Transferrin-targeted polymeric micelles co-loaded with curcumin and paclitaxel: efficient killing of paclitaxel-resistant cancer cells.

PURPOSE: The ability to successfully treat advanced forms of cancer remains a challenge due to chemotherapy resistance. Numerous studies indicate that NF-κB, a protein complex that controls the expression of numerous genes, as being a key factor in producing chemo-resistant tumors. In this study, the therapeutic potential of transferrin (TF)-targeted mixed micelles, made of PEG-PE and vitamin E co-loaded with curcumin (CUR), a potent NF-κB inhibitor, and paclitaxel (PCL), was examined. METHODS: The cytotoxicity of non-targeted and TF-targeted CUR and PCL micelles as a single agent or in combination was investigated against SK-OV-3 human ovarian adenocarcinoma along with its multi-drug resistant (MDR) version SK-OV-3-PCL-resistant (SK-OV-3TR) cells in vitro. RESULTS: Our results indicated that the TF-targeted combination micelles were able to improve the net cytotoxic effect of CUR and PCL to clear synergistic one against the SK-OV-3 cells. In addition, even though the non-targeted combination treatment demonstrated a synergistic effect against the SK-OV-3TR cells, the addition of the TF-targeting moiety significantly increased this cytotoxic effect. While keeping CUR constant at 5 and 10 µM and varying the PCL concentration, the PCL IC50 decreased from ~1.78 to 0.68 µM for the non-targeted formulations to ~0.74 and 0.1 µM for the TF-targeted ones, respectively. CONCLUSION: Our results indicate that such co-loaded targeted mixed micelles could have significant clinical advantages for the treatment of resistant ovarian cancer and provide a clear rational for further in vivo investigation.

In silico, in vitro and in vivo safety evaluation of Limosilactobacillus reuteri strains ATCC PTA-126787 & ATCC PTA-126788 for potential probiotic applications.

The last two decades have witnessed a tremendous growth in probiotics and in the numbers of publications on their potential health benefits. Owing to their distinguishing beneficial effects and long history of safe use, species belonging to the Lactobacillus genus are among the most widely used probiotic species in human food and dietary supplements and are finding increased use in animal feed. Here, we isolated, identified, and evaluated the safety of two novel Limosilactobacillus reuteri (L. reuteri) isolates, ATCC PTA-126787 & ATCC PTA-126788. More specifically, we sequenced the genomes of these two L. reuteri strains using the PacBio sequencing platform. Using a combination of biochemical and genetic methods, we identified the two strains as belonging to L. reuteri species. Detailed in silico analyses showed that the two strains do not encode for any known genetic sequences of concern for human or animal health. In vitro assays confirmed that the strains are susceptible to clinically relevant antibiotics and do not produce potentially harmful by-products such as biogenic amines. In vitro bile and acid tolerance studies demonstrated that the two strains have similar survival profiles as the commercial L. reuteri probiotic strain DSM 17938. Most importantly, daily administration of the two probiotic strains to broiler chickens in drinking water for 26 days did not induce any adverse effect, clinical disease, or histopathological lesions, supporting the safety of the strains in an in vivo avian model. All together, these data provide in silico, in vitro and in vivo evidence of the safety of the two novel candidates for potential probiotic applications in humans as well as animals.

Intra- and trans-cellular delivery of enzymes by direct conjugation with non-multivalent anti-ICAM molecules.

