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Hormones play an important role in normal and diseased breast tissue development. However, they can also disrupt cell-matrix interactions and their role in extracellular matrix reorganization during epithelial morphogenesis remains poorly understood, partly due to a lack of sensitive approaches for matrix characterization. Here, we assess the hormonal regulation of matrix reorganization in a three-dimensional (3D) breast tissue culture model using a novel metric, i.e., 3D directional variance, to characterize the 3D organization of collagen fibers visualized via high-resolution, second harmonic generation imaging. This metric enables resolving and quantifying patterns of spatial organization throughout the matrix surrounding epithelial structures treated with 17β-estradiol (E2) alone, and E2 in combination with either promegestone, a progestogen, or prolactin. Addition of promegestone results in the most disorganized fibers, while the E2 alone treatment leads to the most organized ones. Location-dependent organization mapping indicates that only the prolactin treatment leads to significant heterogeneities in the regional organization of collagen fibers, with higher levels of alignment observed at the end of the elongated epithelial structures. The observed collagen organization patterns for all groups persist for tens of micrometers. In addition, a comparison between 3D directional variance and typical 2D analysis approaches reveals an improved sensitivity of the 3D metric to identify organizational heterogeneities and differences among treatment groups. These results demonstrate that 3D directional variance is sensitive to subtle changes in the extracellular micro-environment and has the potential to elucidate reciprocal cell-matrix interactions in the context of numerous applications involving the study of normal and diseased tissue morphogenesis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A novel biomimetic drug delivery system (BDDS) inspired by the pH-dependent ferric ion-transport and release manner of transferrin (Tf) was developed for combating multidrug-resistant breast cancer. Tf-inspired carrier was synthesized by modifying bovine serum albumin (BSA) with histamine (HA) through amide reaction to provide superior specific coordination sites for ferric ion-drug complexes, and self-assembled into nanoparticles (NPs) induced by coordination bond. Tf-inspired NPs were prepared via environment-friendly method, and well redispersed in saline after lyophilization. When internalized into tumor cells by SPARC (secreted protein acidic and rich in cysteine) mediated endocytosis, Tf-inspired NPs bypassed and decreased the P-glycoprotein-mediated drug efflux and led to more effective treatment of multidrug-resistant breast cancer compared with free drugs both in vitro and in vivo due to the enhanced cellular uptake and rapid pH-responsive drug release. Moreover, Tf-inspired NPs exhibited good biocompatibility and low systemic toxicity. Thus, our results demonstrate that Tf-inspired NPs based on coordination bond represent as a smart drug delivery strategy to combat multidrug-resistant cancer and have great potential for clinical applications in cancer therapy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Photothermal-based combination therapy using functional nanomaterials shows great promise in eradication of aggressive tumors and improvement of drug sensitivity. The therapeutic efficacy and adverse effects of drug combinations depend on the precise control of timely tumor-localized drug release. Here a polymer-dopamine nanocomposite is designed for combination therapy, thermo-responsive drug release and prevention of uncontrolled drug leakage. The thermo-sensitive co-polymer poly (2-(2-methoxyethoxy) ethyl methacrylate-co-oligo (ethylene glycol) methacrylate)-co-2-(dimethylamino) ethyl methacrylate-b-poly (D, l-lactide-co-glycolide) is constructed into core-shell structured nanoparticles for co-encapsulation of two cytotoxic drugs and absorption of small interfering RNAs against survivin. The drug-loaded nanoparticles are surface-coated with polydopamine which confers the nanoformulation with photothermal activity and protects drugs from burst release. Under tumor-localized laser irradiation, polydopamine generates sufficient heat, resulting in nanoparticle collapse and instant drug release within the tumor. The combination strategy of photothermal, chemo-, and gene therapy leads to triple-negative breast cancer regression, with a decrease in the chemotherapeutic drug dosage to about 1/20 of conventional dose. This study establishes a powerful nanoplatform for precisely controlled combination therapy, with dramatic improvement of therapeutic efficacy and negligible side effects., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Antiangiogenesis therapy has been served as a potent cancer treatment strategy for decades, yet disrupting neovasculature would provoke tumor cells into invasive growth and result in distal metastasis. The basic cause of cancer metastasis can be traced down to the presence