Pluronics and MDR Reversal: An Update

1Introduction

Chemotherapy remains themain treatment option for most cancersdespite of its limitations, such as systemic toxicity, severe sideeffects, and limited efficacy. The major reason for chemotherapy failureis poor delivery of drug to cancer cells and/or intracellular targets.There are a number of barriers that have to be overcome for successfultreatment, and multidrug resistance (MDR) is one of them. Tumors ofdifferent origin have different susceptibility to chemotherapy, andfrequently cancers are intrinsically resistant. On the other hand,even though many primary tumors and metastatic lesions, for examplebreast, ovarian, and small cell lung carcinomas initially respondwell to the chemotherapeutic treatment, cancers often relapse anddevelop drug resistance. Moreover, cancer cells simultaneously acquireresistance not only to the drug the patient was treated with but alsoto the broad spectrum of drugs that are structurally and functionallyunrelated to each other. Initially MDR was attributed to the expressionof drug efflux transporters on the cell membrane that actively pumpthe drugs out of the cells.¹ Now it isgenerally recognized that MDR is a complex phenomenon and usuallyis governed by one or more of the following mechanisms: (1) activedrug removal by drug efflux transporters of the ATP-binding cassette(ABC) superfamily, such as P-glycoprotein (Pgp, ABCB1), multidrugresistance-associated protein 1 (MRP1, ABCC1), and breast cancer resistanceprotein (BCRP, ABCG2); (2) loss of cell surface receptors or drugtransporters or alterations in membrane lipid composition that limitdiffusion of the drug into the cells; (3) compartmentalization ofthe drug in cellular vesicles; (4) altered/increased drug targets;(5) increased drug metabolism; (6) alterations in cell cycle; (7)active damage repair; and (8) inhibition of apoptosis (Figure 1).

Figure 1

Mechanisms of MDR in cancer cells: (1) active drug effluxby drugtransporters, such as Pgp, MRP, and BCRP; (2) loss of cell surfacereceptors and/or drug transporters or alterations in membrane lipidcomposition; (3) compartmentalization of the drug in cellular vesicles;(4) altered/increased drug targets; (5) alterations in cell cycle;(6) increased drug metabolism/enzymatic inactivation; (7) active damagerepair; and (8) inhibition of apoptosis.

Despite much effort contributed to overcoming MDR, the successis still very limited in clinical settings. This effort mainly centeredon the following approaches.²⁻⁶ First, the modification of treatment regimens by increasing thedose of the administered drug(s) or using non-cross-resistant drugs.Second, use of small molecule inhibitors of drug efflux transportersto increase the drug uptake in MDR tumors.⁷⁻⁹ Third, use ofantibodies and antibody fragments to target and inhibit drug effluxtransporters.¹⁰⁻¹² Fourth, silencing of the gene expression of the drugefflux transporters¹³⁻¹⁵ or antiapoptotic proteins, such as BCL2^13,16 using antisense oligonucleotides, siRNA, or micro RNA. Fifth, useof small molecules to suppress non-ABC transporter-mediated resistance.^17,18 Finally, use of nanotechnology-based carriers to bypass drug effluxtransporters in MDR cancer cells.² Of theseapproaches the first two were evaluated in clinics. Unfortunately,a simple dose increase has been associated with increased risks ofsystemic toxicity and severe side effects, while finding a propercombination of non-cross-resistant drugs in many cases is complicated.As far as the use of the Pgp inhibitors is concerned, the outcomeswere often poor, and many such inhibitors failed due to toxicity ordrug metabolism associated issues.^8,9 Moreover, mostof the approaches under development face traditional drug deliveryissues, which are especially severe in the cases of nucleic acid orprotein therapeutics.

Nanotechnology offers several advantagesboth for the deliveryof the chemotherapeutic agents, allowing them to bypass drug effluxtransporters, and for the delivery of agents that could inhibit drugresistance mechanisms to increase efficacy of the chemotherapy. First,it allows improving pharmacokinetic parameters of administered compounds.Nanomedicines have longer circulation times and can passively accumulatein the tumors with leaky vasculature and poor lymphatic drainage bythe enhanced permeability and retention (EPR) effect.^19,20 Attaching specific tumor-targeting antibodies, antibody fragments,or other targeting moieties (receptor ligands, peptides, etc.) canresult in active targeting of the nanomedicines to the tumor cells,which can further improve drug delivery. Second, two or more activecompounds can be incorporated into a single carrier allowing simultaneousdelivery of several cytotoxic drugs for combination therapy and/ora cytotoxic drug with a MDR modulator, such as small molecule inhibitor,antibody, or nucleic acid. Third, a nanocarrier can be designed insuch a way that it will release its cargo at the tumor site in responseto specific tumor conditions, such as pH or presence of particularenzymes, therefore limiting other organs and tissues to the exposureto free drug and reducing systemic toxicity. Finally, in contrastto small molecules that mainly utilize diffusion to penetrate thecells, nanocarriers are taken up by either “passive”endocytosis or receptor-mediated endocytosis and, therefore, can bypassdrug efflux transporters on the plasma membrane. In the latter casethe endocytosis is triggered by interaction of targeting ligand withits receptor on plasma membrane, which accelerates the uptake comparedto “passive” endocytosis. If the receptor is predominantlyexpressed on cancer cells, in addition to faster uptake this allowsselective targeting of the nanocarrier to cancer cells.

Additionally,polymeric carriers can have a biological activityof their own. One such example is represented by a class of copolymers,called Pluronic block copolymers or poloxamers, that are widely usedin various drug delivery systems²¹⁻³² and in tissue engineering.³³⁻³⁶ Pluronics are triblock copolymers of poly(ethyleneoxide) (PEO) and poly(propylene oxide) (PPO), arranged in PEO-PPO-PEOstructure. Depending on the length of the blocks the hydrophilic–lipophilicbalance (HLB) of the copolymers changes. In the solution Pluronicsspontaneously form micelles above the critical micelle concentration(CMC). The core of the micelles contains PPO blocks and allows incorporationof hydrophobic drugs. Previously thought to be “inert”,Pluronics display a unique set of biological activities and have beenshown to be potent sensitizers of MDR cancer cells in vitro and in vivo.^{21,23,37−40} Moreover, Pluronics were shown to prevent the development of MDRupon selection with an anthracycline antibiotic, doxorubicin (Dox),both in vitro and in vivo.^41,42 We have also recently demonstrated that Pluronics in combinationwith Dox can deplete tumorigenic cell subpopulations and decreasecancer cells’ tumorigenicity and tumor aggressiveness upontreatment in vivo.²² Inthis review we will discuss each of these mechanisms in more details.

