Eradication of cancer stem cells in triple negative breast cancer using doxorubicin/pluronic polymeric micelles.

The potency of polymeric micelle-based doxorubicin, SP1049C, against cancer stem cells (CSCs) in triple negative breast cancer (TNBC) is evaluated. CSCs with high epithelial specific antigen (ESA), high CD44 and low CD24 expression levels were derived from the TNBC cancer cells, MDA-MB-231 and MDA-MB-468. These CSCs were resistant to free doxorubicin (Dox) and displayed increased colony formation, migration, and invasion in vitro, along with higher tumorigenicity in vivo, compared to the parental and non-CSCs counterparts. SP1049C downregulated the expression and inhibited the functional activity of the breast cancer resistance protein (BCRP/ABCG2) in CSCs. The polymeric micelle drug had higher cytotoxicity and potency in reducing the colony formation of CSCs compared to the free drug. It was also more potent in inhibiting the tumor growth in the orthotopic animal tumor models derived from CSCs. These results indicate that SP1049C is active against CSCs and has potential in treating TNBC.

Pluronic block copolymers enhance the anti-myeloma activity of proteasome inhibitors.

Proteasome inhibitors (PIs) have markedly improved response rates as well as the survival of multiple myeloma (MM) patients over the past decade and have become an important foundation in the treatment of MM patients. Unfortunately, the majority of patients either relapses or becomes refractory to proteasome inhibition. This report describes that both PI sensitive and resistant MM cells display enhanced sensitivity to PI in the presence of synthetic amphiphilic block copolymers, Pluronics (SP1017). SP1017 effectively overcomes both acquired resistance and tumor microenvironment-mediated resistance to PIs. The combination of bortezomib and SP1017 augments accumulation of ubiquitinated proteins, increases markers of proteotoxic and ER stress, and ultimately induces both the intrinsic and extrinsic drug-induced apoptotic pathways in MM cells. Notably, co-treatment of bortezomib and SP1017 intensifies SP1017-induced disorganization of the Golgi complex and significantly reduces secretion of paraproteins. Using a human MM/SCID mice model, the combination of bortezomib and SP1017 exerted enhanced antitumor efficacy as compared to bortezomib alone, delaying disease progression, but without additional toxicity. Collectively, these findings provide proof of concept for the utility of combining PI with SP1017 and present a new approach to enhance the efficacy of current treatment options for MM patients.

Poly(2-oxazoline) based micelles with high capacity for 3rd generation taxoids: preparation, in vitro and in vivo evaluation.

The clinically and commercially successful taxanes, paclitaxel and docetaxel suffer from two major drawbacks, namely their very low aqueous solubility and the risk of developing resistance. Here, we present a method that overcomes both drawbacks in a very simple manner. We formulated 3rd generation taxoids, able to avoid common drug resistance mechanisms with doubly amphiphilic poly(2-oxazoline)s (POx), a safe and highly efficient polymer for the formulation of extremely hydrophobic drugs. We found excellent solubilization of different 3rd generation taxoids irrespective of the drug's chemical structures with essentially quantitative drug loading and final drug to polymer ratios around unity. The small, highly loaded micelles with a hydrodynamic diameter of less than 100nm are excellently suited for parenteral administration. Moreover, a selected formulation with the taxoid SB-T-1214 is about one to two orders of magnitude more active in vitro than paclitaxel in the multidrug resistant breast cancer cell line LCC6-MDR. In contrast, in wild-type LCC6, no difference was observed. Using a q4d×4 dosing regimen, we also found that POx/SB-T-1214 significantly inhibits the growth of LCC6-MDR orthotropic tumors, outperforming commercial paclitaxel drug Taxol and Cremophor EL formulated SB-T-1214.

Pluronics and MDR reversal: an update.

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 2-3 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.

Can nanomedicines kill cancer stem cells?