of circulating tumor cells (CTCs) which detach from primary tumor site and act as 'seeds'. Epithelial cell adhesion molecule (EpCAM) is a potential biomarker for selective capture of epithelium-derived CTCs. Here, we integrated tumor neovessles-targetable ligands K237 peptide with Ep23 aptamer against EpCAM into a single drug-loaded nanoplatform using paclitaxel (PTX) as the model drug, aiming at damaging the primary tumor and neutralizing CTCs simultaneously to achieve a synergistic anti-tumor therapeutic effect. Enhanced cellular uptake, cell apoptosis-induction and cell-viability inhibition efficiency of the peptide and aptamer dual-functionalized nanoparticles (dTNP) were observed in both human umbilical vein endothelial cells (HUVEC) and 4T1 cells in vitro. Using cone-and-plate viscometer to create venous flow velocity, dTNP was also found to be able to capture CTCs under shear stress. The CTC-targeting and neutralization effect of dTNP in bloodstream and 4T1-GFP cell-derived lung metastasis mice model was confirmed via in vivo flow cytometry (IVFC), intravital imaging and confocal microscopy analysis. As a result, the orthotropic breast tumor-bearing mice administrated with PTX-loaded dTNP exhibited the optimal therapeutic effect. Taken together, the findings here provided direct evidence that the tumor neovasculature and CTCs dual-targeting drug delivery system could provide a novel modality for the treatment of highly-invasive breast cancer., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer for which there is no available targeted therapy. TNBC cases contribute disproportionately to breast cancer-related mortality, thus the need for novel and effective therapeutic methods is urgent. We have previously shown that a National Cancer Institute (NCI) investigational drug aminoflavone (AF) exhibits strong growth inhibitory effects in TNBC cells. However, in vivo pulmonary toxicity resulted in withdrawal or termination of several human clinical trials for AF. Herein we report the in vivo efficacy of a nanoformulation of AF that enhances the therapeutic index of AF in TNBC. We engineered a unique unimolecular micelle nanoparticle (NP) loaded with AF and conjugated with GE11, a 12 amino acid peptide targeting epidermal growth factor receptor (EGFR), since EGFR amplification is frequently observed in TNBC tumors. These unimolecular micelles possessed excellent stability and preferentially released drug payload at endosomal pH levels rather than blood pH levels. Use of the GE11 targeting peptide resulted in enhanced cellular uptake and strong growth inhibitory effects in TNBC cells. Further, AF-loaded, GE11-conjugated (targeted) unimolecular micelle NPs significantly inhibit orthotopic TNBC tumor growth in a xenograft model, compared to treatment with AF-loaded, GE11-lacking (non-targeted) unimolecular micelle NPs or free AF. Interestingly, the animals treated with AF-loaded, targeted NPs had the highest plasma and tumor level of AF among different treatment groups yet exhibited no increase in plasma aspartate aminotransferase (AST) activity level or observable tissue damage at the time of sacrifice. Together, these results highlight AF-loaded, EGFR-targeted unimolecular micelle NPs as an effective therapeutic option for EGFR-overexpressing TNBC., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

By its unique advantages over traditional medicine, nanomedicine has offered new strategies for cancer treatment. In particular, the development of drug delivery strategies has focused on nanoscale particles to improve bioavailability. However, many of these nanoparticles are unable to overcome tumor resistance to chemotherapeutic agents. Recently, new opportunities for drug delivery have been provided by oligonucleotides that can self-assemble into three-dimensional nanostructures. In this work, we have designed and developed functional DNA nanostructures to deliver the chemotherapy drug doxorubicin (Dox) to resistant cancer cells. These nanostructures have two components. The first component is a DNA aptamer, which forms a dimeric G-quadruplex nanostructure to target cancer cells by binding with nucleolin. The second component is double-stranded DNA (dsDNA), which is rich in -GC- base pairs that can be applied for Dox delivery. We demonstrated that Dox was able to efficiently intercalate into dsDNA and this intercalation did not affect the aptamer's three-dimensional structure. In addition, the Aptamer-dsDNA (ApS) nanoparticle showed good stability and protected the dsDNA from degradation in bovine serum. More importantly, the ApS&Dox nanoparticle efficiently reversed the resistance of human breast cancer cells to Dox. The mechanism circumventing doxorubicin resistance by ApS&Dox nanoparticles may be predominantly by cell cycle arrest in S phase, effectively increased cell uptake and decreased cell efflux of doxorubicin. Furthermore, the ApS&Dox nanoparticles could effectively inhibit tumor growth, while less cardiotoxicity was observed. Overall, this functional DNA nanostructure provides new insights into the design of nanocarriers to overcome multidrug resistance