2Reversal of ABC Transporter-Mediated Resistanceby Pluronics

2.1Structure and Function of ABC Transporters

The first drug efflux transporter in cancer cells was describedby Juliano and Ling in 1976.¹ They haveshown that drug-resistant Chinese hamster ovary cells express a 170kDa membrane glycoprotein, now known as P-glycoprotein (Pgp, ABCB1),that was unique to the drug-resistant cells.¹ The cells were selected for resistance to colchicine and showedcross-resistance to a wide range of different compounds. The degreeof drug resistance correlated with the amount of Pgp on the cell surface.Later, in early 1990s a second drug efflux transporter, called multidrugresistance-associated protein (MRP1 or ABCC1), was discovered in adrug-resistant lung cancer cell line.⁴³ Pgp and MRP1 show a partial overlap in substrate specificity. NormallyMRP1 plays a major role in cell detoxifying mechanism by transportof exogenous and endogenous compounds conjugated to glutathione (GSH),which for some substrates is required as a cofactor for MRP1 activity.In contrast, Pgp does not require a cofactor and can efflux a widevariety of functionally and structurally diverse but commonly hydrophobicdrugs.⁴⁴ Another important drug effluxtransporter, named breast cancer resistance protein (BCRP, ABCG2),was identified in 1998 by Doyle et al. in human breast cancer cellline selected for Dox resistance.⁴⁵ Itsexpression is associated with resistance to number of drugs, suchas mitoxantrone, camptothecins, anthracyclines, etc.⁴⁶ Pgp, MRP1, and BCRP belong to the large superfamily ofATP-binding cassette (ABC) membrane transporters with 48 members ofthe superfamily that are divided into 7 subgroups (A–G). Theyhave conserved structures and ubiquitously expressed in all formsof living organisms, from bacteria to humans. Pgp is the most studiedABC transporter (Figure 2). It is a productof mdr1 gene and can be found in many normal tissues,like epithelial cells of gastrointestinal tract,⁴⁷ liver, the luminal membrane of proximal tubular epithelialcells in kidney,^48,49 cornea,⁵⁰ and the luminal membrane of the endothelial cells in the blood–brainbarrier.⁵¹ Overall, Pgp is mostly expressedin tissues with barrier functions and its main role is to protectthe organism from toxic compounds. It has a typical structure forABC transporters and comprises two trans-membrane domains (TMDs),each of which has 6 membrane-spanning α helixes, and two intracellularnucleotide-binding domains (NBDs), which bind and hydrolyze ATP providingenergy for transmembrane movement of the drugs (Figure 2). Pgp substrates are mostly hydrophobic (but structurallyunrelated) and partition into a lipid bilayer.⁵² Among these substrates are important anticancer drugs includingseveral anthracyclines (Dox, daunorubicin, mitoxantrone), vincristine,taxanes, etoposide, teniposide, actinomycin D, and others. Understandingthe mechanism of Pgp function is critical for the design of noveleffective MDR modulators. Several models for Pgp-mediated drug transporthave been proposed.⁵³⁻⁵⁶ Recently the crystal structure of mouse Pgp, which has 87% sequenceidentity to human Pgp, was described⁵⁶ (Figure 2). By analyzing the costructures of Pgp complexeswith two cyclopeptide inhibitors the authors elucidated the mechanismof drug efflux by Pgp and provided insight into the transporter’sbroad substrate specificity. The drug-binding pocket of Pgp is localizedin the TM domain of the protein. The inward-open conformation of Pgpallows the substrate access both from cytoplasm and from the innerleaflet of the membrane but not from the upper leaflet or extracellularspace. The upper part of the drug-binding pocket contains predominantlyhydrophobic and aromatic amino acid residues, and the lower half ofthe chamber has more polar and charged residues. The drug-bindingpocket in Pgp is very large and in inward-facing conformation is accessiblethrough two portals that are wide enough to fit hydrophobic drugsand phospholipids and allow Pgp to “scan” the innerleaflet to select and bind specific lipids and hydrophobic drugs beforetransport.⁵⁶ Overall, the authors proposed,that Pgp has broad flexibility and can sample widely open conformationsto accommodate large substrates, explaining the broad substrate specificityof the transporter. Usually the drug enters Pgp’s binding sitefrom the inner leaflet of the membrane, which stimulates the bindingof two molecules of ATP by NBDs followed by their dimerization. Thedimerization of NBDs causes the major conformational change in theprotein and formation of the outward-facing structure, open to theextracellular space. The drug is released due to the change of theaffinity of the protein to it or is facilitated by ATP hydrolysis,which brings the protein back to the initial state.⁵⁶

Figure 2

Structure and localization of Pgp in plasma membrane. (A) Pgp isa transmembrane protein with drug-binding pocket localized in theinner leaflet of the plasma membrane, and two NBD localized in cytoplasm.Functional Pgp is localized in cholesterol, sphingomyelin, and GM1ganglioside-rich membrane microdomains, called lipid rafts, whereit is surrounded by fluid phase of the membrane, containing unsaturatedfatty acids like DPPC. Pgp is pictured in inward-open (outward closed)conformation ready to bind substrate. The model is based on X-rayanalysis⁵⁶ and NMR data from protein databank (http://www.rcsb.org/). (B) Incorporation of Pluronicinto lipid bilayer disrupts lipid rafts, possibly causing conformationalchanges in Pgp, which results in inhibition of Pgp ATPase and transportactivities.

