chemotherapy remains the 
 main treatment option for most cancers 
 despite of its limitations , such as systemic toxicity , severe side 
 effects , and limited efficacy .
the major reason for chemotherapy failure 
 is poor delivery of drug to cancer cells and/or intracellular targets . 

there are a number of barriers that have to be overcome for successful 
 treatment , and multidrug resistance ( mdr ) is one of them .
tumors of 
 different origin have different susceptibility to chemotherapy , and 
 frequently cancers are intrinsically resistant . on the other hand , 

even though many primary tumors and metastatic lesions , for example 
 breast , ovarian , and small cell lung carcinomas initially respond 
 well to the chemotherapeutic treatment , cancers often relapse and 
 develop drug resistance .
moreover , cancer cells simultaneously acquire 
 resistance not only to the drug the patient was treated with but also 
 to the broad spectrum of drugs that are structurally and functionally 
 unrelated to each other .
initially mdr was attributed to the expression 
 of drug efflux transporters on the cell membrane that actively pump 
 the drugs out of the cells .
now it is 
 generally recognized that mdr is a complex phenomenon and usually 
 is governed by one or more of the following mechanisms : ( 1 ) active 
 drug removal by drug efflux transporters of the atp - binding cassette 
 ( abc ) superfamily , such as p - glycoprotein ( pgp , abcb1 ) , multidrug 
 resistance - associated protein 1 ( mrp1 , abcc1 ) , and breast cancer resistance 
 protein ( bcrp , abcg2 ) ; ( 2 ) loss of cell surface receptors or drug 
 transporters or alterations in membrane lipid composition that limit 
 diffusion of the drug into the cells ; ( 3 ) compartmentalization of 
 the 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 ) . mechanisms of mdr in cancer cells : ( 1 ) active drug efflux 
 by drug 
 transporters , such as pgp , mrp , and bcrp ; ( 2 ) loss of cell surface 
 receptors and/or drug transporters or alterations in membrane lipid 
 composition ; ( 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 damage 
 repair ; and ( 8) inhibition of apoptosis . despite much effort contributed to overcoming mdr , the success 
 is still very limited in clinical settings .
first , the modification of treatment regimens by increasing the 
 dose of the administered drug(s ) or using non - cross - resistant drugs . 

second , use of small molecule inhibitors of drug efflux transporters 
 to increase the drug uptake in mdr tumors .
third , use of 
 antibodies and antibody fragments to target and inhibit drug efflux 
 transporters .
fourth , silencing of the gene expression of the drug 
 efflux transporters or antiapoptotic proteins , such as bcl2 using antisense oligonucleotides , sirna , or micro rna .
fifth , use 
 of small molecules to suppress non - abc transporter - mediated resistance . finally , use of nanotechnology - based carriers to bypass drug efflux 
 transporters in mdr cancer cells . of
unfortunately , 
 a simple dose increase has been associated with increased risks of 
 systemic toxicity and severe side effects , while finding a proper 
 combination of non - cross - resistant drugs in many cases is complicated . 

as far as the use of the pgp inhibitors is concerned , the outcomes 
 were often poor , and many such inhibitors failed due to toxicity or 
 drug metabolism associated issues .
moreover , most 
 of the approaches under development face traditional drug delivery 
 issues , which are especially severe in the cases of nucleic acid or 
 protein therapeutics .
nanotechnology offers several advantages 
 both for the delivery 
 of the chemotherapeutic agents , allowing them to bypass drug efflux 
 transporters , and for the delivery of agents that could inhibit drug 
 resistance mechanisms to increase efficacy of the chemotherapy .
nanomedicines have longer circulation times and can passively accumulate 
 in the tumors with leaky vasculature and poor lymphatic drainage by 
 the enhanced permeability and retention ( epr ) effect . attaching specific tumor - targeting antibodies , antibody fragments , 
 or other targeting moieties ( receptor ligands , peptides , etc . )
can 
 result in active targeting of the nanomedicines to the tumor cells , 
 which can further improve drug delivery .
second , two or more active 
 compounds can be incorporated into a single carrier allowing simultaneous 
 delivery of several cytotoxic drugs for combination therapy and/or 
 a cytotoxic drug with a mdr modulator , such as small molecule inhibitor , 
 antibody , or nucleic acid .
third , a nanocarrier can be designed in 
 such a way that it will release its cargo at the tumor site in response 
 to specific tumor conditions , such as ph or presence of particular 
 enzymes , therefore limiting other organs and tissues to the exposure 
 to free drug and reducing systemic toxicity . finally ,
in contrast 
 to small molecules that mainly utilize diffusion to penetrate the 
 cells , nanocarriers are taken up by either  passive  

endocytosis or receptor - mediated endocytosis and , therefore , can bypass 
 drug efflux transporters on the plasma membrane . in the latter case 

the endocytosis is triggered by interaction of targeting ligand with 
 its receptor on plasma membrane , which accelerates the uptake compared 
 to  passive  endocytosis .
if the receptor is predominantly 
 expressed on cancer cells , in addition to faster uptake this allows 
 selective targeting of the nanocarrier to cancer cells .
one such example is represented by a class of copolymers , 
 called pluronic block copolymers or poloxamers , that are widely used 
 in various drug delivery systems and in tissue engineering .
pluronics are triblock copolymers of poly(ethylene 
 oxide ) ( peo ) and poly(propylene oxide ) ( ppo ) , arranged in peo - ppo - peo 
 structure . depending on the length of the blocks the hydrophilic 
lipophilic 
 balance ( hlb ) of the copolymers changes . in the solution pluronics 

the core of the micelles contains ppo blocks and allows incorporation 
 of hydrophobic drugs .
previously thought to be  inert  , 
 pluronics display a unique set of biological activities and have been 
 shown to be potent sensitizers of mdr cancer cells in vitro and in vivo .
moreover , pluronics were shown to prevent the development of mdr 
 upon selection with an anthracycline antibiotic , doxorubicin ( dox ) , 
 both in vitro and in vivo .
we have also recently demonstrated that pluronics in combination 
 with dox can deplete tumorigenic cell subpopulations and decrease 
 cancer cells tumorigenicity and tumor aggressiveness upon 
 treatment in vivo . in 
 this review
the first drug efflux transporter in cancer cells was described 
 by juliano and ling in 1976 .
they have 
 shown that drug - resistant chinese hamster ovary cells express a 170 
 kda membrane glycoprotein , now known as p - glycoprotein ( pgp , abcb1 ) , 
 that was unique to the drug - resistant cells .
the cells were selected for resistance to colchicine and showed 
 cross - resistance to a wide range of different compounds . the degree 
 of drug resistance correlated with the amount of pgp on the cell surface . 