through targeted drug delivery., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Targeting multiple stages in metastatic breast cancer is one of the effective ways to inhibit metastatic progression. To target human metastatic breast cancer as well as improving patient compliance, we developed an orally active low molecular weight heparin (LMWH)-taurocholate conjugated with tetrameric deoxycholic acid, namely LHTD4, which followed by physical complexation with a synthetic bile acid enhancer, DCK. In breast cancer, both transforming growth factor-β1 (TGF-β1) and CXCL12 exhibit enhanced metastatic activity during the initiation and progression stages of breast cancer, thus we direct the focus on investigating the antimetastatic effect of LHTD4/DCK complex by targeting TGF-β1 and CXCL12. Computer simulation study and SPR analysis were performed for the binding confirmation of LHTD4 with TGF-β1 and CXCL12. We carried out in vitro phosphorylation assays of the consecutive receptors of TGF-β1 and CXCL12 (TGF-β1R1 and CXCR4, respectively). Effects of LHTD4 on in vitro cell migration (induced by TGF-β1) and chemotaxis (mediated by CXCL12) were investigated. The in vivo anti-metastatic effect of LHTD4 was evaluated in an accelerated metastasis model and an orthotopic MDA-MB-231 breast cancer model. The obtained KD values of TGF-β1 and CXCL12 with LHTD4 were 0.85 and 0.019 μM respectively. The simulation study showed that binding affinities of LHTD4 fragment with either TGF-β1 or CXCL12 through additional electrostatic interaction was more stable than that of LMWH fragment. In vitro phosphorylation assays of TGF-β1R1 and CXCR4 showed that the effective inhibition of receptor phosphorylation was observed with the treatment of LHTD4. The expressions of epithelial to mesenchymal transition (EMT) marker proteins such as vimentin and Snail were prevented by LTHD4 treatment in in vitro studies with TGF-β1 treated MDA-MB-231 cells. Moreover, we observed that LHTD4 negatively regulated the functions of TGF-β1 and CXCL12 on migration and invasion of breast cancer cell. In several advanced orthotopic and experimental breast cancer metastasis murine models, the treatment with LHTD4 (5 mg/kg daily, p.o.) significantly inhibited metastasis compared to the control. Overall, LHTD4 exhibited anti-metastatic effects by inhibiting TGF-β1 and CXCL12, and the clinically relevant dose of orally active LHTD4 was found to be effective in preclinical studies without any apparent toxicity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Triple-negative breast cancer is a malignant cancer type with a high risk of early recurrence and distant metastasis. Unlike other breast cancers, triple-negative breast cancer is lack of targetable receptors and, therefore, patients largely receive systemic chemotherapy. However, inevitable adverse effects and acquired drug resistance severely constrain the therapeutic outcome. Here we tailor-designed a porphyrin-based micelle that was self-assembled from a hybrid amphiphilic polymer comprising polyethylene glycol, poly (d, l-lactide-co-glycolide) and porphyrin. The bilayer micelles can be simultaneously loaded with two chemotherapeutic drugs with synergistic cytotoxicity and distinct physiochemical properties, forming a uniform and spherical nanostructure. The drug-loaded micelles showed a tendency to accumulate in the tumor and can be internalized by tumor cells for drug release in acidic organelles. Under near-infrared laser irradiation, high density of self-quenched porphyrins in the hydrophobic layer absorbed light efficiently and converted into an excited state, leading to the release of sufficient heat for photothermal therapy. The integration of localized photothermal effect and synergistic chemotherapy conferred great chemosensitivity to cancer cells and achieved tumor regression using about 1/10 of traditional drug dosage. As a result, chemotherapy-associated adverse effects were successfully avoided. Our present study established a novel porphyrin-based nanoplatform with photothermal activity and expanded drug loading capacity, providing new opportunities for challenging conventional chemotherapy and fighting against stubborn triple-negative breast cancer., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Breast cancer stem cells (CSC) are thought responsible for tumor growth and relapse, metastization and active evasion to standard chemotherapy. The recognition that CSC may originate from non-stem cancer cells (non-SCC) through plastic epithelial-to-mesenchymal transition turned these into relevant cell targets. Of crucial importance for successful therapeutic intervention is the identification of surface receptors overexpressed in both CSC and non-SCC. Cell surface nucleolin has been described as overexpressed in cancer cells as well as a tumor angiogenic marker. Herein we have addressed the questions on whether nucleolin was a common receptor among breast CSC and non-SCC and whether it could be exploited for targeting purposes. Liposomes functionalized with the nucleolin-binding F3 peptide, targeted simultaneously, nucleolin-overexpressing