2.2Inhibitionof Pgp Activity by Pluronic: Roleof Pluronic–Membrane Interactions

As was mentionedabove, Pluronic block copolymers are potent sensitizers of MDR cells.The sensitization mechanism is complex and involves multiple eventshappening at different levels in the cell. The polymer–cellinteraction starts in the cell membrane, where drug efflux transportersare localized. Pluronics were shown to be strong inhibitors of ABCtransporters, specifically Pgp, MRP, and BCRP.^39,57−59 They suppress the transporters’ ATPase activityand their interaction with the drug. The inhibition might be in partdue to the alterations of lipid microenvironment of the transportersby Pluronic. Due to their amphiphilic structure, Pluronic block copolymerscan interact with cell membrane and change its properties,⁶⁰ which are critical for proper function of ABCtransporters.

2.2.1Role of Lipid Microenvironment for Pgp Function

Membrane structure and composition play a crucial role in cellphysiology, function, and signaling. Plasma membrane is a heterogeneousstructure composed of various domains with different lipid compositionand packing.⁶¹ In particular, so-called“lipid rafts” are compact membrane microdomains containingpredominantly cholesterol and sphingolipids (mainly sphingomyelin)with long and saturated fatty acids, that are “floating”in more fluid membrane phase that contains glycerophospholipids withshorter and unsaturated acyl chains (Figure 2).⁶² These domains are resistant to lowtemperature solubilization by some detergents, like Triton X100 orBrij 96, and this is used for their isolation. Depending on the cellline and the method used for membrane fractionation Pgp can be foundeither mostly in detergent-resistant membrane fractions or distributedbetween the detergent-resistant and detergent-soluble fractions.⁶³⁻⁶⁶ Furthermore, it was found that Pgp distribution between differentmembrane fractions depends on the transporter’s expressionlevel: the lower the expression of Pgp is, the greater portion ofPgp is localized in detergent-resistant cholesterol-rich membranedomains.⁶⁷ It is well-known that the functionof most membrane proteins is directly linked to the composition andviscosity of their lipid microenvironment. Pgp is a lipid flippase⁶⁸ and requires interaction with phospholipidsfor continuous display drug-mediated ATPase activity⁶⁹ and interaction with the substrate.⁷⁰ Moreover, an increasing number of studies report that Pgplocalization in lipid rafts and precise properties of rafts are essentialfor the transporter’s proper function.⁶² For example depletion of cholesterol with methyl-β-cyclodextrinin drug-resistant VLB human T-cell lymphoblastic leukemia cells ledto disassembly of the lipid rafts, redistribution of Pgp from lipidrafts to other microdomains of plasma membrane, and inhibition ofPgp transporter activity. On the other hand, enrichment of membraneswith cholesterol also resulted in inhibition of Pgp function, althoughthe localization of Pgp did not change compared to control. However,the increase in cholesterol content changed the lipid raft distributionand composition, which most likely accounts for the impairment ofthe Pgp function.⁷¹ It was also shown recentlythat caveolin-1 overexpression decreases plasma membrane cholesterollevels (similar to the effect of methyl-β-cyclodextrin thatdepletes cholesterol from the membrane) and results in the increaseof membrane fluidity and inhibition of Pgp function in drug-resistantHs578T/Dox cells.⁷² Another study by Barakatet al. demonstrated that there are two functionally different populationsof Pgp in drug-resistant human CEM lymphoblastic leukemia cells.⁶³ The first population localized in detergent-resistantmembrane fraction has higher ATPase activity, which is completelyinhibited by orthovanadate and activated by verapamil. The secondpopulation localized in soluble membrane fractions has lower ATPaseactivity and is less sensitive to orthovanadate. Moreover, verapamil,a well-known Pgp activator, inhibits Pgp ATPase activity in this secondpopulation.⁶³ The authors conclude thatinteraction of Pgp with its substrates could be affected by differentlipid microenvironment in soluble membrane fractions, specificallyby lower content of cholesterol compared to the detergent-resistantmembrane fraction.⁶³

2.2.2Pluronic Interaction with Lipid Membranes

Pluronicbinding to the cell membrane depends on Pluronic hydrophobicityand the temperature.⁷³ The binding is drivenby hydrophobic interactions of PPO chain blocks with the fatty acidresidues in the lipid bilayer and by hydrophilic interactions of PEOchain blocks with the polar groups of the lipids at the membrane surface.This binding may lead to either membrane destabilization⁷⁴ or healing of “injured” membranes.^75,76 Pluronics also exhibit ionophoric activity and can facilitate transmembranetransport of low molecular drugs, accelerate phospholipid’sflip-flop rate, and decrease membrane microviscosity.^73,77,78 Pluronic effects on the membranetransport depend on the copolymer HLB, concentration, and the exposuretime. For example, hydrophobic Pluronic L61 ((EO)₄-(PO)₃₀-(EO)₄, HLB 3, MW 2000 g/mol, EO = ethylene oxide;PO = propylene oxide) depending on the level of its aggregation canact either as a transmembrane carrier of drug molecules or as an ionchannel.⁷⁸ Specifically, it was proposedthat L61 monomers and dimers can act as the carriers while L61 oligomersare likely to form the channels.⁷⁸ On theother hand hydrophilic Pluronic F68 (Poloxamer 188, (EO)₇₆-(PO)₃₉-(EO)₇₆, HLB 29, MW 8400 g/mol) with80% PEO content effectively restores damaged cell membranes afterelectroporation, heat shock, or intense radiation.⁷⁹⁻⁸¹ Using X-rayreflection (XR) and grazing-incidence X-ray diffraction (GIXD) methodsin a model Langmuir lipid monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), Wuet al. have shown that F68 interacts with the damaged membrane areas,but does not affect the ordered membrane phase, and gets excludedwhen lipid packing density is restored.⁷⁶ Recently it was demonstrated that F68 molecules do not insert intolipid bilayer nor affect the overall lipid packaging, however, theyfacilitate the membrane sealing activity by diminishing the fluctuationof membrane surface and hydration of the inner part of the bilayer.⁸² However, in another study using giant unilamellarvesicles (GUV) as model membrane system Wang et al. demonstrated thatF68 can incorporate in the membranes, disrupt their integrity, andact as a permeabilizer if it is exposed to the membranes for sufficienttime.⁸³

