Nanomedicinal Strategies to Treat Multidrug-resistant Tumors: Current Progress

Drug Delivery Systems to Overcome P-gp-mediated Drug Resistance & their Possible Mechanisms

As mentioned previously, MDR is a major impediment to the success of cancer chemotherapy. P-gp was the first multidrug transporter discovered, and remains the best characterized and most clinically relevant to date. As such the majority of clinical trials have focused on P-gp, as highlighted in Table 1. P-gp is the product of the MDR1 gene and effluxes drugs without chemical modification. Approximately 50% of the anticancer drugs used clinically today are substrates of P-gp.^[100] The emergence of drug resistance has made many of the chemotherapy drugs ineffective. Different strategies attempting to overcome P-gp-mediated drug resistance have been developed as previously described. Therefore, this section focuses on overcoming P-gp-mediated drug resistance using drug delivery systems. Unlike the potentially more serious effects of the active P-gp inhibitors, drug delivery systems may be inhibitors of P-gp with low pharmacological activity and reduced side effects. The formulations discussed here to overcome MDR are summarized in Table 2 and will be highlighted in the following sections.

P-gp inhibition by surfactant-based formulations

In 1972, a published study demonstrated that Tween 80 enhanced the cytotoxicity of actinomycin D and daunomycin in Chinese hamster resistant cells.^[101] Since this report, a number of lipid and polymeric excipients present in pharmaceutical formulations have been reported to modulate the activity of P-gp. P-gp, an efflux pump located in the apical membranes of intestinal absorptive cells, can reduce the absorption of drugs and consequently decrease the oral bioavailability. Thus, the lipid formulation strategy for enhancing absorption of drugs that are P-gp substrates became an attractive topic and has been extensively reviewed for oral delivery.^{[100,102–104]} Caco-2 and MDCK cells expressing P-gp are the most widely used cell models to study the oral absorption. Most of the surfactants inhibiting P-gp are nonionic. They can be divided into two classes according to the chemical structure.^[102] The first class of surfactants exhibit a hydrophilic head group and a hydrophobic tail responsible for membrane anchoring, including triglycerides, Cremophor EL, Solutol HS-15, vitamin E TPGS, Tween 80 and Brij 35. The second class of surfactants, which lack a typical membrane anchor, includes PEG and polyethylene oxide (EO)–polypropylene oxide (PO) block copolymers (Pluronics or poloxamers). All surfactant inhibitors contain a unit that comprises hydrogen bond acceptor groups such as ester groups and polyoxyethylene sequences, which could form hydrogen bonds with the transmembrane sequences of P-gp, which are rich in hydrogen bond donor groups. Provided that the binding affinity of surfactants is higher than that of the drug, the surfactants may be used to inhibit P-gp and, therefore, enhance drug absorption. The inhibitory effects of surfactants on P-gp efflux are related to the structure of surfactants, such as the length of the hydrophilic chain and hydrophilic–lipophilic balance (HLB). TPGS 1000 has been reported to influence drug efflux well below its reported critical micelle concentration (CMC) of 0.02 wt%.^[105] A structure–activity relationship study was carried out to understand the influence of PEG chain length on apical efflux transporters in Caco-2 cell monolayers.^[106] TPGS analogs containing different PEG chain lengths (molecular weight from 200 to 6000 Da) were synthesized. The results suggested that PEG chain length was essential to influence rhodamine 123 efflux in vitro. TPGS 1000 turned out to be one of the most potent analogs to inhibit P-gp efflux. The effect of HLB values of excipients on P-gp modulation was also evaluated for their effects on the uptake of epirubicin in Caco-2 cells.^[107] Surfactants with enhanced efficacy in this study, including Tween 20, Tween 80, Brij 30 and Myrj 52, consisted of a polyethylene and intermediate hydrocarbon chain. The characteristics of the surfactants allow them to partition between lipid bilayers and P-gp domains. The optimal HLB value to enhance epirubicin uptake was in the range of 10 to 17. A related study on Pluronic block copolymers with varying length of EO and PO segments was performed in bovine brain microvessel endothelial cells.^[108] The most efficacious block copolymers exhibited intermediate length of 30–60 PO blocks and a relatively hydrophobic structure (HLB <20; e.g., Pluronic P85 with 40 PO blocks and an HLB of 16). The common mechanisms of surfactants to inhibit P-gp may include binding competition of drugs with surfactants resulting from an interaction between surfactants and P-gp, and membrane fluidization leading to an indirect protein destabilization.^[108–111] However, the latter mechanism may not be the case for some surfactants. For example, TPGS tends to make lipid bilayers more, rather than less, rigid.^[110]