Intercellular adhesion molecule 1 (ICAM-1) is a cell-surface protein overexpressed in many diseases and explored for endocytosis and transcytosis of drug delivery systems. All previous evidence demonstrating ICAM-1-mediated transport of therapeutics into or across cells was obtained using nanocarriers or conjugates coupled to multiple copies of anti-ICAM antibodies or peptides. Yet, transport of therapeutics linked to non-multivalent anti-ICAM ligands has never been shown, since multivalency was believed to be necessary to induce transport. Our goal was to explore whether non-multivalent binding to ICAM-1 could drive endocytosis and/or transcytosis of model cargo in different cell types. We found that anti-ICAM was specifically internalized by all tested ICAM-1-expressing cells, including epithelial, fibroblast and neuroblastoma cells, primary or established cell lines. Uptake was inhibited at 4°C and in the presence of an inhibitor of the ICAM-1-associated pathway, rather than inhibitors of the clathrin or caveolar routes. We observed minimal transport of anti-ICAM to lysosomes, yet prominent and specific transcytosis across epithelial monolayers. Finally, we coupled a model cargo (the enzyme horseradish peroxidase (HRP)) to anti-ICAM and separated a 1:2 antibody:enzyme conjugate for non-multivalent ICAM-1 targeting. Similar to anti-ICAM, anti-ICAM-HRP was specifically internalized and transported across cells, which rendered intra- and trans-cellular enzyme activity. Therefore, non-multivalent ICAM-1 targeting also provides transport of cargoes into and across cells, representing a new alternative for future therapeutic applications via this route.

Cancer cell spheroids for screening of chemotherapeutics and drug-delivery systems.

Over the last few decades, the most popular platform to perform high-throughput screening for viable anti-neoplastic compounds has been monolayer cell culture. However, cells in monolayer culture lose many of their in vivo characteristics. As a result, this platform provides a limited predictive value in determining the clinical outcome of the compounds of interest. Using a technique known as 3D spheroid culture, may be the answer to this conundrum. Spheroids have been shown to mimic the tissue-like properties of tumors necessary for the proper evaluation of compounds. In this review, production of cancer cell spheroids, utilization of these spheroids in understanding various therapeutic mechanisms and the potential for their use in high-throughput screening of drugs and drug-delivery systems are discussed in detail.

The effect of co-delivery of paclitaxel and curcumin by transferrin-targeted PEG-PE-based mixed micelles on resistant ovarian cancer in 3-D spheroids and in vivo tumors.

Multicellular 3D cancer cell culture (spheroids) resemble to in vivo tumors in terms of shape, cell morphology, growth kinetics, gene expression and drug response. However, these characteristics cause very limited drug penetration into deeper parts of the spheroids. In this study, we used multi drug resistant (MDR) ovarian cancer cell spheroid and in vivo tumor models to evaluate the co-delivery of paclitaxel (PCL) and a potent NF-κB inhibitor curcumin (CUR). PCL and CUR were co-loaded into the polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) based polymeric micelles modified with transferrin (TF) as the targeting ligand. Cytotoxicity, cellular association and accumulation into the deeper layers were investigated in the spheroids and compared with the monolayer cell culture. Comparing to non-targeted micelles, flow cytometry and confocal imaging proved significantly deeper and higher micelle penetration into the spheroids with TF-targeting. Both in monolayers and in spheroids, PCL cytotoxicity was significantly increased when co-delivered with CUR in non-targeted micelles or as single agent in TF-targeted micelles, whereas TF-modification of co-loaded micelles did not further enhance the cytotoxicity. In vivo tumor inhibition studies showed good correlation with the 3D cell culture experiments, which suggests the current spheroid model can be used as an intermediate model for the evaluation of co-delivery of anticancer compounds in targeted micelles.

Polyethylene glycol-phosphatidylethanolamine (PEG-PE)/vitamin E micelles for co-delivery of paclitaxel and curcumin to overcome multi-drug resistance in ovarian cancer.

The therapeutic potential of mixed micelles, made of PEG-PE and vitamin E co-loaded with curcumin and paclitaxel, was investigated against SK-OV-3 human ovarian adenocarcinoma along with its multi-drug resistant version SK-OV-3-paclitaxel-resistant (TR) cells in vitro and in vivo. The addition of curcumin at various concentrations did not significantly enhance the cytotoxicity of paclitaxel against SK-OV-3 in vitro. However, a clear synergistic effect was observed with the combination treatment against SK-OV-3TR in vitro. In vivo, this combination treatment produced a three-fold tumor inhibition with each of these cell lines. Our results indicate that such co-loaded mixed micelles could have significant clinical advantages for the treatment of resistant ovarian cancer.