Overall, the interactionof Pluronics with lipid membranes proceeds in two steps: (1) the absorptionat the membrane and (2) the insertion in the membrane (Figure 3). The first step is common to all Pluronics anddoes not depend much on the copolymer structure. The second step dependsstrongly on the hydrophobicity of the copolymer with the more hydrophobiccopolymers being morel likely to insert.⁸³ Extremely hydrophilic Pluronics absorb on the membrane without penetratinginto the lipid bilayer. Pluronics with longer PPO blocks insert intothe membrane below the polar head groups, loosen the lipid packaging,and, therefore, act as permeabilizers,⁸² They can translocate through the membrane (depending on their HLB).Furthermore, using molecular dynamics simulations Nawaz and coauthorsobserved that membrane bends upon insertion of Pluronics.⁸⁴ They have shown that membrane-disruptive activityof Pluronics is due to interaction of hydrophilic blocks with thepolar head groups of the lipid molecules and depends on the lengthof the PEO block. Short PEO blocks drag the polar groups toward theinner part of the membrane, which results in membrane bending andpermeabilization. Pluronics with longer PEO blocks can temporarilystabilize the local structure of the membrane.

Figure 3

Schematic presentationof interaction of Pluronics with differenthydrophobicity with lipid membranes: (1) absorption of Pluronic moleculeson the surface of the membrane, (2) insertion into the lipid bilayer,and (3) translocation through the membrane.

Pluronic copolymers can significantly increase the antitumoractivityof PEGylated liposomal drugs in vivo, specificallyDOXIL by stimulating the drug release from liposomes at the tumorsite.³⁰ One of the main problems of longcirculating liposomal drugs is insufficient release of the activecompound at the tumor site. We have demonstrated that “post-administration”of Pluronic P85 ((EO)₂₆-(PO)₄₀-(EO)₂₆, HLB 16, MW 4600 g/mol) 48 h after DOXIL results in Dox releaseand redistribution toward tumor bulk along with a marked improvementof antitumor activity. This effect is time-dependent as it is essentialto allow sufficient time for the liposomes to accumulate at the tumorsite before administering Pluronic. It is likely that that the enhancedantitumor effect at least in part is due to facilitated release ofDox from the liposomes in the tumors induced by Pluronic. Furthermore,in addition to permeabilization effect on liposomal membranes thecopolymer could also sensitize the MDR cells and deplete the cancerstem cells (CSCs) (as discussed below).^22,29

Anotherimportant aspect in Pluronic interactions with lipid membranesis the dependence of these interactions on the cell type and the membranecomposition. For example, the membrane microviscosity of murine myelomaSP2/0 cells significantly decreased after treatment with L61, whilethe membrane viscosity in normal mouse splenocytes was less affected.⁷³ Moreover, Pluronic P105 ((EO)₃₇-(PO)₅₆-(EO)₃₇, HLB 15, MW 6500 g/mol) was demonstratedto permeabilize the acidic endosomal vesicles in drug-resistant A2780/ADRcells, while the vesicles in sensitive cells were less affected.⁸⁵ These differences may be attributed to differencesin membrane lipid compositions. Several studies have reported lowerfluidity and higher heterogeneity of plasma membrane in MDR cellscompared to sensitive cells.^86,87 Drug-resistant cellsalso contain smaller amounts of unsaturated fatty acids and have highercontent of esterified cholesterol and triglycerides.^88,89 Using liposomes of different lipid composition and viscosity itwas demonstrated that the L61 effects on lipid flip-flop and membranepermeability toward Dox increase as the membrane viscosity increases.⁹⁰

Pluronics inhibit Pgp and MRP ATPase activitiesby decreasing maximumreaction rate (V_max) and the affinityof the enzyme to ATP as well as to the substrates such as vinblastine(expressed as increase in Michaelis constant, K_m).⁴⁰ Some neutral detergents, suchas Tween-20, Nonidet P-40, and Triton X-100, were also shown to inhibitPgp ATPase activity at concentrations that are required for membranefluidization.⁹¹ Overall, alterations inmembrane structure and fluidity induced by various compounds stronglyaffect Pgp function. Therefore, it was suggested that inhibition ofthe transporter’s activity by Pluronic is at least partly dueto the Pluronic-induced changes in the local membrane environment(Figure 2).

3Effectof Pluronic on Cancer Cells’ Metabolism

To furtherunderstand the mechanism of Pluronic sensitization ofMDR cancer cells one needs to focus on the events at the subcellularlevel, which were characterized in great detail using P85 as an example.²³ This copolymer exhibits evident and profoundselectivity with respect to energy metabolism in MDR cancer cells.It is rapidly taken up by the cells via a caveolae-mediated endocytosispathway⁹² and colocalizes with mitochondriaalready 15 min after exposure to the cells.³⁸ This results in a drastic depletion of intracellular ATP levelsin MDR cancer cells, while non-MDR cells require significantly higherdoses of Pluronic to achieve similar depletion. Noteworthy, the abilityto deplete cellular ATP levels strongly correlates with the chemosensitizationproperties of the copolymers in MDR cells.⁹³ The selectivity of Pluronic copolymers toward MDR phenotype is probablyattributable to innate metabolic and physiological differences betweenMDR and non-MDR cells. In contrast to normal cells, that use oxidativephosphorylation for ATP production, cancer cells mostly rely on glycolysisas an adaptation to hypoxic conditions in the early stages of tumordevelopment.⁹⁴ Drug-resistant cells requiremore ATP to support the drug efflux transporter activity and drugmetabolism. Adaptations leading to MDR therefore in part are associatedwith changes in energy metabolism to meet new energy requirements.It was shown that human breast cancer cells with acquired resistanceto Dox exhibit 3-fold higher glycolysis rate than their sensitivecounterparts.⁹⁵ Another study by Miccadeiet al. found that both respiration and glycolysis rates are increasedin drug-resistant Ehrlich cells, resulting in almost 50% higher ATPproduction compared to the drug sensitive cells.⁹⁶ It was also shown that MDR cells have significantly higheractivity of the respiratory chain complexes in mitochondria wherenearly 50% of ATP was produced, compared to only 35% of ATP producedin mitochondria of sensitive cells. Moreover, it was later demonstratedthat MDR cancer cells have lower mitochondrial membrane potential,use fatty acids for mitochondrial oxidation when glucose becomes limited,and have high levels of expression of uncoupling protein 2 (UCP2),which results in less efficient ATP synthesis.⁹⁷ Overall, the compromised mitochondrial function in MDRcells may be the Achilles’ heel of MDR cells that allows effectiveand selective inhibition of ATP production in drug-resistant cells.