later , in early 1990s a second drug efflux transporter , called multidrug 
 resistance - associated protein ( mrp1 or abcc1 ) , was discovered in a 
 drug - resistant lung cancer cell line .
normally 
 mrp1 plays a major role in cell detoxifying mechanism by transport 
 of 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 wide 
 variety of functionally and structurally diverse but commonly hydrophobic 
 drugs .
another important drug efflux 
 transporter , named breast cancer resistance protein ( bcrp , abcg2 ) , 
 was identified in 1998 by doyle et al . in human breast cancer cell 
 line selected for dox resistance .
its 
 expression is associated with resistance to number of drugs , such 
 as mitoxantrone , camptothecins , anthracyclines , etc .
pgp , mrp1 , and bcrp belong to the large superfamily of 
 atp - binding cassette ( abc ) membrane transporters with 48 members of 
 the superfamily that are divided into 7 subgroups ( a  g ) .
they 
 have conserved structures and ubiquitously expressed in all forms 
 of living organisms , from bacteria to humans .
it is a product 
 of mdr1 gene and can be found in many normal tissues , 
 like epithelial cells of gastrointestinal tract , liver , the luminal membrane of proximal tubular epithelial 
 cells in kidney , cornea , and the luminal membrane of the endothelial cells in the blood 
overall , pgp is mostly expressed 
 in tissues with barrier functions and its main role is to protect 
 the organism from toxic compounds .
it has a typical structure for 
 abc transporters and comprises two trans - membrane domains ( tmds ) , 
 each of which has 6 membrane - spanning  helixes , and two intracellular 
 nucleotide - binding domains ( nbds ) , which bind and hydrolyze atp providing 
 energy for transmembrane movement of the drugs ( figure 2 ) .
pgp substrates are mostly hydrophobic ( but structurally 
 unrelated ) and partition into a lipid bilayer . among these substrates
are important anticancer drugs including 
 several anthracyclines ( dox , daunorubicin , mitoxantrone ) , vincristine , 
 taxanes , etoposide , teniposide , actinomycin d , and others .
understanding 
 the mechanism of pgp function is critical for the design of novel 
 effective mdr modulators .
recently the crystal structure of mouse pgp , which has 87% sequence 
 identity to human pgp , was described ( figure 2 ) . by analyzing the costructures of pgp complexes 
 with two cyclopeptide inhibitors
the authors elucidated the mechanism 
 of drug efflux by pgp and provided insight into the transporter s 
 broad substrate specificity .
the drug - binding pocket of pgp is localized 
 in the tm domain of the protein .
the inward - open conformation of pgp 
 allows the substrate access both from cytoplasm and from the inner 
 leaflet of the membrane but not from the upper leaflet or extracellular 
 space .
the upper part of the drug - binding pocket contains predominantly 
 hydrophobic and aromatic amino acid residues , and the lower half of 
 the chamber has more polar and charged residues .
the drug - binding 
 pocket in pgp is very large and in inward - facing conformation is accessible 
 through two portals that are wide enough to fit hydrophobic drugs 
 and phospholipids and allow pgp to  scan  the inner 
 leaflet to select and bind specific lipids and hydrophobic drugs before 
 transport .
overall , the authors proposed , 
 that pgp has broad flexibility and can sample widely open conformations 
 to accommodate large substrates , explaining the broad substrate specificity 
 of the transporter .
usually the drug enters pgp s binding site 
 from the inner leaflet of the membrane , which stimulates the binding 
 of two molecules of atp by nbds followed by their dimerization .
the 
 dimerization of nbds causes the major conformational change in the 
 protein and formation of the outward - facing structure , open to the 
 extracellular space .
the drug is released due to the change of the 
 affinity of the protein to it or is facilitated by atp hydrolysis , 
 which brings the protein back to the initial state .
( a ) pgp is 
 a transmembrane protein with drug - binding pocket localized in the 
 inner leaflet of the plasma membrane , and two nbd localized in cytoplasm . 

functional pgp is localized in cholesterol , sphingomyelin , and gm1 
 ganglioside - rich membrane microdomains , called lipid rafts , where 
 it is surrounded by fluid phase of the membrane , containing unsaturated 
 fatty acids like dppc .
pgp is pictured in inward - open ( outward closed ) 
 conformation ready to bind substrate .
the model is based on x - ray 
 analysis and nmr data from protein data 
 bank ( http://www.rcsb.org/ ) .
( b ) incorporation of pluronic 
 into lipid bilayer disrupts lipid rafts , possibly causing conformational 
 changes in pgp , which results in inhibition of pgp atpase and transport 
 activities .
as was mentioned 
 above , pluronic block copolymers are potent sensitizers of mdr cells . 