Liposomes

Liposomes have been, and continue to be, the most intensively researched colloidal drug delivery systems even for more than four decades after their discovery. Liposomes are normally composed of phospholipids that spontaneously form multilamellar, concentric bilayer vesicles, with layers of aqueous media separating the lipid layers. The particle size of small unilamellar vesicles, which are comprised of a single, lipid outer layer with an aqueous core, is in the range 20–80 nm. The surface of the liposomes may be charged or uncharged based on the selection of different phospholipids. There are many methods to prepare liposomes, including precipitation.^[112] Liposomes may be used to load both hydrophobic and hydrophilic drugs. Hydrophilic drugs reside in the aqueous core, whereas hydrophobic drugs tend to remain in the lipid layers. Hydrophobic drugs are added during the formation of liposomes. Hydrophilic drugs may also be loaded during formation, but for charged drugs the pH-gradient method may be used wherein a pH gradient between the internal and external aqueous domains drives the drug into the interior of the liposomes by partitioning through the membrane. Liposomes have poor loading capacity for hydrophobic drugs that cannot be dissolved in sufficient amounts in the phospholipid bilayer or sequestered in the liposome core. Furthermore, after intravenous administration, such drugs often rapidly partition from the bilayers into cells, or bind to serum proteins, preventing accumulation at the target site. Several liposomal drugs are now marketed including Ambisome® (amphotericin B), Doxil® (doxorubicin hydrochloride) and Visudyne® (verteporfin) to name a few.

Liposomal delivery systems have been shown to inhibit P-gp efflux.^{[66,113–117]} The proposed mechanisms included bypassing P-gp through an endocytosis pathway^[117] and direct interaction with P-gp. The interaction of liposomes with P-gp was proved by complete inhibition of photoaffinity labeling of P-gp by azidopine.^[114] However, other studies demonstrated that liposomes had limited effectiveness in addressing P-gp-mediated resistance in laboratory in vitro models of cellular resistance and in clinical studies.^[118–121] Liposome formulations containing both an anticancer drug and a P-gp inhibitor have been studied recently. The results showed that liposomal coencapsulated drugs had better responses in both in vitro and in vivo resistant models compared with a single drug.^[122–124] Moreover, liposomal targeting delivery systems have been investigated to overcome P-gp-mediated drug resistance.^[123] For example, doxorubicin and verapamil were coencapsulated into liposomes with 95 and 70% loading efficiency, respectively. To achieve active targeting, human transferrin (Tf) was conjugated to the liposomes to target Tf receptors, which are overexpressed in leukemia cells. In resistant leukemia K562 cells (Tf receptor⁺), Tf-conjugated coloaded liposomes showed 5.2- and 2.8-times greater cytotoxicity than nontargeted coloaded liposomes and Tf-conjugated doxorubicin liposomes, respectively. It was concluded that TfR-targeted liposomes coloaded with doxorubicin and verapamil were effective in selective targeting and reversal of drug resistance in cells.^[123]

Polymer, lipid nancapsules & nanoparticles

Nanocapsules have a liquid core (generally an oil) surrounded by a polymeric membrane structured by polymers or a combination of hydrophilic/lipophilic surfactants. Vegetable oils and triglycerides with medium- and long-chain fatty acids are the common components for the lipid cores. The drugs are confined to the lipid core, which serves as a reservoir to allow a high drug loading for hydrophobic drugs and a slow release profile. Thus, nanocapsules are pharmaceutically attractive for water-insoluble drugs.

