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RICE UNIVERSITY Cisplatin@US-tube Carbon Nanocapsules for Enhanced Chemotherapeutic Delivery by Adem Guven A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE Doctor of Philosophy APPROVED, THESIS COMMITTEE Lon J. Wilson, Committee Chair Professor of Chemistry Vicki L. Colvin, The Kenneth S. PitzerSchlumberger Professor of Chemistry and Chemical & Biomolecular Engineering, Vice Provost of Research Enrique V. Barrera, Professor Mechanical Engineering and Materials Science Michael T. Lewis, Associate Professor Lester & Sue Smith Breast Cancer Center, Baylor College of Medicine HOUSTON, TEXAS December 2013 ABSTRACT Cisplatin@US-tube Carbon Nanocapsules for Enhanced Chemotherapeutic Delivery by Adem Guven The use of chemotherapeutic drugs in cancer therapy is often limited by problems with administration such as insolubility, inefficient biodistribution, lack of selectivelty, and inability of the drug to cross cellular barriers. To overcome these limitations, various types of drug delivery systems have been explored, and recently, carbon nanotube (CNT) materials have garnered special attention in the area. This thesis details the preparation, characterization, and in vitro and in vivo testing of a new, ultra-short single-walled carbon nanotube (US-tube)-based drug delivery system for the treatment of cancer. In particular, the encapsulation of cisplatin (CDDP), a widely-used anticancer drug, within US-tubes has been achieved by a loading procedure that is reproducible, and the resulting CDDP@US-tube material characterized by high-resolution transmission electron microscopy (HR-TEM), energy-dispersive spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and inductively-coupled optical emission spectroscopy (ICP-OES). Dialysis studies performed in phosphate-buffered saline (PBS) at 37 °C have demonstrated that CDDP release from CDDP@US-tubes can be controlled (retarded) by wrapping the CDDP@US-tubes with Pluronic®-F108 surfactant. The anticancer activity of Pluronic-wrapped CDDP@US-tubes (W-CDDP@US-tubes) has been evaluated against two different breast cancer cell lines, MCF-7 and MDA-MB-231, and found to exhibit enhanced cytotoxicity over free CDDP. Moreover, it has been shown that CDDP release from W-CDDP@US-tubes nanocapsules can be stimulated remotely by a radiofrequency (RF) field which disrupts the Pluronic coating to release CDDP. RFinduced release-dependent cytotoxicity of W-CDDP@US-tubes has been evaluated in vitro against two different liver cancer cell lines, Hep3B and HepG2, and found to exhibit superior cytotoxicity compared to W-CDDP@US-tubes not exposed to RF. Finally, in vivo biodistribution and therapeutic efficacy of the CDDP@US-tube material has been evaluated against three different breast cancer xenograft mouse (SCID/Beige) models, and found to exhibit greater efficacy in suppressing tumor growth than free CDDP for both a MCF-7 cell line xenograft model and a BCM-4272 patient-derived xenograft (PDX) model. The CDDP@US-tubes also demonstrated prolonged circulation time compared to free CDDP which enhances permeability and retention (EPR) effects resulting in significantly more CDDP accumulation in tumors, as determined by Platinum (Pt) analysis via inductively-coupled plasma mass-spectrometry (ICP-MS). Acknowledgments Although only my name appears on the cover of this thesis, many people have contributed to its existence. I owe my gratitude to all of the people who have made this journey nothing short of amazing. First and foremost, I would like to express my sincere gratitude to my advisor Prof. Lon J. Wilson for his constant assistance, encouragement, guidance and the tremendous support he provided throughout my PhD studies. I couldn't have asked for more from an advisor. I also like to thank to Professor Vicki L. Colvin, Professor Enrique V. Barrera and Professor Michael T. Lewis for their kindness to take time out of their busy schedules to serve on my thesis committee. I am particularly grateful to my collaborators Professor Michael. T. Lewis and Dr. Gabriel J. Villarreal from Baylor College of Medicine, Professor Steven A. Curley and Dr. Mustafa Raoof from The University of Texas M.D. Anderson Cancer Center. Without their contribution, a substantial part of this thesis would have been blank pages. I have been very fortunate to be surrounded by the wonderful people in Wilson Laboratory who made my years in the lab such a wonderful experience. I feel obliged to thank you whom I have had opportunity to work: Ari Berlin, Ayrat Gizzatov, Brandon Cisneros, Dr. Jeyarama Ananta, Justin Law, Dr. Lesa A. Tran, Matthew Cheney, Matthew Makansi, Mayra Hernandez-Rivera, Meghan Jebb, Dr. vi Michael Matson, Nadia Lara, Dr. Scott Berger, Stuart Corr, Sue Friend, Tawana Robinson, Rafael Lima, Dr. Richa Sethi, Sophia Phounsavath, Vazrik Keshishian, and Yuri Mackeyev)! It has been a joy and a privilege to know you all. In addition to those mentioned above, I am very thankful to many friends in US, Turkey and other parts of the World who helped me with their various forms of support throughout my graduate study and before. Finally, and most importantly, I would like to thank my wife, Esra, for her endless understanding, unconditional support and encouragement that allowed me to finish this journey. vii Contents Acknowledgments .................................................................................................................... v Contents ..................................................................................................................................... vii List of Figures ............................................................................................................................. x List of Tables ............................................................................................................................ xv Nomenclature ......................................................................................................................... xvi Introduction ............................................................................................................................... 1 1.1. Overview................................................................................................................... 1 1.2. Organization of the thesis ........................................................................................ 2 Background and Literature Review .................................................................................. 4 2.1. Cancer Chemotherapy.............................................................................................. 4 2.2. Cisplatin .................................................................................................................... 6 2.3. Carbon nanotubes (CNTs) for biomedical applications ........................................... 8 2.4. CNTs as nanocapsules for drug delivery ................................................................ 10 Cisplatin@US-tube carbon nanocapsules for enhanced chemotherapeutic delivery ...................................................................................................................................... 13 3.1. Introduction ............................................................................................................ 13 3.2. Materials and Methods .......................................................................................... 15 3.2.1. Preparation of US-tubes .................................................................................. 15 3.2.2. Encapsulation of CDDP within US-tubes.......................................................... 15 3.2.3. Characterization of CDDP@US-tubes .............................................................. 16 3.2.4. CDDP release studies from CDDP@US-tubes .................................................. 17 3.2.5. Cell viability studies ......................................................................................... 18 3.2.6. Cell internalization rate studies ....................................................................... 19 3.2.7. Transmission electron microscopy .................................................................. 19 3.2.8. Statistical evaluation........................................................................................ 20 viii 3.3. Results and Discussion ........................................................................................... 21 3.3.1. Encapsulation of CDDP within US-tubes.......................................................... 21 3.3.2. Release studies of CDDP from CDDP@US-tubes ............................................. 27 3.3.3. In vitro cytotoxicity studies .............................................................................. 29 3.4. Conclusions............................................................................................................. 34 Remotely-Triggered Cisplatin Release From Carbon Nanocapsules by Radiofrequency Fields.......................................................................................................... 35 4.1. Introduction ............................................................................................................ 35 4.2. Methods ................................................................................................................. 39 4.2.1. Sample Preperation ......................................................................................... 39 4.2.2. Inductively-coupled plasma optical emission spectrometry (ICP-OES)........... 40 4.2.3. RF generator setup .......................................................................................... 41 4.2.4. CDDP release from W-CDDP@US-tubes.......................................................... 42 4.2.5. Fluorescence measurements ........................................................................... 42 4.2.6. Cell studies ....................................................................................................... 43 4.2.7. Statistical analysis ............................................................................................ 44 4.3. Results .................................................................................................................... 44 4.3.1. RF absorption by US-tubes .............................................................................. 44 4.3.2. Localized heating of US-tubes in a physiological environment ....................... 46 4.3.3. RF-triggered release of CDDP from US-tubes .................................................. 49 4.3.4. RF-triggered release and cytotoxicity .............................................................. 51 4.4. Discussion ............................................................................................................... 53 4.5. Conclusion .............................................................................................................. 57 In vivo Biodistribution and Anticancer Activity of Cisplatin Delivered by Ultrashort Carbon Nanotube Capsules ..................................................................................... 59 5.1. Introduction ............................................................................................................ 59 5.2. Materials and Methods .......................................................................................... 60 5.2.1. Sample preparation: ........................................................................................ 60 5.2.2. Establishment of xenografts and treatment ................................................... 61 5.2.3. Biodistribution and blood circulation studies ................................................. 62 ix 5.2.4. Inductively-coupled plasma-optical emission mass spectrometry (ICP-MS) .. 63 5.2.5. Statistical analysis ............................................................................................ 63 5.3. Results .................................................................................................................... 64 5.3.1. Tumor suppressing effect of CDDP@US-tubes on MCF-7 xenograft in SCID/Beige mice ......................................................................................................... 64 5.3.2. Tumor suppressing effect of CDDP@US-tubes on MDA-MB-231 xenograft in SCID/Beige mice ......................................................................................................... 69 5.3.3. Tumor suppressing effect of CDDP@US-tubes on the BCM-4272 PDX xenograft in SCID/Beige mice .................................................................................... 73 5.3.4. Blood circulation and the biodistribution studies ........................................... 77 5.4. Discussion ............................................................................................................... 83 Conclusion and Future Perspective ................................................................................. 87 References ................................................................................................................................ 90 List of Figures Figure 2.1 − Tumor resistance to cisplatin mediated by inadequate levels of platinum reaching target DNA. Taken from.13 ............................................................... 8 Figure 2.2 − Preperation of US-tubes from SWCNTs. ................................................. 12 Figure 3.1 − Preparation and purification of CDDP@US-tubes.............................. 22 Figure 3.2 − Pt determination by ICP-OES analysis of each collected aliquot of H2O wash solution for a typical CDDP@US-tube sample. The data are expressed as mean ± SD of three independent experiment. Error bars may be smaller than symbols............................................................................................................ 23 Figure 3.3 − HR-TEM images of a) bundled CDDP@US-tubes and b) bundled empty US-tubes c) EDS spectrum of CDDP@US-tubes showing carbon and nickel peaks (metal catalyst) from US-tubes, copper peaks (from the TEM Cugrid), and platinum and chloride peaks (from CDDP). ...................................................................................................................................................... 25 Figure 3.4 − XPS spectrum of a) CDDP@US-tubes (survey), b) Pt 4f peaks for various samples: i) metallic platinum (Pt0), ii) CDDP@US-tubes (Pt2+), iii)CDDP (Pt2+) and iv) Na2PtCI6.6H2O (Pt4+)................................................................................... 27 Figure 3.5 − Comparative time release profiles of CDDP released from CDDP@US-tubes and W-CDDP@US-tubes (Pluronic-F108-wrapped) in PBS at 37 °C. The data are expressed as mean ± SD of three independent experiments. ...................................................................................................................................................... 28 Figure 3.6 − Time and concentration dependency of cell viability studies for MCF-7 and MDA-MB-231 cells incubated with CDDP, W-CDDP@US-tubes, and W-US-tubes. The quantity of US-tubes at each CDDP concentration is equivalent for the CDDP@US-tube and US-tube samples, with the concentration being calculated from their wt % values obtained by ICP-OES for Ni and Y which is contained within US-tube materials as the metal catalysts for carbon nanotube growth. The data are expressed as mean ± SD for three independent experiments. (*) and (#) indicate a statistically significant difference between W-CDDP@US-tube data compared to free CDDP data (*p < 0.01, #p<0.05). ........................................................................................................................ 30 xi Figure 3.7− Comparative internalization rate of CDDP and W-CDDP@US-tubes into MCF-7 cells as a function of incubation time at 10 µM CDDP concentration. The graph presents data for the average number of Pt2+ ions per cell for an average of three trials. Error bars are standard errors for the three different experiments. (ns=not significant, *p-value<0.05) ...................................................... 32 Figure 3.8 − (a, b) TEM images of a single MCF-7 cell from different angles. (c, d, e, f)TEM images of MCF-7 cells incubated with W-CDDP@US-tubes at 10 µM CDDP for 24 h. White arrows point to (a, b) ‘vesicularized’, and (c, d) ‘free’ CDDP@US-tube aggregates in the cytoplasm. Scale bar = 2 µm. ........................... 