Redox responsive cerium oxide nanoparticles and cd44 targeted nan

Wayne State University Wayne State University Theses 1-1-2017 Redox Responsive Cerium Oxide Nanoparticles And Cd44 Targeted Nanomicelles For Selective Cancer Therapy Zhaoxian Wang Wayne State University, Follow this and additional works at: https://digitalcommons.edu/oa_theses Part of the Medicinal Chemistry and Pharmaceutics Commons Recommended Citation Wang, Zhaoxian, "Redox Responsive Cerium Oxide Nanoparticles And Cd44 Targeted Nanomicelles For Selective Cancer Therapy" (2017). Wayne State University Theses.edu/oa_theses/592 This Open Access Thesis is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Theses by an authorized administrator of DigitalCommons@WayneState. REDOX RESPONSIVE CERIUM OXIDE NANOPARTICLES AND CD44 TARGETED NANOMICELLES FOR SELECTIVE CANCER THERAPY by ZHAOXIAN WANG THESIS Submitted to the Graduate School Wayne State University Detroit, Michigan in partial fulfillment of the requirements For the degree of MASTER OF SCIENCE 2017 MAJOR: PHARMACEUTICAL SCIENCE Approved By: Advisor Date © COPYRIGHT BY ZHAOXIAN WANG 2017 All Rights Reserved ACKNOWLEDGMENTS My deepest gratitude goes first and foremost to my advisor Dr.

Arun Iyer for his constant support, encouragement, patience and guidance during my Master of Science (MS) degree program. Without his illuminating instruction and persistent help, I would not have achieved my goal. Besides my advisor, I am also greatly indebted to the rest of my thesis committee members: Dr. Fei Chen and Dr.

Mohammad Mehrmohammadi, for their continuous support, motivation, constructive advice and challenging questions. My sincere thanks also go to Dr. Fei Chen and Dr. Zhengping Yi for the assistance, encouragement and training in the basic research techniques that I learned during lab rotation.

They provided all the freedom to use the equipment in their lab which helped me a lot during the Master’s program. Furthermore, many thanks go to all my lab mates in the Use-Inspired Biomaterials & Integrated Nano Delivery (U-BIND) Systems Laboratory. Special thanks to Dr. Samaresh Sau and Dr.

Prashant Kesharwani for teaching me all the basic research techniques in pharmaceutics and for the encouragement and patience with me. My sincere gratitude towards Dr. Sushil Kashaw, Duy Luong, Shaimaa Yousef, Hashem Alsaab, Kaustubh Gawde, Ketki Bhise, Rami Alzhrani and Katyayani Tatiparti, for the stimulating discussions, collaborations and moral support. I really had a great deal of fun spending time in the lab with my colleagues in the last two years.

Their friendship and collaboration mean a lot to me and my project would not have been successful without their support. ii I would like to thank Dr. Subhash Padhye and Dr. Fazlul Sarkar for the antitumor compound CDF, Dr, Zhi Mei for TEM imaging, Dr.

Asfar Azmi for fluorescent microscopy imaging and Dr. Arun Rishi for the cells lines. Finally, I am especially grateful to all the faculty and staff members of the Department of Pharmaceutical Sciences, and to all the graduate students for most critical and constant support during the past two years. Last but not the least, I am deeply appreciative of the support given to me by my family, and my parents Aiguo Wang and Xianzhen Xu.

Their love provided me inspiration and their affection and constant support was my driving force. I love you both and wish you all the happiness you ensured I had the opportunity to experience. You have all contributed irreversibly to the personality I have become. I cannot thank you enough.

iii TABLE OF CONTENTS ACKNOWLEDGMENTS. ii LIST OF FIGURES. vii CHAPTER 1 INTRODUCTION. Cerium oxide nanoparticles and its anti-cancer effects by reactive oxygen species modulation.

Targeted anticancer drug delivery using hyaluronic acid engineered nanomicelles .1 CDF (3,4-difluorobenzylidene diferuloylmethane) – highly potent but extremely lipophilic anticancer drug .2 Hyaluronic Acid Engineered Vitamin E TPGS nanomicelles in targeted drug delivery. Explore the pH-dependent redox activity in Cerium oxide nanoparticles for selective cancer cell killing .1 Characterization of cerium oxide nanoparticles .2 Cell viability analysis by MTT assay .3 Quantification of intracellular levels of reactive oxygen species (ROS). Hyaluronic acid engineered nanomicelles (HA-SMA-TPGS) for the targeted delivery of CDF to CD44 overexpressing cancer cells .1 Synthesis and Characterization of SMA-TPGS Conjugates (Non- targeted) and HA-SMA-TPGS Conjugates (Targeted) .2 Preparation and Characterization of CDF – loaded Nanomicelles .3 Drug Encapsulation and Loading .4 In vitro Release Profile of CDF-Loaded Nanomicelles .5 Cellular Uptake Study .6 In vitro Cytotoxicity Assay.7 CD44 Receptor Blocking Assay .8 Flow Cytometry Analysis. Explore the pH-dependent redox activity in Cerium oxide nanoparticles for selective cancer cell killing.