the sensitization mechanism is complex and involves multiple events 
 happening at different levels in the cell .
cell 
 interaction starts in the cell membrane , where drug efflux transporters 
 are localized .
pluronics were shown to be strong inhibitors of abc 
 transporters , specifically pgp , mrp , and bcrp .
the inhibition might be in part 
 due to the alterations of lipid microenvironment of the transporters 
 by pluronic . due to their amphiphilic structure
, pluronic block copolymers 
 can interact with cell membrane and change its properties , which are critical for proper function of abc 
 transporters .
membrane structure and composition play a crucial role in cell 
 physiology , function , and signaling .
plasma membrane is a heterogeneous 
 structure composed of various domains with different lipid composition 
 and packing . in particular , so - called 
  lipid rafts  are compact membrane microdomains containing 
 predominantly cholesterol and sphingolipids ( mainly sphingomyelin ) 
 with long and saturated fatty acids , that are  floating  
 in more fluid membrane phase that contains glycerophospholipids with 
 shorter and unsaturated acyl chains ( figure 2 ) .
these domains are resistant to low 
 temperature solubilization by some detergents , like triton x100 or 
 brij 96 , and this is used for their isolation . depending on the cell 
 line and the method used for membrane fractionation pgp can be found 
 either mostly in detergent - resistant membrane fractions or distributed 
 between the detergent - resistant and detergent - soluble fractions .
furthermore , it was found that pgp distribution between different 
 membrane fractions depends on the transporter s expression 
 level : the lower the expression of pgp is , the greater portion of 
 pgp is localized in detergent - resistant cholesterol - rich membrane 
 domains .
it is well - known that the function 
 of most membrane proteins is directly linked to the composition and 
 viscosity of their lipid microenvironment .
pgp is a lipid flippase and requires interaction with phospholipids 
 for continuous display drug - mediated atpase activity and interaction with the substrate .
moreover , an increasing number of studies report that pgp 
 localization in lipid rafts and precise properties of rafts are essential 
 for the transporter s proper function . for example
depletion of cholesterol with methyl--cyclodextrin 
 in drug - resistant vlb human t - cell lymphoblastic leukemia cells led 
 to disassembly of the lipid rafts , redistribution of pgp from lipid 
 rafts to other microdomains of plasma membrane , and inhibition of 
 pgp transporter activity .
on the other hand , enrichment of membranes 
 with cholesterol also resulted in inhibition of pgp function , although 
 the localization of pgp did not change compared to control .
however , 
 the increase in cholesterol content changed the lipid raft distribution 
 and composition , which most likely accounts for the impairment of 
 the pgp function .
it was also shown recently 
 that caveolin-1 overexpression decreases plasma membrane cholesterol 
 levels ( similar to the effect of methyl--cyclodextrin that 
 depletes cholesterol from the membrane ) and results in the increase 
 of membrane fluidity and inhibition of pgp function in drug - resistant 
 hs578t / dox cells .
another study by barakat 
 et al . demonstrated that there are two functionally different populations 
 of pgp in drug - resistant human cem lymphoblastic leukemia cells .
the first population localized in detergent - resistant 
 membrane fraction has higher atpase activity , which is completely 
 inhibited by orthovanadate and activated by verapamil .
the second 
 population localized in soluble membrane fractions has lower atpase 
 activity and is less sensitive to orthovanadate .
moreover , verapamil , 
 a well - known pgp activator , inhibits pgp atpase activity in this second 
 population .
the authors conclude that 
 interaction of pgp with its substrates could be affected by different 
 lipid microenvironment in soluble membrane fractions , specifically 
 by lower content of cholesterol compared to the detergent - resistant 
 membrane fraction .
pluronic 
 binding to the cell membrane depends on pluronic hydrophobicity 
 and the temperature .
the binding is driven 
 by hydrophobic interactions of ppo chain blocks with the fatty acid 
 residues in the lipid bilayer and by hydrophilic interactions of peo 
 chain blocks with the polar groups of the lipids at the membrane surface . 

this binding may lead to either membrane destabilization or healing of  injured  membranes .
pluronics also exhibit ionophoric activity and can facilitate transmembrane 
 transport of low molecular drugs , accelerate phospholipid s 
 flip - flop rate , and decrease membrane microviscosity .
pluronic effects on the membrane 
 transport depend on the copolymer hlb , concentration , and the exposure 
 time .
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 can 
 act either as a transmembrane carrier of drug molecules or as an ion 
 channel .
specifically , it was proposed 
 that l61 monomers and dimers can act as the carriers while l61 oligomers 
 are likely to form the channels . on the 
 other hand hydrophilic pluronic f68 ( poloxamer 188 , ( eo)76-(po)39-(eo)76 , hlb 29 , mw 8400 g / mol ) with 