33 Figure 4.1 − Schematic describing synthesis, purification, and RF-triggered disruption of Pluronic®-F108 and subsequent release of CDDP from WCDDP@US-tubes. .................................................................................................................... 39 Figure 4.2 − Heating rates of W-CDDP@US-tubes and W-US-tubes suspended in 0.17% pluronic F108 in comparison with filtrate alone as a function of concentration. ......................................................................................................................... 45 Figure 4.3 − Panel A. Schematic demonstrating the principle of the temperature-dependent Pluronic disruption assay. Panel B and C. Temperature-dependent fluorescence quenching with a transition temperature close to the melting point of Pluronic F108 is indicative of Pluronic disruption from the w-CDDP-US-tube surface. Panel D. RF durationdependent quenching of fluorescence demonstrates localized heating of WCDDP@US-tubes in PBS. Water bath temperature controls (HT) fail to demonstrate quenching of APC fluorescence suggesting lack of Pluronic disruption due to bulk temperature. The RF-dependent quenching is therefore not a result of non-thermal denaturation of APC. ...................................................... 47 Figure 4.4 − CDDP release from W-CDDP@US-tubes with or without RF. Panel A. Release of CDDP from W-CDDP@US-tubes with or without RF exposure for 2.5 or 5 min is shown. In addition, bulk temperature controls are demonstrated to evaluate the contribution of bulk ionic heating to CDDP release from W-CDDP@US-tubes. Panel B. Relative areas-under-curve depicts cumulative CDDP release over 168 h relative to untreated control. ................... 51 Figure 4.5 − Release-dependent cytotoxicity of W-CDDP@US-tubes. Hep3B and HepG2 human liver cancer cells were plated in the bottom well of the trans-well plate and treated with PBS, W-US-tubes or W-CDDP@US-tubes followed with or without RF exposure for 3 min once, twice, or three times at xii 24 h intervals. The cell viability was assessed 120 h after treating with WCDDP@US-tubes. .................................................................................................................... 52 Figure 5.1 − Suppression of tumor growth by intraperitoneal injection of CDDP@US-tubes to an MCF-7 breast cancer xenograft. (A), Tumor growth of MCF-7 tumor-bearing mice after receiving different treatments as indicated. The mean body weight changes is also plotted in A. Fold increase in tumor volume (B) and Kaplan-Meier survival curves (C) depicting the tumor doubling times for different treatment groups. .......................................................... 66 Figure 5.2 − Platinum and yttrium concentration in tissue from mice treated over a period of ~ 45 d with weekly saline, w-US-tubes, CDDP, CDDP@UStubes, and W-CDDP@US-tubes administration via i.p. injection. (A) Concentration of platinum (from cisplatin) and (B) yttrium (from US-tubes) in tissue (mean ± s.e., n = 8). Statistically significant different (p < 0.05) are marked with symbols as indicated above (unpaired, two-sided t-test). ........... 68 Figure 5.3 − Suppression of tumor growth by intraperitoneal injection of CDDP@US-tubes to an MDA-MB-231 breast cancer xenograft. (A), Tumor growth of MDA-MB-231 tumor-bearing mice after receiving different treatments as indicated. (* indicates p < 0.05, unpaired, two-sided t-test, n = 8; error bars = s.e.) The mean body weight changes is also plotted in A. Fold increase in tumor volume (B) and Kaplan-Meier survival curves (C) depicting the tumor tribling times for different treatment groups. ........................................ 71 Figure 5.4 − Platinum and yttrium concentration in tissue from mice treated over a period of ~ 45 d with weekly saline, w-US-tubes, CDDP, CDDP@UStubes, and W-CDDP@US-tubes administration via i.p. injection. (A) Concentration of platinum (from cisplatin) and (B) yttrium (from US-tubes) in tissue (mean ± s.e., n = 8). Statistically significant different (p<0.05) are marked with symbols as indicated above (unpaired, two-sided t-test). ........... 72 Figure 5.5 − Suppression of tumor growth by intraperitoneal injection of CDDP@US-tubes to an Patient-derived, 4272 transplant, xenograft. (A), Tumor growth of Patient-derived tumor-bearing mice after receiving different treatments as indicated. (unpaired, two-sided t-test, n=9; error bars= s.e.) The mean body weight changes is also plotted in A. Fold increase in tumor volume (B) and Kaplan-Meier survival curves (C) depicting the tumor doubling times for different treatment groups. ......................................................................................... 75 xiii Figure 5.6 − Platinum and yttrium concentration in tissue from mice treated over a period of ~ 45 d with weekly saline, W-US-tubes, CDDP, CDDP@UStubes, and W-CDDP@US-tubes administration via i.p. injection. (A) Concentration of platinum (from cisplatin) and (B) yttrium (from US-tubes) in tissue (mean ± s.e., n = 8). Statistically significant different (p < 0.05) are marked with symbols as indicated above (unpaired, two-sided t-test). ........... 77 Figure 5.7 − Blood circulation for CDDP, W-CDDP@US-tubes, CDDP@US-tubes in MDA-MB-231 tumor-bearing mice as determined by measuring Pt (from CDDP) and Y (from US-tubes) concentration in blood via ICP-MS at different time points post injection (mean ± s.e., n = 3). CDDP@US-tubes showed prolonged blood circulation compared to free CDDP. .............................................. 78 Figure 5.8 − Time-dependent in vivo biodistribution of intraperitoneally injected CDDP, W-CDDP@US-tubes, and CDDP@US-tubes in MDA-MB-231 tumor bearing mice. (A) Concentration of platinum (from CDDP), (B) Concentration of yttrium (from US-tubes) in tissue (mean ± s.e., n = 3). Statistically significant different (p < 0.05) are marked with symbols as indicated above (unpaired, two-sided t-test). ............................................................. 83 Figure 6.1 − Hydrolysis of the peptide sequence on a CDDP@US-tube by PSA and/or MMP-2 . ....................................................................................................................... 89 xiv List of Tables Table 3.1 – Pt content by ICP-OES of CDDP@US-tubes under different loading solvent and washing conditions. EtOH is ethyl alcohol and DMF is dimethylformamide. Table presents the wt% Pt for an average of three trials under each condition and the standard deviation. .................................................... 24 Nomenclature °C Degrees Celsius ANOVA One-Way Analysis of Variance APC Allophycocyanin AUC Area-Under-Curve AMF Alternating Magnetic Field BCM Baylor College of Medicine BSA Bovine Serum Albumin CCD Charge-Coupled Device CDDP Cisplatin (Cis-diamminedichloroplatinum(II)) CDDP@US-tube CDDP within US-tube CNT Carbon Nanotube CO2 Carbon Dioxide DNA Deoxyribonucleic acid DMF Dimethyformamide DMSO Dimethyl Sulfoxide EDS Energy Dispersive X-ray Spectroscopy EDTA Ethylenediaminetetraacetic Acid EtOH Ethanol FBS Fetal Bovine Serum HNO3 Nitric Acid HCIO3 Chloric Acid xvii HR-TEM High-Resolution Transmission Electron Microscopy ICP-OES Inductively-Coupled Plasma Optical Emission Spectroscopy ICP-MS Inductively-Coupled Plasma Mass Spectrometry MAbs Monoclonal Antibodies MEM Minimal Essential Medium MMP-2 Metalloproteinase-2 MRI Magnetic Resonance Imaging MTT 3-(4,5 dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide MWCNT Multi-Walled Carbon Nanotube NIR Near-Infrared PBS Phosphate Buffered Saline PDX Patient-Derived Xenograft PSA Prostate Specific Antigen RES Reticuloendothelial System RF Radiofrequency RNA Ribonucleic acid RT Room Temperature SWCNT Single-Walled Carbon Nanotube SD Standard Deviation siRNA Small Interfering RNA xviii TEM Transmission Electron Microscopy THF Tetrahydrofuran US-tube Ultra-Short SWCNT W-CDDP@US-tube Pluronic® F108-wrapped CDDP@US-tubes XPS X-ray Photo Electron Spectroscopy Chapter 1 Introduction 1.1. Overview Although chemotherapy is one of the major approaches for the treatment of cancer, a vast majority of existing chemotherapeutic (anticancer) drugs cannot be employed to full clinical benefit due to problems associated with administration, such as low water solubility, rapid elimination, nonspecific biodistribution, rapid breakdown in vivo, insufficient accumulation in tumor tissue, inability to cross cellular barriers, and inability to differentiate between healthy cells and cancer cells, leading to systemic toxicity and adverse side effects that greatly limit the dosage level. These limitations necessitate the development of more efficient ways to administer anticancer drugs systemically, yet more selectively target tumor tissue, thereby improving efficacy while minimizing undesirable side effects. 1 2 Over the last few years, with the advancement in nanotechnology, numerous nanotechnology-based drug delivery systems have been developed in an effort to maximize therapeutic effectiveness of conventional drug delivery, while limiting undesirable side effects. Among different type of nanomaterials, carbon nanotubes (CNTs) are of special interest in the area of drug delivery as potential drug delivery agents due to their unique physical and chemical properties. This thesis details the development of a new CNT-based drug delivery platform for the treatment of cancer which is comprised of ultra-short single-walled carbon nanotubes (hereafter called US-tubes) loaded with the cisplatin (CDDP) chemotherapeutic drug (CDDP@UStubes) that has demonstrated enhanced chemotherapeutic efficacy for breast cancer both in vitro and in vivo. 1.2. Organization of the thesis Chapter 1: Introduction and overview. Chapter 2: Background and literature review. This chapter provides brief background information about conventional cancer chemotherapy and the cisplatin. It also reviews recent progress about the development and use of CNTs-based drug delivery system. Chapter 3: Cisplatin@US-tube carbon nanocapsules for enhanced chemotherapeutic drug delivery. This chapter describes the synthesis, characterization and in vitro anticancer activity evaluation of CDDP@UStubes in different breast cancer cell lines. 3 Chapter 4: Remotely-triggered cisplatin release from carbon nanocapsules by radiofrequency fields. This chapter demonstrates CDDP release from CDDP@US-tubes nanocapsules as achieved by remote radiofrequency (RF) field activation. Release-dependent in vitro cytotoxicity data is also demonstrated for human hepatocellular carcinoma cell lines. Chapter 5: In vivo biodistribution and anticancer activity of cisplatin delivered by ultra-short carbon nanotube capsules. This chapter is an extension of Chapter 3 and provides the first in vivo biodistribution behaviors and efficacy information for CDDP@US-tubes after the systematic administration in three different breast cancer tumor models. Chapter 6: Conclusion and future perspective. This chapter summarizes the important conclusions of the thesis and proposes future research directions. 4 Chapter 2 Background and Literature Review 2.1. Cancer Chemotherapy Cancer is a multifactorial cell disease, characterized by cells with unrestricted division potential, evasion of apoptosis, and invasive expansion to other body parts through blood and lymph systems.1 It is known to be one of the most devastating diseases, accounting for 7.6 million deaths in 2008 which is expected to continue to rise to over 13.1 million by 2030.2 In 2012, nearly 577,190 Americans died of cancer. In other words, about one in every three women and one in every two men in the United States will be affected by cancer in their lifetime. Despite centuries of intensive research and progress, the development of effective and favorable treatments for cancer continues as an elusive goal. Surgery, radiation, and chemotherapy are the most common treatment modalities for cancer which are performed on the basis of clinical and pathologic staging that is determined using 5 morphologic diagnostic tools, such as conventional radiological and histopathological examinations.3 In contrast to surgery, radiation therapy, and other forms of “local" treatment, chemotherapy offers a type of “systemic" treatment because chemotherapeutic (anticancer) drugs circulate throughout the body in the bloodstream to reach cancer cells wherever they may have spread. Despite some advances over the last few decades in conventional chemotherapy, the efficacy of the vast majority of cancer therapeutics, while being potent, is often limited for many reasons. A major problem limiting the success of many anticancer agents is their inability to selectively target tumor cells and tissues. Generally, chemotherapeutic agents target cells that are actively propagating, a characteristic hallmark of cancer cells. Unfortunately, these agents are unable to differentiate cancer cells from actively growing normal cells (e.g., cells in the blood, mouth, intestines, hair, and particularly in bone marrow). Severe side effects occur when chemotherapeutic agents suppress dividing immune and somatic cells in bone marrow and peripheral blood lymphoid tissues, resulting in fatal side effects such as suppression of bone marrow activity. Moreover, even if the anticancer drugs selectively accumulate in the tumor interstitium, they can have limited efficacy against numerous tumor types because cancer cells are able to develop mechanism of resistance, which is a significant hurdle to overcome for successful chemotherapy. In addition, rapid elimination and widespread distribution of drugs in the body require high doses or continuous infusion in order to induce sufficient antitumor effects, resulting in dose-limiting side effects. There are many chemotherapeutics 6 drugs available, and the choice of which one to use depends on the cancer type, stage, and grade. 