Nanoparticle-mediated Drug Delivery System Research Articles

Abstract 890: Human TCF-4 isoforms regulate apoptosis through upregulating Bcl-xL expression in a SxxSS motif-dependent manner.

Abstract Background: The T-cell factor (TCF)-4 is a key transcriptional protein activated by Wnt/β-catenin signaling. Previously we identified 14 TCF-4 isoforms derived from human HCC cell lines. The TCF-4J and K pair have been characterized based on the presence (K) or absence (J) of a SxxSS motif. Furthermore, we have shown that TCF-4J predominantly expressed in poorly differentiated HCC tissues and conferred high tumorigenic potential to HCC cells (Koga et al., PLoS ONE 2012). In contrast, TCF-4K showed low tumorigenic potential, which is an important feature of cancer stem cells (CSCs). Another characteristic of CSC is robust resistance to anticancer agents. Among the agents, doxorubicin (DXR) has been a key drug applied to HCC treatment through a unique nanoparticle-mediated drug delivery system. Thus, we aimed to assess if TCF-4J and K differentially contributed to resistance to DXR-induced HCC cell apoptosis, focusing on the expression profile of Bcl-xL, since the β-catenin/TCF-4 complex is known to up-regulate Bcl-xL gene expression. Methods: The human HCC cell line HAK-1A was used. TCF-4K mutants (269A, 272A, and 273A) were prepared with conversion of serine (S) in the SxxSS motif to alanine (A) by site-directed mutagenesis. Stable cell clones overexpressing TCF-4J (J cells), K (K cells), and K-mutants (269A, 272A, and 273A cells, respectively) were established. The cells were incubated with DXR at 0, 100, 500, and 1000 ng/mL over 4 days. A MTS assay and Western blot analysis were performed to evaluate cell proliferation and the apoptosis-related protein levels of cleaved PARP, cleaved caspase-3, and Bcl-xL, respectively. Flow cytometric analysis was employed to determine the sub-G1 population. Results: Dose-dependent apoptotic effects were verified by PARP cleavage, caspase activation, and increase in the sub-G1 population. Unexpectedly, K cells, harboring the SxxSS motif, were most resistant to DXR. Under 1000 ng/mL of DXR treatment, the absorbance ratio of viable cells at Day 3 to that at Day 0 in control cells, J cells, and K cells were 1.15, 1.53, and 1.76, respectively. However, the K-mutant 269A cells lost high resistance, and the ratio fell to 1.11. Consistent with the results, the expression level of Bcl-xL was highest in K cells. The Bcl-xL protein level of 269A mutant cells was found similar to that of mock DNA transfected cells. Conclusions: The findings suggest that the SxxSS motif of TCF-4 is involved in resistance to DXR, by modulating the expression level of Bcl-xL. We suggest that phosphorylation at serine 269 in the TCF-4K isoform is critical to promote anti-apoptotic potential through increasing the Bcl-xL expression in HCC. Citation Format: Hironori Koga, Miran Kim, Yoshito Tomimaru, Toru Nakamura, Mitsuhiko Abe, Osamu Hashimoto, Yu Ikezono, Hiroshi Masuda, Hirohisa Yano, Takato Ueno, Takuji Torimura, Jack R. Wands, Michio Sata. Human TCF-4 isoforms regulate apoptosis through upregulating Bcl-xL expression in a SxxSS motif-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 890. doi:10.1158/1538-7445.AM2013-890

Liposomes and nanotechnology in drug development: focus on ocular targets

Poor drug delivery to lesions in patients’ eyes is a major obstacle to the treatment of ocular diseases. The accessibility of these areas to drugs is highly restricted by the presence of barriers, including the corneal barrier, aqueous barrier, and the inner and outer blood–retinal barriers. In particular, the posterior segment is difficult to reach for drugs because of its structural peculiarities. This review discusses various barriers to drug delivery and provides comprehensive information for designing nanoparticle-mediated drug delivery systems for the treatment of ocular diseases. Nanoparticles can be designed to improve penetration, controlled release, and drug targeting. As highlighted in this review, the therapeutic efficacy of drugs in ocular diseases has been reported to be enhanced by the use of nanoparticles such as liposomes, micro/nanospheres, microemulsions, and dendrimers. Our recent data show that intravitreal injection of targeted liposomes encapsulating an angiogenesis inhibitor caused significantly greater suppression of choroidal neovascularization than did the injection of free drug. Recent progress in ocular drug delivery systems research has provided new insights into drug development, and the use of nanoparticles for drug delivery is thus a promising approach for advanced therapy of ocular diseases.