80% peo content effectively restores damaged cell membranes after 
 electroporation , heat shock , or intense radiation . using x - ray 
 reflection ( xr ) and grazing - incidence x - ray diffraction ( gixd ) methods 
 in 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 ) , wu 
 et al .
have shown that f68 interacts with the damaged membrane areas , 
 but does not affect the ordered membrane phase , and gets excluded 
 when lipid packing density is restored .
recently it was demonstrated that f68 molecules do not insert into 
 lipid bilayer nor affect the overall lipid packaging , however , they 
 facilitate the membrane sealing activity by diminishing the fluctuation 
 of membrane surface and hydration of the inner part of the bilayer .
however , in another study using giant unilamellar 
 vesicles ( guv ) as model membrane system wang et al . demonstrated that 
 f68 can incorporate in the membranes , disrupt their integrity , and 
 act as a permeabilizer if it is exposed to the membranes for sufficient 
 time .
overall , the interaction 
 of pluronics with lipid membranes proceeds in two steps : ( 1 ) the absorption 
 at the membrane and ( 2 ) the insertion in the membrane ( figure 3 ) .
the first step is common to all pluronics and 
 does not depend much on the copolymer structure .
the second step depends 
 strongly on the hydrophobicity of the copolymer with the more hydrophobic 
 copolymers being morel likely to insert .
pluronics with longer ppo blocks insert into 
 the membrane below the polar head groups , loosen the lipid packaging , 
 and , therefore , act as permeabilizers , they can translocate through the membrane ( depending on their hlb ) . 
 furthermore , using molecular dynamics simulations nawaz and coauthors 
 observed that membrane bends upon insertion of pluronics .
they have shown that membrane - disruptive activity 
 of pluronics is due to interaction of hydrophilic blocks with the 
 polar head groups of the lipid molecules and depends on the length 
 of the peo block .
short peo blocks drag the polar groups toward the 
 inner part of the membrane , which results in membrane bending and 
 permeabilization .
pluronics with longer peo blocks can temporarily 
 stabilize the local structure of the membrane .
schematic presentation 
 of interaction of pluronics with different 
 hydrophobicity with lipid membranes : ( 1 ) absorption of pluronic molecules 
 on the surface of the membrane , ( 2 ) insertion into the lipid bilayer , 
 and ( 3 ) translocation through the membrane .
pluronic copolymers can significantly increase the antitumor 
 activity 
 of pegylated liposomal drugs in vivo , specifically 
 doxil by stimulating the drug release from liposomes at the tumor 
 site .
one of the main problems of long 
 circulating liposomal drugs is insufficient release of the active 
 compound at the tumor site .
post - administration  
 of pluronic p85 ( ( eo)26-(po)40-(eo)26 , hlb 16 , mw 4600 g / mol ) 48 h after doxil results in dox release 
 and redistribution toward tumor bulk along with a marked improvement 
 of antitumor activity .
this effect is time - dependent as it is essential 
 to allow sufficient time for the liposomes to accumulate at the tumor 
 site before administering pluronic .
it is likely that that the enhanced 
 antitumor effect at least in part is due to facilitated release of 
 dox from the liposomes in the tumors induced by pluronic .
furthermore , 
 in addition to permeabilization effect on liposomal membranes the 
 copolymer could also sensitize the mdr cells and deplete the cancer 
 stem cells ( cscs ) ( as discussed below ) .
another 
 important aspect in pluronic interactions with lipid membranes 
 is the dependence of these interactions on the cell type and the membrane 
 composition .
for example , the membrane microviscosity of murine myeloma 
 sp2/0 cells significantly decreased after treatment with l61 , while 
 the membrane viscosity in normal mouse splenocytes was less affected . moreover , pluronic p105 ( ( eo)37-(po)56-(eo)37 , hlb 15 , mw 6500 g / mol ) was demonstrated 
 to permeabilize the acidic endosomal vesicles in drug - resistant a2780/adr 
 cells , while the vesicles in sensitive cells were less affected .
several studies have reported lower 
 fluidity and higher heterogeneity of plasma membrane in mdr cells 
 compared to sensitive cells .
drug - resistant cells 
 also contain smaller amounts of unsaturated fatty acids and have higher 
 content of esterified cholesterol and triglycerides . using liposomes of different lipid composition and viscosity
it 
 was demonstrated that the l61 effects on lipid flip - flop and membrane 
 permeability toward dox increase as the membrane viscosity increases .
pluronics inhibit pgp and mrp atpase activities 
 by decreasing maximum 
 reaction rate ( vmax ) and the affinity 
 of the enzyme to atp as well as to the substrates such as vinblastine 
 ( expressed as increase in michaelis constant , km ) .
some neutral detergents , such 
 as tween-20 , nonidet p-40 , and triton x-100 , were also shown to inhibit 
 pgp atpase activity at concentrations that are required for membrane 
 fluidization .
overall , alterations in 
 membrane structure and fluidity induced by various compounds strongly 
 affect pgp function .
therefore , it was suggested that inhibition of 
 the transporter s activity by pluronic is at least partly due 
 to the pluronic - induced changes in the local membrane environment 
 ( figure 2 ) .
to further 
 understand the mechanism of pluronic sensitization of 
 mdr cancer cells one needs to focus on the events at the subcellular 
 level , which were characterized in great detail using p85 as an example .
this copolymer exhibits evident and profound 
 selectivity with respect to energy metabolism in mdr cancer cells . 

it is rapidly taken up by the cells via a caveolae - mediated endocytosis 
 pathway and colocalizes with mitochondria 
 already 15 min after exposure to the cells .
this results in a drastic depletion of intracellular atp levels 
 in mdr cancer cells , while non - mdr cells require significantly higher 
 doses of pluronic to achieve similar depletion .
noteworthy , the ability 
 to deplete cellular atp levels strongly correlates with the chemosensitization 
 properties of the copolymers in mdr cells .
the selectivity of pluronic copolymers toward mdr phenotype is probably 
 attributable to innate metabolic and physiological differences between 
 mdr and non - mdr cells .
in contrast to normal cells , that use oxidative 
 phosphorylation for atp production , cancer cells mostly rely on glycolysis 
 as an adaptation to hypoxic conditions in the early stages of tumor 
 development .
drug - resistant cells require 
 more atp to support the drug efflux transporter activity and drug 
 metabolism .
adaptations leading to mdr therefore in part are associated 
 with changes in energy metabolism to meet new energy requirements . 