Characterization of cerium oxide nanoparticles. Cell viability analysis by MTT assay. Quantification of intracellular levels of reactive oxygen species (ROS). Hyaluronic acid engineered nanomicelles (HA-SMA-TPGS) for the targeted delivery of CDF to CD44 overexpressing cancer cells .1 Synthesis and Characterization of TPGS-SMA Conjugates (Non- targeted) and HA-TPGS-SMA Conjugates (Targeted) .2 Characterization of CDF-loaded nanomicelles .3 In vitro release profile of CDF-loaded nanomicelles .4 Cellular uptake study .5 In vitro Cytotoxicity Assay.6 CD44 receptor blocking assay.7 Fluorescence activated cell sorting (FACS) analysis .1 Explore the pH-dependent redox activity in Cerium oxide nanoparticles for selective cancer cell killing .2 Hyaluronic acid engineered nanomicelles (HA-SMA-TPGS) for the targeted delivery of CDF to CD44 overexpressing cancer cells.

Summary and future direction. 57 vi LIST OF FIGURES Scheme 1. Schematic illustration of synthesis HA-SMA-TPGS conjugate and self- assembly of SMA-TPGS-CDF and HA-SMA-TPGS-CDF to form nanomicelles in overexpressed CD44 receptor cancer cells. Transmission electron microscopy(TEM) image of CNs, PEG-CNs and GLY-CNs.

Scale bar: 100 nm; Figure 2. In vitro cell viability assay showing % live cells at 24h after treating MCF10A (normal breast cells) and A549 (lung cancer cells) with NPs at pH 6.4 at various concentrations. Data represent mean ± SD, n=5. Quantification of intracellular ROS (H2O2) in A549 cells at pH 6.

The results show all the treatments CNs, PEG-CNs and GLY-CNs at pH 6.5 produce more intracellular ROS (H2O2) compare to pH 7.4 and untreated control experiment. Data represent mean ± SD, n=3. Fourier transform infrared spectroscopy (FTIR) of native HA, SMA polymer, TPGS and SMA-TPGS conjugates, HA-SMA-TPGS conjugates are shown. Characterization of HA, SMA, TPGS and SMA-TPGS conjugates and HA-SMA-TPGS conjugates by proton nuclear magnetic resonance spectroscopy (1H NMR).

(A) Plots of the fluorescence of excitation wavelengths ration of I335 nm/ I332 nm from pyrene vs. the concentrations of SMA-TPGS and HA-SMA-TPGS in vii aqueous solution. (B) Hydrodynamic size of SMA-TPGS-CDF nanomicelles and HA-SMA-TPGS nanomicelles by DLS. (C) The morphology of SMA-TPGS-CDF nanomicelles and HA-SMA-TPGS nanomicelles characterized by TEM.

Scale bar: 500 nm. In vitro drug release study of SMA-TPGS-CDF nanomicelles and HA- SMA-TPGS-CDF nanomicelles incubated in PBS at pH 5. Data are presented as mean ± SD, n=3. Fluorescence microscopic images of (A) MDA-MB-231, and (B) MDA- MB-468 cells after 3h incubation with Rhodamine B labeled nanomicelles and free Rhodamine B.

Blue and red colors fluorescence light indicate cell nuclei and Rhodamine B, respectively. 24h and 48h viability assay on MDA-MB-231 and MDA-MB-468 treated with (A) Free CDF, SMA-TPGS-CDF nanomicelles and HA-SMA-TPGS-CDF nanomicelles at various total drug concentrations. (B) HA, SMA, TPGS, SMA- TPGS copolymer, SMA-TPGS copolymer at various total drug concentrations. Data represent mean ± SD, n=6.

In vitro cytotoxicity assay observed at 24h and 48h after CD44 receptor blockade and treating of MDA-MB-231 and MDA-MB-468 with free CDF, SMA- TPGS-CDF nanomicelles and HA-SMA-TPGS-CDF nanomicelles at various total drug concentrations. Data represent mean ± SD, n=6. Free CDF, SMA-TPGS-CDF nanomicelles and HA-SMA-TPGS-CDF nanomicelles with an increasing apoptosis measured by FACs using staining of viii Annexin V-FITC and PI in (A) MDA-MB-231 and (B) MDA-MB-468. SMA-TPGS copolymer, SMA-TPGS copolymer set as control.

Western blot showing the expression downregulation of PTEN level and upregulation of NF-κB level in protein level after treating with the CDF, SMA- TPGS-CDF nanomicelles and HA-SMA-TPGS-CDF nanomicelles in (A) MDA-MB- 231 and (B) MDA-MB-468 cells. ix 1 CHAPTER 1 INTRODUCTION 1. Background Cancer is known as a group of diseases characterized by cellular mutation and uncontrolled growth. If the spread of the cancer cells is out of control, eventually It can cause death.

