovascularization (Li et al., 2013b). Doxorubicin-CTAB micelles co-loaded pH responsive MSNPs overcome multi-drug resistance in both drug-resistant MCF-7/ADR cells and drugsensitive MCF-7 cells due to chemosensitization potential of CTAB arresting the cell cycle and inducing apoptosis . Manganese oxide-based MSNPs loaded with Doxorubicin multifunctionalized as theranostics circumvented MDR, restored drugs anti-proliferative effect by endocytosis, P-gp inhibition and ATP depletion in cancer cells . Anticancer drug loaded magnetic MSNPs were internalized by A549 cells through an energy-dependent clathrin induced endocytosis pathway and inhibited cancer cell growth under magnetic field Sekhon, 2012). Exposure of Doxorubicin loaded zinc doped iron oxide nanocrystals in mesoporous silica framework surface-modified with pseudorotaxanes to AC field caused death of (MDA-MB-231) breast cancer cells (Thomas et al., 2010). Hyperthermia stimulated the intracellular GSH level in A549 human lung cancer cells and enhanced anti-cancer efficacy of Doxorubicin MSNPs by inducing cell death and apoptosis . Lejiao Jia et al. developed Paclitaxel MSNPs and revealed that anti-tumor activity of Paclitaxel in breast cancer cells (MCF-7) was dependent on pore-size and apoptosis increased with increased nanoparticle pore size (Jia et al., 2013). Galactose functionalized Camptothecin MSNPs with photosensitizer (porphyrin) enhanced anti-cancer activity in human cell lines of colorectal (HCT-116), pancreatic (Capan-1) and breast cancer (MDA-MB-231) (Gary-Bobo et al., 2012).

OTHER INORGANIC NANOPARTICLES
Inorganic nanoparticles for cancer therapy include quantum dots, carbon nanotubes, silica nanoparticles, gold nanoparticles, iron oxide magnetic nanoparticles and ceramic nanoparticles. Doxorubicin covalently bounded to polyethylenimine via pH sensitive hydrazone linkage and conjugated to iron oxide nanoparticles functionalized with polyethylene glycol circumvented MDR and reduced cell viability in DOX-resistant cells over-expressed in rat glioma C6 cells compared to free drug  (Zhao et al., 2013). Lectin functionalized Paclitaxel magnetic nanoparticles lowered the IC 50 with higher cellular uptake and cytotoxic effect on Bcr-Abl positive K562 cells in chronic myelogenous leukemia (Singh et al., 2011). Cisplatin magnetic nanoparticles enhanced inhibition of A549 cells and cisplatin-resistant A549 cells in MDR lung cancer cells, lowered the levels of MRP1, lung resistance-related protein, Akt and Bad pathways and increased the levels of caspase-3 genes and proteins (Li et al., 2013a). Single drug Tetrandrine loaded magnetic nanoparticles revealed a 100-fold lowering in mdrl mRNA level but no reduction in total P-gp content while magnetic nanoparticles loaded with Adriamycin and Tetrandrine synergistically reversed multidrug resistant in K562/A02 resistant cell lines (Chen et al., 2008). Heparin coated Doxorubicin superparamagnetic iron oxide nanoparticles promoted apoptosis due to regulation of anti-apoptotic genes including caspase-3, bax, bcl-2 and surviving in human ovarian cancer cell lines A2780 (Javid et al., 2011). Gold nanoparticles (AuNPs) are versatile platform for cancer drug delivery (Kumar and Liang, 2011;Kumar et al., 2013) and have recently entered cancer clinical trials phase I and II (Thakor et al., 2011;Vigderman and Zubarev, 2012). Gu et al. successfully synthesized doxorubicin grafted-PEGylated gold nanoparticles to overcome Doxorubicin resistant in cell lines . Oxaliplatin grafted on PEGylated AuNPs rapidly distributed in the nucleus and enhanced the chemotherapeutic efficacy (Brown et al., 2010). AuNPs surface conjugated with therapeutic peptide (PMI or p12) and targeted peptide (CRGDK) was rapidly internalized for better efficacy in overcoming breast cancer (Kumar et al., 2012). AuNPs covalently grafted with doxorubicin through thioctic acid-PEG linker inhibited growth of drug resistant breast cancer cells due to high drug concentrations inside cancer cells due to acid sensitive release from endosomes (Wang et al., 2011b). Zhang et al. observed similar effects with gold nanoparticle-DNA-paclitaxel conjugate (Zhang et al., 2011d). Gold nanorods functionalized with gastrinreleasing peptide (Bombesin) showed uptake via GRP receptormediated endocytosis with high binding affinity to breast cancer cells (Chanda et al., 2009(Chanda et al., , 2010. Selenium nanoparticles significantly enhanced the expression of pp38, Bax and cytochrome C in estrogen receptor-α positive cells (MCF-7) but not in estrogen receptor-α-negative cells (MDA-MB-231) and prevented mammary tumor growth by inducing cell death (Vekariya et al., 2012).
Quantum dots are semiconductor inorganic fluorescent nanocrystals with small and uniform sizes (1-20 nm), high surface to volume ratio, surface conjugation with multiple ligands and biocompatibility (Zhang et al., 2008a; Geszke-Moritz and Moritz, 2013). Water-soluble cadmium telluride (CdTe) quantum dots capped with negatively charged 3-mercapitalpropionic acid combined with Daunorubicin as a biomarker for simultaneous cellular imaging and inhibition of MDR for treatment of drug-sensitive leukemia K562 and drug-resistant leukemia K562/A02 cell lines was developed by Yanyan Zhou et al. The study revealed significant drug uptake in target cancer cells and cytotoxicity suppression in both cell lines (Zhou et al., 2010). Further Zhang et al. demonstrated rapid uptake and increased apoptosis rate which activated apoptosis-related caspases protein expression in drug-resistant human hepatoma HepG2/ADM cells with Daunorubicin-3-mercaptopropionic acid-capped Cadmium telluride quantum dots . Paclitaxel-loaded PLGA quantum dots were more cytotoxic than free Paclitaxel in paclitaxel-resistant KB paclitaxel-50 cells than paclitaxelsensitive KB, however treatment with Verapamil reversed the MDR activity and reduced viability of KB paclitaxel-50 cell (Kuo et al., 2009). Doxorubicin conjugated via pH-sensitive hydrazone bond and aptamer to quantum dots when targeted to mutated MUC1 mucin over expressed in ovarian carcinoma revealed higher cytotoxicity than free drug with preferential accumulation in ovarian tumor and drug release in acidic environment of cancer cells (Savla et al., 2011).