2.2. Cisplatin Cisplatin, cis-diamminedichloroplatinum(II), (CDDP) is one of the most potent, and widely-used, chemotherapeutic agents in cancer therapy for the treatment of variety of solid tumors, including testicular, ovarian, bladder, cervical, head and neck, oesophageal, small cell lung and breast cancer.4–6 The biological activity of CDDP is derived from its interaction with DNA, with which it forms stable adducts that interfere with the normal transcription and replication processes of the cell which results in cell death. Despite its success, the clinical use of CDDP faces a number of serious problems. CDDP has limited bioavailability due to its reactive nature. Upon administration, CDDP rapidly binds to albumin and other plasma proteins and spontaneously degrades in the bloodstream, leading to inactivation of nearly 90% of the injected dose,5 and it rapidly cleared from the blood by glomerular excretion.7 In addition, the lack of selectivity of CDDP causes many dose-limiting severe adverse reactions including renal toxicity from renal tubular damage, gastrointestinal toxicity, peripheral neuropathy, asthenia (lack or loss of strength), and ototoxicity (damage to the hearing or balance functions of the ear).8 Moreover, the resistance of tumors to CDDP remains one of the main limitation of CDDP in the clinical. Certain cancer types are intrinsically resistance to CDDP (e.g., colon cancer, non-small-cell lung cancer), while others acquire resistance after exposure to the drug over time 7 (e.g., ovarian cancer, small cell lung cancer).9 CDDP resistance is known to be a multifactorial phenomenon, involving reduced drug uptake, inactivation by thiol containing species, increased repair/tolerance of platinum−DNA adducts, and alterations in proteins involved in apoptosis. Among these, decreased intracellular concentration of CDDP is generally considered the major cause of CDDP resistance in cancer cells.6,10,11 Because CDDP is highly polar, it enters cells relatively slowly compared to other small-molecule cancer drugs, using either transporters such as the copper transporter CTR1 or by passive diffusion (Figure 2.1).12,13 The uptake of CDDP is influenced by several factors, such as pH, sodium and potassium ion content, and the presence of a reducing agent, transporters or gated channels.5,13 Loss of chloride groups is required before CDDP binds to DNA. After CDDP is internalized within a cell, it replaces one or both chloride groups with water molecules which is facilitated by the low concentration of chloride that is found within cells, resulting in the more reactive platinum species, [Pt(H2O)Cl(NH3)2]+ and [Pt(H2O)2(NH3)2]2+. Binding of these activated species to thiol-containing peptides such as metalothionein and tripeptide glutathione, consume the activated CDDP in the cytoplasm before DNA binding occurs, thereby causing resistance.5,13 Because CDDP is one of the most potent anticancer drug in the clinic, its potential use in cancer therapy is very attractive. With the emerging trends and recent advances in nanotechnology, it has become increasingly possible to address some of the shortcomings associated with CDDP as an anticancer drug. 8 Figure 2.1 − Tumor resistance to cisplatin mediated by inadequate levels of platinum reaching target DNA. Taken from.13 2.3. Carbon nanotubes (CNTs) for biomedical applications In recent years, the application of nanotechnology in medicine, defined as nanomedicine, has garnered tremendous attention for the purpose of cancer diagnosis and therapy, and it is currently the subject of an intense research effort by many scientists worldwide. Among diverse classes of nanomaterials, carbon nanotubes (CNTs), an allotropic form of carbon which consists of graphite sheets rolled up into tubular form, have been extensively explored in many fields. Regardless whether they contain one (SWCNTs) or multiple (MWCNTs) graphene 9 sheets, CNTs exhibit many unique physical, mechanical and chemical properties that make them potentially desirable for many application, including nanomedicine.14–18 In recent years, research toward applying CNTs for biomedical applications has been progressing rapidly which has resulted in numerous publication in the field. In particular, recent studies have shown that CNTs use in nanomedicine as a platform holds great promise, especially for the detection and treatment of cancer due to their many intrinsic unique properties such as high surface area, high aspect ratio, high thermal conductivity, remarkable optical and electronic properties.19–26 Owing to their high surface area, CNTs enable the engineering of surface modification for a variety of therapeutics molecules either by specific adsorption or by covalent bioconjugation.20–22,27–30 Due to their hollow cylindrical structure, CNTs provide internal cavities which are capable of accommodating small molecules or ions.17 While the toxicological effects of CNTs have been widely debated in the literature,31– 34it has been shown recently that appropriately functionalized35 and highly-purified SWCNTs can be nontoxic and well-tolerated in vivo.36 In addition, CNTs are considered to be relatively bioinert, even though one study has reported that they can be degraded in vivo by neutrophils.37 Several reports have demonstrated that CNTs readily cross cell membranes due to their intrinsic lipophilic character and high aspect ratio (needle-like structure) and are able to shuttle biological molecules including drug molecules, proteins, plasmid DNA, and siRNA into cells.27,28,38,39 Relying on their optical properties including strong near-infrared (NIR) optical absorption, resonance Raman scattering, and photoluminescence, SWCNTs have been exploited as optical imaging or contrast agents for tracking, detecting, and 10 imaging purposes.22,40–43 Moreover, optical or radiofrequency wavelength irradiation of SWCNTs can also selectively be used for photothermal and photoacoustic therapy.24,44,45 Finally, CNT materials, whether derivatized or underivatized, have been observed to be eliminated from animals via the kidneys.36,46 Taken together accumulating evidence suggests that, CNTs have the potential to play a significant role in the diagnosis and therapy of cancer. 2.4. CNTs as nanocapsules for drug delivery Drug delivery is one of the most promising biomedical applications of CNTs, and over the years, several CNT-based systems have been developed with the intention of obtaining better results for drug delivery. In these systems, drug molecules have been mainly attached to the surface or sidewalls of CNTs either by specific adsorption or by covalent bonding.20,21,29,30,47–49 In addition to surface attachment, it has been shown that such molecules can be encapsulated within the interior cavity of CNTs.50–52 Encapsulation of drug molecules within the interior of CNTs may to be more beneficial than attachment to the external surface for several reasons. First, the cytotoxicity of drug molecules can be sequestered within CNTs interior by providing an insulating environment for the drug that prevents degradation and leakage during circulation before the drug reaches its target sites. Secondly, attaching a drug molecule to the external walls of CNTs typically involves covalent or non-covalent conjugations which may alter the pharmacological activity of the drug. Moreover, the interior of CNTs has a favorable binding energy for the adsorption of lipophilic molecules, making it possible to encapsulate lipophilic drug 11 molecules by simple adhesive forces. Finally, encapsulation of drug molecules within the CNT interior space also preserves the external surface of CNTs for further chemical modification for desired cancer cell targeting with peptides and/or antibodies. Although the ideal length of CNTs for biomedical application is uncertain, but US-tubes (~1.4 nm × 20 - 80 nm), which are produced from full-length SWCNTs via a fluorination and pyrolysis procedure53 (Figure 2.2), may be best suited due to their short and relatively uniform-lengths (ca. 95% ≤ 50 nm) which might help them avoid the reticuloendothelial system (RES), while enhancing their cellular uptake properties and eventual elimination profiles. US-tubes, with sidewall defects from the chemical cutting process, have proven a convenient platform as nanocapsules for the loading and containment of ions, molecules, and drugs whose cytotoxicity is sequestered within the US-tubes.54–58 Additionally, it has been demonstrated that the exterior surface of US-tubes can be modified with chemical moieties for enhanced solubilization, with peptides for biological targeting purposes and with monoclonal antibodies (MAbs) for the specific targeting of cancer cells.59,60 Moreover, a medical imaging agents derived from US-tubes (gadonanotubes) has been shown to translocate into cells without exhibiting significant toxicity.61 Finally, one recent study36 has shown that highly-purified SWCNTs and US-tubes are welltolerated in vivo by Swiss mice, even at very high doses (~0.5 g kg-1 b.w.). 12 Figure 2.2 − Preperation of US-tubes from SWCNTs. Chapter 3 Cisplatin@US-tube carbon nanocapsules for enhanced chemotherapeutic delivery1 3.1. Introduction Chemotherapy is one of the main treatments for both localized and metastasized cancer, and it is typically-used in conjunction with surgery and/or radiotherapy. For chemotherapy, it is desirable to deliver a sufficient quantity of drug to malignant cells, while minimizing undesirable side effects. However, the efficacy of many drugs is hindered due to complications with administration such as limited solubility, rapid elimination, inefficient distribution, inability to cross 1 Major portions of this chapter have been published in the following research article: Guven, A.; Rusakova, I. A.; Lewis, M. T.; Wilson, L. J. Cisplatin@US-tube Carbon Nanocapsules for Enhanced Chemotherapeutic Delivery. Biomaterials 2012, 33, 1455–1461. 13 14 cellular barriers, and the inability to differentiate between normal cells and cancer cells. CDDP is one of the most potent and widely-used anticancer drugs for the treatment of a variety of solid tumors, including testicular, ovarian, bladder, cervical, head and neck, oesophageal, small-cell lung and breast cancers.4,5 The biological activity of CDDP is derived from its interactions with DNA since CDDP interferes with the normal transcription and replication processes of the cell which results in cell death.5,11 Despite its success, the clinical use of CDDP and its derivatives are limited due to severe side effects including nephrotoxicity, neurotoxicity, ototoxicity, nausea, and vomiting.8 Thus, the development of efficient drug delivery systems that selectively increase the concentration of chemotherapeutics, such as CDDP, in diseased cells while limiting their concentration in normal cells continues to be of great interest. Although a variety of drug delivery systems, including microspheres,62,63 silica nanoparticles,64,65 dendrimers,66 polymeric micelles,67–69 liposomes,70–72 and carbon nanohorns,73,74 can be used as drug delivery agents, CNTs offer a number of special properties which suggest that they too might be engineered into desirable drug delivery platforms to compliment other more typically-used materials. To date, a number of CNT based drug delivery systems have been explored. In these systems, drug molecules mainly have been attached onto the surface or sidewalls of the nanotubes either by specific adsorption or by covalent bonding.21,29 In addition to surface attachment, it has been shown that small drug molecules can also be embedded75 or encapsulated50 within SWNT materials. In this chapter, we describe the preparation, characterization, and in vitro testing of a new US-tube-based drug 15 delivery platform for the treatment of cancer, which allows for the possible design of a cancer-specific enzyme-activatable US-tube-based drug delivery platform that releases its drug cargo within cancer cells only. 3.2. Materials and Methods 3.2.1. Preparation of US-tubes US-tubes were prepared from full-length SWCNTs (purchased from Carbon Solutions Inc., CA, USA), which were produced by the electric-arc discharge method, as previously reported.53 To remove amorphous carbon and metal catalyst impurities, US-tubes were first bath-sonicated in concentrated HCI for 1 h, then collected by filtration on a glass filter and finally washed several times with distilled water. These purified US-tubes were then chemically reduced by a metallic Na° /THF reduction procedure to create individual US-tubes.76 3.2.2. Encapsulation of CDDP within US-tubes Chemically debundled US-tubes (20 mg) were dispersed in deionized water (20 mL) via bath-sonication for 1 h. CDDP (20 mg) was then added to the US-tube suspension and vigorously stirred for 24 h. After stirring, the solution was left undisturbed overnight whereupon CDDP-loaded US-tubes (CDDP@US-tubes) flocculated from solution. After removing the supernatant solution, CDDP@US-tubes were collected on a glass filter. To remove the excess CDDP from the outer surface of the US-tubes, the CDDP@US-tubes were washed forty times with deionized water 16 (total of 60 mL) until all exterior CDDP was removed, as judged from Pt analysis on the filtrate aliquots by ICP-OES (see below). The collected CDDP@US-tubes were then dried in an oven at 80 ˚C. 3.2.3. Characterization of CDDP@US-tubes To confirm the encapsulation of CDDP within the US-tubes, HR-TEM studies were performed using a JEOL 2000 FX microscope equipped with an energydispersive spectrometer (EDS) and operated at 200 kV. Conventional and HR-TEM imaging methods were used. Precautions were taken to minimize structural changes in the sample caused by electron-beam heating effects. One such precaution was the use of a cold sample stage which kept the sample temperature in the range of -70 ˚C to -50 ˚C. The HR-TEM samples were prepared by placing a small amount of CDDP@US-tube sample on a copper grid coated with amorphous carbon holey film. No solvents were used during the procedure. To confirm that CDDP was removed from the exterior surface of US-tubes by washing, washings were collected and analyzed by ICP-OES. Elemental analyses of CDDP@US-tube samples were carried out by multiple analytical techniques: XPS, EDS, and ICP-OES. For XPS analysis, solid samples were placed on indium foil (Sigma-Aldrich) and then examined using a PHI Quantera Spectrometer with photo and Auger emissions produced via a monochromatic Al Ka X-ray source with a pass energy of 140 eV for survey and 30 eV for high-resolution scans. EDS measurements were carried out using an OXFORD EDS system installed in a JEOL-2100F TEM operating at 200 kV. Samples for EDS measurements were prepared via bath- 17 sonication in methanol for a few min, and a drop of dispersed solution was then placed on a TEM copper grid. ICP-OES analyses were made using a Perkin Elmer Optima 4300 DV instrument with a CDD detector. The quantity of CDDP internalized within the US-tubes was quantified by measuring the Pt concentration of each sample. Samples for ICP-OES measurements were prepared by digesting the CDDP@US-tubes with 26% HCIO3, as previously described.61 The platinum signal was observed at 265.95 nm. Five scans were performed for each sample, and the concentration was determined from the average of scans. Yttrium was used as the internal standard. 3.2.4. CDDP release studies from CDDP@US-tubes CDDP release studies from US-tubes were performed by enclosing the CDDP@US-tubes inside a dialysis-membrane (20,000 MW) cylinder, which was immersed in PBS at 37 ˚C for one week. During immersion, the solid CDDP@US-tube sample in the dialysis-membrane cylinder was sampled periodically by filtration and collection on a glass frit filter, followed by washing with distilled water to remove PBS. The Pt concentrations of solid CDDP@US-tube samples were then determined by ICP-OES. CDDP release with time from CDDP@US-tube samples was also determined after dispersing the CDDP@US-tube samples in a 0.17% (w/v) Pluronic®-F108 solution via probe sonication for 2 min to produce Pluoronic®F108- wrapped CDDP@US-tubes (W-CDDP@US-tubes). Pluronic®-F108 is a neutrally charged, non-cytotoxic surfactant that is commonly used to administer 18 carbon nanotube materials for in vitro and in vivo testing.61 Release of CDDP from W-CDDP@US-tubes and CDDP@US-tubes was then compared. 3.2.5. Cell viability studies The cytotoxic activity of CDDP released from W-CDDP@US-tubes was evaluated against two different breast cancer cell lines (MCF-7 and MDA-MB-231) by using MTT cell proliferation assay (Invitrogen). This colorimetric assay quantifies cell proliferation and viability by measuring the reduction of the tetrazolium component (MTT) into an insoluble formazan product by the mitochondria of viable cells. The cells were cultured in Minimum Essential Medium (MEM) supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.15% insulin (10 mg mL -1) at 37 ˚C under a humidifying atmosphere containing 5% CO2. The cells were then seeded onto 96-well plates at a density of 5 × 103 cells well-1 in 150 µL MEM. After 24 h of incubation to allow cell adhesion, the medium in each well was replaced with 150 µL of new medium containing the serial dilution of either CDDP,W-CDDP@US-tubes, or W-US-tubes at various concentrations (5 - 50 µM), and cells were then incubated at 37 ˚C in 5% CO2 for 24 h, and 48 h. An equivalent US-tube concentration of CDDP@US-tubes and US-tubes was used for each of the concentration point by calculating the US-tubes concentration of CDDP@US-tubes from Pt wt % values obtained by ICP-OES analysis. After incubation for each time period, the medium in each well containing CDDP, W-CDDP@US-tubes, or W-US-tubes was aspirated and the cells washed with phosphate-buffered saline (PBS). For cell viability measurements, 100 µL of fresh medium (free of phenol red) and 10 µL of MTT 19 reagent (5 mg mL-1 in PBS) was added to each well and incubated for 4 h to allow the formation of formazan crystals. After removing all but 25 µL of medium from the wells, 50 µL of dimethyl sulfoxide (DMSO) was added to each well (to solubilize the formazan) and mixed thoroughly and incubated at 37 ˚C for an additional 10 min. The absorbance values were measured using a microplate reader (Bio-Rad Model 680) at 570 nm to determine relative viabilities. 3.2.6. Cell internalization rate studies To determine the comparative cell internalization rate of free CDDP and WCDDP@US-tubes into MCF-7 cells, the cells were plated on a cell culture dishes at 5 × 105 cell/dish and allowed to reach up to 70 - 80% confluence. After removing the medium in each dishes, the attached cells were first washed with PBS and then incubated with free CDDP and W-CDDP@US-tube samples in MEM at 10 mm CDDP concentration for various times (3, 6, 12, 24, and 48 h). After each incubation time, the cells were washed with PBS, collected from the dishes by lifting up with trypsinEDTA, counted with a cell counter (Cellometer Auto T4), and prepared for ICP-OES analysis to quantify Pt in each sample. 