When Pluronic reaches mitochondria of MDR cells, it inhibits complexesI and IV of the respiratory chain and depletes mitochondrial membranepotential.³⁸ The mechanism of Pluronicinhibition of respiratory chain complexes’ activities is notfully understood. In mitochondria Pluronic may undergo chemical reactionand provide peroxides to respiratory chain. In other words Pluronicmay act as a prooxidant, which were shown to induce apoptosis in cancercells.⁹⁸ Noteworthy, the effects of Pluronicon Pgp activity, ATP levels, and cytotoxicity are reversible. Pgpfunction is restored 1 h after the removal of Pluronic. At the sametime, the amount of cell-bound Pluronic rapidly decreases. The sensitizationeffect of Pluronic is abolished in the same time frame, while it takesabout 10 h to restore ATP levels.³⁸ Interestingly,Pgp expression seems to be essential for Pluronic effects on respirationand ATP levels. Inhibition of oxygen consumption as well as ATP depletionby Pluronic was observed not only in drug-selected resistant cellsbut also in cells stably transfected with mdr1 gene, encoding Pgp.^38,39 Inhibition of Pgp with highly specific inhibitor GF120918 abolishedthe Pluronic-induced ATP depletion, while the inhibitor itself didnot affect ATP levels in MDR cells.³⁸

Pluronic effects in MDR cancer cells exhibit remarkably simpleand clear structure–functional relationships.⁵⁷ The studies of the concentration dependence of the Pluronicin MDR cells effect suggested that these effects are produced mainlyby the copolymer single chains as they leveled up or decreased abovethe CMC. Hydrophilic Pluronics with HLB 20 and above have little ifany sensitization effect in MDR cells. Using Pgp expressing brainmicrovessel endothelial cells (BMECs) it was demonstrated that suchPluronics do not decrease membrane microviscosity, do not inhibitPgp ATPase activity, practically do not internalize in the cells,and do not induce ATP depletion.⁹⁹ Of allother Pluronics with HLB fewer than 20 the most active in MDR cellsare the copolymers with intermediate lengths of the hydrophobic PPOblock from about 30 to about 60 PO units.¹⁰⁰ Such copolymers include L61, P85, and P105 discussed above. Thesecopolymers bind with the cell membranes, decrease membrane microviscosity,and inhibit Pgp ATPase activity.⁹⁹ Moreover,they internalize into cells and produce ATP depleting effects. Thecopolymers with shorter PPO blocks, fewer than 30 PO units, also internalizein cells. However, they do not decrease membrane microviscosity, donot inhibit Pgp ATPase, and do not deplete ATP. Presumably, they arenot sufficiently “disruptive” to the membrane structuresto produce all these effects. The copolymers with longer PPO blocksproduce strong effects decreasing membrane microviscosity and inhibitingPgp ATPase. But they do not penetrate inside the cells and do notreach mitochondria remaining stuck in the cell membranes, presumablydue to their extreme hydrophobicity. Accordingly, such hydrophobiccopolymers do not induce ATP depletion.⁹⁹ Notably, it was demonstrated that both ATP depletion and inhibitionof Pgp ATPase activity are essential for the sensitization of Pgpoverexpressing cells.^39,93 When one of these factors wasexcluded, the drug efflux pump remained functional in both MDR cancerand Pgp-expressing BMECs.^39,93

4Effectof Pluronic on Proapoptotic Signaling

Oxidative stress isa condition in which the balance between theproduction of reactive oxygen species (ROS) by cells and the abilityto detoxify them is impaired. If oxidative stress persists, the formedperoxides and free radicals will damage all components of the cell,including membranes, proteins, and DNA. Accumulation of significantdamage, which a cell fails to repair, will lead to apoptosis. Generally,oxidative stress is associated with increased production of ROS and/ordecreased ability of the cell to eliminate these species. Glutathioneis a major cellular antioxidant that protects the cells against ROS,toxins, and drugs. It is a tripeptide that exists in reduced (GSH)and oxidized (GSSG) states, and normally more than 90% of cellularglutathione is in a reduced state. An accurate ratio between GSH andGSSG is important to maintain the intracellular redox state, witha decrease in GSH/GSSG ratio indicative of oxidative stress. GSH isalso a cofactor of glutathione S-transferase (GST),the major cellular detoxifying enzyme. Furthermore, several membersof the MRP family of ABC transporters require GSH for transport activity.Pluronic was shown to deplete the GSH levels and inhibit the GST activityin several MDR cell lines.⁵⁷ Inhibitionof the GSH/GST detoxifying system in turn decreases the MRP-mediatedefflux. The decrease of cellular GSH is also an early sign of apoptosisinduced by oxidative stress, death receptor activation, or mitochondrialapoptotic signaling.¹⁰¹