DENDRIMERS
Novel delivery systems comprising of Doxorubicin, dendrimer and vector protein rAFP3D to bind alpha-fetoprotein receptors on tumor cell surface accumulated in the cells by receptor mediated endocytosis and demonstrated high cytotoxicity against human ovarian adenocarcinoma cell lines -Doxorubicin-sensitive SKOV3 cells and Doxorubicin-resistant SKVLB cells revealed low toxicity against human peripheral blood lymphocytes reversing the MDR in Doxorubicin-resistant cells (Yabbarov et al., 2013). The cancer-targeting potential of folate/dextran/galactose ligands anchored on poly(propylene imine) dendrimers evaluated on HeLa and SiHa cell lines indicated an IC 50 values of 0.05, 0.2, 0.8 and 0.08 μM for folate, dextran, galactose formulations and free paclitaxel respectively on HeLa cells while the IC 50 values of 0.6, 0.8, 10 and 6 μM with folate, dextran and galactose formulations and free PTX respectively with SiHa cells. The study revealed the targeting potential of ligands in the order folate > dextran > galactose (Kesharwani et al., 2011). Dendrimer phthalocyanineencapsulated polymeric micelle combined with doxorubicin and mediated by photochemical internalization showed doxorubicin release from endo-lysosomes to nuclei after photoirradiation and nuclear accumulation of doxorubicin, higher antitumor activity than DPc/m-PDT alone in drug-resistant MCF-7 cells and xenograft model (Lu et al., 2011). Biotin, a cell growth promoter is required for rapid proliferation of cancer cells and is overexpressed on cancer cell surface than normal tissue. Bifunctional dendrimer conjugated with biotin a targeting moiety and fluorescein isothiocyanate an imaging moiety exhibited higher cellular uptake by an energy-dependent process in HeLa cells than conjugate without biotin. Conjugation of targeting moieties such as sugar, folic acid, antibody, peptide and epidermal growth factor to dendrimers leads to preferential accumulation of drug in the targeted tissue or cells. Similar biotin-conjugate carriers have been reported to increase uptake of anti-cancer drugs in tumor cells (Yang et al., 2009a). Cytotoxicity of dendrimerschlorambucil conjugate and inhibition of [3H] thymidine incorporated in DNA on both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that the conjugate had more potent antiproliferative activity and actively inhibited collagen biosynthesis than chlorambucil (Bielawski et al., 2011). Similar cytotoxicity effects have been reported by Khandare et al. for conjugation of paclitaxel to linear PEG polymers and PAMAM dendrimers. PAMAM dendrimer-paclitaxel conjugate showed significantly higher toxicity while linear PEG-paclitaxel conjugate showed more than 25-fold lower toxicity compared to free drug with increased IC 50 dose (Khandare et al., 2006). Surface modified G3 PAMAM dendrimers with permeation enhancing lauryl chains conjugated with Paclitaxel via glutaric anhydride linker revealed the potential to cross cellular barriers in cell monolayers indicated by increased apparent permeability coefficient and increased cytotoxicity in both human colon adenocarcinoma cell line (Caco-2) and primary cultured porcine brain endothelial cells (PBECs). The interactions of hydrophobic lauryl moieties of L6-G3glu-pac dendrimer conjugate with plasma membrane revealed 12-fold greater permeability across both cell monolayers than free Paclitaxel (Teow et al., 2013). Dendrimer conjugated with methotrexate a dual-acting molecule showed cytotoxicity due to its potent inhibitory activity against dihydrofolate reductase and binds folic acid receptor, upregulated on cancer cell surface (Li et al., 2012b).