3.2.7. Transmission electron microscopy MCF-7 cells were routinely cultured as described earlier and grown on a cell culture dish, and incubated with W-CDDP@US-tubes (10 μM CDDP) at 37 ˚C. After 24 h incubation, the cell monolayers were briefly rinsed with PBS, and fixed in situ with a primarily fix solution (2% PF + 2.5% glutaraldehyde + 0.1M cacodylate + 20 2mM CaCl2 in sodium cacodylate buffer, pH7.4) for 1 h at room temperature (RT) on a tissue rotator, washed with buffer (3x5min in 0.1M sodium cacodylate buffer + 2mM CaCl2, pH 7.4), post-fixed with 1% OsO4 in 0.1M sodium cacodylate buffer for 1 hour, rinsed with H2O (3x5min), saturated (stained) with aq. uranyl acetate for 45 min in the dark (foil wrapped), washed with H2O (3x5min), dehydrated through a series of graded alcohol washes in the following order: 2x10min in 30%, 50%, 70%, and 90%, 3x15min in 95%, and 3x20min in 100% ethanol, and changed 3 times in Epon (1 h each). After removing the last change of Epon, the cell monolayers were embedded in a fresh change of Epon plastic and polymerized at 70 ˚C over night. For comparison, in a separated dish, MCF-7 cells without incubating with W-CDDP@UStubes were also grown and prepared for TEM as a control group. Several 1 μm sections were cut and stained with 1% methylene blue and 1% basic fuchsin. Ultrathin sections (80 nm) were cut from the sample block using an RMC MTXL ultra microtome and mounted on 100-mesh copper grids. The grids were stained with 2% alcoholic uranyl acetate and Reynold’s lead citrate. The samples were prepared by the Integrated Microscopy Core at Baylor College of Medicine, and examined with a JEOL 1230 TEM at 120kV equipped with a digital imaging system at Rice University. 3.2.8. Statistical evaluation Unless otherwise noted, all data are expressed as mean ± standard deviation(SD) of at least three independent experiments. Wherever appropriate, the statistical significance of data were analyzed using the Microsoft Excel statistical 21 package. A one-tail homoscedastic unpaired t-test was used to determine statistical significance; a p-value of less than 0.05 (p < 0.05) was considered statistically significant. 3.3. Results and Discussion 3.3.1. Encapsulation of CDDP within US-tubes The encapsulation of CDDP within US-tubes has been achieved as detailed in the Materials and Methods Section and as outlined in Figure 3.1. After an extensive washing procedure (>20×) with deionized water, 10 mL aliquots of each washing were collected and analyzed for Pt content by inductively-coupled plasma optical emission spectrometry (ICP-OES) to determine CDDP release over time. As shown in Figure 3.2, the majority of CDDP was removed by the first few washings with water. Since no additional removal of CDDP was observed after 20 washings, it was concluded that all exterior CDDP was removed by 20 washings. 22 Figure 3.1 − Preparation and purification of CDDP@US-tubes. 23 P t c o n c e n t r a t io n ( m g / L ) 2 .0 1 .5 1 .0 0 .5 0 .0 0 10 20 30 40 N u m b e r o f w a s h in g s Figure 3.2 − Pt determination by ICP-OES analysis of each collected aliquot of H2O wash solution for a typical CDDP@US-tube sample. The data are expressed as mean ± SD of three independent experiment. Error bars may be smaller than symbols. After the extensive washing procedure, the CDDP@US-tubes were collected by filtration and dried in an oven at 80 ˚C. To determine the quantity of CDDP within the US-tubes, the Pt concentration was determined via ICP-OES. Because of the limited solubility of CDDP in water, CDDP@US-tube preparation was also attempted in a few different solvent mixtures in which CDDP is more soluble, and the results are summarized in Table 3.1. Although CDDP has greater solubility in ethanol (EtOH) and dimethylformamide (DMF), greater loading was achieved when water 24 alone was used. For this reason, further preparations were performed in water to enhance CDDP@US-tube yields. Material Loading Solvent wt% Pt (after one washing with H2O) wt% Pt (after 40 washings with H2O) CDDP@US-tubes H2O 8.70 ± 0.43 6.37 ± 0.57 CDDP@US-tubes H2O:EtOH (1:1) 6.66 ± 0.31 4.42 ± 0.35 CDDP@US-tubes H2O:DMF (1:1) 8.51 ± 0.34 3.38 ± 0.30 CDDP@SWCNTs H2O 6.27 ± 0.89 0.48 ± 0.11 Table 3.1 − Pt content by ICP-OES of CDDP@US-tubes under different loading solvent and washing conditions. EtOH is ethyl alcohol and DMF is dimethylformamide. Table presents the wt% Pt for an average of three trials under each condition and the standard deviation. A CDDP@US-tube sample has been characterized by high-resolution transmission electron microscopy (HR-TEM). As seen in Figure 3.3a, CDDP clusters are observed as dark spots encapsulated along the interior axis of the US-tubes, with both Pt and Cl content confirmed by electron-dispersion spectroscopy (EDS). For comparison, empty US-tube images do not reveal such dark spots (Figure 3.3b). Since the dimension of an individual CDDP molecule is about 0.4 nm × 0.6 nm × 0.1 nm,77 CDDP molecules have the ability to easily fit within US-tubes (ca. 1.4 nm in diameter) and cluster at the sidewall defect sites. Based on the CDDP aggregate sizes 25 (black spots) which ranged in length from 0.75 to 1.0 nm, up to four or five CDDP molecules exist in each aggregate. Earlier theoretical calculations have also concluded that CDDP can easily fit within the confines of SWCNTs.78 C c) a) Cl Cu Cu b) Pt Cl 0 2 Cu Pt Ni 8 10 Energy (keV) Figure 3.3 − HR-TEM images of a) bundled CDDP@US-tubes and b) bundled empty US-tubes c) EDS spectrum of CDDP@US-tubes showing carbon and nickel peaks (metal catalyst) from US-tubes, copper peaks (from the TEM Cugrid), and platinum and chloride peaks (from CDDP). EDS analysis was also performed on a solid CDDP@US-tube sample (Figure 3.3c). EDS detected carbon, nickel (metal catalyst) from US-tubes, copper (from the TEM grid), and platinum and chloride (from CDDP). EDS also determined the same Pt:Cl (1:1) mole ratio as that of the XPS data. To determine whether the CDDP Pt center remains in its original oxidation state (Pt2+) after encapsulation within US-tubes, the X-ray photoelectron 26 spectroscopy (XPS) spectra of the Pt 4f peaks of CDDP@US-tubes, metallic platinum (Pt0), and sodium hexachloroplatinate hexahydrate, (Na2PtCl6.6H2O; Pt4+), were obtained. Figure 3.4a shows the survey XPS spectrum of CDDP@US-tubes, confirming the presence of platinum, chloride, and nitrogen from CDDP and carbon and oxygen from US-tubes. Figure 3.4b shows that the Pt 4f peaks of the CDDP@UStube sample were positioned between the Pt 4f peaks of metallic platinum and Na2PtCl6.6H2O, confirming that the CDDP@US-tube sample contains Pt2+ and not reduce Pt0 or oxidize Pt4+. For comparison, we also obtained the Pt 4f spectrum of free CDDP (Strem Chemicals) which displayed peaks at 72.30 eV and 75.65 eV. On the other hand, the CDDP@US-tube sample exhibited Pt 4f peaks at 72.90 eV and 76.25 eV, corresponding to a +0.60 eV shift from that of free CDDP sample. This difference apparently arises from the environment of the encapsulating US-tubes. XPS also confirmed a 2:1 N:Cl ratio and a 2:1 N:Pt ratio, suggesting that the extensive washing process with water to prepare the CDDP@US-tube sample nearly halved the amount of chloride ion present in the original CDDP sample, likely replacing them with water molecules.79 Since the loss of chloride ions from CDDP is desirable before CDDP binds to DNA and other cellular targets,13 the loss of chloride ions in the CDDP@US-tube sample should not affect the efficacy of the Pt2+ species released from the US-tubes. 27 0 m e t a l lic p l a t in u m ( P t ) a) b) 4 4 1 0 4 - C 1s 6 1 0 C D D P (P t 2+ 2+ ) ) N a 2 P t C l 6 .6 H 2 O ( P t 4+ ) 68 70 72 74 76 78 80 B in d i n g E n e r g y ( e V ) 82 - O KLL - O 1s - N 1s - Pt 4d 5 - Pt 4d - Cl 2s 4 - Cl 2p 2 1 0 - Pt 4f 7 c /s 3 8 1 0 4 C D D P @ U S -tu b e s (P t 0 0 200 400 600 800 1000 B in d i n g E n e r g y ( e V ) Figure 3.4 − XPS spectrum of a) CDDP@US-tubes (survey), b) Pt 4f peaks for various samples: i) metallic platinum (Pt0), ii) CDDP@US-tubes (Pt2+), iii)CDDP (Pt2+) and iv) Na2PtCI6.6H2O (Pt4+). 3.3.2. Release studies of CDDP from CDDP@US-tubes Release of CDDP from a drug delivery system is an important parameter that should occur in a controlled fashion. To test the release of CDDP from CDDP@UStubes, we prepared a sample in which the CDDP@US-tube sample was dispersed via probe sonication for 2 min in an aqueous 1.7 mg mL-1 Pluronic®-F108 surfactant solution. The pluronic-wrapped CDDP@US-tube sample (W-CDDP@US-tubes) was expected to slow CDDP release since the sidewall defects and ends of US-tubes should be, at least, somewhat blocked by surfactant. Dialysis was performed on both CDDP@US-tubes and W-CDDP@US-tubes in phosphate-buffered saline (PBS) at 37 ˚C. The CDDP release rate for these two samples was investigated by determining the Pt content of samples over time by ICP-OES analysis. Figure 3.5 displays the 28 comparative release profiles of CDDP from CDDP@US-tubes and W-CDDP@UStubes. From the obtained data, we observed that the W-CDDP@US-tube system leaked only 6.1% CDDP in 24 h and only 16.9% over one week. However, unwrapped CDDP@US-tubes showed a much greater rate of release, with almost 50% of CDDP released in the first 3 h and about 80% after one week. From this data, we infer that the release of CDDP from W-CDDP@US-tubes is retarded because the pluronic wrapping around the US-tubes helps prevent drug release. C D D P @ U S -tu b e s 100 R e le a s e d C D D P ( % ) 80 60 W -C D D P @ U S -tu b e s 40 20 0 0 50 100 150 200 250 T im e ( h ) Figure 3.5 − Comparative time release profiles of CDDP released from CDDP@US-tubes and W-CDDP@US-tubes (Pluronic-F108-wrapped) in PBS at 37 °C. The data are expressed as mean ± SD of three independent experiments. 29 3.3.3. In vitro cytotoxicity studies A series of in vitro studies have been conducted to determine the cytotoxicity profiles of W-CDDP@US-tubes using the well-established MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay. The performance of W-CDDP@US-tubes was evaluated against two different breast cancer cell lines (MCF-7 and MDA-MB-231), which are known to be resistant to CDDP treatment,80 at 24 h and 48 h incubation times, and their efficacies compared to that of free CDDP. Figure 3.6 displays the comparative cytotoxicities of CDDP, WUS-tubes, and W-CDDP@US-tubes in a dose- and time-dependent manner. The cytotoxicity of CDDP and W-CDDP@US-tubes both increased with increasing CDDP concentration. While the US-tubes alone have a relatively small effect on cell viability for concentrations ≤20 mM, the W-CDDP@US-tubes clearly exhibited greater cytotoxicity for both cell lines and even greater cytotoxicity when compared to free CDDP at all CDDP concentrations. Although, the W-CDDP@UStubes displayed greater efficacy for each incubation time than free CDDP at an equivalent dose, the efficacy of the W-CDDP@US-tube sample was greater at 24 h than 48 h. To investigate this result, we decided to determine the Pt concentration per cell for the MCF-7 cell line treated with CDDP and W-CDDP@US-tubes as a function of time. 30 CDDP W -C D D P @ U S -tu b e s 120 120 100 100 80 * 60 * * 40 20 * C e l l v i a b il i t y ( % ) C e l l v i a b il i t y ( % ) M C F - 7 c e ll s W -U S - t u b e s 80 60 40 * 24h # 0 0 10 20 30 40 50 0 C D D P c o n c e n tr a tio n ( M ) 120 120 100 100 80 * 60 * 40 * * 20 10 20 30 40 50 C D D P c o n c e n tr a tio n ( M ) C e l l v i a b il i t y ( % ) C e l l v i a b il i t y ( % ) # 48h 0 M D A - M B - 2 3 1 c e lls * 20 80 60 # 40 20 24h 48h 0 0 0 10 20 30 40 50 C D D P c o n c e n tr a tio n ( M ) 0 10 20 30 40 50 C D D P c o n c e n tr a tio n ( M ) Figure 3.6 − Time and concentration dependency of cell viability studies for MCF-7 and MDA-MB-231 cells incubated with CDDP, W-CDDP@US-tubes, and W-US-tubes. The quantity of US-tubes at each CDDP concentration is equivalent for the CDDP@US-tube and US-tube samples, with the concentration being calculated from their wt % values obtained by ICP-OES for Ni and Y which is contained within US-tube materials as the metal catalysts for carbon nanotube growth. The data are expressed as mean ± SD for three independent experiments. (*) and (#) indicate a statistically significant difference between W-CDDP@US-tube data compared to free CDDP data (*p < 0.01, #p<0.05). 31 These results are given in Figure 3.7 which demonstrate that the cells treated with W-CDDP@US-tubes have significantly more Pt than cells treated with free CDDP over the first 24 h time period, however by 48 h, the Pt concentration in per cell is about the same for both W-CDDP@US-tubes and free CDDP. Thus, the US-tube structure apparently assists the delivery of free CDDP from W-CDDP@US-tubes into the cells over the first 24 h. In addition, the release rate of CDDP from W-CDDP@UStubes in vitro is likely greater than in only PBS (ca. 6% after 24 h) because previously observed aggregation of US-tubes within mesenchymal stem cells has indicated the removal of the Pluronic coating from US-tubes upon passage through the cell membrane.61 All of the above results taken together demostrate that WCDDP@US-tubes are more cytotoxic than free CDDP at the 24 h time point (p < 0.05). 32 CDDP@US-tube ns CDDP 8E+10 Number of Pt ions per cell ns * 6E+10 * 4E+10 * 2E+10 0 3 6 12 24 48 Time (h) Figure 3.7− Comparative internalization rate of CDDP and W-CDDP@US-tubes into MCF-7 cells as a function of incubation time at 10 µM CDDP concentration. The graph presents data for the average number of Pt2+ ions per cell for an average of three trials. Error bars are standard errors for the three different experiments. (ns=not significant, *p-value<0.05) We have also confirmed the uptake of CDDP@US-tubes into the cytoplasm of MCF-7 cells (Figure 3.8), although the uptake mechanism pathway or pathways have not been fully investigated yet. The cellular uptake of W-CDDP@US-tubes, which appears as irregular electron-dense aggregates within the cytoplasm as opposed to free MCF-7 cells (Figure 3.8), has been confirmed by TEM analysis; however, both vesicularized and free CDDP@US-tube aggregates have been observed to exist in the cells. To determine the dominant intracellular uptake mechanism of CDDP@US- 33 tubes within MCF-7 cells will require more extensive studies, which are currently underway and will be reported subsequently. (a) (c) (e) (b) (d) (f) Figure 3.8 − (a, b) TEM images of a single MCF-7 cell from different angles. (c, d, e, f)TEM images of MCF-7 cells incubated with W-CDDP@US-tubes at 10 µM CDDP for 24 h. White arrows point to (a, b) ‘vesicularized’, and (c, d) ‘free’ CDDP@US-tube aggregates in the cytoplasm. Scale bar = 2 µm. Since both cell lines used in this study are resistant to CDDP, and it is wellknown that reduced drug uptake is a major cause of CDDP resistance in cancer cell lines,11,13 the present data suggests that CDDP@US-tube materials help to overcome CDDP resistance by increasing drug accumulation in resistant cells. 34 3.4. Conclusions In this study, CDDP encapsulation within US-tubes has been confirmed by HR-TEM. Elemental analysis (XPS, and EDS) of CDDP@US-tubes has revealed that about half of the chloride ions of the encapsulated CDDP are replaced with water molecules during the aqueous washing procedure to prepare CDDP@US-tubes. It has also been shown that CDDP@US-tubes release CDDP upon dialysis in PBS at 37 °C much more slowly when CDDP@US-tubes are wrapped with Pluronic-F108 surfactant. Finally, the CDDP@US-tube platform exhibited greater cytotoxicities against MCF-7 and MDA-MB-231 cells at 24 h, when compared to free CDDP. These results suggest that by wrapping CDDP@US-tubes with cancer-specific enzymeactivatable peptide sequences, that cancer-cell-specific chemotherapeutics might be developed using the carbon nanocapsule platform of this study. Chapter 4 Remotely-Triggered Cisplatin Release From Carbon Nanocapsules by Radiofrequency Fields2 4.1. Introduction Maximizing therapeutic efficacy and minimizing adverse effects remain the fundamental goals of drug delivery in cancer therapy. A vast majority of cancer chemotherapeutics, while potent agents, cannot be employed to full clinical benefit because of toxicity concerns. Over the past decade, drug-loaded nanocarriers have been widely fabricated and studied to enhance tumor specific delivery. Most 2 This chapter has been published in the following research article: M. Raoof, B. T. Cisneros, A. Guven, S. Phounsavath, S. J. Corr, L. J. Wilson, S. A. Curley. “Remotely triggered cisplatin release from carbon nanocapsules by radiofrequency fields” Biomaterials 2013, 34, 1862-1869. 