it was shown that human breast cancer cells with acquired resistance 
 to dox exhibit 3-fold higher glycolysis rate than their sensitive 
 counterparts .
another study by miccadei 
 et al . found that both respiration and glycolysis rates are increased 
 in drug - resistant ehrlich cells , resulting in almost 50% higher atp 
 production compared to the drug sensitive cells .
it was also shown that mdr cells have significantly higher 
 activity of the respiratory chain complexes in mitochondria where 
 nearly 50% of atp was produced , compared to only 35% of atp produced 
 in mitochondria of sensitive cells .
moreover , it was later demonstrated 
 that 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 .
overall , the compromised mitochondrial function in mdr 
 cells may be the achilles heel of mdr cells that allows effective 
 and selective inhibition of atp production in drug - resistant cells .
when pluronic reaches mitochondria of mdr cells , it inhibits complexes 
 i and iv of the respiratory chain and depletes mitochondrial membrane 
 potential .
the mechanism of pluronic 
 inhibition of respiratory chain complexes activities is not 
 fully understood . in mitochondria
in other words pluronic 
 may act as a prooxidant , which were shown to induce apoptosis in cancer 
 cells .
noteworthy , the effects of pluronic 
 on pgp activity , atp levels , and cytotoxicity are reversible .
pgp 
 function is restored 1 h after the removal of pluronic . at the same 
 time
the sensitization 
 effect of pluronic is abolished in the same time frame , while it takes 
 about 10 h to restore atp levels .
interestingly , 
 pgp expression seems to be essential for pluronic effects on respiration 
 and atp levels .
inhibition of oxygen consumption as well as atp depletion 
 by pluronic was observed not only in drug - selected resistant cells 
 but also in cells stably transfected with mdr1 gene , encoding pgp .
inhibition of pgp with highly specific inhibitor gf120918 abolished 
 the pluronic - induced atp depletion , while the inhibitor itself did 
 not affect atp levels in mdr cells .
pluronic effects in mdr cancer cells exhibit remarkably simple 
 and clear structure  functional relationships .
the studies of the concentration dependence of the pluronic 
 in mdr cells effect suggested that these effects are produced mainly 
 by the copolymer single chains as they leveled up or decreased above 
 the cmc .
hydrophilic pluronics with hlb 20 and above have little if 
 any sensitization effect in mdr cells . using pgp expressing brain 
 microvessel endothelial cells ( bmecs )
it was demonstrated that such 
 pluronics do not decrease membrane microviscosity , do not inhibit 
 pgp atpase activity , practically do not internalize in the cells , 
 and do not induce atp depletion . of all 
 other pluronics with hlb fewer than 20
the most active in mdr cells 
 are the copolymers with intermediate lengths of the hydrophobic ppo 
 block from about 30 to about 60 po units .
these 
 copolymers bind with the cell membranes , decrease membrane microviscosity , 
 and inhibit pgp atpase activity .
moreover , 
 they internalize into cells and produce atp depleting effects . the 
 copolymers with shorter ppo blocks , fewer than 30 po units , also internalize 
 in cells . however , they do not decrease membrane microviscosity , do 
 not inhibit pgp atpase , and do not deplete atp .
presumably , they are 
 not sufficiently  disruptive  to the membrane structures 
 to produce all these effects . the copolymers with longer ppo blocks 

but they do not penetrate inside the cells and do not 
 reach mitochondria remaining stuck in the cell membranes , presumably 
 due to their extreme hydrophobicity . accordingly
notably , it was demonstrated that both atp depletion and inhibition 
 of pgp atpase activity are essential for the sensitization of pgp 
 overexpressing cells .
when one of these factors was 
 excluded , the drug efflux pump remained functional in both mdr cancer 
 and pgp - expressing bmecs .
oxidative stress is 
 a condition in which the balance between the 
 production of reactive oxygen species ( ros ) by cells and the ability 
 to detoxify them is impaired .
if oxidative stress persists , the formed 
 peroxides and free radicals will damage all components of the cell , 
 including membranes , proteins , and dna .
accumulation of significant 
 damage , which a cell fails to repair , will lead to apoptosis .
generally , 
 oxidative stress is associated with increased production of ros and/or 
 decreased ability of the cell to eliminate these species .
glutathione 
 is 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 cellular 
 glutathione is in a reduced state .
an accurate ratio between gsh and 
 gssg is important to maintain the intracellular redox state , with 
 a decrease in gsh / gssg ratio indicative of oxidative stress .
gsh is 
 also a cofactor of glutathione s - transferase ( gst ) , 
 the major cellular detoxifying enzyme .
furthermore , several members 
 of the mrp family of abc transporters require gsh for transport activity . 

pluronic was shown to deplete the gsh levels and inhibit the gst activity 
 in several mdr cell lines .
inhibition 
 of the gsh / gst detoxifying system in turn decreases the mrp - mediated 
 efflux .
the decrease of cellular gsh is also an early sign of apoptosis 
 induced by oxidative stress , death receptor activation , or mitochondrial 
 apoptotic signaling .
one of the 
 major sources of ros in the cells is electron transport 
 chain in mitochondria . in normal conditions oxygen
is reduced in mitochondria 
 by cytochrome c oxidase ( complex iv ) to produce water . 
 however , a small amount of electrons passing through the electron 
 transfer chain reduce oxygen to produce superoxide radical .
the main 
 superoxide 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 inhibitors 
 like rotenone , piericidin a , and rolliniastatin increases the ros 
 production . as was mentioned above , pluronic quickly reaches mitochondria 
 and inhibits complexes i and iv in mdr cells ( figure 4 ) .
moreover , it stimulates the production of ros and release 
 of cytochrome c , which are the early signs of mitochondrial 
 apoptotic pathway . if ros are not neutralized , 
 they induce damage of mitochondrial membrane , proteins , and dna .
this 
 leads to permeabilization of outer mitochondrial membrane , swelling 
 of mitochondria , and release of proapoptotic proteins , like cytochrome c , apoptosis inducing factor ( aif ) , and endonuclease g. in cytoplasm 
 cytochrome c binds to apoptosis protease activating 
 factor ( apaf-1 ) and forms apoptosome .
the activated caspase 9 in turn 
 activates the effector caspases , which all together contribute to 
 the completion of apoptosis .
similar to atp depletion and inhibition 
 of respiration , pluronic induced the ros formation and cytochrome c release selectively in mdr cells , while non - mdr cells 
 did not respond in that manner .
pluronic quickly enters the cells , reaches mitochondria , 
 and induces mitochondrial membrane depolarization ( 1 ) , inhibition 
 of 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 significantly 
 enhanced the proapoptotic signaling compared to the drug alone and 
 inhibited the antiapoptotic defense mechanisms in vitro .
it was demonstrated that dox / pluronic treatment 
 of tumor - bearing mice significantly increased levels of caspases 8 
 and 9 compared to dox alone .
overall , 
 pluronic induces early as well as late stages of proapoptotic 
 signaling in mdr cells in vitro and in vivo .
inhibition of mitochondria respiratory chain complexes is most 
 likely the main reason for increased ros production in mdr cells after 
 treatment with pluronic .
additionally , depletion of major intrinsic 
 cellular antioxidant gsh would increase cell sensitivity to the ros . 