NANOSTRUCTURED LIPID CARRIERS (NLCs)
Mitoxantrone hydrochloride nanostructured lipid-dextran sulfate hybrid carriers enhanced cytotoxicity and invaded cells by clathrin-mediated endocytosis with high drug accumulation in BCRP overexpressing MCF-7/MX cells and overcome MDR compared to solution . Oral bioavailability of Etoposide was enhanced 1.8-, 3.0-, and 3.5-fold in NLCs, PEG40-NLCs and DSPE-NLCs respectively compared to suspension. Etoposide DSPE-NLCs and NLCs revealed highest cytotoxicity, lower cellular viability and strong inhibitory effects against human epithelial-like lung carcinoma cells (A549) than etoposide with IC 50 values of 40.61 ± 6.15 nM, 61.78 ± 7.49 nM, and 210.87 ± 0.76 nM respectively after 24 h (Némati et al., 1996;Zhang et al., 2011c). Oleh Taratula et al. developed dual targeting NLCs loaded with an anticancer drug (Doxorubicin or Paclitaxel) to induce cell death and siRNA to target MRP1 mRNA and BCL2 mRNA to suppress pump and nonpump cellular resistance in lung cancer cells respectively and overcome resistance. Further conjugation of targeting moiety Luteinizing Hormone Releasing Hormone (LHRH peptide) to NLCs enhanced the targeting specificity to cancer cells overexpressing LHRH receptors (Taratula et al., 2013). Folate decorated Paclitaxel and Doxorubicin loaded NLCs designed by Xing-Guo Zhang et al. exhibited high cytotoxicity against human breast cancer (MCF-7) cells and multi-drug resistant (MCF-7/ADR) cells with Paclitaxel NLCs and in MCF-7/ADR cells with Doxorubicin NLCs with MDR reversal potential of 34.3-fold for Paclitaxel NLCs and 6.4-fold for Doxorubicin NLCs. Similar cytotoxicity trend was observed against human ovarian cancer (SKOV3) cells and multidrug resistant (SKOV3 TR ) cells with reversal power of 31.3 and 2.2-fold for Paclitaxel NLCs and Doxorubicin NLCs respectively compared to Taxol and Doxorubicin solution (Zhang et al., 2008b). Potential of active targeting the low density lipoprotein (LDL) receptors over expressed on cancer cells was utilized by Jaber Emami et al. and developed Paclitaxel loaded cholesterol NLCs which were taken by human colorectal cancer cell line (HT-29) through LDL receptor endocytic pathway and revealed IC 50 values of 5.24 ± 0.96 ng/mL compared to 8.32 ± 1.35 ng/mL of free Paclitaxel solubilized in Cremophor-EL after 72 h exposure (Emami et al., 2012). Folate decorated Paclitaxel and Doxorubicin NLCs exhibited high cytotoxicity in MCF-7 and MCF-7/ADR cells while Doxorubicin NLCs revealed high cytotoxicity only in MCF-7/ADR cells compared to Taxol and Doxorubicin solution, while Paclitaxel and Doxorubicin NLCs revealed same cytotoxicity trends against human ovarian cancer cells (SKOV3) and their multidrug resistant (SKOV3 TR ) cells. The reversal power of Paclitaxel and Doxorubicin NLCs were 34.3-and 6.4-folds, respectively (Zhang et al., 2008b).
Nanovehicles enhance chemotherapeutics solubility, bioavailability, therapeutic index and overcome dose-limiting toxicity, non-specific biodistribution, non-targeting and emerging drug resistance in cancer therapy. Multifunctional nanocarriers along with distinct size and surface characteristics are able to target tumor cells through active and passive targeting approaches. Nanocarrier's ability to down regulate ABC transporters or carry gene expression modulator/inhibitor enhance drugs intracellular tumor concentrations improving the chemotherapeutic efficacy. Thus nanotechnology is a novel approach for specific delivery of chemotherapeutics with potential to overcome complexity of MDR in tumors treatments.

SILENCING OF DRUG RESISTANCE GENES
RNAi technology has been explored as a therapeutic strategy to overcome MDR by silencing drug efflux transporter genes such as P-gp/MDR1 and MRP1. RNAi mediated silencing through siRNA, through transfection with shRNA (Saad et al., 2008;MacDiarmid et al., 2009;Chen et al., 2010;Patil et al., 2010) and decreased MDR1 expression with antisense oligodeoxynucleotides  are strategies to overcome P-gp associated MDR using RNAi. Targeting transferrin receptors with PEG coated siRNA nanoparticles silenced target gene M2 ribonucleotide reductase in refractory metastatic melanoma (Davis et al., 2010). Cationic and anionic liposome polycation-DNA nanoparticles loaded with C-Myc siRNA and Doxorubicin suppressed MDR1 gene expression via silencing the transcription level by targeting transcription factors, intercalation of Doxorubicin, topoisomerase II inhibition, transcription inhibition of resistant tumors and tumor regression . Sigma receptors overexpressed on non-small cell lung cancer, breast tumor and prostate cancer targeted with anisamide decorated nanoparticles reduced tumor growth of C-Myc siRNA, down-regulated MDR1 expression and increased Doxorubicin accumulation in xenograft model of NCI/ADR-RES (OVCAR-8 derived) tumor (Banerjee et al., 2004). Bacterially derived minicells encapsulating siRNA targeting MDR1 gene transcripts with cytotoxic drugs downregulated P-gp and increased survival of mice bearing human tumor xenografts (MacDiarmid et al., 2009).

INHIBITION OF DRUG RESISTANCE PROTEINS
To overcome MDR, colloidal carries inhibiting drug resistance proteins P-gp includes polymeric nanoparticles (Khdair et al., 2009;Kuo et al., 2009;Patil et al., 2009a;Song et al., 2009), quantum dots (Kuo et al., 2009), liposomes , nanoemulsions (Ganta and Amiji, 2009) etc. which contains combination of P-gp inhibitors with anticancer drugs such as Paclitaxel, Vincristine, or Doxorubicin. Biotin or folic acid functionalized PLGA nanoparticles encapsulating Tariquidar and Paclitaxel resulted in higher cytotoxicity and inhibited tumor growth in human MDR tumor xenografts compared to Paclitaxel nanoparticles alone (Robey et al., 2008;Patil et al., 2009b). Paclitaxel loaded theragnostic PLGA nanoparticles conjugated to quantum dots were more effective than free Paclitaxel in Paclitaxel-sensitive nasopharyngeal KB carcinoma cells and Paclitaxel-resistant KB PTX-50 while cytotoxicity enhanced in presence of Paclitaxel-loaded nanoparticles with Verapamil (Kuo et al., 2009). Transferrin coated liposomes co-encapsulating Doxorubicin and Verapamil exhibited 5 and 3-fold cytotoxicity in Doxorubicin-resistant human erythroleukemia K562 cells compared to non-targeted liposomes and transferrin targeted liposomes with Doxorubicin alone respectively .