35 36 strategies that involve either active or passive targeting have been successful in demonstrating tumor-specific accumulation of these nanocarriers to achieve anticancer toxicity in pre-clinical models. However, recent studies demonstrate that intra-tumoral distribution of nanocarriers is patchy rather than homogenous.81–84 Nanocarriers extravasate from the vascular space and tend to accumulate in the peri-vascular space through fenestrations in tumor vasculature (also called enhanced permeation and retention effect).85 There are a multitude of factors that limit subsequent permeation of nanocarriers from the peri-vascular space to tumor cells, including the size of the nanocarriers, interstitial fluid pressure within the tumor, variations in pH in the microenvironment, composition of the extracellular matrix, and lymphatic clearance of nanocarriers.84,86–89 The spatial separation between drug-loaded nanocarriers and tumor cells often renders this strategy futile. To take advantage of intra-tumoral drug depots that are formed by these nanocarriers, environmentally- or remotely-triggered drug release mechanisms are being developed. The premise is based on the notion that free drug, because of its smaller molecular size, can rapidly diffuse along its concentration gradient to cells distant from the nanocarrier depots. Environmentally sensitive nanocarriers exploit pH,90 temperature,91–93 or the presence of tumor-specific enzymes (such as matrix metalloproteinases94) to trigger drug release. This strategy is inherently limited by the nonhomogeneous microenvironment.95 pH and enzyme Temperature-sensitive concentrations nanocarriers in (such the tumor as certain liposomes) depend on a strategy that raises bulk tumor temperature above a critical transition temperature to accomplish a phase change.96 However, raising bulk 37 tumor temperature selectively and safely remains a significant challenge. Because of these limitations, nanocarriers that can be remotely-activated or disrupted by focused ultrasound, light, or alternating magnetic fields to trigger drug release are under investigation. In each case, the nanocarrier is comprised of a nano-susceptor that absorbs energy and dissipates it locally to actuate release. Alternating magnetic fields offer full-body-penetration but acoustic and photo-triggered drug release are less ideal because of the non-uniformity and limited depth of penetration. These studies highlight the need for innovative strategies to enhance efficacy of nanocarriers to increase drug or biologic agent release after tumoral delivery. In developing remotely-triggered nanocarriers, carbon nanotubes are of particular interest to us. SWCNTs have been derivatized, functionalized, and loaded or conjugated with chemotherapeutics.97 More importantly, SWCNTs exhibit unique properties in radiofrequency (RF) electric fields such as enhancement of local Efields, absorption and dissipation of RF energy as heat, and alignment parallel to the incident E-field.24,98,99 Because RF fields at low megahertz frequency offer the advantage of full-body-penetration, nanotube-RF coupling can potentially be exploited to triggered release. However, full-length SWCNTs have a very high aspect ratio and, therefore, may not be ideal for drug delivery applications. To address this problem, US-tubes have been fabricated using a fluorination and pyrolysis process.53 The defects created in the US-tube sidewalls by this procedure allow for easy loading of cytotoxic chemotherapeutics, e.g. cisplatin (CDDP), contrast agents (Gd3+, I2), or radioisotope agents (211AtCl).54,58,100 Moreover, in vivo studies have demonstrated that US-tubes are non-toxic and well tolerated by mice even at very 38 high doses (∼0.5 mg/kg) making them a suitable candidate for nanocarrier development.36 We hypothesized that we could produce US-tubes loaded with CDDP and wrapped with a Pluronic coating to prevent CDDP release from the US-tubes. We further hypothesized that heating of the surface of US-tubes by a non-invasive RF field would lead to loss of Pluronic coating and CDDP release (Figure 4.1). Our studies were designed to confirm and characterize Pluronic-wrapped CDDP@UStubes (W-CDDP@US-tubes), to measure CDDP release from W-CDDP@US-tubes following RF field exposure, and to demonstrate RF-induced CDDP release at levels relevant to produce cancer cell cytotoxicity. 39 Figure 4.1 − Schematic describing synthesis, purification, and RF-triggered disruption of Pluronic®-F108 and subsequent release of CDDP from WCDDP@US-tubes. 4.2. Methods 4.2.1. Sample Preperation Pluoronic-F108-wrapped CDDP-US-tubes (W-CDDP-US-tubes) were prepared as described previously.100 In brief, full-length SWCNTs (Carbon Solution Inc., Riverside, CA), produced by an electric-arc discharge method, were cut into UStubes by fluorination followed by pyrolysis at 1000 °C under an inert atmosphere. 40 US-tubes were first purified in concentrated HCl by bath-sonication to remove amorphous carbon and metal catalyst impurities (nickel and yttrium), then chemically reduced by a metallic Na°/THF reduction procedure to produce individual US-tubes.76 The debundled US-tubes were dispersed in deionized water via bath-sonication for 60 min, and then CDDP was added to the US-tube suspension and vigorously stirred for 24 h. The reaction vessel was then left undisturbed overnight whereupon CDDP@US-tubes flocculated from solution. The CDDP@UStubes were collected by filtration on a glass filter, washed with excess deionized water to remove all exterior CDDP from the outer surface of the US-tubes (as judged from Pt analysis on the filtrate aliquots by ICP-OES), and dried at 80 °C. WCDDP@US-tubes were prepared by suspending dry CDDP-US-tube samples in a 0.17% (w/v) Pluronic-F108 solution via probe sonication for 2 min, followed by centrifugation at 3200 rpm for 10 min (3×) to remove unsuspended CDDP@UStubes. Extensive characterization of this CDDP@US-tubes material has been reported previously.100 4.2.2. Inductively-coupled plasma optical emission spectrometry (ICP-OES) ICP-OES analyses were carried out by a Perkin Elmer Optima 4300 DV instrument with a CCD detector. The quantity of CDDP in each sample was determined by measuring the Pt concentration, which was detected at 265.95 nm. For all ICP-OES measurements, the collected samples were transferred into glass vials, then digested with 26% HClO3. The samples were then diluted with 2% trace 41 metal grade HNO3. The Pt concentrations were determined from an average of five scans for each sample. Yttrium (371.029 nm) was used as the internal standard. 4.2.3. RF generator setup We employed the Kanzius External RF (13.56 MHz) Generator System (ThermMed, LLC, Erie, PA) as described previously.101 The generator utilizes a capacitively-coupled transmitter and receiver head to generate a uniform RF field within an air-gap of 10 cm. The generator input power was arbitrarily set at 950 W for all experiments. To demonstrate heating of US-tubes or CDDP@US-tubes, a suspension was created using 0.17 % (w/v) Pluronic® F-108 in deionized water or in PBS. Varying concentrations were loaded in a 1.3 mL quartz cuvette. The cuvette was placed in a Teflon holder and positioned at 5/16 inch from the transmitter head. The incident E-field intensity at this position was estimated to be 700 kV/m. The temperature of the cuvette was recorded using an infrared thermal camera (FLIR Systems, Boston, MA). Heating rates were calculated between 5 and 120 s of RF exposure where the Time vs. Temperature plots were noted to be linear. In order to expose 12-well cell culture plates to the RF field for the CDDP release studies and cell studies, the 12-well plate was positioned at an arbitrary point in the RF field on a Teflon holder. At this point the ionic heating of 1 mL PBS in a well of a 12-well plate was noted to be (∼1 °C/min). This was used as an internal control before, during, and after RF field exposures. The 12-well plates loaded with samples were equilibrated to 37 °C in a cell culture incubator prior to each exposure. The plates were then removed from the incubator and placed on the Teflon holder at an 42 ambient temperature of (20–22 °C). The plates were allowed to cool to 30 °C before starting the RF exposure experiments. This step was necessary to distinguish bulk from local heating effects. 4.2.4. CDDP release from W-CDDP@US-tubes CDDP release studies from US-tubes were performed as previously described but with some modification.100 Briefly, lyophilized CDDP@US-tubes were suspended in PBS with 0.17% (w/v) Pluronic F108 at a US-tube concentration of approximately 700 mg/L. In order to expose the CDDP@US-tubes to various experimental conditions, 1 mL of sample was placed in each well of a 12-well plate. The plate was then exposed to the RF field or to constant temperature water bath hyperthermia at appropriate experimental parameters as detailed in the results section. The samples were then placed inside a dialysis-membrane (20,000 MW) cylinder, which was immersed in 800 mL PBS at room temperature (22–24 °C) for 168 h. During immersion, the W-CDDP-US-tube sample in the dialysis-membrane cylinder was sampled periodically. The PBS solution in the container was under constant circulation and was replaced at least 4 h before each sampling to maintain a favorable concentration gradient. 4.2.5. Fluorescence measurements For fluorescence studies, samples were exposed to varying parameters in a 12-well cell culture plate and after exposure, were transferred to a 96-well culture plate at 0.2 mL per well. Fluorescence was measured using a Fluostar Omega (BMG 43 Labtech, Ortenberg, Germany) apparatus with the appropriate excitation and emission filter sets for allophycocyanin. To demonstrate temperature-dependent Pluronic disruption from the W-CDDP-US-tube surface, fluorescence was measured using a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA). 4.2.6. Cell studies Both human liver cancer cell lines (Hep3B and HepG2) were purchased from American Type Culture Collection (Manassas, VA) and maintained as per the instructions of the supplier. Media, i.e., MEM (for HepG2 or Hep3B) was supplemented with 10% (v/v) fetal bovine serum. Additional supplementation was performed with sodium pyruvate, non-essential amino acids, Penicillin G and Streptomycin. Cells were cultured in T-75 or T-150 tissue culture flasks (Corning Inc., Corning, NY). For each cell line, the short tandem repeat fingerprint was confirmed by the Cell Line Characterization Core Service (M. D. Anderson Cancer Center, Houston, TX) within one year of all experiments. All media and supplements were purchased from Gibco (Life Technologies, Grand Island, NY). The cells were passaged approximately every three to five days before reaching confluency. Media was replaced every three days. Release-dependent cytotoxicity was assessed in a 0.4-micron trans-well system (Corning Inc., Corning, NY). The W-CDDP@US-tubes were loaded in the top well and sub-confluent monolayers were grown in the bottom well. Aggregates of W-CDDP@US-tubes failed to pass through the filter as confirmed by UV–Vis (not shown). Released CDDP could, however, diffuse through the filter and enter the 44 bottom compartment containing adherent cells with resultant CDDP-induced cytotoxicity. Viability was assessed 120 h after drug exposure using a standard MTT assay. 4.2.7. Statistical analysis Unless otherwise stated, data points represent average from 3 to 6 independent experiments and error bars represent standard error of the mean. For inferential statistics, a p-value < 0.05 was considered significant. Two group comparisons were made using two-tailed t-test for an unpaired sample. Multiple group comparisons were made using one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. 4.3. Results 4.3.1. RF absorption by US-tubes We prepared US-tubes loaded with and without CDDP as detailed in the methods. Heating of full-length SWCNTs has been demonstrated and theoretically predicted under the influence of RF irradiation.40 It was desirable to determine if US-tubes dissipate heat when exposed to low-frequency radiowaves. The Kanzius RF generator was used for this purpose. US-tubes loaded with or without CDDP were subjected to gentle filter centrifugation through a 10 kDa centrifugation filter and resuspended in a solution of 0.17% (w/v) Pluronic-F108. This was repeated several times until the filtrate heating rate was about the same as a solution of 45 0.17% Pluronic® F-108. This was necessary to eliminate ionic heating effects in the RF field that have been previously reported.101 The heating rates for the two suspensions and their background are shown in Figure 4.2. Here, it can be noted that both US-tube and CDDP-US-tube suspensions heat in the RF field significantly above the background ionic heating. We also observed that the heating rates were concentration-dependent. Comparing the relative heating of US-tubes to CDDP@US-tubes, we found that drug loading somewhat attenuated the heating of US-tubes under RF field exposure. Figure 4.2 − Heating rates of W-CDDP@US-tubes and W-US-tubes suspended in 0.17% pluronic F108 in comparison with filtrate alone as a function of concentration. 46 4.3.2. Localized heating of US-tubes in a physiological environment In order to understand the heating of CDDP@US-tubes in a more physiologically-relevant environment, CDDP@US-tubes in 0.17% (w/v) Pluronic® F-108 were suspended in phosphate buffered saline (PBS, pH 7.4). Significant ionic heating under RF exposure was observed when the W-CDDP@US-tubes were suspended in PBS, however, there were no statistically significant differences in the heating rates of filtrate and suspension. We conclude that RF exposure of CDDP@US-tubes in PBS does not result in significant enhancement of the bulk temperature. Furthermore, these findings suggest that the ionic heating of PBS either overshadows or abrogates the heating contribution due to CDDP@US-tubes alone. To address this hypothesis, an experiment was designed to probe localized heating of CDDP@US-tubes in PBS. It has previously been observed that uncoated SWCNTs have non-specific affinity for proteins and it is also known that Pluronicwrapping attenuates the binding of proteins to the surface of SWCNTs.102 Using this information, a relatively thermostable fluorescent protein (allophycocyanin (APC)) was utilized to probe the disruption of Pluronic on CDDP@US-tubes surfaces. The principle of this assay is demonstrated in Figure 4.3A. W-CDDP@US-tubes prevent APC binding and a fluorescent signal can be detected. Local heating of US-tubes or bulk ionic heating should disrupt Pluronic-wrapping above the melting point of Pluronic F-108 (∼57.5 °C) allowing APC to bind to US-tube surfaces and thus produce quenching of APC fluorescence. Similar fluorescence quenching behavior 47 has been described previously for other carbon-based nanomaterials such as carbon nanotubes103 fullerenes104 and nanographene sheets.105 Figure 4.3 − Panel A. Schematic demonstrating the principle of the temperature-dependent Pluronic disruption assay. Panel B and C. Temperature-dependent fluorescence quenching with a transition temperature close to the melting point of Pluronic F108 is indicative of Pluronic disruption from the w-CDDP-US-tube surface. Panel D. RF durationdependent quenching of fluorescence demonstrates localized heating of WCDDP@US-tubes in PBS. Water bath temperature controls (HT) fail to demonstrate quenching of APC fluorescence suggesting lack of Pluronic disruption due to bulk temperature. The RF-dependent quenching is therefore not a result of non-thermal denaturation of APC. 48 In order to test this hypothesis further, APC was added to a suspension of WCDDP@US-tubes in PBS and a temperature-dependent decrease in fluorescence was observed. This was normalized to fluorescence measurements from a control solution of APC in 0.17% (w/v) Pluronic-F108 to exclude effects from photobleaching, temperature-dependent fluorescence quenching, or thermal denaturation of APC. The normalized curve demonstrated quenching of APC due to the presence of Pluronic-wrapped W-CDDP@US-tubes (Figure 4.3B). The first derivative of this curve revealed the peak transition at 59.7 °C (Figure 4.3C), which is consistent with the transition temperature of Pluronic-F108 (∼57.5 °C). Therefore, we attribute the quenching of APC fluorescence to the temperaturedependent disruption of Pluronic-F108 on CDDP@US-tube surface due to the rise in bulk temperature. Next, this system was used to study localized heating of W-CDDP@US-tubes in the RF field while suspended in PBS. The results, shown in Figure 4.3D, demonstrated that RF field exposure resulted in a dose-dependent quenching of fluorescence of APC, suggesting Pluronic disruption. We monitored the bulk temperature during this exposure, which reached a maximum of 42.5 °C after 5 min of RF field exposure. This experiment was then repeated by exposing the CDDP-UStube suspension to 42.5 °C in a water bath for 5 min instead of exposing it to RF. Fluorescence measurements did not show significant quenching of APC in the water bath experiment. This implies that the Pluronic disruption and APC fluorescence quenching is not due to a rise in bulk temperature. It is possible that fluorescence quenching of APC could be due to non-thermal denaturation of APC in the RF field 49 but it was found that exposing APC in 0.17% (w/v) Pluronic-F108 solution to the RF field for 5 min does not result in significant fluorescence quenching, thus, excluding this possibility. We conclude that these data demonstrate localized heating at the surface of W-CDDP@US-tubes in a physiologic salt solution as well as disruption of Pluronic on the W-CDDP@US-tube surface. 