putative breast CSC and non-SCC, which was paralleled by OCT4 and NANOG mRNA levels in cells from triple negative breast cancer (TNBC) origin. In murine embryonic stem cells, both nucleolin mRNA levels and F3 peptide-targeted liposomes cellular association were dependent on the stemness status. An in vivo tumorigenic assay suggested that surface nucleolin overexpression per se, could be associated with the identification of highly tumorigenic TNBC cells. This proposed link between nucleolin expression and the stem-like phenotype in TNBC, enabled 100% cell death mediated by F3 peptide-targeted synergistic drug combination, suggesting the potential to abrogate the plasticity and adaptability associated with CSC and non-SCC. Ultimately, nucleolin-specific therapeutic tools capable of simultaneous debulk multiple cellular compartments of the tumor microenvironment may pave the way towards a specific treatment for TNBC patient care., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Poly(ethylene glycol) (PEG) is usually used to protect nanoparticles from rapid clearance in blood. The effects are highly dependent on the surface PEG density of nanoparticles. However, there lacks a detailed and informative study in PEG density and in vivo drug delivery due to the critical techniques to precisely control the surface PEG density when maintaining other nano-properties. Here, we regulated the polymeric nanoparticles' size and surface PEG density by incorporating poly(ε-caprolactone) (PCL) homopolymer into poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-PCL) and adjusting the mass ratio of PCL to PEG-PCL during the nanoparticles preparation. We further developed a library of polymeric nanoparticles with different but controllable sizes and surface PEG densities by changing the molecular weight of the PCL block in PEG-PCL and tuning the molar ratio of repeating units of PCL (CL) to that of PEG (EG). We thus obtained a group of nanoparticles with variable surface PEG densities but with other nano-properties identical, and investigated the effects of surface PEG densities on the biological behaviors of nanoparticles in mice. We found that, high surface PEG density made the nanoparticles resistant to absorption of serum protein and uptake by macrophages, leading to a greater accumulation of nanoparticles in tumor tissue, which recuperated the defects of decreased internalization by tumor cells, resulting in superior antitumor efficacy when carrying docetaxel., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In this study we examined the efficacy of our micellar system in xenograft models of triple negative breast cancers and explored the effect of the micelles on post-treatment tumours in order to elucidate the mechanism underlying the nanomedicine treatment in oncology. Here, we developed docetaxel-loaded vitamin E D-α-tocopheryl polyethylene glycol succinate (TPGS) micelles, of which the surface modified with cetuximab ligands for targeting epidermal growth factor receptors (EGFR) that are overexpressed in MDA-MB-231 breast cancer cells. The targeting micelles accumulated in the tumours immediately after the intravenous injection and retained for at least 24 h. The successful delivery of docetaxel into the tumours by the targeting micelles was shown by the greater degree of tumour growth inhibition than that for Taxotere(®) after the 15-day treatment. Furthermore, the explanted tumour culture study involving gene analysis and immunohistochemistry staining indicated that the in vivo micelle treatment induced cell cycle arrest and attenuated cell proliferation. In addition, the targeting and non-targeting micellar formulations brought about anti-angiogenesis and anti-migration effects. Overall, both the in vivo and ex vivo data increased the confidence that our micellar formulations effectively targeted and inhibited EGFR-overexpressing MDA-MB-231 tumours., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The combination of chemotherapy with photothermal therapy, which may lead to improved therapeutic efficacies and reduced side effects of conventional chemotherapy, would require safe drug delivery systems (DDSs) with strong near-infrared (NIR) absorbance, efficient drug loading, and effective tumor homing ability. Herein, we fabricate nano-assemblies containing J-aggregates of a NIR dye, IR825, for drug delivery and combined photothermal & chemotherapy of cancer. It is found that IR825 could be complexed with a low-molecular-weight cationic polymer polyethylenimine (PEI), forming IR825@PEI J-aggregates with greatly enhanced NIR absorbance red-shifted to 915 nm. Those nano-assemblies of J-aggregates are further modified with polyethylene glycol (PEG), obtaining IR825@PEI-PEG nano-complex which exhibits great dispersity in physiological solutions, excellent photostability, and is able to efficiently load chemotherapeutic drug doxorubicin (DOX) via a unique strategy different from drug loading in conventional amphiphilic polymer-based DDSs. In vivo animal experiments uncover that IR825@PEI-PEG/DOX upon intravenous injection into tumor-bearing mice shows rather