Development of drug conjugates in cancer therapy and evaluation of dual siRNA silencing effect on breast cancer growth and invasion

Title from PDF of title page, viewed on March 15, 2013Dissertation advisor: Kun ChengVitaIncludes bibliographic references (p. 152-169)Thesis (Ph.D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2012The objective of this dissertation is to present various approaches for treatment cancer, which is the leading cause of death worldwide. Compared to other disease, cancer has many unique biological characteristics that can be exploited for its therapy. In chapter 1 and 2, its molecular characteristics and microenvironments, as well as the corresponding therapeutic strategies, are summarized. In chapter 3, we developed a peptide drug conjugate to specifically deliver TGX-221 to HER2 overexpressed prostate cancer cells. TGX-221 is a highly potent phosphoinositide 3- kinases β (PI3Kβ) inhibitor that holds great promise as a novel chemotherapy agent for prostate cancer. However, poor solubility and lack of targetability limit its therapeutic applications. The peptide drug conjugate was proven to be gradually cleaved by PSA to release TGX-D1 (TGX- 221 analogue). Both the peptide drug conjugate and its cleaved products demonstrate a comparable activity to the parent drug, TGX-D1. Moreover, cellular uptake of the peptide drug conjugate and its cleaved product SL-TGX were significantly higher in prostate cancer cells compared to the parent drug. The high cellular uptake of dipeptide drug conjugate SL-TGX might be mediated by peptide transporters in prostate cancer cells. However, the expression of peptide transporters in prostate cancer cell lines has not been reported before. Therefore, in Chapter 4, the expression profile and functional activity of peptide transporters were investigated in the prostate cancer cell lines LNCaP, PC-3 and DU145. Peptide transporter 1 (PEPT1) is found overexpressed in PC-3 cells, and peptide transporter 2 (PEPT2) is upregulated in LNCaP cells. We also developed another approach to enhance water solubility and targetability of hydrophobic drugs. In Chapter 5, we developed a polymer-rapamycin conjugate using a novel, linear and PEG based multiblock copolymer (Mw ~ 32 kDa). Rapamycin has demonstrated potent anti-tumor activity in preclinical and clinical studies. However, the clinical development of its formulations has been hampered due to its poor solubility and undesirable distribution in vivo. The polymer-rapamycin conjugate provided enhanced solubility in water compared with free rapamycin and shows a profound activity against a panel of human cancer cell lines. This polymer-rapamycin conjugate also presented high drug loading capacity (wt% ~ 28%) when GlyGlyGly was used as a linker. The uptake study further indicated that the lysosome is the major site of intracellular localization of polymer drug conjugate. Thus, these preclinical data suggested that polymer rapamycin conjugate is a novel anti-cancer agent that holds great promising for treatment of a wide variety of tumors. Macromolecules such as siRNA can also be used as anticancer drugs. In chapter 6, we designed nine HER2 siRNAs and ten VEGF siRNAs and identified potent siRNA that can silence the target gene up to 75-83%. The most potent HER2 and VEGF siRNAs were used to conduct functional studies in HER2 positive breast cancer cells. Combination of HER2 and VEGF siRNAs demonstrated synergistic silencing effect on VEGF. Both HER2 siRNA and VEGF siRNA showed significant inhibition on cell migration and proliferation. HER2 siRNA also demonstrated dramatic suppression of cell spreading and adhesion to ECM, as well as induction of apoptosis. Dual silencing of HER2 and VEGF led to significant cell morphology change and substantial suppression on migration, spreading, cell adhesion, and proliferation.Introduction -- Review of literature -- Development of a peptide drug conjugate for prostate cancer therapy -- Expression profile and functional activity of peptide transporters in prostate cancer cell lines -- Design and synthesis of a rapamycin conjugate using a novel poly(ethylene glycol) multiblock copolymer -- Inhibition of breast cancer cell growth and invasion by dual silencing of her2 and vegf -- Letters of permissio

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ATP-triggered anticancer drug delivery

Nanoparticles can deliver drugs to tumours but improvements in selectively targeting tumour cells are required. Here, Mo et al. develop nanocarriers that take advantage of high ATP levels in tumour cells and show that these