One of themajor sources of ROS in the cells is electron transportchain in mitochondria. In normal conditions oxygen is reduced in mitochondriaby cytochrome c oxidase (complex IV) to produce water.However, a small amount of electrons passing through the electrontransfer chain reduce oxygen to produce superoxide radical. The mainsuperoxide radical producing complexes in mitochondria are NADH dehydrogenase(complex I) and cytochrome bc1 complex (complex III).It is well-known that inhibition of complex I by certain inhibitorslike rotenone, piericidin A, and rolliniastatin increases the ROSproduction. As was mentioned above, Pluronic quickly reaches mitochondriaand inhibits complexes I and IV in MDR cells (Figure 4). Moreover, it stimulates the production of ROS and releaseof cytochrome c, which are the early signs of mitochondrialapoptotic pathway.³⁸ If ROS are not neutralized,they induce damage of mitochondrial membrane, proteins, and DNA. Thisleads to permeabilization of outer mitochondrial membrane, swellingof mitochondria, and release of proapoptotic proteins, like cytochrome c, apoptosis inducing factor (AIF),¹⁰² and endonuclease G.¹⁰³ In cytoplasmcytochrome c binds to apoptosis protease activatingfactor (APAF-1) and forms apoptosome. The apoptosome cleaves and activatesthe procaspase-9 and forms caspase 9. The activated caspase 9 in turnactivates the effector caspases, which all together contribute tothe completion of apoptosis. Similar to ATP depletion and inhibitionof respiration, Pluronic induced the ROS formation and cytochrome c release selectively in MDR cells, while non-MDR cellsdid not respond in that manner.³⁸

Figure 4

Effect of Pluronicon mitochondrial electron transport chain inMDR cancer cells. Pluronic quickly enters the cells, reaches mitochondria,and induces mitochondrial membrane depolarization (1), inhibitionof complexes I (2) and IV (3), release of cytochrome c (4), and ATP depletion (5).

In addition to induction of ROS production and cytochrome c release in MDR cells, Pluronic promotes drug-induced apoptosis.Treatment of MDR cells with Dox/Pluronic P85 formulation significantlyenhanced the proapoptotic signaling compared to the drug alone andinhibited the antiapoptotic defense mechanisms in vitro.¹⁰⁴ Similar effects were observed in vivo. It was demonstrated that Dox/Pluronic treatmentof tumor-bearing mice significantly increased levels of caspases 8and 9 compared to Dox alone.¹⁰⁵

Overall,Pluronic induces early as well as late stages of proapoptoticsignaling in MDR cells in vitro and in vivo. Inhibition of mitochondria respiratory chain complexes is mostlikely the main reason for increased ROS production in MDR cells aftertreatment with Pluronic. Additionally, depletion of major intrinsiccellular antioxidant GSH would increase cell sensitivity to the ROS.It has been shown that drug-induced ROS production may be directlylinked to their cytotoxic activity^106,107 and thatdetoxification of free radicals by GSH/GST is very important in MDRcells to facilitate drug resistance.¹⁰⁸ Therefore, when combined with Dox, Pluronic not only drasticallyincreases the drug accumulation in the cells but also promotes theapoptosis in the MDR cells. This in combination with the Dox effectsresults in significantly increased cell death.

5PluronicsPrevent Development of MDR and SuppressCSCs

The mechanism of development of MDR in cancer remainsa highlydebated subject, and most likely there is no uniform theory that willapply to all cancers.^109−114 It is now widely accepted that CSCs play an important role in cancerdevelopment, metastasis, and development of drug resistance. CSCscomprise a small cell subpopulation within the tumor with distinctfunctional and phenotypical characteristics. First, CSCs overexpressspecific markers. However, these markers differ from cancer to cancerand to date there is no uniform marker that can be used to isolateCSCs from every tumor.¹⁰⁹ Second, CSCshave unlimited ability to divide and produce cells of all other phenotypesin the tumor. Third, CSCs are able to form tumors when transplantedinto mice and to form so-called tumorspheres when grown in anchorageindependent conditions. Finally, CSCs are intrinsically drug resistant:they overexpress drug efflux transporters, such as Pgp and BCRP, haveactive antiapoptotic pathways, and spend most of their time in theG₀ nondividing cell cycle state, which makes them insensitiveto cytostatic drugs often used in chemotherapy.¹¹⁵ Therefore, CSCs can avoid classical chemotherapy and repopulatethe tumor, possibly leading to MDR development. Moreover, there arereports suggesting that CSCs’ phenotype is dynamic and canbe acquired by non-CSCs under certain conditions.^109,110 Overall, successful therapy needs to be equally efficient in eliminatingboth bulk tumor cells and CSCs.

In addition to MDR chemosensitizationproperties, Pluronics alsoprevent the development of MDR upon selection with cytotoxic drugs in vitro and in vivo.^41,42 Specifically, in one study human breast carcinoma MFC7 cells wereselected with Dox for drug resistance in the presence or absence ofP85 at concentration below CMC (0.001 wt %).⁴¹ The cells cultured with Dox/P85 were not able to grow at concentrationsof the drug exceeding just 10 ng/mL. In contrast, cells cultured withDox alone eventually developed MDR and could tolerate up to 10,000ng/mL Dox in the culture media. Further analysis has shown that cellstreated with Dox/P85 did not overexpress Pgp and, therefore, remainedsensitive to the drug. In contrast, cells exposed to Dox alone exhibitedsignificant overexpression of Pgp. This developed drug resistancecan be resensitized by Pluronic to the initial level of the drug sensitivecells. Interestingly, when the cells were selected with lower concentrationof Dox, they were not sensitized by Pluronic, even though they displayedlow levels of Pgp expression and detectable levels of mdr1 mRNA. Functionalanalysis of Pgp activity using accumulation of Pgp substrate (Rhodamine123) showed that Pgp in those cells was not or nearly not functionalcompared to more resistant cells.^41,42 Even thoughcells selected with lower concentrations of Dox were not sensitizedwith Pluronic, they showed strong ATP depletion in response to Pluronictreatment.⁴¹ Moreover, it was demonstratedthat selection of cells with Dox and Dox/P85 resulted in very differentchanges in the gene expression patterns in these cells. P85 alone,however, had little if any effect on the gene expression.⁴¹ Similar results were observed in P388 murineleukemia tumor cells selected for Dox resistance with or without P85both in vitro and in vivo.⁴² Overall, this suggests that simple additionof “inert” polymer excipient to the drug drasticallychanges pharmacogenomic responses of cancer cells to this drug.