high tumor uptake as illustrated by photoacoustic imaging. In vivo combined photothermal & chemotherapy is then carried out, demonstrating great synergistic anti-tumor therapeutic effect remarkably superior to those achieved by the respective mono-therapies. Hence, we present a novel type of nanoscale DDSs based on nano-assemblies of small molecules without involving amphiphilic polymers, promising for imaging-guided combination cancer therapy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The combination therapy has exhibited important potential for the treatment of cancers, especially for drug-resistant cancers. In this report, bi-functional nanoprobes based on doxorubicin (DOX)-loaded NaYF4:Yb/Tm-TiO2 inorganic photosensitizers (FA-NPs-DOX) were synthesized for in vivo near infrared (NIR)-triggered inorganic photodynamic therapy (PDT) and enhanced chemotherapy to overcome the multidrug resistance (MDR) in breast cancers. Using the up-conversion luminescence (UCL) performance of NaYF4:Yb/Tm converting near-infrared (NIR) into ultraviolent (UV) lights, reactive oxygen species (ROS) were triggered from TiO2 inorganic photosensitizers for PDT under the irradiation of a 980 nm laser, by which the deep-penetration and low photo-damage could be reached. Moreover, nanocarrier delivery and folic acid (FA) targeting promoted the cellular uptake, and accelerated the release of DOX in drug-sensitive MCF-7 and resistant MCF-7/ADR cells. The toxicity assessment in vitro and in vivo revealed the good biocompatibility of the as-prepared FA-NPs-DOX nanocomposites. By the combination of enhanced chemotherapy and NIR-triggered inorganic PDT, the viability of MCF-7/ADR cells could decrease by 53.5%, and the inhibition rate of MCF-7/ADR tumors could increase up to 90.33%, compared with free DOX. Therefore, the MDR of breast cancers could be obviously overcome by enhanced chemotherapy and NIR-triggered inorganic PDT of FA-NPs-DOX nanocomposites under the excitation of a 980 nm laser., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Recent advances in nanomedicine provide promising alternatives for cancer treatment that may improve the survival of patients with metastatic disease. The goal of the present study was to evaluate graphene oxide (GO) as a potential anti-metastatic agent. For this purpose, GO was modified with polyethylene glycol (PEG) to form PEG-modified GO (PEG-GO), which improves its aqueous stability and biocompatibility. We show here that PEG-GO exhibited no apparent effects on the viability of breast cancer cells (MDA-MB-231, MDA-MB-436, and SK-BR-3) or non-cancerous cells (MCF-10A), but inhibited cancer cell migration in vitro and in vivo. Analysis of cellular energy metabolism revealed that PEG-GO significantly impaired mitochondrial oxidative phosphorylation (OXPHOS) in breast cancer cells; however, PEG-GO showed no effect on OXPHOS in non-cancerous cells. To explore the underlying mechanisms, a SILAC (Stable Isotope Labeling by Amino acids in Cell culture) labeling strategy was used to quantify protein expression in PEG-GO-exposed breast cancer versus non-cancerous cells. The results indicated that PEG-GO selectively down-regulated PGC-1α in breast cancer cells and thus modified the expression of diverse energy generation-related proteins, which accounts for the inhibition of OXPHOS. The inhibition of OXPHOS by PEG-GO significantly reduced ATP production and impaired assembly of the F-actin cytoskeleton in breast cancer cells, which is required for the migratory and invasive phenotype of cancer cells. Taken together, these effects of PEG-GO on cancer cell metastasis may allow the development of a new approach to treat metastatic breast cancer., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Despite the exciting advances in cancer therapy over past decades, tumor metastasis remains the dominate reason for cancer-related mortality. In present work, DNA-wrapped gold nanorods with doxorubicin (DOX)-loading (GNR@DOX) were developed for treatment of metastatic breast cancer via a combination of chemotherapy and photothermal ablation. The GNR@DOX nanoparticles induced significant temperature elevation and DOX release upon irradiation with near infrared (NIR) light as shown in the test tube studies. It was found that GNR@DOX nanoparticles in combination with laser irradiation caused higher cytotoxicity than free DOX in 4T1 breast cancer cells. Animal experiment with an orthotropic 4T1 mammary tumor model demonstrated that GNR@DOX nanoplatform significantly reduced the growth of primary tumors and suppressed their lung metastasis. The Hematoxylin and Eosin (H&E) and immunohistochemistry (IHC) staining assays confirmed that the tumor growth inhibition and metastasis prevention of GNR@DOX nanoparticles were attributed to their abilities to induce cellular apoptosis/necrosis and ablate intratumoral blood vessels. All these results suggested a considerable potential of GNR@DOX nanoplatform for treatment of metastatic breast cancer., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Refractoriness of invasive breast cancer is closely related with the vasculogenic