SILENCING OF Bcl-2 AND HIF1α GENE EXPRESSION
Nanotechnology approaches suppressing drug resistance mechanisms independent of drug efflux pumps are silencing of B-cell lymphoma 2 (Bcl-2) (Indran et al., 2011) (Figure 3) and hypoxiainducible factor alpha (HIF1-α) genes. Bcl-2 family proteins are regulators of apoptosis and HIF1-α gene encodes a transcription factor in cellular response to hypoxia (Rapisarda and Melillo, 2009). Two isoforms of Bcl-2, Isoform 1 (1G5M) and Isoform 2 (1G5O/1GJH) exhibit similar fold antiapoptotic activity, however their ability to bind the BAD and BAK proteins suggest differences in antiapoptotic activity of the isoforms. Bcl-2 gene damage is a major cause of cancer and resistance to cancer treatments because over-expression of anti-apoptotic genes and under-expression of pro-apoptotic genes results in lack of cell death. Hypoxia regions present in solid tumors are indicators of malignant progression, metastatic development and chemoresistance. The degree of intra-tumoral hypoxia depends on expression of HIF-1 which is composed of 2 sub-units HIF-1α and HIF-1β and is major factor for cell survival in hypoxic environment (O'Donnell et al., 2006). Matrine (active component of Sophora flavescence dry roots) in human gastric cancer MKN45 tumor cells activates caspase-3, 7 and up-regulates pro-apoptotic molecules Bok, Bak, Bax, Puma, Bim and induces apoptosis via Bcl-2 (Noguchi et al., 2003; FIGURE 3 | Mitochondrial pathway.

Frontiers in Pharmacology | Pharmacology of Anti-Cancer Drugs
July 2014 | Volume 5 | Article 159 | 12 Luo et al., 2007). Cationic cholesterol derivative with hydroxyethylamino head group, cholesteryl-3bcarboxyamidoethylene-N-hydroxyethylamine (I) on liposome significantly promoted gene transfection, Bcl-2 antisense phosphorothioate oligonucleotides complexed with cationic liposomes suppressed human cancer cell growth and induced apoptosis in human cervix epithelial carcinoma cell lines HeLa and mouse fibroblast NIH3T3 cells (Okayama et al., 1997). Positively charged chitosan coated PLGA nanoparticles with siRNA increased transfection and blocked the expression of anti-apoptotic Bcl-2 gene with significant cellular uptake and tumor regression (Jagani et al., 2013). Dong-feng Yu et al. developed cationic liposomes to downregulate the expression of Bcl-2 gene with siRNA transfection with enhanced apoptosis and sensitivity of 5-Fluorouracil in gastric adenocarcinoma SGC-7901 cell . Suppression of Bcl-xL gene through co-delivery of Doxorubicin and small hairpin RNA (shRNA) in polyplexes conjugated with an anti-PSMA aptamer specifically binds the prostate-specific membrane antigen expressed on prostate cancer cell surface. Aptamer polyplexes revealed excellent tumoricidal efficacy and significantly lowered the IC 50 values by 17-fold compared to mixture of shRNA and Doxorubin (Kim et al., 2010a). Gene silencing capability of siRNA loaded magnetic MSNPs coated with polyethyleneimine effectively knock downed both exogenous enhanced green fluorescent protein (EGFP) gene and endogenous Bcl-2 gene with negligible cytotoxicity and released siRNA in cancer cells . Glycoprotein transferrin (Tf) is a ligand for transferrin receptors (TfR) overexpressed on cancer cells and internalized by receptor-mediated endocytosis. Novel transferrin receptor-targeted liposomes delivered phosphorothioate antisense oligodeoxyribonucleotide (ODN-G3139) in TfR positive K562 leukemia cells and downregulated Bcl-2 protein in K562 cells 2-fold greater than non-targeted liposomes and 10-fold greater than free G3139. Tf-conjugated liposomes with G3139 reduced Bcl-2 transcription by >80%, lowered IC 50 from 1.8 to 0.18 μM and sensitized K562 cells to Daunorubicin (Chiu et al., 2006).