4.3.3. RF-triggered release of CDDP from US-tubes Previously it was demonstrated that CDDP can be loaded into US-tubes through sidewall defects and wrapping the CDDP@US-tubes with Pluronic can significantly retard subsequent release of CDDP.100 Thus, based on these previous as well as present studies, it was reasoned that Pluronic disruption caused by localized heating of US-tubes should enhance release of CDDP from W-CDDP@US-tubes in an RF field-triggered fashion. In order to test this hypothesis, W-CDDP@US-tubes were suspended in PBS and subjected to varying durations of RF exposure. Samples not exposed to RF were used as a negative control. In order to differentiate release of CDDP due to bulk temperature and that due to localized heating of W-CDDP@UStubes, water bath temperature controls were used. These samples were incubated in a water bath for a duration that matched the RF experiments. The incubation temperature for the water bath experiments was chosen as the final average temperature reached after 2.5 or 5 min of RF exposure. After the appropriate exposure, samples were immediately loaded in dialysis cassettes and CDDP release at room temperature was determined by ICP measurements. 50 Data shown in Figure 4.4 demonstrated that RF field exposure approximately doubles the release of CDDP from W-CDDP@US-tubes after 2.5 or 5 min of RF exposure at each time point measured (relative to no-RF controls). Relative areaunder-curve (AUC) depicts cumulative release relative to the no-RF control. Regardless of the duration (2.5 or 5 min), RF exposure enhanced cumulative release of CDDP by 1.9 fold after 168 h. Samples exposed to RF reached an average bulk temperature of 38.7 °C after 2.5 min and 49.5 °C after 5 min of RF exposure. We then exposed a W-CDDP@US-tubes suspension to these temperatures and durations in a water bath and measured the release at room temperature similar to RF exposed samples. For the sample exposed at 49.5 °C for 5 min, we found significant enhancement of release that was similar to but less than the RF exposed samples (cumulative release, 1.7 fold), suggesting bulk temperatures lower than Pluronic's transition temperature of 57.5 C can allow release of CDDP. However, the temperature control sample exposed at 38.7 C for 2.5 min demonstrated far less enhancement in cumulative release (1.3-fold). These results demonstrate and distinguish between CDDP release from W-CDDP@US-tubes caused by bulk and local (at the surface of the CDDP@US-tubes) temperature rise, when exposed to the RF field. 51 Figure 4.4 − CDDP release from W-CDDP@US-tubes with or without RF. Panel A. Release of CDDP from W-CDDP@US-tubes with or without RF exposure for 2.5 or 5 min is shown. In addition, bulk temperature controls are demonstrated to evaluate the contribution of bulk ionic heating to CDDP release from W-CDDP@US-tubes. Panel B. Relative areas-under-curve depicts cumulative CDDP release over 168 h relative to untreated control. 4.3.4. RF-triggered release and cytotoxicity To evaluate the significance of these findings, liver cancer cells were exposed to W-CDDP@US-tubes in a trans-well system such that the CDDP@US-tubes nanocarriers were spatially separated from the cellular compartment. Only released CDDP could diffuse through the trans-well membrane (pore size <400 nm). This system is designed to model the limitations of in vivo drug delivery using nanocarriers where the drug-loaded carriers can be spatially separated from their 52 intended site of delivery in cancerous tissue because of multi-scale barriers to transport. The experimental scheme is represented in Figure 4.5. Figure 4.5 − Release-dependent cytotoxicity of W-CDDP@US-tubes. Hep3B and HepG2 human liver cancer cells were plated in the bottom well of the trans-well plate and treated with PBS, W-US-tubes or W-CDDP@US-tubes followed with or without RF exposure for 3 min once, twice, or three times at 24 h intervals. The cell viability was assessed 120 h after treating with WCDDP@US-tubes. Approximately 5000 human liver cancer cells (Hep3B or HepG2) were plated on the bottom well of a 12-well trans-well system. After 24 h, the cells were found to be adherent at which point a second compartment was placed on top and the cells were treated with PBS, W-US-tubes (13.7 mg/L) or W-CDDP@US-tubes (CDDP 2.5 μM, US-tube 13.7 mg/L). This concentration was the approximate IC50 for WCDDP@US-tubes for both cells lines at 120 h. The trans-well system was or was not 53 subjected to a 3-min RF field exposure 1, 2, or 3 times at 24 h intervals. Viability was assessed at 120 h when the PBS treated control samples reached confluence. For both cell lines, it was noted that even the US-tubes alone are slightly cytotoxic at this concentration. It was also noted that this slight cytotoxicity was not enhanced by multiple RF exposures. The data in Figure 4.5 demonstrated that Hep3B and HepG2 cells possess different sensitivities to RF field exposure. As shown, Hep3B cells are not affected by multiple 3-min RF field exposures while HepG2 cells demonstrate slight toxicity to RF. Interestingly, this toxicity is not enhanced after repeated RF exposures. The W-CDDP@US-tube treatment reduced the viability of both cell lines to 50% due to the spontaneous release of CDDP. This cytotoxicity was significantly enhanced by a single 3-min RF exposure (p < 0.01). For Hep3B cells, repeated 3-min RF exposure demonstrated slightly higher cytotoxicity but this was not statistically significant. For the HepG2 cells, multiple RF exposures did not further enhance the observed cytotoxicity. Overall, these data clearly demonstrated cytotoxicity attributable to RF-triggered CDDP release from CDDP@US-tubes. 4.4. Discussion Nanoparticle-mediated drug delivery is widely studied as it may help to overcome drug resistance and enhance tumor-selective toxicity. However, several challenges remain before this potential can be fully realized. While studies have demonstrated successful accumulation of nanocarriers within tumors, it can be argued that the therapeutic efficacy of these nanomaterials is limited by their sequestration in the peri-vascular space. The slow passive release of drugs from 54 nanocarriers is therefore often not sufficient to allow therapeutic levels to be achieved within the tumor. Indeed, this argument is supported by recent studies that demonstrate patchy distribution of nanocarriers in the vascularized areas of the tumor, while certain avascular areas are completely devoid of the chemotherapeutic agent.82–84,87 The barriers to transport are incompletely understood and are likely to be multifaceted, involving both host and nanoparticle properties. While host parameters (interstitial fluid pressure, extracellular matrix composition, pH, and cellular stroma components) are difficult to modulate, nanoparticle design can be readily altered.88,89 It is well known that smaller nanoparticles permeate through the tumor interstitium better than larger nanoparticles.106 However, small nanoparticles are less likely to take advantage of the EPR effect and could be readily cleared by the renal excretion. Hence, nanoparticle properties that govern optimal tumor accumulation are different from those that allow optimal tumor-wide permeation. An alternative approach provides a practical solution to this problem and utilizes remotely-triggered release of drugs after intra-tumoral accumulation of nanocarriers. In this study, we have described a carbon nanocapsule-based system that can readily release its drug cargo in response to a non-invasive RF-trigger. Carbon nanotube interaction with an RF field has been previously studied.24 It was noted that under RF field exposure, full-length nanotubes dissipate energy as heat. Furthermore, this energy is sufficient to raise the bulk temperature of the surrounding environment allowing the use of SWCNTs as remotely-activated thermal agents. The heating of US-tubes by an RF field has not been previously 55 described. As for SWCNTs, the remote heating of W-CDDP@US-tubes and W-UStubes in deionized water by an RF field has been demonstrated here. However, bulk heating of W-US-tubes or W-CDDP@US-tubes has only been observed in a non-ionic environment. When suspended in a physiologically relevant ionic environment (PBS), the bulk temperature rises due to ionic heating of PBS and the local heating of US-tubes cannot be measured. Theoretical studies have predicted that only metallic SWCNT can elevate bulk temperatures of ionic solvents through a “lightning-rod” effect where the metallic SWCNTs polarize in the presence of an E-field and enhance E-field strength by several orders of magnitude.99 This can lead to a dissipation mechanism in the bulk ionic solvent that increases with increasing conductivity of the solvent. Contrary to metallic SWCNTs, the power dissipation by semi-conducting SWCNTs is thought to occur predominantly in the nanotube core and therefore is unlikely to enhance bulk temperature in a conductive host such as PBS. Defects created in the sidewalls of US-tubes during the fabrication process can alter the electronic properties of SWCNTs from which they are derived. Based on the experimental observations of this study, it is infer that the US-tubes behave as a poor dielectric with predominant energy dissipation within the core of the US-tube. This heating can be somewhat abrogated by the CDDP loading, the reason for which is not clear at this time but could be due to basic changes in the dielectric properties of the US-tube. Furthermore, it has been demonstrated here that despite the lack of contribution to temperature change in the bulk ionic media, W-CDDP@US-tubes can dissipate energy at the molecular level that is sufficient to disrupt the Pluronic 56 coating in an RF dose-dependent manner, which subsequently allows CDDP release. Several studies have demonstrated remotely-triggered drug release. For instance, ultrasound-triggered release has been demonstrated from acoustically-responsive gold nanocages, drug-loaded liposomes, or microbubbles.107–109 While significant tumor regression was noted in these studies, small animal models do not recapitulate the limits imposed by depth and variability of penetration of ultrasound. Similarly, light has been extensively studied as a trigger to drug release with reports dating back to the 1980s. Photo-activation utilizes photoisomerization, photo crosslinking, photosensitization-induced oxidation, or photo-thermal activation of gold nanoparticles (See Ref.110 for a review). Regardless of the mechanism of light-triggered drug release, penetration of light even at THz frequencies is limited to a few centimeters, and cannot be utilized for deep-seated tumors, thus necessitating alternative deep-penetrating strategies. Recently, Peiris et al. utilized an alternating magnetic fields (AMF) at radiowave frequencies (10 kHz) to trigger drug release from iron oxide nanoparticles cross-linked to drugloaded liposome in a serial chain.81 Similar to W-CDDP@US-tubes, they noted that drug release could be accomplished without raising bulk ionic temperature. They attributed this behavior to nanoscale vibration of magnetic iron oxide particles in an AMF. In contrast, W-CDDP@US-tubes are a less magnetic material but a material that can be activated by the incident electric field of RF exposure as demonstrated by the present work. Similar to an AMF, RF electric fields can easily penetrate to deep-seated tumors and have well-documented safety. The present study has demonstrated that RF exposure can significantly enhance drug release of CDDP from 57 carbon nanocapsules to enhance cytotoxicity. We have also found that RF-triggered drug release from the carbon nanocapsules exhibits cytotoxicity in a drug releasedependent manner. Increasing the duration of RF exposure or frequency of RF exposure does not further enhance drug release suggesting an irreversible change in the Pluronic coating on the W-CDDP@US-tube surface. Unlike iron oxide crosslinked liposomes, US-tubes serve the dual purpose of an encapsulating agent and that of a susceptor in an RF field, thereby eliminating the need for complex preparation methods. While it has been demonstrated here that a Pluronic coating significantly retards drug release from the nanocapsules, up to 30% of CDDP can be passively released despite Pluronic coating. Future studies already underway are evaluating alternative temperature-sensitive coatings to allow stealth nanoparticles that only permit drug release in the presence of an RF field. In addition, preliminary data in our laboratory has demonstrated that the US-tube platform can be functionalized with antibodies, which will allow tumor specific targeting in conjunction with the known EPR effect. 4.5. Conclusion A carbon-nanocapsule system has been developed that can be remotely triggered by RF irradiation to release CDDP. Mechanistic insight into the process has revealed thermal disruption of a Pluronic coating by US-tube heating in the RF field without appreciable rise in bulk temperature at an RF dose that is minimally toxic to cancer cells. Furthermore, a release-dependent cytotoxicity in human liver cancer 58 cell lines in a trans-well system has been observed that mimics spatial limitations in delivery of chemotherapeutics by drug-loaded nanocarriers in vivo. Chapter 5 In vivo Biodistribution and Anticancer Activity of Cisplatin Delivered by Ultra-short Carbon Nanotube Capsules3 5.1. Introduction In our previous studies (Chapter 3),100 we have shown that the CDDP@US-tubes exhibited greater cytotocities against MCF-7 and MDA-MB-231 breast cancer cell lines in vitro, when compared to free CDDP. It was also demonstrated that the US-tube platform apparently assists the delivery of encapsulated CDDP which suggested that CDDP@US-tube materials help to overcome CDDP resistance by increasing drug accumulation in otherwise resistant 3 A. Guven, G. J. Villares, L. J. Wilson and M. T. Lewis. Manuscript in preparation. 