mimicry (VM) channels, which exhibit highly drug resistance to conventional chemotherapies. In the present study, the nanostructured targeting epirubicin plus celecoxib liposomes were developed by modifying a human immunodeficiency virus peptide lipid-derivative conjugate (DSPE-PEG2000-PTDHIV-1) for elimination of invasive breast cancer cells along with their VM channels. The studies were undertaken on invasive human breast cancer MDA-MB-435S cells and MDA-MB-435S xenografts in nude mice. The constructed targeting epirubicin plus celecoxib liposomes were approximately 100 nm in size. In vitro results showed that the targeting liposomes exhibited strong transport ability across cell and nuclei membranes of invasive breast cancer, were able to penetrate and destruct the invasive breast cancer spheroids, initiated apoptosis via activating apoptotic enzymes (caspase 8, 3), and destroyed the VM channels via down-regulating the protein indicators (MMP-9, VE-Cad, FAK, EphA2 and HIF-1α) in invasive breast cancer cells. In vivo results demonstrated that the targeting liposomes displayed a prolonged circulation time in blood system, accumulated more in tumor location, were able to eliminate the VM channels and angiogenesis in tumor tissues, and resulted in a robust overall anticancer efficacy in invasive breast cancer MDA-MB-435S xenografts in nude mice. In conclusion, the nanostructured targeting epirubicin plus celecoxib liposomes could eliminate invasive breast cancer along with the VM channels, hence providing a promising strategy for treatment of invasive breast cancer., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Metastasis is one of the major obstacles for the successful therapy of breast cancer. Although increased candidate drugs targeting cancer metastasis are tested, their clinical translation is limited by either serve toxicity or low efficacy. In present work, a nano-drug delivery system loading atorvastatin calcium (Ator) was developed for the efficient suppression of the metastasis of breast cancer. The nano-drug delivery system was constructed by a amphiphilic copolymer of methoxy polyethylene glycol-s-s-vitamin E succinate (mPEG-s-s-VES, PSV), which was consisted of a hydrophilic mPEG1k segment and a hydrophobic VES head, which were conjugated with a linker bearing amide and disulfide groups simultaneously. Self-assembly of PSV and Ator formed Ator-loaded PSV micelles (ASM) with good colloidal stability, high drug loading content (up to 50%) and great encapsulation efficiency (99.09 ± 0.28%). In cellular level, it was found that the ASM could efficiently release the Ator payload into cytosol due to detachment of PEG shell at high intracellular glutathione condition. ASM could significantly inhibit the migration and invasion of 4T1 breast cancer cells with inhibitory rates of 79.2% and 88.5%, respectively. In a 4T1 orthotropic mammary tumor metastatic cancer model, it was demonstrated that ASM could completely blocked the lung and liver metastasis of breast cancer with minimal toxicity owing to enhanced Ator accumulation in tumor and lung as compared with that of free Ator. The down-regulations of metastasis-promoting MMP-9, Twist and uPA proteins were demonstrated as the main underlying mechanism. As a result, ASM could be a promising drug delivery system for the efficient therapy of breast cancer metastasis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Tumor progression is coincident with mechanochemical changes in the extracellular matrix (ECM). We hypothesized that tumor stroma stiffening, alongside a shift in the ECM composition from a basement membrane-like microenvironment toward a dense network of collagen-rich fibers during tumorigenesis, confers resistance to otherwise powerful chemotherapeutics. To test this hypothesis, we created a high-throughput drug screening platform based on our poly(ethylene glycol)-phosphorylcholine (PEG-PC) hydrogel system, and customized it to capture the stiffness and integrin-binding profile of in vivo tumors. We report that the efficacy of a Raf kinase inhibitor, sorafenib, is reduced on stiff, collagen-rich microenvironments, independent of ROCK activity. Instead, sustained activation of JNK mediated this resistance, and combining a JNK inhibitor with sorafenib eliminated stiffness-mediated resistance in triple negative breast cancer cells. Surprisingly, neither ERK nor p38 appears to mediate sorafenib resistance, and instead, either ERK or p38 inhibition rescued sorafenib resistance during JNK inhibition, suggesting negative crosstalk between these signaling pathways on stiff, collagen-rich environments. Overall, we discovered that β1 integrin and its downstream effector JNK mediate sorafenib resistance during tumor stiffening. These results also highlight the need for more advanced cell culture platforms, such as our high-throughput PEG-PC system, with which to screen chemotherapeutics., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Resistance to single or multiple chemotherapeutic drugs is a major complication in clinical oncology and is one of the most common treatment limitations in patients