Most tumors are heterogeneous and many cancers contain small population of highly tumorigenic and intrinsically drug resistant cancer stem cells (CSCs). Like normal stem cell, CSCs have the ability to self-renew and differentiate to other tumor cell types. They are believed to be a source for drug resistance, tumor recurrence and metastasis. CSCs often overexpress drug efflux transporters, spend most of their time in non-dividing G0 cell cycle state, and therefore, can escape the conventional chemotherapies. Thus, targeting CSCs is essential for developing novel therapies to prevent cancer relapse and emerging of drug resistance. Nanocarrier-based therapeutic agents (nanomedicines) have been used to achieve longer circulation times, better stability and bioavailability over current therapeutics. Recently, some groups have successfully applied nanomedicines to target CSCs to eliminate the tumor and prevent its recurrence. These approaches include 1) delivery of therapeutic agents (small molecules, siRNA, antibodies) that affect embryonic signaling pathways implicated in self-renewal and differentiation in CSCs, 2) inhibiting drug efflux transporters in an attempt to sensitize CSCs to therapy, 3) targeting metabolism in CSCs through nanoformulated chemicals and field-responsive magnetic nanoparticles and carbon nanotubes, and 4) disruption of multiple pathways in drug resistant cells using combination of chemotherapeutic drugs with amphiphilic Pluronic block copolymers. Despite clear progress of these studies the challenges of targeting CSCs by nanomedicines still exist and leave plenty of room for improvement and development. This review summarizes biological processes that are related to CSCs, overviews the current state of anti-CSCs therapies, and discusses state-of-the-art nanomedicine approaches developed to kill CSCs.

Effect of doxorubicin/pluronic SP1049C on tumorigenicity, aggressiveness, DNA methylation and stem cell markers in murine leukemia.

PURPOSE: Pluronic block copolymers are potent sensitizers of multidrug resistant cancers. SP1049C, a Pluronic-based micellar formulation of doxorubicin (Dox) has completed Phase II clinical trial and demonstrated safety and efficacy in patients with advanced adenocarcinoma of the esophagus and gastroesophageal junction. This study elucidates the ability of SP1049C to deplete cancer stem cells (CSC) and decrease tumorigenicity of cancer cells in vivo. EXPERIMENTAL DESIGN: P388 murine leukemia ascitic tumor was grown in BDF1 mice. The animals were treated with: (a) saline, (b) Pluronics alone, (c) Dox or (d) SP1049C. The ascitic cancer cells were isolated at different passages and examined for 1) in vitro colony formation potential, 2) in vivo tumorigenicity and aggressiveness, 3) development of drug resistance and Wnt signaling activation 4) global DNA methylation profiles, and 5) expression of CSC markers. RESULTS: SP1049C treatment reduced tumor aggressiveness, in vivo tumor formation frequency and in vitro clonogenic potential of the ascitic cells compared to drug, saline and polymer controls. SP1049C also prevented overexpression of BCRP and activation of Wnt-ß-catenin signaling observed with Dox alone. Moreover, SP1049C significantly altered the DNA methylation profiles of the cells. Finally, SP1049C decreased CD133(+) P388 cells populations, which displayed CSC-like properties and were more tumorigenic compared to CD133(-) cells. CONCLUSIONS: SP1049C therapy effectively suppresses the tumorigenicity and aggressiveness of P388 cells in a mouse model. This may be due to enhanced activity of SP1049C against CSC and/or altered epigenetic regulation restricting appearance of malignant cancer cell phenotype.

A simple way to enhance Doxil® therapy: drug release from liposomes at the tumor site by amphiphilic block copolymer.

The antitumor efficacy of Doxil® is hindered by the poor release of the active drug from the liposome at the tumor sites. This study investigates a possibility to enhance drug release from the liposomes and increase therapeutic efficacy of Doxil® by administering Pluronic block copolymers once the liposomal drug accumulates in the tumor sites. In our study, the fluorescence de-quenching experiments were designed to investigate the drug release from liposome by Pluronic P85. MTT cytotoxicity assay and confocal microscopy images were carried out to determine whether Pluronic P85 could facilitate release of Dox from Doxil®. Anti-tumor growth and distribution of drug were evaluated when Pluronic P85 was injected 1h, 48h, or 96h after the Doxil® administration in A2780 human ovarian cancer xenografts. Addition of Pluronic P85 resulted in release of Dox from the liposomes accompanied with significant increases of Dox delivery and cytotoxic effect in cancer cells. The greatest anti-tumor effect of single injection of Doxil® was achieved when Pluronic P85 was administered 48h after Doxil®. The confocal tile scanning images of tumor section showed that copolymer treatment induced the release of the drug in the tumors from the vessels regions to the bulk of the tumor. No release of the drug remaining in circulation was observed. Our study has demonstrated a simple approach for localized release of Dox from liposome by Pluronic P85 at the tumor site, which was therapeutically beneficial.