MODULATION OF CERAMIDE LEVELS
Ceramide lipids are endogenous lipids and potent mediators of cellular responses in cancer including apoptosis, cell growth suppressor, differentiation, cell migration and adhesion. Ceramides are located in cell membranes and mitochondrial outer membrane, releasing pro-apoptotic factors Cytochrome c by forming permeable channels. Few sphingolipids are vital signal transducer and cell regulator in growth suppression and apoptosis. Extracellular agent such as tumor necrosis factor α activates sphingomyelinase and cleaves membrane sphingomyelin to generate cellular ceramide. Ceramide are converted to sphingolipids in presence of P-gp and accelerates cancer cell death by coadministration of P-gp antagonists with short-chain ceramides (C 6 -ceramide) (Hannun and Obeid, 1995;Pettus et al., 2002;Boddapati et al., 2008). Exposure to chemotherapy and/or anticancer drugs increase intracellular ceramide levels in cancer cells and is involved in membrane clustering of the death receptor. Most anticancer chemotherapeutics stimulate ceramide accumulation through increased ceramide synthesis or inhibition of ceramide catabolism. Neutralization of ceramide via glycosylation or phosphorylation in malignant cells is linked to MDR. New therapeutic strategies to overcome resistance focus on increasing endogenous ceramide levels by stimulating ceramide synthesis, inhibiting ceramide neutralization, or direct delivery of exogenous ceramide (Barth et al., 2011). Cytotoxicity of C 6 -ceramide nanoliposomes with P-gp antagonist (Tamoxifen, Cyclosporine-A, VX-710 (Biricodar), Verapamil) in human CRC cell lines (HCT-15, HT-29, LoVo) revealed synergistic effect of caspase dependent apoptosis, poly ADP ribose polymerase(PARP) cleavage, DNA fragmentation, cell cycle arrest, increased mitochondrial membrane permeability and enhanced protein expression of tumor suppressor p53 (Morad et al., 2013). Shabbits and Mayer (2003) revealed that cytotoxicity and cellular uptake of ceramides are dependent on acyl chain length with the most active C 6 -ceramide (IC 50 value = 3-14 μM) and least active C16-ceramide (IC 50 value = 100 μM) in MDA435/LCC6 human breast cancer and J774 mouse macrophage cell lines (Shabbits and Mayer, 2003). Cisplatin-Fe 3 O 4 magnetic nanoparticles reversed resistance of ovarian carcinoma cell line SKOV3/DDP by 2.2fold and down-regulated mRNA levels of Bcl-2 and survivin expression with increased cell apoptosis (Jiang et al., 2009). Similarly Daunorubicin-Fe 3 O 4 magnetic nanoparticles lowered the transcriptions of Mdr-1 and Bcl-2 gene and increased the transcriptions and expressions of Bax and caspase-3 in K562n and K562-n/VCR cells in nude mice to overcome MDR (Chen et al., 2009b). Lonidamine and Paclitaxel dual loaded PLGA/PEG/EGFR-peptide targeted nanoparticles at 1 μM paclitaxel/10 μM lonidamine dose revealed <10% cell viability for all hypoxic cell lines and <5% cell viability for all normoxic cell lines overexpressing EGFR in human breast and ovarian cancer cell lines. EGFR-peptide targeted nanoparticles promoted mitochondrial binding of Bcl-2 proteins (Lonidamine) and hyperstabilizing microtubules (Paclitaxel) to overcome MDR (Milane et al., 2011). siRNA cationic polymeric nanoparticles downregulated Bcl-2 mRNA expression levels (<10%) in HepG2, HeLa and MDA-MB-231 cell lines and sensitized HeLa cells to Paclitaxel (Beh et al., 2009). Transferrin targeted protamine lipid nanoparticles of antisense oligonucleotide (G3139) down-regulated Bcl-2 to overcome resistance in K562, MV4-11 and Raji leukemia cell lines and was more effective than non-targeted lipid nanoparticles and frees G3139 and induced caspase-dependent apoptosis (Yang et al., 2009b). Co-administration of Paclitaxel (20 mg/kg) and C 6 -ceramide (100 mg/kg) in poly(ethylene oxide)-modified poly(epsilon-caprolactone) nanoparticles revealed > 4.3-and 3-fold increase in tumor growth delay and 3.6-and 3-fold increase in tumor volume doubling time in wild-type SKOV-3 and multidrug resistant (MDR-1 positive) SKOV-3 TR models respectively compared to individual agents (Devalapally et al., 2007). Tumor accumulation of Paclitaxel from Paclitaxel-C 6 -ceramide poly (beta-amino ester) nanoparticles was high compared to free drug in sensitive MCF-7 and multidrug resistant MCF-7TR (MDR-1 positive) human breast adenocarcinoma (van Vlerken et al., 2008). C 6 -ceramide nanoliposomes revealed caspase-dependent apoptosis and diminished survivin protein expression in treatment of human and rat natural killer-large granular lymphocytic leukemia cells . C 6 -ceramide loaded temperature-sensitive lineardendritic nanoparticle revealed preferential uptake of fluorescein isothiocyanate-labeled linear-dendritic nanoparticles into human MDA-MB-231 breast adenocarcinoma cells with growth inhibition and solid tumor apoptosis with hyperthermia (Stover et al., 2008). Cytotoxicity of cetyltrimethyl ammonium bromide stabilized SLNs loaded MBO-asGCS oligonucleotide with or without C 6 -ceramide evaluated in NCI/ADR-RES human ovary cancer cells revealed enhanced uptake of MBO-asGCS oligonucleotide with downregulation of GCS reversing resistance of cells to Doxorubicin (Siddiqui et al., 2010). Docetaxel loaded hyaluronic acid-ceramide nanoparticles enhanced intracellular uptake in CD44-overexpressing cell line (MCF-7) and revealed MDR effect in MCF-7/ADR cells (Cho et al., 2011). Doxorubicin loaded polyethylene glycol conjugated hyaluronic acid-ceramide revealed greater uptake in CD44 receptor expressed in SCC7 cell line (Cho et al., 2012). C 6 -ceramide nanoliposomal with Gemcitabine or an inhibitor of glucosylceramide synthase [D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP)] in Gemcitabine resistant human pancreatic cancer cell line revealed cytotoxicity and inhibited tumor growth . Transferrin modified ceramide liposomes initiated lysosomal membrane permeabilization resulting in leakage of hydrolytic enzymes (cathepsins) into cytoplasm, induced cancer cells apoptosis and revealed antitumor and pro-apoptotic effects in A2780-ovarian carcinoma xenograft mouse model compared to ceramide-free and ceramide-loaded non-modified liposomes (Koshkaryev et al., 2012). Co-administration of Tamoxifen and Paclitaxel in poly (ethylene oxide) modified poly (epsilon-caprolactone) polymeric nanoparticles enhanced antitumor efficacy, lowered IC 50 of Paclitaxel by 10-and 3-fold in SKOV3 cells and P-gp over-expressing SKOV3 TR cells respectively (Devalapally et al., 2008). Polymeric nanoparticles co-encapsulating Paclitaxel and C 6 -ceramide enhanced apoptotic signaling and reduced tumor volume 2-fold over standard Paclitaxel monotherapy (van Vlerken et al., 2010). Ceramide-generating properties of 4-HPR (Fenretinide) are being evaluated in phase II study of recurrent ovarian cancer and C 6 -ceramide nanoliposomes are being evaluated as neoplastic-selective agent (Chapman et al., 2010).