with reoccurring cancers. Nanoparticle (NP)-based drug delivery systems (DDS's) have been shown to overcome drug resistance in cancer cells mainly by avoiding the activation of efflux pumps in these cells. We demonstrate in this work that polyester-based hyperbranched dendritic-linear (HBDL)-based NPs carrying doxorubicin (Dox) can effectively overcome microsomal glutathione transferase 1 (MGST1)-mediated drug resistance in breast cancer cells. Our DDS was much more effective at considerably lower intracellular Dox concentrations (IC50 6.3 μm vs. 36.3 μm) and achieved significantly greater reductions in viability and induced higher degrees of apoptosis (31% vs. 14%) compared to the free drug in the resistant cells. Dox-loaded HBDL NPs were found to translocate across the membranes of resistant cells via active endocytic pathways and to be transported to lysosomes, mitochondria, and the endoplasmic reticulum. A significantly lower amount of Dox accumulated in these cytoplasmic compartments in resistant cells treated with free Dox. Moreover, we found that Dox-HBDL significantly decreased the expression of MGST1 and enhanced mitochondria-mediated apoptotic cell death compared to free Dox. Dox-HBDL also markedly activated the JNK pathway that contributes to the apoptosis of drug-resistant cells. These results suggest that HBDL NPs can modulate subcellular drug distribution by specific endocytic and trafficking pathways and that this results in drug delivery that alters enzyme levels and cellular signaling pathways and, most importantly, increases the induction of apoptosis. Our findings suggest that by exploiting the cell transport machinery we can optimize the polymeric vehicles for controlled drug release to overcome drug resistance combat drug resistance with much higher efficacy., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In this study, thioridazine (THZ), which was reported to kill cancer stem cells, was used in a combination therapy with doxorubicin (DOX) to eradicate both cancer cells and DOX-resistant cancer stem cells to mitigate the reoccurrence of the disease. Both THZ and DOX were loaded into micelles with sizes below 100 nm, narrow size distribution and high drug content. The micelles were self-assembled from a mixture of acid-functionalized poly(carbonate) and poly(ethylene glycol) diblock copolymer (PEG-PAC) and urea-functionalized poly(carbonate) (PUC) and PEG diblock copolymer (PEG-PUC). The drug-loaded mixed micelles (MM) were used to target both cancer cells and stem cells via co-delivery. Cancer stem cells were sorted by a side population assay from BT-474 and MCF-7 human breast cancer cell lines, and identified by CD44+/CD24- phenotype. The cytotoxicity of various formulations was evaluated on the sorted cancer stem cells (side population SP cells), sorted non-stem-like cancer cells (non-side population NSP cells) and unsorted cancer cells. Antitumor activity was also evaluated on BT-474 xenografts in nude mice. As compared with NSP cells, DOX suppressed SP cell growth less effectively, while THZ and THZ-MM were more effective in the inhibition of SP cells. A stronger inhibitory effect was observed on SP cells with the co-delivery of free DOX and THZ or DOX-MM and THZ-MM as compared to free DOX or DOX-MM. THZ and THZ-MM were capable of lowering the population of SP cells in unsorted cells. In the BT-474 xenografts, the co-delivery of DOX-MM and THZ-MM produced the strongest antitumor efficacy, and both THZ and THZ-MM showed strong activity against cancer stem cells. This combination therapy may provide a promising strategy for breast cancer treatment by targeting both cancer cells and cancer stem cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We report a reducible copolymer self-assembled with superparamagnetic iron oxide nanoparticles (SPIONs) to deliver doxorubicin (DOX) for cancer therapy. The copolymer of reducible polyamidoamine (rPAA) with poly(ethylene glycol)(PEG)/dodecyl amine graft was synthesized by Michael addition. rPAA@SPIONs were formed by the alkyl grafts of reducible copolymers intercalated with the oleic acid layer capped on the surface of magnetite nanocrystals. The intercalating area formed a reservoir for hydrophobic anti-cancer drug (DOX), whilst the PEG moiety in the copolymers helped the nanoparticle well-dispersible in aqueous solution. We employed two-photon excited fluorescence (TPEF) and coherent anti-Stokes Raman (CARS) to investigate drug delivery in intra-cellular structures of live cells, and used Vivaview(®) technique to show real-time inhibition efficacy of nanoparticles in live cells. rPAA@SPIONs present efficiently drug loading with reducible responsibility in vitro tests. Finally, rPAA@SPIONs were tested in mice with xenograft MDA-MB-231 breast tumor though i.v. injection and inhibited tumor growth efficiently. MRI was used to monitor nanoparticles aggregation in tumor site. Histology and Prussian blue on kidney, liver, and heart in mice indicated that DOX/rPAA@SPIONs showed no significant toxicity for mice organs after 24 days treatment., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Lung