Solid lipid nanoparticles made from biodegradable or biocompatible solid lipids were developed in the beginning of the 1990s as an alternative colloidal carrier system for controlled drug delivery. Solid lipid nanoparticles are matrix systems in which the drug is physically and uniformly dispersed. The release of a drug incorporated in the lipid matrix occurs due to degradation of the particles by lipases present at the site of injection, leading to a prolonged release of drugs from the solid lipid nanoparticles.^[125] A comprehensive review on solid lipid nanoparticles can be found in the literature.^[126]

A number of studies have investigated encapsulation of anticancer drugs into polymer nanoparticles and lipid nanoparticles (or nanocapsules) to overcome P-gp-mediated drug resistance.^[127–132] Among them, polyalkylcyanoacrylate nanoparticles were the earliest ones investigated in resistant cell lines.^[130] The results showed that nonbiodegradable polymethacrylate nanoparticles can be internalized by an endocytosis process and reverse P-gp-mediated drug resistance in vitro.^[133] For in vivo studies, biodegradable doxorubicin-loaded polyisohexylcyanoacrylate (PIHCA) nanoparticles were developed. These PIHCA nanoparticles showed more cytotoxicity than free doxorubicin in doxorubicin-resistant C6 cells. Later on, more rapidly biodegradable PIBCA nanoparticles were formulated to load doxorubicin. Doxorubicin uptake from PIBCA nanoparticles was different than that from PIHCA nanoparticles, as doxorubicin-loaded PIBCA nanoparticles caused higher cellular doxorubicin uptake than free doxorubicin. In addition, it was demonstrated that PIBCA nanoparticles did not enter cells via an endocytosis pathway and efflux of doxorubicin in nanoparticles had a similar profile with free doxorubicin. Mechanistic studies found that nanoparticles could deliver a high concentration of doxorubicin close to or adhered onto the cell membrane, resulting in saturation of P-gp; the formation of an ion pair between cyanoacrylic acid (a nanoparticle degradation product) and doxorubicin could also mask the positive charge of doxorubicin and facilitate diffusion of doxorubicin across cell membranes.^[134,135] However, in vivo studies using MDR tumors demonstrated that these nanoparticles were not efficacious in vivo, perhaps due to poor delivery to the tumors.^[136] A new polymer–lipid hybrid nanoparticle (PLN) system was used to increase the cytotoxicity of doxorubicin in resistant cells.^[132] Doxorubicin uptake and retention from doxorubicin-loaded nanoparticles were significantly enhanced compared with free doxorubicin. Blank PLNs did not improve doxorubicin uptake and retention in resistant MDA-MB-435/LCC6MDR1 cells. These results indicated that the PLNs did not influence P-gp activity by themselves. The results also revealed that phagocytosis was an important pathway for PLN to enter the cells. Based on this pathway, doxorubicin-loaded PLNs could bypass P-gp, leading to enhanced doxorubicin uptake in resistant cells.^[137] Recently, sodium bis(2-ethylhexyl) sulfosuccinate (AOT)–alginate nanoparticles were evaluated for their potential to overcome P-gp-mediated drug resistance. Doxorubicin-loaded AOT–alginate nanoparticles enhanced the cytotoxicity of doxorubicin in resistant NCI/ADR-RES cells. It was observed that: the uptake of rhodamine was significantly increased using rhodamine-entrapped nanoparticles in resistant cell; blank nanoparticles improved rhodamine accumulation in a dose-dependent manner in resistant cells; and the enhancement in rhodamine accumulation was not due to membrane permeabilization. However, the mechanism of AOT–alginate nanoparticles to overcome P-gp-mediated drug resistance has not been established.