However, our understanding of the mechanism behind the preventionof MDR development by Pluronic and alterations in gene expressionprofiles is very limited. In view of CSC theory a small populationof tumor cells is guiding tumor progression, metastasis, and MDR development.Since CSCs share certain characteristics of MDR cells, such as overexpressionof drug efflux transporters (Pgp, BCRP) and altered metabolic pathways,^116−118 we proposed that Pluronics can sensitize CSCs to chemotherapeuticdrugs similar to MDR cells. In a recent study using the same P388leukemia ascitic tumor model as before,⁴² we demonstrated that Dox/Pluronic combination, SP1049C, comprisingmixed micelles of Pluronic F127 ((EO)₁₀₀-(PO)₆₅-(EO)₁₀₀, HLB 22, MW 12 600 g/mol) and L61, effectivelydecreases frequency of tumor initiating cells and, as a result, suppressestumorigenicity and tumor aggressiveness in vivo.²² In agreement with previous findings, SP1049Calso prevented the development of MDR by inhibition of BCRP overexpression.In contrast to Dox alone, SP1049C depleted the tumorigenic CD133+and ALDH+ cell subpopulations. Furthermore, in vitro pretreatment of ascitic cells with SP1049C significantly reducedthe in vitro colony forming potential of the cellsalready at 10 ng/mL Dox, while Dox alone had the same effect at 10times higher concentration. As mentioned above, Dox/Pluronic combinationdrastically changes the gene expression profiles in cancer cells comparedto Dox or Pluronic alone upon continuous exposure. In this work wehave shown that DNA methylation patterns also change drastically upon in vivo treatment of cancer cells with SP1049C comparedto saline control, polymers, or Dox alone. It is well-known that misregulationof DNA methylation/demethylation plays an important role in cancerorigin, progression, angiogenesis, metastasis, and MDR development.^119−122 SP1049C not only induced the strongest epigenetic changes but alsoshowed very small overlap of affected genes with other treatment groups.Functional analysis of affected genes done using ingenuity pathwayanalysis (IPA) has shown that the top affected biological functionsand canonical pathways affected by SP1049C treatment relate to cellularfunction, growth, and maintenance, as well as regulation of stem celldifferentiation and pluripotency. Altogether, on top of MDR sensitization,the prevention of MDR development by Pluronics, depletion of tumorigeniccell subpopulations, and decrease of tumorigenicity and tumor aggressivenessoffer significant advantages for the development of new formulationsof approved and/or experimental therapeutics.

6RecentExamples of Pluronic-Based and SimilarDrug Delivery Systems

Pluronic copolymers attracted a lotof attention in drug deliveryand tissue engineering applications. Pluronic-based micellar formulationof Dox, SP1049C, was the first in class polymeric micelle drug toadvance to clinical stage¹²³ and has successfullycompleted phase II clinical trial in advanced esophageal cancer patients.¹²⁴ In studies in rodent and nonrodent animal modelsit has been demonstrated, as well as in patients, that MTD and pharmacokineticprofiles of Dox alone and SP1049C are very similar.³⁷ SP1049C did enhance the tumor accumulation of the drugin tumor bearing mice. Moreover, animal studies using MDR overexpressingtumors have shown that Pluronic formulations in vivo exhibit key effectsobserved in mechanistic studies in vitro.¹⁰⁵ First, noninvasive single photon emission computed tomography (SPECT)and tumor tissue radioactivity sampling demonstrated that intravenouscoadministration of Pluronic P85 with a Pgp substrate, ⁹⁹Tc-sestamibi, greatly increases the tumor uptake of this substratein the MDR tumors. Second, ³¹P magnetic resonance spectroscopy(³¹P-MRS) in live animals and tumor tissue sampling forATP suggest that P85 and Dox formulations induce pronounced ATP depletionin MDR tumors. Finally, these formulations were also shown to increasetumor apoptosis in vivo by terminal deoxynucleotidyl transferase dUTPnick end labeling (TUNEL) assay and reverse transcription polymerasechain reaction (RT-PCR) for caspases 8 and 9.

In phase I clinicalstudy of SP1049C in 26 patients, maximum tolerateddose (MTD) and dose-limiting toxicity (DLT) were determined as 70and 90 mg/m² respectively. SP1049C also showed slower clearancecompared to conventional Dox. In phase II study 21 patients (19 evaluablefor response) with metastatic or locally advanced unresectable adenocarcinomaof the esophagus and gastroesophageal junction (GEJ) were treatedwith 75 mg/m² SP1049C every 3 weeks until disease progressionor unacceptable toxicity. In this study SP1049C demonstrated prominentsingle agent antitumor activity (47% objective response rate in theevaluable population, 9 partial responders, 10 month median overallsurvival, and 6.6 month progression free survival) with toxicity profilesimilar to that of Dox at equivalent dose and administration schedule.

Unique biological activities of Pluronics in addition to theirdrug solubilization properties make Pluronics a very attractive platformfor drug delivery. For example, in recent work Chen and coauthorsused mixed micelles of P105 and F127 to overcome Pgp-mediated MDRto methotrexate (MTX) in vitro and in vivo.¹²⁵ This system has shown relativelyhigh drug loading and pH-dependent drug release, improved pharmacokinetics,biodistribution and antitumor activity in human lung (A549) and oralepidermoid carcinoma (KBv) MDR xenograft tumor models, and reducedsystemic toxicity (Table 1). The same grouphas also used Pluronic P105/F127 mixed micelles to deliver docetaxel(DTX) to Taxol-resistant non-small cell lung cancer.¹²⁶ While in drug sensitive cells the micelles had similarIC50 to Taxotere, in drug-resistant A549/Taxol cells they demonstrated10-fold lower IC50 compared to Taxotere control (0.059 μg/mLvs to 0.593 μg/mL). In in vivo A549/Taxol drug-resistant tumormodel DTX loaded mixed Pluronic micelles showed 69.05% tumor inhibition,versus 34.43% for Taxotere control (Table 1).¹²⁶