TARGETING NF-κB
Transcriptional factor nuclear factor-kappa B (NF-κB) plays vital role in cancer development and resistance. Degradation of inhibitor κB after phosphorylation by inhibitor κB kinases activates NF-κB translocating into nucleus and initiating transcription contributing to tumor development, progression, chemoresistance, inflammation, and autoimmune diseases (Zingarelli et al., 2003;Li and Sethi, 2010). NF-κB is involved in multiple cellular processes including stress, cytokine gene expression, free radicals, cellular adhesion, cell cycle activation, apoptosis, and oncogenesis (Baud and Karin, 2009). NF-κB is activated via two distinct signal transduction pathways in cancer, the canonical and non-canonical pathways. NF-κB regulates expression of key proteins such as Bcl-2, Bcl-XL, cellular inhibitors of apoptosis, survivin, TRAF, Cox-2, MMP9, iNOS, and cell cycle regulatory components. Thus, NF-κB is a potential target for cancer therapeutics since inhibitors of NF-κB mediates antitumor responses and enhances tumor sensitivity to anticancer drugs (Luqman and Pezzuto, 2010). Activation of NF-κB affects cancer cell survival while inhibition of NF-κB enhances sensitivity of cancer cells to antineoplastic agents (Schwartz et al., 1999). NF-κB is important in tumorigenic process due to its strong anti-apoptotic functions in cancer cells (Magné et al., 2006). Polyethylene glycol-5000 coated Curcumin PLGA nanoparticles induced apoptosis of leukemic cells, inhibited TNF-induced NF-κB activation and suppressed NF-κB-regulated proteins involved in cell proliferation (cyclin D1), invasion (MMP-9) and angiogenesis (VEGF) (Nair et al., 2010). Micellar aggregates of cross-linked copolymers N-isopropylacrylamide with N-vinyl-2-pyrrolidone and poly(ethyleneglycol) monoacrylate encapsulating Curcumin induced cellular apoptosis, blocked NF-κB activation and downregulated proinflammatory cytokines (IL-6, IL-8 and TNFα) in human pancreatic cancer cell lines (Bisht et al., 2007). Silica nanoparticles (50-200 μg/mL) generated reactive oxygen species, mitochondrial depolarization and apoptosis in human umbilical vein endothelial cells (HUVECs), activated c-Jun N-terminal kinase (JNK), c-Jun, p53, caspase-3 and NF-κB, increased Bax expression and suppressed Bcl-2 protein while the highest concentration significantly increased the necrotic rate, LDH leakage, expression of CD54 and CD62E and release of TF, IL-6, IL-8 and MCP-1 (Liu and Sun, 2010). Potential of gene therapy for targeting NF-κB has recently been explored as a new strategy in cancer (Tas et al., 2009). Degradation of TSP [Tween 85s-s-polyethyleneimine (TSP)] a non-viral gene vector for p65 (shRNA) from TSP/p65 shRNA nanoparticles with release of shRNA blocked NF-κB signaling pathway, induced cell apoptosis and down-regulated p65 expression in breast cancer cells (Xiao et al., 2013). Inhibition of NF-κB with pyrrolidine dithiocarbamate (PDTC) an antioxidant and heavy metals chelator suppressed release of IκBα from NF-κB and induced cell death in neuroblastoma cells (Schreck et al., 1992). Doxorubicin and NF-κB inhibitor PDTC entrapped folic acid conjugated chitosan nanoparticles enhanced intracellular targeting of tumor cells via folic acid receptor mediated endocytosis and lowered IC 50 values compared to free drug to overcome resistance (Fan et al., 2010).

NANOCARRIERS ADDRESSING EFFLUX PUMP DEPENDENT AND INDEPENDENT DRUG RESISTANCE MECHANISMS
Simultaneously delivery of single/multiple anticancer agents in nanocarriers addressing both efflux pump dependent (P-gp) and independent (NF-κB) drug resistance mechanisms enhances cell apoptosis and induce cancer cell death. Paclitaxel and Curcumin (NF-κB and P-gp inhibitor) co-encapsulated in flaxseed oil nanoemulsion enhanced cancer cell sensitivity to Paclitaxel and cytotoxicity in SKOV3 and drug resistant SKOV3 TR human ovarian adenocarcinoma cells (Ganta and Amiji, 2009). Doxorubicin and siRNA containing cationic liposomes simultaneously silenced MRP1 and Bcl-2 (Saad et al., 2008). Doxorubicin/Mitomycin/5-Fluorouracil loaded hydroxyapatite nanoparticles acted synergistically with recombinant mutant human tumor necrosis factor-α (rmhTNFα) reduced P-gp levels of mRNA, increased intracellular concentration in human hepatoma xenografts of HepG2/ADM cells and suppressed tumor cell growth by apoptosis (Al-Bataineh et al., 2010;Ronaldson et al., 2010).

PHYSICAL APPROACHES TO OVERCOME MDR DRUG DELIVERY WITH THERMAL THERAPY
In hyperthermia therapy, cells undergo heat stress (41-46 • C) resulting in activation and/or initiation of intracellular and extracellular degradation mechanisms like protein denaturation, protein folding, aggregation and DNA cross linking, changing tumor cell physiology and leading to apoptosis or making cancer cells more sensitive to anti-cancer drugs. Hyperthermia increases blood flow to the tumor cells and enhances delivery of nanocarriers and thus used as an adjunct treatment to increase efficacy of chemotherapy and enhance radiation induced tumor damage. Depending on the degree of temperature, hyperthermia is classified (i) in-thermo ablation; tumor subjected to >46 • C (upto 56 • C) causes cells to undergo direct tissue necrosis, coagulation or carbonization (ii) moderate hyperthermia (41-46 • C) affects both cellular and tissue (iii) diathermia (<41 • C) for rheumatic diseases. Cellular effects of moderate hyperthermia include induction and regulation of apoptosis, signal transduction and MDR. Super-paramagnetic iron oxide particles induced therapeutic hyperthermia; liposomal nanocarrier revealed high intra-tumoral accumulation of magnetic particles on application of magnetic field (100-120 kHz) to attain temperatures 40-45 • C. Folate receptor targeted Doxorubicin liposomes with hyperthermia reduced IC 50 in cervical carcinoma cells. Temperature sensitive poly(N-isopropylacrylamide) nanocarriers release anticancer drugs in presence of specific temperature triggers. Hyperthermia enables magnetic nanoparticles to enter tumor cells by generating heat in tissues/cells and is utilized for selective targeting through cancer-specific binding agents and controlled drug delivery over conventional hyperthermia (Chicheł et al., 2007;Kumar and Faruq, 2011).