^[129] As mentioned previously, the surfactant solutol HS-15, a mixture of free PEG 660 and PEG 660 hydroxystearate, could inhibit P-gp. Solutol HS-15-based lipid nanocapsules (LNC) containing paclitaxel or etoposide were studied for their potential to overcome MDR.^[138,139] Paclitaxel-loaded LNCs were shown to significantly reduce cancer cell survival in comparison with Taxol in 9L cells and F98 cells. Solutol HS-15 on its own did not improve the effects of paclitaxel. Similarly, paclitaxel-loaded LNCs significantly reduced tumor mass in vivo, whereas Taxol did not have a significant effect in an MDR-expressing F98 subcutaneous glioma model. The mixture of solutol HS-15 and paclitaxel did not improve tumor responses in this in vivo model. This indicated the importance of the nanocarrier itself for the anticancer effect on MDR. The study also showed that LNC internalization was not mediated by clathrin-dependent endocytosis. It was assumed that LNC endocytosis involves one or both of the two known cholesterol-enriched membrane microdomains. The consecutive intracellular cholesterol movements would constitute the core of the LNC inhibitory effects on MDR. Thus, according to these mechanism studies, the investigators proposed that the inhibition of the MDR efflux pump by LNCs could result from the interaction of the released intracellular free Solutol HS-15 with MDR efflux pump and redistribution of intracellular cholesterol.^[138] Recently, doxorubicin and paclitaxel-loaded lipid-based nanoparticles containing Brij 78 as a surfactant were used to overcome P-gp-mediated drug resistance. These drug-loaded nanoparticles showed six- to nine-fold lower IC₅₀ values in P-gp overexpressing human cancer cells than those of free drugs.^[140] Paclitaxel nanoparticles showed marked anticancer efficacy in a nude mouse HCT-15 xenograft model via intratumoral injection^[141] and in a nude mouse NCI/ADR-RES xenograft model after intravenous injection^[140] as compared to all control groups. A series of in vitro cell assays were used to understand the underlining mechanisms. Enhanced uptake and prolonged retention of doxorubicin were observed with nanoparticle-based formulations in P-gp-overexpressing cells. Calcein acetoxymethylester assays and ATP assays confirmed that Brij 78 and blank nanoparticles inhibited P-gp and transiently depleted ATP. The change in the mitochondrial potential and mitochondrial swelling were observed to be dominant in MDR cells, suggesting Brij 78 and nanoparticles influence the mitochondrial respiratory chain. It was concluded that nanoparticles may be used to target both drug and biological mechanisms to overcome MDR via P-gp inhibition and ATP depletion.^[140] It is noteworthy that the drugs delivered into MDR cells by PLGA nanoparticles are susceptible to efflux by P-gp.^[142] PLGA nanoparticles were taken up by cells via endocytosis, resulting in an increase of cellular concentration of the drug encapsulated into the nanoparticles. Following entry, nanoparticles were retained in the cytoplasm for a sustained period of time and slowly released the drug in the cellular cytoplasm. However, paclitaxel-loaded PLGA nanoparticles did not show significant cytotoxicity in resistant NCI/ADR-RES cells. It was proved that P-gp activity did not affect the uptake and retention of nanoparticles themselves. Thus, the inefficiency of paclitaxel-loaded PLGA nanoparticles could result from the active efflux of the drug in cytoplasm by P-gp. P-gp could not only extract the drug when the drug diffused into the cell through the lipid bilayer, but also pump out the drug present in the cytoplasm. Consequently, the efficiency of overcoming P-gp based on endocytosis of nanoparticles may be limited.