In another work Shen et al.developed novel Pluronic-polyethyleneimine (PEI)/d-α-tocopheryl polyethylene glycol 1000succinate (TPGS) nanoparticles to overcome paclitaxel (PTX) drug resistanceand codeliver survivin shRNA.¹²⁷ TPGS wasused to improve micelle stability and drug loading, P85 was used toform micelles and inhibit GST activity, and PEI was used to bind shRNA.These complex nanoparticles have shown a synergistic effect in cytotoxicityexperiments in A549/T PTX resistant cells, but not in parental A549drug sensitive cells, and displayed effective antitumor activity in vivo in MDR tumor model. Furthermore, the authors haveshown that GST isolated from MDR cells was 3.8 times more active thanextracted from sensitive cells and that both P85 and P85–PEIconjugate effectively inhibited only GST of MDR cells but not of non-MDRcells. This is an important observation, since GST plays an importantrole in PTX metabolism and its inhibition would increase accumulationof PTX in the cells. Other examples that use Pluronic MDR reversalproperties for overcoming MDR include poly(caprolactone)-modifiedPluronic P105 (P105-CL) PTX loaded micelles developed by Wang et al.¹²⁸ to overcome ovarian cancer PTX drug resistance.These polymers displayed ATP depletion, inhibition of mitochondrialfunction, and membrane fluidization activities, similar to what wasreported before for other Pluronics.^57,99 A few yearsearlier the same group developed folate-targeted Pluronic micellesfor delivery of PTX and circumvention of MDR.¹²⁹ The authors have shown that folate conjugated PluronicP105 or L101 PTX loaded micelles better accumulate in MCF7/ADR cellsand have significantly higher efficiency compared to nontargeted micellesof PTX alone (Table 1).

The biological response-modifying propertiesare, however, notunique to Pluronics. A number of other natural and synthetic polymershave been reported to inhibit drug efflux transporters.^130,131 For example, polymers developed by Cambon and colleagues with similararchitecture to Pluronics, but with poly(styrene oxide) (PSO) insteadof PPO, also form micelles which have shown efficient drug loadingand pH-dependent release, as well as Pgp inhibition activity.¹³⁰ Furthermore, in another study from the samegroup the authors evaluated the structure–activity relationshipsof nearly 30 copolymers with structures similar to Pluronics, butcontaining different hydrophobic blocks, including propylene oxide,lactide, methylene, butylene oxide, valerolactone, caprolactone, styreneoxide, and glycidyl.¹³² Many of the screenedcopolymers induced increase of Dox accumulation in the Pgp overexpressingMDR cells, as well as inhibition of Pgp ATPase activity. Notably,the most active copolymers had longer hydrophobic chains comparedto what is considered optimal for Pluronics,⁹⁹ that is, Pluronics with intermediate length of hydrophobic blockand relatively low HLB.

Furthermore, TPGS was also reportedto inhibit Pgp.¹³³ TPGS is a common formof vitamin E, and ithas been recognized as a potent enhancer of oral absorption of drugsdue to inhibition of drug efflux transporters. Collnot et al. comparedTPGS with different PEG lengths (200–6000) and have found thatcommercial TPGS-1000 is one the most potent analogues in the seriesof polymers. Other pharmaceutical excipients, including some Tweens(PEGylated sorbitanes), Brij (Alkyl-PEO surfactants), and Myrj (PEO-stearates),also demonstrated Pgp inhibition, that strongly depends on HLB ofthe polymer,¹³⁴ albeit they generally remainless potent than Pluronics.

Altogether, there are number ofpolymers that possess the advantageousproperties of inhibition of drug efflux transporters and can be usedto overcome cancer MDR or to improve oral drug bioavailability. Pluronics,however, represent the most studied group of potent polymers withrespect to molecular mechanism of Pgp inhibition and MDR sensitization.Considering similar activities observed in other groups of polymers,it is likely that some general patterns of structure–activityrelationships of Pluronics (HLB, architecture, etc.) and spectrumof biological effects can be extrapolated to other amphiphilic polymers.

7Conclusions

Intrinsicand acquired drug resistance represents the great obstaclefor successful treatment of cancer. Numerous approaches have beenutilized in attempts to overcome drug resistance with limited success.In this review we have discussed the biological properties of Pluronicblock copolymers and other polymers with similar biological activities,which, in addition to carrier function, make them an attractive platformfor drug delivery. The MDR chemosensitization activity of Pluronics(and other surfactants) has been known for a while now, and the mechanismshave been extensively studied (Figure 5). However,we are still far from complete understanding of how exactly Pluronicsinteract with MDR cells and why these effects are specific to MDRphenotype. Recent studies have shown that combination of chemotherapeuticdrug (Dox) with Pluronic effectively depletes tumorigenic cell subpopulationand decreases tumorigenicity and tumor aggressiveness.²² This finding being so simple by nature drasticallychanges the whole concept from Pluronics being just another MDR modulatorto a class of agents that might help to combat cancer at its rootby killing CSCs. On the other hand, we now have even more questionsregarding the mechanism of action of Pluronic than we had before.We believe that thorough understanding of these mechanisms will allowbetter design of Pluronic (and similar polymers)-based drug deliverysystems for effective cancer therapy.

Figure 5

Summary of Pluronic effects in cancercells. (A) Pluronic bindingwith plasma membrane of MDR cancer cells (1) induces membrane fluidization,disruption of membrane microdomains, and inhibition of drug effluxtransporters’ activity (Pgp shown as an example). Pluronicalso reaches mitochondria where it (2, 3) inhibits complexes I andIV of mitochondria respiratory chain and (3) induces inner mitochondrialmembrane depolarization. This (4) results in ATP depletion and (5)promotes cytochrome c release and ROS generationin MDR cells. Altogether, the MDR cells respond to a Dox/Pluroniccombination by (6) an increased proapoptotic signaling and decreasedantiapoptotic defense. (B) Moreover, Dox/Pluronic combination effectivelydepletes tumorigenic subpopulation of CSCs, prevents development ofMDR, and significantly alters DNA methylation and gene expressionprofiles.