DRUG DELIVERY WITH ULTRASOUND THERAPY
Ultrasound induces thermal effects and helps nanocarrier's extravasation in tumor, enhance drug diffusion through tumor interstitium, release drug from nanocarriers within tumor and increase intracellular drug accumulation on irradiation to enhance treatment of MDR cancer. Howard et al. demonstrated that sonication enhanced uptake of Paclitaxel 20-fold from micellar system in breast cancer tumor cell line and inhibited 90% cell proliferation. Doxorubicin-pluronic® P105 micelles with ultrasound resulted in high intracellular drug accumulation in promyelocytic leukemia HL-60 cells, ovarian carcinoma drug sensitive and multidrug resistant cells (A2780 and A2780/ADR) and breast cancer (MCF-7) cells (Marin et al., 2002). Paclitaxel micelles of methoxy poly (ethylene glycol)-block-poly (D, Llactide) enhanced intracellular drug accumulation 2-fold and cytotoxicity in drug-sensitive (MDCKII and MCF-7) and Pgp expressing (MDCKII-MDR and NCI-ADR) cell lines with ultrasound (Wan et al., 2012). Pure and mixed micelles of pluronic® P105, PEG2000-diacylphospholipid and poly (ethylene glycol)-co-poly(β-benzyl-L-aspartate) loaded Doxorubicin with ultrasound treatment enhanced intracellular drug accumulation in ovarian carcinoma tumor model in nu/nu mice and inhibited tumor growth rate (Gao et al., 2005). Ultrasound therapy downregulated levels of P-gp, MRP and lung resistance protein to 62.84 ± 3.42%, 10.26 ± 1.18%, and 3.05 ± 0.37% in HepG2/ADM cells from 96.97 ± 2.41%, 20.84 ± 3.12%, and 1.16 ± 0.59% levels, respectively. Ultrasound increased percent Bax in HepG2/ADM cells leading to cellular apoptosis and MDR reversal (Liu et al., 2001;Rapoport, 2004;Howard et al., 2006;Kedar et al., 2010;Milane et al., 2011;Wu et al., 2011;Gao et al., 2012). The studies indicate potential of ultrasound waves to disrupt nanocarrier core, form micropores in cell membrane allowing diffusion of drugs and modulate membrane drug efflux pumps function. However which mechanisms of ultrasound (heat, cavitation or microstreaming) are predominantly involved in modulating drug efflux transporter on cell membrane is still unclear. The studies on these aspects are underway in our lab.

DRUG DELIVERY WITH PHOTODYNAMIC THERAPY
Photodynamic therapy (PDT) has wide applications in cancer therapy due to its specificity and selectivity. PDT involves three components light, oxygen, and a photosensitizer (non-toxic drug) to achieve photocytotoxicity. PDT includes administration of a photosensitizer which specifically accumulates in cancer cells and when illuminated with red visible light (620-690 nm) generates reactive oxygen species in presence of tissue oxygen and causes cell death. Photofrin-2 (hematoporphyrin derivative) is the only PDT drug approved for clinical application in treatment of bladder, lung and esophageal cancer. Folic acid coated phospholipid-capped protoporphyrin IX (PpIX) loaded FITCsensitized mesoporous silica nanocarriers (NanoPDT) effectively targeted receptors overexpressed on HeLa cells with high intracellular PpIX concentrations compared to free PpIX. NanoPDT on irradiation generated oxygen species, enhanced photocytotoxicity and inhibited 65% tumor growth in nude mice inoculated with B16F10 melanoma (Teng et al., 2013). Dendrimer phthalocyanine encapsulated polymeric micelle showed higher PDT efficacy in mice bearing human lung adenocarcinoma A549 cells, enhanced photocytotoxicity upon photoirradiation and accumulated in endolysosomes than Photofrin-2 (Nishiyama et al., 2009). However, polyethylene-glycol(PEG)-grafted transferrinconjugated liposomes of second-generation photosensitizer 5, 10, 15, 20-tetra (m-hydroxyphenyl) chlorin (Foscan) did not improved the photocytotoxicity or intracellular accumulation of Foscan in esophageal cancer cell line when compared to unmodified liposomes (Paszko et al., 2013). Chitosan functionalized pluronic nanogel containing gold nanorod as photothermal therapy agent and chlorine e6 (Ce6) as photosensitizer for PDT enhanced tumor ablation in-vivo by combination of PDT followed by photothermal therapy compared to single therapy (Kim et al., 2013). Co-delivery of Docetaxel and photosensitizer zinc-phthalocyanine (ZnPc) loaded nanoparticles on irradiation decreased viability in HeLa cells after 72 h and enhanced antitumor activity in orthotopic amelanotic melanoma animal model compared to Docetaxel nanoparticles alone (Conte et al., 2013). Hyperbranched poly(ether-ester) (HPEE) loaded photosensitizer chlorin (e6) nanoparticles revealed better up taken by human oral tongue cancer CAL-27 cells after 4 h with cytoplasmic localization and higher phototoxicity compared to free ce6 after irradiation (Li et al., 2012c). Efficiency of second generation photosensitizers [2, 9, 17, 23-tetrakis-(1,6-hexanedithiol) phthalocyaninato] zinc (II) in PDT either free or encapsulated in gold nanoparticles/liposomes on photodamage of fibroblast and breast cancer cells revealed breast cancer cell damage with phthalocyanine liposomes while gold nanoparticles improved the effect with PDT (Nombona et al., 2012). Hematoporphyrin loaded liposomes revealed higher intratumoral accumulation of photosensitizer compared to free drug in MS-2 fibrosarcoma mouse model. Necrosis or apoptosis contributes to PDT mediated cell death in cancer and is due to generation of reactive oxygen species causing oxidative damage of cellular organelles. Thus, drug delivery in combination with PDT is a novel technique to selectively target cancer cells leading to cell lysis and overcome MDR in cancer therapy improving the efficacy.