It is estimated that approximately 1,700,000 of new cancer cases occurred and over 600,000 patients are expected to die of cancer in the US, which translates to more than 1,600 people per day in 2017 [1]. Most of the current treatments for cancer are surgery [2,3] which is often combined with chemotherapy [4–6], hormonal therapy [7], radiation [8] and targeted therapy [9]. Currently, chemotherapy is the first line therapy for patients after having some type of surgery for cancer [10,11]. However, the major limitations of neoadjuvant chemotherapy is the non-specific distribution in the human body which often cause unexpected side effects to normal cells [12].

Multiple drug resistance (MDR) of cancer cells is another limitation of chemotherapeutic drugs[13,14]. The severe non-target and multiple drug resistance could be overcome if drugs could be delivered to targeted site towards cancer cells. Targeted therapeutics have a great clinical potential in increasing the cytotoxicity of cancer cells and decreasing side effects to normal cells [15]. Cerium oxide nanoparticles and its anti-cancer effects by reactive oxygen species modulation Nanotechnology using organic and inorganic materials can play a meaningful role in addressing the selective therapy of cancers.

The application of nanotechnology has seen rapid growth in many areas such as, [16], nanomedicine products[17], 2 imaging[18] and drug delivery[19]. Different metal oxide nanoparticles, including iron oxide nanoparticles[20–22], zinc oxide nanoparticles[23], gallium oxide nanoparticles[24], have been widely investigated for their anti-cancer effects. Cerium oxide nanoparticles have the ability to undergo oxidation-reduction cycles between valence state of Ce+3 and Ce+4 which is related to redox reactions[25,26]. Cerium oxide displays a unique pH-dependent antioxidant activity.

At normal pH, it shows antioxidant properties which can protect the cells by scavenging reactive oxygen species [27], whereas in acidic pH (cancer cells environment) it presents more cytotoxicity by mediating oxidative stress to the cancer cells[28]. Along these lines, redox responsive cerium oxide nanoparticles can play a versatile role in cancer therapy due to reactive oxygen species. The physicochemical characterization, cytotoxicity of cerium oxide nanoparticles and the quantification of intracellular levels of reactive oxygen species were evaluated in detail. Targeted anticancer drug delivery using hyaluronic acid engineered nanomicelles 1.1 CDF (3,4-difluorobenzylidene diferuloylmethane) – highly potent but extremely lipophilic anticancer drug In our previous study, 3,4-difluorobenzylidene diferuloylmethane or in short CDF was synthesized that showed better bioavailability than its natural analog, diferuloylmethane, in various type of cell lines, include pancreatic, breast, lung, cervical and ovarian cancers.

It has several folds higher stability with better half- life compare with its natural analog, curcumin[29]. CDF can cause inactivation of carcinomas signaling pathways consistent with miR-21 down-regulation of 3 transcription of DNA, NF-kb, and up-regulation of MiR-200 and phosphatase and tensin homolog (PTEN)[30–32]. However, the major issue limiting CDF for preclinical and clinical translation is its highly hydrophobicity. Therefore, In our earlier studies, we successful synthesized different formulation including dendrimer, nanoparticles and liposomes to overcome CDF’s solubility issue that resulted in significant increase in chemotherapeutic efficacy[12,18,33–37].2 Hyaluronic Acid Engineered Vitamin E TPGS nanomicelles in targeted drug delivery 1.1 What are nanomicelles? Nanomicelles are constructed using amphiphilic polymers which can self- assemble into particles with the core-shell architecture having nano-sized dimension.

The inner core is composed of the hydrophobic domain which can encapsulate the hydrophobic chemotherapeutic agents, and the outer shell mostly consists of the hydrophilic group which can improve the solubility of the nanocarriers and protect the chemotherapeutic drugs from premature degradation[38–41]. The nano-range of the micelles helps the formulation effectively to target tumor through the enhanced permeability and retention (EPR- ) effect[42,43]. The hydrophilic part of amphiphilic polymers can be modified with different targeting ligand such as folic acid[44,45], hyaluronic acid[34], and transferrin[46–48] to achieve active targeting. In recent years, nanomicelles have received growing scientific attention due to their effectiveness in safety and efficacy for cancer therapy.

Currently, several polymeric nanomicelles-based formulations have been moved into clinical trials including Genexol- 4 PM[49,50]NK105[51,52], SP1049[53,54], Docetaxel-loaded targeted polymeric nanoparticles (DTXL-TNP)[55].2 Tumor passive targeting strategy of nanomicelles Targeted nano-sized chemotherapeutic strategies are generally classified into passive targeting and active targeting.