Polymer–drug conjugates

Poly(N-[2-hydroxypropyl]methacrylamide)(polyHPMA) and HPMA copolymers have been proposed by several groups as potential drug delivery systems. HPMA is a water-soluble, nonimmunogenic synthetic polymer. HPMA copolymer–doxorubicin conjugates have shown the potential to overcome drug resistance.^[143–145] A series of studies on HPMA–doxorubicin conjugates addressed multiple mechanisms of MDR in addition to P-gp-mediated drug resistance. After the HPMA–doxorubicin conjugate was internalized by an endocytosis pathway, the spacer between the polymer and the drug was cleaved by an enzymatic hydrolysis reaction in the lysosomal compartment of the cells, resulting in the release of the drug from the conjugate. Chronic exposure of sensitive A2780 cells to HPMA–doxorubicin conjugates did not induce MDR as measured by quantification of MDR1 gene expression; inhibition of MPR gene expression and a decrease of resistance against Taxol was evident.^[146] By contrast, repeated exposure to free doxorubicin led to an increased resistance to doxorubicin and Taxol, and upregulation of the MDR1 gene. Further in vitro mechanistic studies on overcoming MDR revealed that HPMA–doxorubicin conjugates inhibited: drug detoxification systems by suppressing the expression of genes encoding glutathione and UDP; and cellular defensive mechanism by activating apoptosis signaling pathways and downregulating the expression of the bcl-2 protein family and mechanisms of DNA repair, replication and synthesis leading to more DNA damage.^[147,148] These results indicated that underlying mechanisms triggered by macromolecular carriers can modulate the biological response of the cell at a molecular level, resulting in an overall increased cytotoxicity. The ability of HPMA–doxorubicin conjugates to overcome MDR in vivo was demonstrated in solid tumor mouse models of sensitive human ovarian carcinoma A2780 and resistant A2780/AD tumors.^[149] HPMA–doxorubicin conjugates significantly decreased tumor size by 28-fold in sensitive tumors and 18-fold in resistant tumors, whereas free doxorubicin only reduced tumor size by 2.8-fold in sensitive tumors and had no effect in the resistant tumor model as compared with the control. The underlying mechanisms were also investigated for the in vivo study. An enhanced accumulation of HPMA–doxorubicin conjugates in the tumor was observed and attributed to the EPR effect. The permeability of blood vessels decreased concomitantly with the downregulation of vascular growth and permeability (VEGF) gene in polymer-treated tumors. The other proposed in vitro mechanisms, such as downregulation of the expression of genes responsible for the activity of efflux pumps, detoxification and apoptosis, were also demonstrated in the in vivo studies.

Pluronic micelles

Micelles are the most basic colloidal drug delivery systems and are formed spontaneously in nature. In the body, colloidal micellar species comprising endogenous surfactants and lipid digestion products (i.e., bile salt mixtures) facilitate the absorption of highly insoluble fatty acids and fat soluble vitamins. Micelles have a particle size normally within a 5–100 nm range, are thermodynamically stable and form spontaneously by association of amphiphilic molecules, such as surfactants, under defined concentrations and temperatures. The concentration of a monomeric amphiphile at which micelles appear is termed the critical micelle concentration (CMC). The formation of micelles is driven by the decrease of free energy in the system owing to the removal of hydrophobic fragments from the aqueous environment and the re-establishment of the hydrogen bond network in water. Hydrophobic fragments of the amphiphilic molecules form the core of a micelle, while hydrophilic moieties form the shell of the micelle. When used as drug carriers in aqueous media, micelles solubilize molecules of poorly soluble nonpolar drugs within the micelle core, while polar drugs could be adsorbed on the micelle surface, and substances with intermediate polarity distribute along surfactant molecules in intermediate positions. One limitation of micellar systems is the relatively low hydrophobic volume of the interior of micelles, leading to limited drug loading. Another limitation of conventional micellar systems is the danger of drug precipitation upon the dilution of the solubilized drug with physiological fluids after parenteral administration. The dilution of the formulation by physiological fluids may cause the disassociation of the micelles as the concentration of the surfactants used to solubilize the drugs may be lower than their CMC.

More recently, polymer micelles prepared from amphiphilic copolymers for solubilization of poorly soluble drugs as an alternative to lipid-based surfactant systems have attracted much attention. Polymer micelles offer a more versatile structure, biodegradability and lower CMC, which may lead to better in vivo stability and more conjugation chemistries for linking ligands to the surface of the colloidal system. Polymer micelles are self-assembled from block copolymers comprising a hydrophobic block (e.g., polylactic acid) with a hydrophilic block. As a result of a common progression of development of 'stealth' systems for intravenous administration, PEGylation approaches were used to form stealth micelles to enhance circulation time. Furthermore, the PEG corona can act as a diffusion barrier for hydrophobic drugs to reduce the burst release characteristic of drug-loaded micelles.^[150] Thus, the hydrophilic block on the copolymer typically contains PEG segments with a molecular weight from 1 to 15 kDa. Similar to micelles formed with conventional surfactants, polymeric micelles comprise the core of the hydrophobic blocks stabilized by the corona of hydrophilic chains in water. However, compared with the conventional micelles, polymeric micelles are more stable upon the relatively high dilution conditions experienced in vivo. For example, some amphiphilic copolymers have CMC values as low as 10⁻⁶ M.^[151]