59 60 cell. In this chapter, we report the in vivo biodistribution behaviors and therapeutic efficacy of the CDDP@US-tubes material after the systematic administration on three different human breast cancer xenograft models in mice. 5.2. Materials and Methods 5.2.1. Sample preparation: US-tubes, CDDP@US-tubes and W-CDDP@US-tubes were prepared as previously reported.100 Briefly, full-length SWCNTs (Carbon Solution Inc.), produced by the electric-arc discharge method, were cut into US-tubes by fluorination followed by pyrolysis at 1000 °C under an inert atmosphere.53 US-tubes were first purified in concentrated HCl for 1h by bath-sonication to remove amorphous carbon and metal catalyst impurities (nickel and yttrium), then chemically reduced by a Na0/THF reduction procedure to produce individual US-tubes.76 Next, the US-tubes were refluxed for 5 min in 6N HNO3 and repeatedly washed with deionized water. The debundled US-tubes were dispersed in deionized water via bath-sonication for 60 min, and then CDDP was added to the US-tube suspension and vigorously stirred for 24 h, which was then left undisturbed overnight whereupon CDDP-loaded UStubes (CDDP@US-tubes) flocculated from solution. The CDDP@US-tubes were collected by filtration on a glass filter, washed excessively with deionized water to remove all exterior CDDP from the outer surface of the US-tubes, as judged by platinum analysis on the filtrate aliquots by inductive-coupled plasma optical emission spectroscopy (ICP-OES) and then dried at 80 °C. Extensive characterization 61 of this CDDP@US-tube material has been also reported previously.100 W-US-tube and W-CDDP@US-tube samples were prepared by suspending either dry US-tubes or CDDP@US-tube samples in a 0.17% (w/v) Pluronic® F-108 solution via probe sonication for 2 min, followed by centrifugation at 3200 rpm for 10 min (3x) to remove unsuspended CDDP@US-tubes. 5.2.2. Establishment of xenografts and treatment All animal experiments were performed under a protocol approved by Baylor College of Medicine Institutional Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Experimental Animals. MCF-7 and MDA-MB-231 human breast xenografts were generated by inoculation of 1×106 of either MCF-7 or MDA-MB-231 cells suspended in 20 µL Matrigel-PBS into the epithelium-free “cleared” fat pads of SCID/Beige mice. Human, patient-derived breast cancer xenografts (PDX) (BCM-4272 transplant) were established as described previously.111 For each xenograft experiment, a total of forty human breast-tumor-bearing mice were used. The tumor volumes were monitored weekly by caliper measurement. When the average tumor volume reached at least 100 mm3, mice were randomly divided into treatment or vehicle groups for each experiment. For treatment, 100 µL of different formulations of CDDP, CDDP@US-tubes, W-US-tubes or W-CDDP@US-tubes in saline was administered via i.p. once a week. The administration dose was normalized to be 2.5 mg CDDP per kg (2.5 mg/kg). Tumor size was measured by a caliper two times a week and calculated as the volume = [(tumor length) × (tumor width) 2] / 2. At the 62 end of the experiment, the animals were sacrificed and tissues collected to obtain a quantitative measurement of the biodistribution of CDDP and US-tube samples in each tissue by quantifying platinum (Pt) and yttrium (Y) concentration in each sample via inductively-coupled plasma mass spectrometry (ICP-MS). Pieces of tissue (heart, liver, lung, spleen, kidney, and tumor) were taken from animals of each group, formalin fixed, paraffin embedded, frozen, and processed to 4-µm slides by a tissue-processing core laboratory at BCM. One slide from each tissue sample was stained with hematoxylin and eosin. 5.2.3. Biodistribution and blood circulation studies For biodistribution studies, a total of 45 MDA-MB-231 tumor-bearing mice (tumor size, ~200 mm3) were randomly divided into three groups (CDDP, CDDP@US-tubes, and W-CDDP@US-tubes) of 15 mice each. Three mice from each treatment group were sacrificed at 4, 24, 48, 72, and 168 h after receiving 100 µL of either CDDP, CDDP@US-tube or W-CDDP@US-tube samples in saline at a dose of 2.5 mg CDDP per kg mouse via an i.p injection. The blood samples were collected by cardiac puncture for the blood circulation studies. After harvest, the organs/tissues were immediately frozen in liquid nitrogen, lyophilized to dryness and weighted. Blood and the dried tissue samples were digested by first heating with concentrated HNO3 and then with 26% HClO3. The concentration of Pt and Y were then determined by ICP-MS for blood circulation and biodistribution analysis. 63 5.2.4. Inductively-coupled plasma-optical emission mass spectrometry (ICP-MS) ICP-MS analyses were carried out using a Perkin Elmer Elan Optima 9000 DV instrument with a CDD detector. The quantity of CDDP in each sample was determined by measuring the platinum concentration, which was detected at 265.95 nm. For all ICP-MS measurements, the collected samples were transferred into glass vials and digested first using 10 ml of concentrated HNO3 at elevated temperature (~ 230 °C), and then with 2 ml 26% HClO3. Following digestion, the samples were diluted with 2% v/v trace metal grade HNO3. The concentrations were determined from an average of five scans for each sample. Germanium (371.029 nm) was used as the internal standard. 5.2.5. Statistical analysis All the data are expressed as mean values ± standard error (SE). Graphs were constructed and all statistical analyses were done using GraphPad© Prism 6.0 (GraphPad Software, Inc.). Statistical comparison between two experimental groups was done using a t test (unpaired, two-sided), comparison of multiple groups was performed with two-way ANOVA using Tukey's multiple comparison test. A value of less than 0.05 (p < 0.05) was used for statistical significance. 64 5.3. Results 5.3.1. Tumor suppressing effect of CDDP@US-tubes on MCF-7 xenograft in SCID/Beige mice In previous in vitro studies,100 we determined the efficacy of CDDP@US-tubes against two different breast cancer cell lines (MCF-7 and MDA-MB-231), which are known to be CDDP resistant and showed that US-tubes helped increase the accumulation of the drug in cells compared to free CDDP. To examine the ability of US-tubes to deliver anticancer drugs and to determine the efficacy of CDDP@UStubes on tumor growth suppression in mice in vivo, a series of treatment studies were conducted using three different breast cancer xenograft models (MCF-7, MDAMB-231, and BCM-4272). To determine the in vivo efficacy of the CDDP@US-tubes, we first tested MCF7 xenografts established in SCID/Beige mice. A total of forty MCF-7 tumor bearing mice (weighing 20 ± 2 g) with established xenografts (as detailed in the Materials and Methods Section) were randomly divided into five groups of eight mice each and intraperitoneal (i.p.) injections were started when the average tumor volume reached 150 mm3. Animals in Group 1 (G1) received saline solution (0.9 % w/v NaCl) as a control. Group 2 (G2) was treated with empty Pluronic-wrapped US-tubes (W-US-tubes) as a vehicle control. Group 3 (G3) was treated with CDDP dissolved and diluted in saline solution. Group 4 (G4) was treated with Pluronic-wrapped CDDP@US-tubes (W-CDDP@US-tubes). Finally, Group 5 (G5) was treated with CDDP@US-tubes dispersed in saline solution via bath-sonication. The final 65 concentration of CDDP in G3, G4, and G5 was normalized to be 2.5 mg/kg mouse body weight. The quantity of US-tubes was the same for G2, G4, G5, with the concentration being calculated from their weight% values obtained by ICP-OES for Ni and Y which remains within US-tubes as remnant metal catalysts from the growth of the original full-lengths CNTs from which the US-tubes are derived. Treatment was performed once a week until the end of the experiment. 66 C o n tro l W -C D D P @ U S -tu b e s 20 16 R e la t iv e t u m o r v o lu m e 4 3 2 * 0 0 10 D a y s a fte r 1 * * *# 20 st T u m o r d o u b lin g tim e s C 1 .0 P r o p o r t io n e v e n t - f r e e m ic e 24 5 C D D P @ U S -tu b e s B o d y w e ig h t (g ) A 1 CD D P W -U S -tu b e s 0 .5 0 0 .0 30 0 10 tre a tm e n t 20 30 T im e (d a y s ) *p<0.05 CDDP vs. CDDP@US-tubes 8 p < 0 .0 0 5 6 ns 4 p < 0 .0 5 2 es b es P D C w -C D D D P w @ @ U U S- S- tu tu b D D C -U S- Sa tu li b n P es 0 e B F o ld i n c r e a s e in t u m o r v o l u m e #p<0.05 CDDP vs. W-CDDP@US-tubes Figure 5.1 − Suppression of tumor growth by intraperitoneal injection of CDDP@US-tubes to an MCF-7 breast cancer xenograft. (A), Tumor growth of MCF-7 tumor-bearing mice after receiving different treatments as indicated. The mean body weight changes is also plotted in A. Fold increase in tumor volume (B) and Kaplan-Meier survival curves (C) depicting the tumor doubling times for different treatment groups. 67 Figure 5.1 displays the comparative in vivo therapeutics efficacy of the given treatment groups. As shown in Figure 5.1A, a time-related increase in tumor volume was observed in all treatment groups except for the CDDP@US-tube treated group. Compared to the control injection, W-US-tube injected mice showed similar tumor growth rate, although it was not as effective as CDDP alone or the W-CDDP@UStubes. As showed in Figure 5.1A, there was also no significant difference between the mean body weight changes in all treatment groups over the course of the experiment. The mean fold increase in tumor volume (from day 0 to day 28) of the CDDP@US-tube treated mice was significantly lower compared to CDDP alone or WCDDP@US-tubes (Figure 5.1B). Five out of eight tumors from mice treated with CDDP@US-tubes failed to grow at all, while all control groups got larger. Determination of the tumor growth doubling time (Figure 5.1C) revealed that CDDP@US-tubes significantly improved mouse survival. A n g P t / m g d r y tis s u e 68 50 40 10 30 5 20 n g Y / m g d r y tis s u e C o n tro l w -U S -tu b e s CDDP w -C D D P @ U S -tu b e s C D D P @ U S -tu b e s * 0 * Tum or 10 # 0 Tum or B * H eart Lung S p le e n 15 0 .8 10 K id n e y S to m a ch C o n tro l w -U S -tu b e s CDDP w -C D D P @ U S -tu b e s C D D P @ U S -tu b e s 0 .6 0 .4 0 .2 5 L iv e r 0 .0 Tum or 0 Tum or * p < 0 .0 5 H eart Lung C D D P v s C D D P @ U S -tu b e s, S p le e n # L iv e r s S to m a ch p < 0 .0 5 C D D P v s w - C D D P @ U S - t u b e s , p < 0 .0 5 w - C D D P @ U S - t u b e s v s C D D P @ U S - t u b e s , K id n e y p < 0 .0 5 w - U S - t u b e s v s C D D P @ U S - t u b e s , p < 0 .0 5 w - U S - t u b e s v s w - C D D P @ U S - t u b e s Figure 5.2 − Platinum and yttrium concentration in tissue from mice treated over a period of ~ 45 d with weekly saline, w-US-tubes, CDDP, CDDP@UStubes, and W-CDDP@US-tubes administration via i.p. injection. (A) Concentration of platinum (from cisplatin) and (B) yttrium (from US-tubes) in tissue (mean ± s.e., n = 8). Statistically significant different (p < 0.05) are marked with symbols as indicated above (unpaired, two-sided t-test). At the end of the experiment, animals from each treatment group were sacrificed and tissues were collected to obtain a quantitative measurement of the accumulation of CDDP and US-tubes samples in each tissue by quantifying Pt (for CDDP) and Y (for US-tubes) in each tissue sample via ICP-MS analysis. ICP-MS analysis revealed that the CDDP@US-tube treated mice have significantly more Pt in 69 their tumor, spleen and stomach compare to other treatment groups (Figure 5.2A). By analyzing the Y concentration in tissue, it has also been determined that the level of Y detected in spleen, liver, kidneys, and stomach was found to be greatest, while the amount found in other organs was much lower (Figure 5.2B). The analysis showed that CDDP@US-tube treated mice had significantly more Y in their spleen and stomach compare to mice treated with empty W-US-tube, and W-CDDP@UStubes. 5.3.2. Tumor suppressing effect of CDDP@US-tubes on MDA-MB-231 xenograft in SCID/Beige mice In the second experiment, the in vivo anticancer efficacy of CDDP@US-tube materials was tested in mice bearing MDA-MB-231 human breast cancer xenograft in a manner similar to that described above for MCF-7 xenograft. The tumors were established as described in Materials & Method section and i.p. injections were started when the average tumor volume reached ~100 mm3. Changes in relative tumor volume and body weight are shown in Figure 5.3A. As plotted in Figure 5.3A, the mean body weights of mice in all treatment groups showed similar an increase and there was no significant difference between the treatment groups over course of the experiment. As seen in Figure 5.3A, the control and the empty W-US-tube treated group showed a fast growth pattern, resulting in an approximately 12-fold increase in their tumor volume (from day 0 to day 28). No significant difference in the relative tumor growth ratio (V/V0) of these two treatment group confirmed that the empty W-US-tubes themselves do not affect the tumor growth in mice. On the 70 other hand, the relative tumor growth ratios (V/V0) of CDDP, W-CDDP@US-tubes, and CDDP@US-tubes were similar (Figure 5.3A) and there was no statistically significant difference between the treatment groups. However, the mean fold increase in tumor volume (from day 0 to day 28) of CDDP@US-tube (7.45 ± 0.77) treated mice was significantly lower than mice in control treatment group (13.03 ± 2.02, p = 0.03) (Figure 5.3B), while the fold increase in mice treated with free CDDP (9.09 ± 1.73), and W-CDDP@US-tube (9.57 ± 1.88) was not significant different (p = 0.16, and p = 0.24, respectively). The determination of tumor growth tripling time showed that the median survival of the CDDP@US-tube, and W-CDDP@US-tube treatment groups, (10 d and 14 d, respectively) was longer than that of mice treated with free CDDP, 7 d (Figure 5.3C). 71 w -C D D P @ U S -tu b e s 16 15 12 9 6 3 0 T u m o r t r ib lin g t im e s 1 .0 0 .5 0 10 0 .0 20 0 tre a tm e n t 10 15 20 25 p < 0 .0 5 B ns ns 25 20 15 10 5 es b es tu b S- tu U SP D C w -C D D D P w @ @ U -U S- Sa tu li b n P es 0 e F o ld i n c r e a s e in t u m o r v o l u m e 5 T im e (d a y s ) D D a y s a fte r 1 st D 0 C R e l a t iv e t u m o r r a t io ( V / V o ) 20 C P r o p o r tio n E v e n t - F r e e 24 C D D P @ U S -tu b e s B o d y w e ig h t (g ) A CDD P w -U S -tu b e s C o n tr o l Figure 5.3 − Suppression of tumor growth by intraperitoneal injection of CDDP@US-tubes to an MDA-MB-231 breast cancer xenograft. (A), Tumor growth of MDA-MB-231 tumor-bearing mice after receiving different treatments as indicated. (* indicates p < 0.05, unpaired, two-sided t-test, n = 8; error bars = s.e.) The mean body weight changes is also plotted in A. Fold increase in tumor volume (B) and Kaplan-Meier survival curves (C) depicting the tumor tribling times for different treatment groups. 72 Although the efficacy of free CDDP, W-CDDP@US-tube, and CDDP@US-tube on the MDA-MB-231 xenograft was found to be similar (Figure 5.3A), the ICP-MS analysis of tissue collected from the animals at the end of the experiment revealed that the mice treated with CDDP@US-tubes (4.28 ± 1.17, p = 0.02) and WCDDP@US-tubes (2.16 ± 0.16, p = 0.01) had significantly more Pt in their tumor than B 30 8 # 6 20 * # * # 4 2 * S a lin e W -U S -tu b e s CDDP W -C D D P @ U S -tu b e s C D D P @ U S -tu b e s 0 10 Tum or *# 0 Tum or n g Y / m g d r y tis s u e A n g P t / m g d r y t is s u e mice treated with free CDDP (1.17 ± 0.18) (Figure 5.4A). H eart Lung S p le e n 20 0 .6 15 K id n e y 0 .2 0 .0 5 S to m a ch S a lin e W -U S -tu b e s CDDP W -C D D P @ U S -tu b e s C D D P @ U S -tu b e s 0 .4 10 L iv e r Tum or 0 Tum or * p < 0 .0 5 H eart Lung S p le e n C D D P v s C D D P @ U S -tu b e s, # K id n e y L iv e r S to m a ch p < 0 .0 5 C D D P v s w - C D D P @ U S - t u b e s , p < 0 .0 5 w - C D D P @ U S - t u b e s v s C D D P @ U S - t u b e s , p < 0 .0 5 w - U S - t u b e s v s C D D P @ U S - t u b e s Figure 5.4 − Platinum and yttrium concentration in tissue from mice treated over a period of ~ 45 d with weekly saline, w-US-tubes, CDDP, CDDP@UStubes, and W-CDDP@US-tubes administration via i.p. injection. (A) Concentration of platinum (from cisplatin) and (B) yttrium (from US-tubes) in tissue (mean ± s.e., n = 8). Statistically significant different (p<0.05) are marked with symbols as indicated above (unpaired, two-sided t-test). 73 However, the tumor Pt levels as stated above were considerably lower in tumors than those of CDDP, W-CDDP@US-tubes, and CDDP@US-tubes observed with MCF-7 tumor-bearing mice (2.77 ± 0.35, 3.09 ± 0.57, 9.74 ± 0.83, respectively). This was apparently not sufficient to display tumor suppressing efficacy. Consistent with MCF-7 xenograft study, the level of Y in mice treated with W-US-tubes, WCDDP@US-tubes, and CDDP@US-tubes was found highest in spleen followed by stomach, liver, and kidney, confirming the higher retention of these materials in these organs. 