The role of cell cycle in the efficiency and activity of cancer nanomedicines.

INTRODUCTION: With a wealth of knowledge on the effect of nanoparticle properties, including size, shape, charge and composition, on intracellular delivery, little has been reported on the effect of the cell cycle on the intracellular delivery and activity of nanomedicines including non-viral gene delivery systems. The aim of this review is to shed a light on this topic. AREAS COVERED: It is now evident that nanoparticle cell uptake varies with the cell cycle phase. This review addresses this variation by dissecting the effect of cell population heterogeneity on the intracellular delivery and activity of nanomedicines with a special focus on non-viral gene delivery and combination therapy modalities that utilize cell cycle inhibitors as co-targets for therapy. In addition, the importance of three-dimensional (3D) culture systems in the drug delivery field within the context of the cell cycle will be addressed. EXPERT OPINION: The understanding of the cell cycle machinery has improved dramatically over the last few decades. Developing combination therapy modalities that target the cell cycle to achieve better cancer patient outcome should now be the focus. Furthermore, more effort should be placed on developing a reliable, consistent, high throughput 3D cell culture system since these systems more closely resemble the cell cycle status of in vivo tumors. A switch from 2D to 3D culture systems, to more accurately predict the in vivo efficacy of nanoparticle drug delivery systems, is desirable.

Anti-cancer activity of anti-GLUT1 antibody-targeted polymeric micelles co-loaded with curcumin and doxorubicin.

BACKGROUND: Treatment of late stage cancers has proven to be a very difficult task. Targeted therapy and combinatory drug administration may be the solution. PURPOSE: The study was performed to evaluate the therapeutic efficacy of PEG-PE micelles, co-loaded with curcumin (CUR) and doxorubicin (DOX), and targeted with anti-GLUT1 antibody (GLUT1) against HCT-116 human colorectal adenocarcinoma cells both in vitro and in vivo. METHODS: HCT-116 cells were treated with non-targeted and GLUT1-targeted CUR and DOX micelles as a single agent or in combination. Cells were inoculated in female nude mice. Established tumors were treated with the micellar formulations at a dose of 4 mg/kg CUR and 0.4 mg/kg DOX every 2 d for a total of 7 injections. RESULTS: CUR + DOX-loaded micelles decorated with GLUT1 had a robust killing effect even at low doses of DOX in vitro. At the doses chosen, non-targeted CUR and CUR + DOX micelles did not exhibit any significant tumor inhibition versus control. However, GLUT1-CUR and GLUT1-CUR + DOX micelles showed a significant tumor inhibition effect with an improvement in survival. CONCLUSION: We showed a dramatic improvement in efficacy between the non-targeted and GLUT1-targeted formulations both in vitro and in vivo. Hence, we confirmed that GLUT1-CUR + DOX micelles are effective and deserve further investigation.

Nanopreparations to overcome multidrug resistance in cancer.

Multidrug resistance is the most widely exploited phenomenon by which cancer eludes chemotherapy. Broad variety of factors, ranging from the cellular ones, such as over-expression of efflux transporters, defective apoptotic machineries, and altered molecular targets, to the physiological factors such as higher interstitial fluid pressure, low extracellular pH, and formation of irregular tumor vasculature are responsible for multidrug resistance. A combination of various undesirable factors associated with biological surroundings together with poor solubility and instability of many potential therapeutic small & large molecules within the biological systems and systemic toxicity of chemotherapeutic agents has necessitated the need for nano-preparations to optimize drug delivery. The physiology of solid tumors presents numerous challenges for successful therapy. However, it also offers unique opportunities for the use of nanotechnology. Nanoparticles, up to 400 nm in size, have shown great promise for carrying, protecting and delivering potential therapeutic molecules with diverse physiological properties. In this review, various factors responsible for the MDR and the use of nanotechnology to overcome the MDR, the use of spheroid culture as well as the current technique of producing microtumor tissues in vitro are discussed in detail.