Endocytosis of nanomedicines.

Novel nanomaterials are being developed to improve diagnosis and therapy of diseases through effective delivery of drugs, biopharmaceutical molecules and imaging agents to target cells in disease sites. Such diagnostic and therapeutic nanomaterials, also termed "nanomedicines", often require site-specific cellular entry to deliver their payload to sub-cellular locations hidden beneath cell membranes. Nanomedicines can employ multiple pathways for cellular entry, which are currently insufficiently understood. This review, first, classifies various mechanisms of endocytosis available to nanomedicines including phagocytosis and pinocytosis through clathrin-dependent and clathrin-independent pathways. Second, it describes the current experimental tools to study endocytosis of nanomedicines. Third, it provides specific examples from recent literature and our own work on endocytosis of nanomedicines. Finally, these examples are used to ascertain 1) the role of particle size, shape, material composition, surface chemistry and/or charge for utilization of a selected pathway(s); 2) the effect of cell type on the processing of nanomedicines; and 3) the effect of nanomaterial-cell interactions on the processes of endocytosis, the fate of the nanomedicines and the resulting cellular responses. This review will be useful to a diverse audience of students and scientists who are interested in understanding endocytosis of nanomedicines.

Effect of pluronic p85 on amino acid transport in bovine brain microvessel endothelial cells.

A synthetic amphiphilic block copolymer Pluronic P85 (P85) was shown to be among the most potent inhibitors of Pgp efflux system in the blood-brain barrier (BBB) and capable of enhancing delivery of Pgp substrates to the brain. The purpose of this work is to evaluate the effects of P85 on amino acid transport in BBB. Primary bovine brain microvessel endothelial cells (BBMEC) grown on membrane inserts were used as an in vitro BBB model. Expression of amino acid transporters, like large neutral amino acid transporter 1, cationic amino acid transporter 1, and small neutral amino acid transporter 1, were confirmed by reverse transcriptase polymerase chain reaction. Effects of P85 on amino acid transporters were examined using their substrates: (3)H-phenylalanine, (3)H-lysine, and (3)H-methylaminoisobutyric acid, respectively. BBMEC permeability studies were carried out in apical (AP) to basolateral (BL) and BL to AP directions. P85 added at the AP side had little, if any, effect on AP to BL ("blood to brain") transport for all examined amino acids in BBMEC monolayers. However, 0.1% P85 added at the BL side significantly increased the BL to AP transport of these substrates. Furthermore, the effective concentrations of P85 were also shown to induce plasma membrane depolarization and increase intracellular sodium concentration in BBMEC, which can contribute to the effects of the copolymer on the energy-dependent transport systems. All together, despite profound effects on transport system(s) at the brain side of cell monolayers, P85 had no effect on AP to BL transport of amino acids in brain microvessel endothelial cell model.

Alteration of genomic responses to doxorubicin and prevention of MDR in breast cancer cells by a polymer excipient: pluronic P85.

Polymer therapeutics has emerged as a new clinical option for the treatment of human diseases. However, little is known about pharmacogenetic responses to drugs formulated with polymers. In this study, we demonstrate that a formulation containing the block copolymer Pluronic P85 and antineoplastic drug doxorubicin (Dox) prevents the development of multidrug resistance in the human breast carcinoma cell line, MCF7. Specifically, MCF7 cells cultured in the presence of Pluronic were unable to stably grow in concentrations of Dox that exceeded 10 ng of Dox/mL of culture medium. In sharp contrast, MCF7 cells cultured in the absence of the block copolymer resulted in the selection and stable growth of cells that tolerated a 1000 times higher concentration of the drug (10 000 ng of Dox/mL of culture medium). Detailed characterization of the isolated sublines demonstrated that those cells selected in the polymer-drug formulation did not show amplification of the MDR1 gene, likely resulting in their high sensitivity to the drug. Conversely, cells selected with Dox alone showed an elevated level in the expression of the MDR1 gene along with a corresponding increase in the expression level of the drug efflux transporter, Pgp, and likely contributing to the high resistance of the cells to Dox. Global analysis of the expression profiles of 20K genes by DNA microarray revealed that the use of Pluronic in combination with Dox drastically changed the direction and magnitude of the genetic response of the tumor cells to Dox and may potentially enhance therapeutic outcomes. Overall, this study reinforces the need for a thorough assessment of pharmacogenomic effects of polymer therapeutics.