it has been shown that drug - induced ros production may be directly 
 linked to their cytotoxic activity and that 
 detoxification of free radicals by gsh / gst is very important in mdr 
 cells to facilitate drug resistance .
therefore , when combined with dox , pluronic not only drastically 
 increases the drug accumulation in the cells but also promotes the 
 apoptosis in the mdr cells .
the mechanism of development of mdr in cancer remains 
 a highly 
 debated subject , and most likely there is no uniform theory that will 
 apply to all cancers .
it is now widely accepted that cscs play an important role in cancer 
 development , metastasis , and development of drug resistance .
cscs 
 comprise a small cell subpopulation within the tumor with distinct 
 functional and phenotypical characteristics .
first , cscs overexpress 
 specific markers . however , these markers differ from cancer to cancer 
 and to date there is no uniform marker that can be used to isolate 
 cscs from every tumor .
second , cscs 
 have unlimited ability to divide and produce cells of all other phenotypes 
 in the tumor .
third , cscs are able to form tumors when transplanted 
 into mice and to form so - called tumorspheres when grown in anchorage 
 independent conditions .
finally , cscs are intrinsically drug resistant : 
 they overexpress drug efflux transporters , such as pgp and bcrp , have 
 active antiapoptotic pathways , and spend most of their time in the 
 g0 nondividing cell cycle state , which makes them insensitive 
 to cytostatic drugs often used in chemotherapy .
therefore , cscs can avoid classical chemotherapy and repopulate 
 the tumor , possibly leading to mdr development
. moreover , there are 
 reports suggesting that cscs phenotype is dynamic and can 
 be acquired by non - cscs under certain conditions .
overall , successful therapy needs to be equally efficient in eliminating 
 both bulk tumor cells and cscs .
in addition to mdr chemosensitization 
 properties , pluronics also 
 prevent the development of mdr upon selection with cytotoxic drugs in vitro and in vivo .
specifically , in one study human breast carcinoma mfc7 cells were 
 selected with dox for drug resistance in the presence or absence of 
 p85 at concentration below cmc ( 0.001 wt % ) .
the cells cultured with dox / p85 were not able to grow at concentrations 
 of the drug exceeding just 10 ng / ml . in contrast , cells cultured with 
 dox alone eventually developed mdr and could tolerate up to 10,000 
 ng / ml dox in the culture media .
further analysis has shown that cells 
 treated with dox / p85 did not overexpress pgp and , therefore , remained 
 sensitive to the drug .
in contrast , cells exposed to dox alone exhibited 
 significant overexpression of pgp . this developed drug resistance 
 can be resensitized by pluronic to the initial level of the drug sensitive 
 cells .
interestingly , when the cells were selected with lower concentration 
 of dox , they were not sensitized by pluronic , even though they displayed 
 low levels of pgp expression and detectable levels of mdr1 mrna .
functional 
 analysis of pgp activity using accumulation of pgp substrate ( rhodamine 
 123 ) showed that pgp in those cells was not or nearly not functional 
 compared to more resistant cells . even though 
 cells selected with lower concentrations of dox were not sensitized 
 with pluronic , they showed strong atp depletion in response to pluronic 
 treatment .
moreover , it was demonstrated 
 that selection of cells with dox and dox / p85 resulted in very different 
 changes in the gene expression patterns in these cells .
p85 alone , 
 however , had little if any effect on the gene expression .
similar results were observed in p388 murine 
 leukemia tumor cells selected for dox resistance with or without p85 
 both in vitro and in vivo .
overall , this suggests that simple addition 
 of  inert  polymer excipient to the drug drastically 
 changes pharmacogenomic responses of cancer cells to this drug .
however , our understanding of the mechanism behind the prevention 
 of mdr development by pluronic and alterations in gene expression 
 profiles is very limited . in view of csc theory
a small population 
 of tumor cells is guiding tumor progression , metastasis , and mdr development . 

since cscs share certain characteristics of mdr cells , such as overexpression 
 of drug efflux transporters ( pgp , bcrp ) and altered metabolic pathways , we proposed that pluronics can sensitize cscs to chemotherapeutic 
 drugs similar to mdr cells . in a recent study using the same p388 
 leukemia ascitic tumor model as before
, we demonstrated that dox / pluronic combination , sp1049c , comprising 
 mixed micelles of pluronic f127 ( ( eo)100-(po)65-(eo)100 , hlb 22 , mw 12  600 g / mol ) and l61 , effectively 
 decreases frequency of tumor initiating cells and , as a result , suppresses 
 tumorigenicity and tumor aggressiveness in vivo . in agreement with previous findings
, sp1049c 
 also 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 reduced 
 the in vitro colony forming potential of the cells 
 already at 10 ng / ml dox , while dox alone had the same effect at 10 
 times higher concentration .
as mentioned above , dox / pluronic combination 
 drastically changes the gene expression profiles in cancer cells compared 
 to dox or pluronic alone upon continuous exposure . in this work we 
 have shown that dna methylation patterns also change drastically upon in vivo treatment of cancer cells with sp1049c compared 
 to saline control , polymers , or dox alone .
it is well - known that misregulation 
 of dna methylation / demethylation plays an important role in cancer 
 origin , progression , angiogenesis , metastasis , and mdr development .
sp1049c not only induced the strongest epigenetic changes but also 
 showed very small overlap of affected genes with other treatment groups . 
 functional analysis of affected genes done using ingenuity pathway 
 analysis ( ipa ) has shown that the top affected biological functions 
 and canonical pathways affected by sp1049c treatment relate to cellular 
 function , growth , and maintenance , as well as regulation of stem cell 
 differentiation and pluripotency .
altogether , on top of mdr sensitization , 
 the prevention of mdr development by pluronics , depletion of tumorigenic 
 cell subpopulations , and decrease of tumorigenicity and tumor aggressiveness 
 offer significant advantages for the development of new formulations 
 of approved and/or experimental therapeutics .
pluronic copolymers attracted a lot 
 of attention in drug delivery 
 and tissue engineering applications .
pluronic - based micellar formulation 
 of dox , sp1049c , was the first in class polymeric micelle drug to 
 advance to clinical stage and has successfully 
 completed phase ii clinical trial in advanced esophageal cancer patients . in studies in rodent and nonrodent animal models 