Nanoparticle-Mediated Delivery of Anticancer Agents to Tumor Angiogenic Vessels

Nanoparticle-mediated drug delivery systems targeting tumor angiogenic vessels have been studied for antineovascular cancer therapy achieved by induction of apoptosis of angiogenic endothelial cells. Nanoparticles such as liposomes are considered to accumulate in tumors due to the enhanced permeability and retention effect. The delivery efficiency of this system appears to be affected by the density of tumor angiogenic vessels regardless of modification with tumor-targeting ligands on the surface of nanoparticles. It remains a challenging problem to deliver sufficient amounts of anticancer drugs to hypovascular tumors using nanoparticles. On the other hand, the strategy of angiogenic vessel-targeting is theoretically different from that of tumor cell-targeting since target angiogenic endothelial cells face the circulating blood. In addition, this strategy is expected to cause indirect tumor regression by disrupting angiogenic vessels. In this review, our recent studies are summarized to show the actual efficacy of angiogenic vessel-targeting delivery. We have developed various angiogenic vessel-targeted liposomes and evaluated them in experimental cancer models such as drug-resistant and hypovascular tumors. Our data indicate that increased apoptosis of angiogenic endothelial cells can be achieved by the targeted liposomes encapsulating cytotoxic drugs, resulting in enhanced anticancer effects. The advantages of angiogenic vessel-targeting are discussed based on our recent findings to provide an insight into why angiogenic vessels are a promising target for advanced cancer therapy.

Nanodiamond delivery circumvents tumor resistance to doxorubicin

Intrinsic and acquired drug resistance is a common problem causing the recurring of malignant tumors, such as breast and liver cancers1, 2. Successful chemotherapy is often limited due to significant side effects and development of drug resistance3. With the rapid development of nanotechnology, overcoming drug resistance and improving the efficacy of antitumor drugs with scalable and biocompatible nanotherapies have been extensively explored. Up to date many attempts have been made. Nanoparticles that stimulate the endocytosis of drug resistant cells to raise intracellular drug concentration have been proved an effective way to reverse the acquired resistance of cisplatin-resistant cells4. For the resistant tumor cells that pump drugs out through ATP-binding cassette (ABC) transporters, which is considered as a common mechanism for drug resistance5, one of current approaches is to seek nanoparticle-mediated drug delivery system with prolonged retention time to prevent drug efflux and retain therapeutic concentration for maximized tumor inhibition. Doxorubicin (Dox) has been established as one of the most useful chemotherapeutics with a broad spectrum of activity against human neoplasms. In Dox-resistant cells, the drug can be actively pumped out by ABC transporters6. Nanometer-sized diamonds (NDs) have been used for fluorescent cellular biolabeling7, biomedical imaging8, and as drug carriers9. Chow and colleagues recently developed a biocompatible nanodiamond drug carrier system at 2 to 8 nm in diameter with Dox binding to its surfaces electrostatically, forming NDX complexes with an average size of ∼80 nm. The complexes not only reversed the drug efflux-based chemoresistance to Dox, but also avoided the toxic side effects to healthy tissues often occurred with unconjugated free Dox10. The authors used various Dox-resistant tumor cell lines to determine the ability of NDX in maintaining intracellular drug concentration. The cells retained more Dox when treated with NDX as compared to unmodified Dox treatment. While free Dox was intensively pumped out of these cells by ABC-transporters, sustained release of functional drugs from NDs counteracted the efflux of Dox from the tumor cells. Furthermore, the killing of tumor cells by NDs was not affected by the ABC transporter inhibitor verapamil, suggesting NDs delivery may be used as a nonspecific method for overcoming drug efflux. They also demonstrated in vivo that NDX treatment effectively improved tumor inhibition rate, survival probability and increased apoptosis specifically in murine LT2-Myc liver and 4T1 mammary tumors without affecting adjacent normal tissues (Figure 1). Figure 1 ND delivery of Dox inhibits tumor growth in murine liver tumor models and mammary carcinoma model10. (A) Images of livers/tumors from PBS, Dox, ND, or NDX treated LT2-Myc liver tumor bearing mice. (B) Representative images of excised tumors from PBS, ... Effective chemotherapy requires maintaining chemotherapeutic agents at therapeutic concentration for a prolonged period, maximizing tumor inhibition and minimizing normal tissue toxicity11, 12. The reason for the advantages of NDX over free Dox is likely due to the gradual release of the drug from nanodiamonds, which allows for increased drug retention in blood circulation and tumor, and therefore maintained the Dox concentration below a toxic level to normal tissues. It prevents the “burst effect” — a majority of Dox released in a rapid and uncontrollable fashion, which creates unpredictable pharmacokinetics, and thereby making effective dose regimens difficult to predict and acute toxicity to normal organs/tissues13. Although high-dosage of chemotherapeutic administered as a single dose may exert some effect on tumor, it is also accompanied by significant toxicity. For a solid tumor with a long doubling time, prolonged exposure to an active drug at a minimally effective concentration can affect a greater number of tumor cells while minimizing toxicity to normal organs11. Chow and colleagues' work demonstrates that nanodiamond delivery systems may greatly increase the efficacy of traditional anticancer drugs by preventing drugs from being pumped out of tumor cells, allowing anticancer drugs to kill drug-resistant cells at a lower dosage, thus lowering the drug toxicity to normal organs and tissues. Compared to other nanoparticles, nanodiamond is small in diameter and can be cleared out of body within a certain period after administration. Meanwhile, no immune response or liver toxicity was observed with NDs even at high dosage, demonstrating its good biocompatibility. Moreover, surface modification with different functionalities would allow NDs binding with different types of therapeutics and biomolecules (such as imaging contrast agent, therapeutic siRNA, proteins and some other hydrophilic/hydrophobic drugs) through either covalent or noncovalent bonds. This implies that NDs have a great potential to be used for a wide range of drug delivery in treatment of a variety of diseases. Diamonds at nanoscale may entertain new expectation for treatment of malignant tumors in future.