Pluronics are inert block copolymers comprising of poly(EO) (hydrophilic) and poly(PO) (hydrophobic). Pluronics are different from HPMA copolymers due to their amphiphilic nature. Their surfactant properties allow them to self-assemble into micelles with a hydrophobic PO inner core and a hydrophilic EO outer shell. However, similar to HPMA copolymers, Pluronic block copolymer micelles have also been shown to overcome drug resistance. Extensive studies with Pluronic block copolymer micelles have been reviewed.^[152–154] SP1049C-containing doxorubicin in the mixed micelles of Pluronic L61 and F127 is in clinical trials to treat metastatic adenocarcinoma of the upper GI tract. In addition, SP1049C showed more efficient accumulation in tumors than free doxorubicin, while distribution of SP1049C in normal tissues was similar to that of doxorubicin.^[155] Efficacy of SP1049C was confirmed in in vivo experiments in both sensitive and resistant tumor models, including P388 and P388/ADR murine leukemia, Sp2/0 and Sp2/0-Dnr murine myeloma, 3LL-M27 Lewis lung carcinoma, MCF-7 and MCF-7/ADR human breast carcinomas, and KBv human oral epidermoid carcinoma.^[153,155] However, the toxicity of SP1049C was similar to that of free doxorubicin. This suggested that SP1049C did not improve the toxicity profile of free doxorubicin (e.g., cardiotoxicity), which was improved by liposomal doxorubicin. However, there were no additional side effects reported for SP1049C. Disintegration of micelles in biological fluids upon dilution to a concentration below its CMC is a common concern regarding using micelles for drug delivery. The biodistribution and pharmacokinetics of Pluronic P85 micelles suggested that the elimination of P85 was controlled by the renal elimination of P85 unimers and not by the rate of micelle disintegration.^[156] However, micelle disintegration had been reported with other Pluronic micelles. To further address the potential of micelle disintegration, Pluronic L121 and Pluronic P-105 micelles were chemically modified to form a network or crosslink. Therefore, the CMC of the micelles was greatly reduced and the stability was enhanced, while their ability to inhibit P-gp function still remained.^[157,158]

The complex mechanisms associated with the effects of Pluronic block copolymers on MDR cells have been thoroughly studied. These mechanisms include altering membrane microviscosity.^[159,160] It was suggested that unimers (single block copolymer molecules) are responsible for biological modification as the effect of Pluronic copolymer occurred at concentrations below their CMC. The hydrophobic PO chains of Pluronic unimers insert into the hydrophilic regions of the membrane, resulting in alterations of the membrane structure, and decrease in its microviscosity (membrane fluidization); inhibiting drug efflux transporters, such as P-gp and MRPs, through inhibition of P-gp ATPase activity; and depleting intracellular ATP levels.^[159–161] As these pumps are energy dependent, attenuation of these pumps was related to energy deprivation and abolishment of pump-associated ATPase activity. Thus, it can be surmized that Pluronic-mediated direct and indirect energy depletion leads to cessation of the operation of efflux pumps, and consequently sensitizes resistant cell lines to chemotherapeutic agents. A linear correlation between the extent of ATP depletion and chemosensitization elicited was established, further influencing cell apoptosis signaling. Doxorubicin-loaded Pluronic block copolymer P85 significantly enhanced the pro-apoptotic activity of the drug and prevented the activation of the antiapoptotic cellular defense;^[162] decreasing glutathione (GSH) intracellular levels and glutathione-S-transferase (GST) activity, indicating the inhibition of the GSH–GST detoxification system;^[163] and inhibited mitochondria respiratory chain and decreased oxygen consumption in MDR cells, accompanied by a decrease in mitochondria membrane potential, production of reactive oxygen species and release of cytochrome C. Eventually, this results in Pluronic-enhanced drug-induced apoptosis.^[164]

Nanomedicine. 2010;5(4):597-615. © 2010  Future Medicine Ltd.

Cite this: Nanomedicinal Strategies to Treat Multidrug-resistant Tumors: Current Progress - Medscape - Jun 01, 2010.