it has been demonstrated , as well as in patients , that mtd and pharmacokinetic 
 profiles of dox alone and sp1049c are very similar .
moreover , animal studies using mdr overexpressing 
 tumors have shown that pluronic formulations in vivo exhibit key effects 
 observed in mechanistic studies in vitro .
first , noninvasive single photon emission computed tomography ( spect ) 
 and tumor tissue radioactivity sampling demonstrated that intravenous 
 coadministration of pluronic p85 with a pgp substrate , tc - sestamibi , greatly increases the tumor uptake of this substrate 
 in the mdr tumors .
second , p magnetic resonance spectroscopy 
 ( p - mrs ) in live animals and tumor tissue sampling for 
 atp suggest that p85 and dox formulations induce pronounced atp depletion 
 in mdr tumors .
finally , these formulations were also shown to increase 
 tumor apoptosis in vivo by terminal deoxynucleotidyl transferase dutp 
 nick end labeling ( tunel ) assay and reverse transcription polymerase 
 chain reaction ( rt - pcr ) for caspases 8 and 9 . in phase
i clinical 
 study of sp1049c in 26 patients , maximum tolerated 
 dose ( mtd ) and dose - limiting toxicity ( dlt ) were determined as 70 
 and 90 mg / m respectively .
ii study 21 patients ( 19 evaluable 
 for response ) with metastatic or locally advanced unresectable adenocarcinoma 
 of the esophagus and gastroesophageal junction ( gej ) were treated 
 with 75 mg / m sp1049c every 3 weeks until disease progression 
 or unacceptable toxicity . in this study
sp1049c demonstrated prominent 
 single agent antitumor activity ( 47% objective response rate in the 
 evaluable population , 9 partial responders , 10 month median overall 
 survival , and 6.6 month progression free survival ) with toxicity profile 
 similar to that of dox at equivalent dose and administration schedule .
unique biological activities of pluronics in addition to their 
 drug solubilization properties make pluronics a very attractive platform 
 for drug delivery . for example
, in recent work chen and coauthors 
 used mixed micelles of p105 and f127 to overcome pgp - mediated mdr 
 to methotrexate ( mtx ) in vitro and in vivo .
this system has shown relatively 
 high drug loading and ph - dependent drug release , improved pharmacokinetics , 
 biodistribution and antitumor activity in human lung ( a549 ) and oral 
 epidermoid carcinoma ( kbv ) mdr xenograft tumor models , and reduced 
 systemic toxicity ( table 1 ) . the same group 
 has also used pluronic p105/f127 mixed micelles to deliver docetaxel 
 ( dtx ) to taxol - resistant non - small cell lung cancer . while in drug sensitive cells the micelles had similar 
 ic50 to taxotere , in drug - resistant a549/taxol cells they demonstrated 
 10-fold lower ic50 compared to taxotere control ( 0.059 g / ml 
 vs to 0.593 g / ml ) . in in vivo a549/taxol drug - resistant tumor 
 model dtx loaded mixed pluronic micelles showed 69.05% tumor inhibition , 
 versus 34.43% for taxotere control ( table 1 ) . in another work shen et al . 

developed novel pluronic - polyethylene 
 imine ( pei)/d--tocopheryl polyethylene glycol 1000 
 succinate ( tpgs ) nanoparticles to overcome paclitaxel ( ptx ) drug resistance 
 and codeliver survivin shrna .
tpgs was 
 used to improve micelle stability and drug loading , p85 was used to 
 form micelles and inhibit gst activity , and pei was used to bind shrna . 