metastasis is one of the greatest challenges for breast cancer treatment. Here, a nanodiamonds (NDs)-mediated doxorubicin (DOX) delivery system was first designed to inhibit the lung metastasis of breast cancer effectively. DOX was non-covalently bound to NDs via physical adsorption in an aqueous solution, then DSPE-PEG 2K was coated to the NDs-DOX complex (NDX) to increase the dispersibility and prolong the circulation time. DSPE-PEG 2K coating NDX (DNX) displayed high drug loading and excellent ability to deliver DOX to the nucleus, thereby significantly enhancing cytotoxicity and inducing cell apoptosis. Furthermore, DNX showed good histocompatibility and could improve drug accumulation in lung, as a result, markedly inhibited the lung metastasis of breast cancer. The high anti-metastasis efficacy with the decreased systemic toxicity suggested that DNX could be a promising drug delivery system for the therapy of lung metastasis of breast cancer., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Pluronics have been demonstrated as excellent multidrug resistance (MDR) reversal agent in the form of unimers rather than micelles. However, the effective intracellular delivery of Pluronic(®) unimers to MDR cancer cells still remains a big challenge. To address this issue, a mixed micellar system based mainly on the pH-sensitive copolymer of poly (L-histidine)-poly (D,L-lactide)-polyethyleneglycol-poly (D,L-lactide)-poly (L-histidine) (PHis-PLA-PEG-PLA-PHis) and Pluronic(®) F127, some of which was conjugated with folate, was constructed to intracellularly deliver the unimers of Pluronic(®) P85 to MDR cells. The folate-mediated endosomal pH-sensitive mixed micelles (pHendoSM-P85/f) were prepared by a thin-film hydration method, by which Pluronic(®) P85 unimers and doxorubicin (DOX) were incoporated into the mixed micelles. The incorporation of Pluronic(®) P85 unimers was investigated by the surface tension test. The results indicated that the Pluronic(®) P85 unimers probably first inserted into the binary mixed micelles and then formed a triple-component mixed micelles with Pluronic(®) F127 and PHis-PLA-PEG-PLA-PHis as the loading content increased. Further analyzed with flow cytometry, confocal laser scanning microscopy (CLSM) and MTT assay, the micelles with inserted Pluronic(®) P85 unimers demonstrated much more cellular uptake and higher cytotoxicity against MDR cells than the triple-component mixed micelles and plain Pluronic(®) micelles. The enhanced MDR reversal effect was attributed to the successful intracellular delivery of Pluronic(®) P85 unimers to the MDR cells, which was confirmed by the subcellular colocalization of Pluronic(®) P85 unimers with mitochondria, the decreased ATP energy and mitochondrial membrane potential (MP) in the MCF-7/ADR cells. The pHendoSM-P85/f/DOX also demonstrated more dramatic antitumor efficiency and remarkable reduction of ATP energy in the MDR cells in tumors than the control formulations. The intracellular delivery of Pluronic(®) P85 unimers to the MDR cells based on the targeted and endosomal pH triggerd release mixed micelles has been demonstrated as a promising approach to reverse MDR., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A mineral (calcium phosphate, CaP)-reinforced core-shell-corona micelle was evaluated as a nanocarrier of doxorubicin (DOX) for cancer therapy. The polymer micelles of poly(ethylene glycol)-b-poly(L-aspartic acid)-b-poly(L-phenylalanine) (PEG-PAsp-PPhe) in the aqueous phase provided the three distinct functional domains: the hydrated PEG outer corona for prolonged circulation, the anionic PAsp middle shell for CaP mineralization, and the hydrophobic PPhe inner core for DOX loading. CaP mineralization was performed by initial electrostatic localization of calcium ions at anionic PAsp shells, and the consequent addition of phosphate anions to trigger the growth of CaP. The mineralization did not affect the micelle size or the spherical morphology. The CaP-mineralized micelles exhibited enhanced serum stability. The DOX release from the DOX-loaded mineralized micelles (DOX-CaP-PM) at physiological pH was efficiently inhibited, whereas at an endosomal pH (pH 4.5), DOX release was facilitated due to the rapid dissolution of the CaP mineral layers in the middle shell domains. The in vivo tissue distribution and tumor accumulation of the DOX-CaP-PM that were labeled with a near-infrared fluorescent (NIRF) dye, Cy5.5, were monitored in MDA-MB231 tumor-bearing mice. Non-invasive real-time optical imaging results indicated that the DOX-CaP-PM exhibited enhanced tumor specificity due to the prolonged stable circulation in the blood and an enhanced permeation and retention (EPR) effect compared with the DOX-loaded nonmineralized polymer micelles (DOX-NPM). The DOX-CaP-PM exhibited enhanced therapeutic efficacy in tumor-bearing mice compared with free DOX and DOX-NPM. The CaP mineralization on assembled nanoparticles may serve as a useful guide for enhancing the antitumor therapeutic efficacy of various polymer micelles and nano-aggregates., (Copyright © 2012 Elsevier Ltd. All rights reserved.)