Bio-Inspired Synthetic Nanovesicles for Glucose-Responsive Release of Insulin

A new glucose-responsive formulation for self-regulated insulin delivery was constructed by packing insulin, glucose-specific enzymes into pH-sensitive polymersome-based nanovesicles assembled by a diblock copolymer. Glucose can passively transport across the bilayer membrane of the nanovesicle and be oxidized into gluconic acid by glucose oxidase, thereby causing a decrease in local pH. The acidic microenvironment causes the hydrolysis of the pH sensitive nanovesicle that in turn triggers the release of insulin in a glucose responsive fashion. In vitro studies validated that the release of insulin from nanovesicle was effectively correlated with the external glucose concentration. In vivo experiments, in which diabetic mice were subcutaneously administered with the nanovesicles, demonstrate that a single injection of the developed nanovesicle facilitated stabilization of the blood glucose levels in the normoglycemic state (<200 mg/dL) for up to 5 days

Self-folded redox/acid dual-responsive nanocarriers for anticancer drug delivery

Self-folded redox/acid dual-responsive nanocarriers (RAD-NCs) are developed for physiologically triggered delivery of anticancer drug. The evidenced redox/acid responsiveness, facile decoration of ligands, and active tumor-targeting capability of RAD-NCs suggest their potential as a promising formulation for tumor-targeted chemotherapy

Bio-Inspired Synthetic Nanovesicles for Glucose-Responsive Release of Insulin

A new glucose-responsive formulation for self-regulated insulin delivery was constructed by packing insulin, glucose-specific enzymes into pH-sensitive polymersome-based nanovesicles assembled by a diblock copolymer. Glucose can passively transport across the bilayer membrane of the nanovesicle and be oxidized into gluconic acid by glucose oxidase, thereby causing a decrease in local pH. The acidic microenvironment causes the hydrolysis of the pH sensitive nanovesicle that in turn triggers the release of insulin in a glucose responsive fashion. In vitro studies validated that the release of insulin from nanovesicle was effectively correlated with the external glucose concentration. In vivo experiments, in which diabetic mice were subcutaneously administered with the nanovesicles, demonstrate that a single injection of the developed nanovesicle facilitated stabilization of the blood glucose levels in the normoglycemic state (<200 mg/dL) for up to 5 days

Stably Doped Conducting Polymer Nanoshells by Surface Initiated Polymerization

Despite broad applications ranging from electronics to biomedical sensing and imaging, a long-standing problem of conducting polymers is the poor resistance to dedoping, which directly affects their signature electrical and optical properties. This problem is particularly significant for biomedical uses because of fast leaching of dopant ions in physiological environments. Here, we describe a new approach to engineer multimodal core–shell nanoparticles with a stably doped conductive polymer shell in biological environments. It was achieved by making a densely packed polymer brush rather than changing its molecular structure. Polyaniline (PANI) was used as a model compound due to its concentrated near-infrared (NIR) absorption. It was grafted onto a magnetic nanoparticle via a polydopamine intermediate layer. Remarkably, at pH 7 its conductivity is ca. 2000× higher than conventional PANI nanoshells. Similarly, its NIR absorption is enhanced by 2 orders of magnitude, ideal for photothermal imaging and therapy. Another surprising finding is its nonfouling property, even outperforming polyethylene glycol. This platform technology is also expected to open exciting opportunities in engineering stable conductive materials for electronics, imaging, and sensing

Clickable Protein Nanocapsules for Targeted Delivery of Recombinant p53 Protein

Encapsulating anticancer protein therapeutics in nanocarriers is an attractive option to minimize active drug destruction, increase local accumulation at the disease site, and decrease side effects to other tissues. Tumor-specific ligands can further facilitate targeting the nanocarriers to tumor cells and reduce nonspecific cellular internalization. Rationally designed non-covalent protein nanocapsules incorporating copper-free “click chemistry” moieties, polyethylene glycol (PEG) units, redox-sensitive cross-linker, and tumor-specific targeting ligands were synthesized to selectively deliver intracellular protein therapeutics into tumor cells via receptor-mediated endocytosis. These nanocapsules can be conjugated to different targeting ligands of choice, such as anti-Her2 antibody single-chain variable fragment (scFv) and luteinizing hormone releasing hormone (LHRH) peptide, resulting in specific and efficient accumulation within tumor cells overexpressing corresponding receptors. LHRH-conjugated nanocapsules selectively delivered recombinant human tumor suppressor protein p53 and its tumor-selective supervariant into targeted tumor cells, which led to reactivation of p53-mediated apoptosis. Our results validate a general approach for targeted protein delivery into tumor cells using cellular-responsive nanocarriers, opening up new opportunities for the development of intracellular protein-based anticancer treatment