these complex nanoparticles have shown a synergistic effect in cytotoxicity 
 experiments in a549/t ptx resistant cells , but not in parental a549 
 drug sensitive cells , and displayed effective antitumor activity in vivo in mdr tumor model .
furthermore , the authors have 
 shown that gst isolated from mdr cells was 3.8 times more active than 
 extracted from sensitive cells and that both p85 and p85pei 
 conjugate effectively inhibited only gst of mdr cells but not of non - mdr 
 cells .
this is an important observation , since gst plays an important 
 role in ptx metabolism and its inhibition would increase accumulation 
 of ptx in the cells .
other examples that use pluronic mdr reversal 
 properties for overcoming mdr include poly(caprolactone)-modified 
 pluronic p105 ( p105-cl ) ptx loaded micelles developed by wang et al .
these polymers displayed atp depletion , inhibition of mitochondrial 
 function , and membrane fluidization activities , similar to what was 
 reported before for other pluronics .
a few years 
 earlier the same group developed folate - targeted pluronic micelles 
 for delivery of ptx and circumvention of mdr .
the authors have shown that folate conjugated pluronic 
 p105 or l101 ptx loaded micelles better accumulate in mcf7/adr cells 
 and have significantly higher efficiency compared to nontargeted micelles 
 of ptx alone ( table 1 ) .
the biological response - modifying properties 
 are , however , not 
 unique to pluronics .
a number of other natural and synthetic polymers 
 have been reported to inhibit drug efflux transporters .
for example , polymers developed by cambon and colleagues with similar 
 architecture to pluronics , but with poly(styrene oxide ) ( pso ) instead 
 of ppo , also form micelles which have shown efficient drug loading 
 and ph - dependent release , as well as pgp inhibition activity .
furthermore , in another study from the same 
 group the authors evaluated the structure  activity relationships 
 of nearly 30 copolymers with structures similar to pluronics , but 
 containing different hydrophobic blocks , including propylene oxide , 
 lactide , methylene , butylene oxide , valerolactone , caprolactone , styrene 
 oxide , and glycidyl .
many of the screened 
 copolymers induced increase of dox accumulation in the pgp overexpressing 
 mdr cells , as well as inhibition of pgp atpase activity .
notably , 
 the most active copolymers had longer hydrophobic chains compared 
 to what is considered optimal for pluronics , that is , pluronics with intermediate length of hydrophobic block 
 and relatively low hlb . furthermore , tpgs was also reported 
 to inhibit pgp .
tpgs is a common form 
 of vitamin e , and it 
 has been recognized as a potent enhancer of oral absorption of drugs 
 due to inhibition of drug efflux transporters .
compared 
 tpgs with different peg lengths ( 2006000 ) and have found that 
 commercial tpgs-1000 is one the most potent analogues in the series 
 of 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 of 
 the polymer , albeit they generally remain 
 less potent than pluronics . altogether ,
there are number of 
 polymers that possess the advantageous 
 properties of inhibition of drug efflux transporters and can be used 
 to overcome cancer mdr or to improve oral drug bioavailability .
pluronics , 
 however , represent the most studied group of potent polymers with 
 respect 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  activity 
 relationships of pluronics ( hlb , architecture , etc . ) and spectrum 
 of biological effects can be extrapolated to other amphiphilic polymers .
intrinsic 
 and acquired drug resistance represents the great obstacle 
 for successful treatment of cancer .
numerous approaches have been 
 utilized in attempts to overcome drug resistance with limited success . 
 in this review
we have discussed the biological properties of pluronic 
 block copolymers and other polymers with similar biological activities , 
 which , in addition to carrier function , make them an attractive platform 
 for drug delivery .
the mdr chemosensitization activity of pluronics 
 ( and other surfactants ) has been known for a while now , and the mechanisms 
 have been extensively studied ( figure 5 ) . however , 
 we are still far from complete understanding of how exactly pluronics 
 interact with mdr cells and why these effects are specific to mdr 
 phenotype .
recent studies have shown that combination of chemotherapeutic 
 drug ( dox ) with pluronic effectively depletes tumorigenic cell subpopulation 
 and decreases tumorigenicity and tumor aggressiveness .
this finding being so simple by nature drastically 
 changes the whole concept from pluronics being just another mdr modulator 
 to a class of agents that might help to combat cancer at its root 
 by killing cscs . on the other hand
, we now have even more questions 
 regarding the mechanism of action of pluronic than we had before . 

we believe that thorough understanding of these mechanisms will allow 
 better design of pluronic ( and similar polymers)-based drug delivery 
 systems for effective cancer therapy .
( a ) pluronic binding 
 with plasma membrane of mdr cancer cells ( 1 ) induces membrane fluidization , 
 disruption of membrane microdomains , and inhibition of drug efflux 
 transporters activity ( pgp shown as an example ) .
pluronic 
 also reaches mitochondria where it ( 2 , 3 ) inhibits complexes i and 
 iv of mitochondria respiratory chain and ( 3 ) induces inner mitochondrial 
 membrane depolarization .
this ( 4 ) results in atp depletion and ( 5 ) 
 promotes cytochrome c release and ros generation 
 in mdr cells .
altogether , the mdr cells respond to a dox / pluronic 
 combination by ( 6 ) an increased proapoptotic signaling and decreased 
 antiapoptotic defense .
( b ) moreover , dox / pluronic combination effectively 
 depletes tumorigenic subpopulation of cscs , prevents development of 
 mdr , and significantly alters dna methylation and gene expression 
 profiles .
 multidrug 
 resistance ( mdr ) remains one of the biggest obstacles 
 for effective cancer therapy . 
 currently there are only few methods 
 that are available clinically that are used to bypass mdr with very 
 limited success . in this review 
 we describe how mdr can be overcome 
 by a simple yet effective approach of using amphiphilic block copolymers . 
 
 triblock copolymers of poly(ethylene oxide ) ( peo ) and poly(propylene 
 oxide ) ( ppo ) , arranged in a triblock structure peo - ppo - peo , pluronics 
 or  poloxamers  , raised a considerable interest in the 
 drug delivery field . 
 previous studies demonstrated that pluronics 
 sensitize mdr cancer cells resulting in increased cytotoxic activity 
 of dox , paclitaxel , and other drugs by 23 orders of magnitude . 
 
 pluronics can also prevent the development of mdr in vitro and in vivo . 
 additionally , promising results of 
 clinical studies of dox / pluronic formulation reinforced the need to 
 ascertain a thorough understanding of pluronic effects in tumors . 
 
 these effects are extremely comprehensive and appear on the level 
 of plasma membranes , mitochondria , and regulation of gene expression 
 selectively in mdr cancer cells . 
 moreover , it has been demonstrated 
 recently that pluronics can effectively deplete tumorigenic intrinsically 
 drug - resistant cancer stem cells ( csc ) . 
 interestingly , sensitization 
 of mdr and inhibition of drug efflux transporters is not specific 
 or selective to pluronics . 
 other amphiphilic polymers have shown similar 
 activities in various experimental models . 
 this review summarizes 
 recent advances of understanding the pluronic effects in sensitization 
 and prevention of mdr . 
Introduction
Reversal of ABC Transporter-Mediated Resistance
by Pluronics
Effect
of Pluronic on Cancer Cells Metabolism
Effect
of Pluronic on Proapoptotic Signaling
Pluronics
Prevent Development of MDR and Suppress
CSCs
Recent
Examples of Pluronic-Based and Similar
Drug Delivery Systems
Conclusions