NANOTHERANOSTICS
Nanotheranostics or quadrugnostic are third generation integrated nanovehicles comprising of four elements for diagnosis of tumor location/s, specific targeting to cancer cells, eradication of malignant cells with cytotoxic drug/s and neutralize drug resistance mechanism (Figure 4). Nanotheranostics serve dual roles as diagnostics and therapeutics thereby reducing chemotherapy dose, toxicity and side-effects to healthy tissues and increase therapeutic index (Ahmed et al., 2012). Superparamagnetic ironoxide nanoparticles (SPIONs) with magnetic properties have excellent potential in tumor-targeting, diagnosis, monitoring and therapy (Santhosh and Ulrih, 2013). Polysorbate 80 coated Temozolomide-loaded PLGA superparamagnetic nanoparticles revealed higher intracellular uptake with antiproliferative effect on malignant brain glioma C6 cells compared to non-polysorbate 80 coated nanoparticles . Diagnostic, targeting and therapeutic potential of Doxorubicin loaded SPION and plasmonic gold nanoparticles have been evaluated in cancer treatment (Maeng et al., 2010). PEG and PEI-coated SPIONs increased intracellular concentration of Doxorubicin in resistant rat glioma C6-ADR cell line with IC 50 values 3-5-fold lower FIGURE 4 | Theragnostic agent.
compared to free Doxorubicin (Kievit et al., 2011 reported 3-fold lower IC 50 with PBAE-coated SPIONs than free Doxorubicin in C6-ADR cells in accordance with Kievit et al. Gu et al. revealed that Doxorubicin loaded PEGylated gold nanoparticles were more cytotoxic on MDR cells compared to free Doxorubicin but less effective on sensitive cell lines (human hepatoma cells HepG2 and HepG2-R-a MDR subline). PEGylated Doxorubicin gold nanoparticles increased intracellular uptake and nuclear localization significantly up to 6 h by endocytosis . Theragnostic nanoparticles incorporating Doxorubicin or Paclitaxel for imaging and targeted chemotherapy were developed by Ahmed et al. (2012) and Kelkar and Reineke (2011). Paclitaxel loaded chitosan nanoparticles labeled with Cy5.5 (NIR fluorescence dye) were developed for imaging and cancer therapy in SCC7 tumor-bearing mouse models (Min et al., 2008;Kim et al., 2010b;Na et al., 2011;Ryu et al., 2011). Fluorescence property of Doxorubicin and photoluminescence of gold was utilized for imaging, monitoring drug uptake and tumor cells localization with folic acid decorated Doxorubicin gold nanorods (Newell et al., 2012). Chen et al. developed Doxorubicin encapsulated pH-responsive theragnostic nanoparticles with Cy5 for tissue targeting and imaging. Folate coated Doxorubicin SPIONs made of poly(ethylene oxide)trimellitic anhydride chloride-folate increased anticancer efficacy in liver cancer, lowered expression of CD34 and Ki-67 markers of angiogenesis and cell proliferation respectively with 2-and 4-fold decrease in tumor volume compared to free Doxorubicin and Doxil® respectively (Maeng et al., 2010). Acetylated dendrimerentrapped gold nanoparticles were taken by cell lysosomes and detected under X-ray after incubation in-vitro and in xenograft tumor model after intratumoral and intraperitoneal administration for imaging human lung adenocarcinoma cell line (SPC-A1 cell) (Wang et al., 2011b). Cetuximab conjugated magnetofluorescent silica nanoparticles for targeting EGFR receptor and in-vivo colon cancer imaging revealed high tumor uptake with application of an external magnetic field and MRI signal changes in human colon cancer xenograft mouse model . Folic acid-conjugated PEG-SPIONs labeled with Cy5.5 for imaging and active targeting to lung cancer resulted in higher intracellular uptake in KB cells and lung cancer model compared to non-folic acid coated nanoparticles (Yoo et al., 2012). Butyl rhodamine B fluorescent nanoparticles conjugated with anti-Her-2 monoclonal antibody were developed successfully for imaging and targeting ovarian cancer (Hun et al., 2008). Paclitaxel SPIONs significantly increased intracellular uptake and induced regrowth delay in-vivo in CT-26 cells with no toxicity (Schleich et al., 2013). Folic acid decorated Tamoxifen magnetic nanoparticles were developed for imaging and detection of human breast cancer cells that over express folic acid receptors (Majd et al., 2013). Immuno-targeted gold-iron oxide nanoparticles selectively accumulated in SW1222 xenograft colorectal tumors compared to non-antigen-expressing tumor xenografts. Photothermal treatment with IR irradiation revealed >65% of antigen-expressing tumor cells presented corrupt extracellular matrix and cytoplasmic acidophilia suggesting effectiveness of nanoparticle-assisted thermal therapy (Xie et al., 2010;Lu et al., 2012;Luk et al., 2012;Kirui et al., 2013).

CONCLUSION
Nanodrug delivery systems are versatile platform for delivery of anticancer drugs and have been utilized successfully toward overcoming cancer drug resistance mechanisms, maximizing chemotherapeutic efficacy. Nanocarriers have effectively overcome challenges of limited aqueous solubility, low bioavailability, lack of targeting cancer tissues, increase drug therapeutic index, preferential accumulation by EPR effect and divert ABCtransporter mediated drug efflux MDR with potential to be multi-functionalized for cancer treatment. Nanocarriers promise to alleviate many challenges in clinical cancer therapy to benefit patients in future. Cancer science is progressing rapidly and understanding the molecular basis of drug resistance in cancer promises more effective treatments.