5.3.3. Tumor suppressing effect of CDDP@US-tubes on the BCM-4272 PDX xenograft in SCID/Beige mice To extend our studies beyond cell line xenografts, we evaluated the efficacy of the CDDP@US-tube material with a human breast cancer patient-derived xenograft (PDX) model, where human tumor tissue is surgically resected and transplanted directly into immune-deficient mice. Because traditional cell lines have been cultured for many years, and have accumulated many genetic/epigenetic alterations, there is some argument as to whether their response to treatment accurately reflects patient responses. It has been shown that PDX models display a high degree of similarity in response as the tumor of origin in the patient when challenged with a comparable agent.111–113 Because of their ability to predict clinical tumor response, PDXs are increasingly being considered to be more relevant in vivo preclinical models, and thus, are expected to increase the success of identifying new active antitumor agents for clinical trials.112 BCM-4272 xenografts were established 74 as described previously.111 As shown in Figure 5.5A, the tumors in the control and the empty W-US-tube groups showed a slow but stable growth. Compared with the control group, the CDDP treatment group slowed down tumor growth, but it was not as effective as the W-CDDP@US-tube or CDDP@US-tube treatment groups with both of these treatment groups showing considerable tumor suppression (regression) (p<0.005) compared to CDDP treatment. There were no significant body weight changes between treatment groups until the end of the experiment. 75 C o n tr o l w -U S -tu b e s w -C D D P @ U S -tu b e s 22 20 6 1 .0 5 4 3 2 1 * * * *# *# *# 0 T u m o r d o u b l in g t im e s P r o p o r tio n E v e n t - F r e e R e l a t iv e t u m o r r a t io ( V / V o ) 24 C B o d y w e ig h t (g ) A 0 .5 0 .0 0 0 10 D a y s a fte r 1 20 st CDD P C D D P @ U S -tu b e s 0 30 10 20 30 T im e (d a y s ) tre a tm e n t p < 0 .0 5 12 p < 0 .0 0 5 ns 8 4 s e s P D C w -C D D D P w @ @ U U S- S- tu tu b b e P D D -U S- C tu li b n e e s 0 Sa B F o ld i n c r e a s e in t u m o r v o l u m e *p < 0.05 CDDP vs. CDDP@US-tubes #p < 0.05 CDDP vs. W-CDDP@US-tubes Figure 5.5 − Suppression of tumor growth by intraperitoneal injection of CDDP@US-tubes to an Patient-derived, 4272 transplant, xenograft. (A), Tumor growth of Patient-derived tumor-bearing mice after receiving different treatments as indicated. (unpaired, two-sided t-test, n=9; error bars= s.e.) The mean body weight changes is also plotted in A. Fold increase in tumor volume (B) and Kaplan-Meier survival curves (C) depicting the tumor doubling times for different treatment groups. 76 The mean fold increase in tumor volume (from day 0 to day 28) of control (4.88 ± 0.32) and W-US-tubes treated group (5.32 ± 0.49) were similar, however the CDDP@US-tube (2.57 ± 0.25) treated group displayed a significantly lower fold increase than those of free CDDP treated mice (4.02 ± 0.48, p = 0.01) which was also not significantly different than the control group (4.88 ± 0.32, p = 0.15). The increase in tumor volume for the W-CDDP@US-tubes (3.21 ± 0.26) was found to be significantly lower than the control group (p = 0.001), while it was not statistically different from the free CDDP treatment group (Figure 5.5B). As a result, an increased survival of animals was observed in mice treated with CDDP@US-tubes or W-CDDP@US-tubes compared to mice treated with free CDDP (Figure 5.5C). ICP-MS analysis of the tissues showed that CDDP@US-tube and WCDDP@US-tube treated mice had significantly more Pt in their tumor, spleen and kidney than the mice treated with free CDDP, while the free CDDP treated mice displayed higher Pt accumulation in heart compare to other treatment groups (Figure 5.6A). Consistent with results from the above MCF-7 experiment, for the MDA-MB-231 xenograft studies, the Y concentration was found highest in the spleen followed by kidney, liver and stomach, indicating higher retention of US-tube materials in these organs (Figure 5.6B). Compared with W-CDDP@US-tube and WUS-tube treated mice, the level of Y in the spleen was found significantly higher in mice treated with CDDP@US-tubes. B 30 8 # 6 20 * # * # 4 * 2 S a lin e W -U S -tu b e s CDDP W -C D D P @ U S -tu b e s C D D P @ U S -tu b e s 0 10 Tum or *# 0 Tum or n g Y / m g d r y tis s u e A n g P t / m g d r y t is s u e 77 H eart Lung S p le e n 20 0 .6 15 K id n e y 0 .2 0 .0 5 S to m a ch S a lin e W -U S -tu b e s CDDP W -C D D P @ U S -tu b e s C D D P @ U S -tu b e s 0 .4 10 L iv e r Tum or 0 Tum or * p < 0 .0 5 H eart Lung S p le e n C D D P v s C D D P @ U S -tu b e s, # K id n e y L iv e r S to m a ch p < 0 .0 5 C D D P v s w - C D D P @ U S - t u b e s , p < 0 .0 5 w - C D D P @ U S - t u b e s v s C D D P @ U S - t u b e s , p < 0 .0 5 w - U S - t u b e s v s C D D P @ U S - t u b e s Figure 5.6 − Platinum and yttrium concentration in tissue from mice treated over a period of ~ 45 d with weekly saline, W-US-tubes, CDDP, CDDP@UStubes, and W-CDDP@US-tubes administration via i.p. injection. (A) Concentration of platinum (from cisplatin) and (B) yttrium (from US-tubes) in tissue (mean ± s.e., n = 8). Statistically significant different (p < 0.05) are marked with symbols as indicated above (unpaired, two-sided t-test). 5.3.4. Blood circulation and the biodistribution studies To understand the treatment efficacy of CDDP@US-tube materials, it was important to investigate the biodistribution of the CDDP@US-tubes in a timedependent manner. The biodistribution studies has been conducted after a single i.p. administration of CDDP, W-CDDP@US-tubes, and CDDP@US-tubes (CDDP concentration 2.5 mg/kg) for MDA-MB-231 tumor-bearing mice by determining the 78 Pt (from CDDP) and the Y (from US-tubes) concentrations in plasma and in main organs/tissues (tumor, liver, spleen, kidney, heart, lungs, and stomach) via ICP-MS analysis at different time points (4, 24, 48, 72, and 168 h). The mice from each treatment groups were sacrificed and harvested for blood circulation and biodistribution analysis (see Materials and Methods Section). P t c o n c e n t r a t io n A in b l o o d ( n g / m L ) CD D P w -C D D P @ U S -tu b e s C D D P @ U S -tu b e s 600 * 400 * # * 200 # * 0 4h 24h 48h 72h 168h 4h 24h 48h 72h 168h Y c o n c e n t r a t io n in b l o o d ( n g / m L ) B 60 40 20 0 Figure 5.7 − Blood circulation for CDDP, W-CDDP@US-tubes, CDDP@US-tubes in MDA-MB-231 tumor-bearing mice as determined by measuring Pt (from CDDP) and Y (from US-tubes) concentration in blood via ICP-MS at different time points post injection (mean ± s.e., n = 3). CDDP@US-tubes showed prolonged blood circulation compared to free CDDP. Figure 5.7A illustrates the comparative blood circulation data of free CDDP, W-CDDP@US-tubes and CDDP@US-tubes for MDA-MB-231 tumor-bearing mice as a 79 function of time. ICP-MS analysis of the blood samples revealed that CDDP@UStubes and W-CDDP@US-tubes exhibited a much longer blood circulation time than CDDP which was quickly cleared from the blood. At 4 h, the Pt concentration in blood for mice treated with CDDP, W-CDDP@US-tubes, and CDDP@US-tubes was 271.55 ± 21.51 ng/mL, 250.40 ± 57.03 ng/mL, and 456.00 ± 71.40 ng/mL, respectively. In comparison, CDDP-treated mice showed a significantly lower Pt concentration in the plasma at 24 h (102.78 ± 9.92) which gradually declined until the end of the experiment, while the mice treated with W-CDDP@US-tube and CDDP@US-tube materials maintained relatively higher Pt concentrations (221.11 ± 33.26 and 327.03 ± 80.01, respectively) at 24 h, and continued to be relatively higher until the end of the study. The concentration of Pt in blood at 168 h p.i., the last point of evaluation, in mice treated with W-CDDP@US-tubes and CDDP@UStubes was 2.3-fold (48.27 ± 7.41) and 3.8-fold (78.62 ± 12.83), respectively, higher than in mice treated with free CDDP (20.88 ± 5.14) (p < 0.05). ICP-MS analysis showed that the Pt concentration of CDDP-treated mice in each tissue gradually declined over time. The Pt levels detected in liver, spleen, and kidney was highest, while the amount measured for other organs was much lower. On the contrary, mice treated with W-CDDP@US-tubes and CDDP@US-tubes displayed a significantly different biodistribution than mice treated with free CDDP (Figure 5.8), with much higher Pt concentrations in tumor, spleen, and kidney, while being similar in lung, stomach, heart, and liver (Figure 5.8A). 80 As shown in Figure 5.8, the concentration of Pt in tumors of all treatment groups was similar at 4 h, and then started to increase, and became significantly higher at 48 h and peaked at 72 h in CDDP@US-tube treated mice (3.09 ± 0.48 ng/mg dry tissue), which was more than 5-fold higher than in mice treated with free CDDP (0.56 ± 0.08 ng/mg dry tissue) and approximately 3-fold higher than WCDDP@US-tube treated mice (1.08 ± 0.12). There was also a significant difference between the CDDP and W-CDDP@US-tube treatment groups at 72 h (p = 0.024). At 168 h, the Pt concentration levels in tumors of all treatment groups decreased; however, the accumulation of Pt for the CDDP@US-tubes (1.96 ± 0.44 ng/mg dry tissue) was still significantly higher than that in the CDDP treated groups (0.34 ± 0.1 ng/mg dry tissue, p = 0.022). The Pt concentrations detected in kidney, liver, and spleen after free CDDP administration was found highest, while the amount found in other organs was much lower. The Pt concentration in kidney from mice treated with free CDDP (9.27 ± 1.26), and W-CDDP@US-tubes (7.64 ± 0.18) were highest and found significantly higher than mice treated with CDDP@US-tubes (4.87 ± 0.05) at 4 h, and then gradually declined until the end of the study. Contrary to this, CDDP@US-tube treated mice displayed a relatively consistent Pt concentration in kidney up to 48 h, followed by a decreased and significantly higher Pt concentration at 72 h (4.14 ± 0.3) compare with both the free CDDP (1.38 ± 0.49, p = 0.005), and W-CDDP@US-tube (2.16 ± 0.82, p = 0.02) treatment groups. Although the Pt levels detected in the liver were lower than those measured in the kidneys in the first 24 h, in the long-term (at 168 h), the Pt levels were greater in the liver than in the kidneys. The liver Pt levels in all treatment groups displayed a similar trend at all 81 evaluated time points except 4 h, with greater Pt concentration for CDDP@US-tubes (6.05 ± 0.32) compared to free CDDP (4.87 ± 0.11, p = 002). Figure 5.8 shows that the CDDP@US-tubes and W-CDDP@US-tubes afforded considerably higher Pt accumulation in the spleen compare to free CDDP at 48 h and 72 h, while the mice treated with free CDDP exhibited higher Pt accumulation only at 4 h, and then gradually decreased over time. As shown in Figure 5.8B, ICP-MS analysis confirmed the high retention of CDDP@US-tube and W-CDDP@US-tube materials in the spleen, where the Y concentration was higher than for all other organs. 82 CDDP n g P t / m g d r y tis s u e A w -C D D P @ U S -tu b e s 4 * * * * 3 * 2 # # * 1 0 4h 24h 48h 72h 168h 4h 24h n g P t / m g d r y tis s u e Tum or 4h 24h 48h 72h 168h Lung # 10 5 0 24h 48h 72h 168h 4h 24h 48h 72h 168h 4h 24h 48h 72h 168h K id n e y S p le e n CDD P n g Y / m g d r y tis s u e 72h 168h 15 L iv e r w -C D D P @ U S -tu b e s C D D P @ U S -tu b e s 0 .6 0 .4 0 .2 0 .0 4h 24h 48h 72h 168h 4h 24h 48h 72h 168h 4h 24h 48h 72h 168h H eart Tum or n g Y / m g d r y tis s u e 48h H eart 20 4h B C D D P @ U S -tu b e s 5 Lung 4 3 2 1 0 4h 24h 48h 72h 168h L iv e r * p < 0 .0 5 4h 24h 48h 72h 168h 4h 24h 48h 72h 168h S p le e n C D D P v s C D D P @ U S -tu b e s, # K id n e y p < 0 .0 5 C D D P v s w - C D D P @ U S - t u b e s , p < 0 .0 5 w - C D D P @ U S - t u b e s v s C D D P @ U S - t u b e s 83 Figure 5.8 − Time-dependent in vivo biodistribution of intraperitoneally injected CDDP, W-CDDP@US-tubes, and CDDP@US-tubes in MDA-MB-231 tumor bearing mice. (A) Concentration of platinum (from CDDP), (B) Concentration of yttrium (from US-tubes) in tissue (mean ± s.e., n = 3). Statistically significant different (p < 0.05) are marked with symbols as indicated above (unpaired, two-sided t-test). 5.4. Discussion The in vivo biodistribution and pharmacokinetic studies of CNTs have been extensively studied by a number of research groups using different CNT materials, different surface functionalizations, and tracking techniques, after different administration technique (i.p. vs. i.v.) resulting in varying and sometimes controversial results.22 However, the present study is the first to explore the in vivo delivery of a chemotherapeutic agent (CDDP) encapsulated within a CNT material. Although CDDP is one of the most potent anticancer drugs, its potential use in cancer therapy is hampered due to its severe side effects caused by the lack of selectivity and resistance issues which is mainly attributed to reduced drug accumulation in tumor cells. Because of its reactive nature, CDDP has limited bioavailability. Upon administration, CDDP rapidly binds to albumin and other plasma proteins leading to irreversible deactivation of nearly 90% of the CDDP dose before it accumulates in the tumor by the EPR effect. Because this form of CDDP is inactive, it has no biological effects.114 Thus, an ideal drug delivery systems is expected to not only increase the concentration of CDDP in tumor cells, but also protect it from plasma deactivation. 84 Recently, we developed a new CNT-based drug delivery system that is comprised of CDDP encapsulated within the US-tubes (CDDP@US-tubes) that showed enhanced in vitro antitumoral efficacy against two different breast cancer cell lines, MCF-7 and MDA-MB-231, which are known to be CDDP resistant, compared to free CDDP.100 In the present study, we have investigated the biodistribution and in vivo efficacy of the CDDP@US-tube material in mice with three different breast cancer xenograft models. Previous in vivo toxicity assessment studies have demonstrated that high doses of US-tubes (up to 1000 mg/kg b.w.) after i.p. administration are well tolerated by Swiss mice and showed that a large portion of small aggregates and well-individualized US-tubes reached the systemic circulation and subsequently accumulated in a variety of tissues and cells. 36 From this study, it was reasoned that i.p. administration of CDDP@US-tube materials would also avoid the biodistribution complications associated with i.v. injection such as possible mechanical blockage of the vasculature system. In addition, i.p. administration of free CDDP has been also studied extensively, and the dose of free CDDP that can be given safely by i.p. is substantially higher compared to i.v. administration. Moreover, the concentration of active cisplatin in the plasma is not decreased after i.p. administration comprised to i.v. injection. To determine pharmacokinetics and tissue specific distribution of CDDP vs. US-tubes, we quantified the Pt (from CDDP) and Y (within the US-tubes as remnant metal catalyst) concentrations in samples via ICP-MS analysis which allows to measurement of exact Pt and Y concentration in tissue samples because of its high sensitivity (1 ppb) and specificity due to the absence of background Pt and Y in 85 samples. To our knowledge, this is the first study that measures the remnant metal catalyst, such as Y in our study, to trace and quantitatively determine the blood clearance, and biodistribution behaviors of a CNTs material in vivo. To date, most of the in vivo biodistribution and pharmacokinetic studies of CNT materials reported in the literature have been carried out using radiolabels or spectroscopic tags for indirect detection of CNTs, 26,46,115 which may gradually dissociate, decay, and/or lose activity over time.116 In other studies, rather insensitive CNT Raman scattering intensities have been sometimes employed as a tracer.41 The results of the present study demonstrate that CDDP@US-tubes have greater tumor suppression efficacy than free CDDP in both MCF-7 and BCM-4272 PDX breast tumor models, while it was found to be similar in a MDA-MB-231 xenograft. In all three xenograft studies, the CDDP@US-tubes have afforded higher CDDP uptake in tumors compared to free CDDP. The similar tumor suppression effects of CDDP@US-tubes and CDDP on the MDA-MB-231 xenograft is possibly due to greater aggressive tumor growth of the MDA-MB-231 xenograft compared to MCF-7 and BCM-4272 PDX xenografts, which resulted in considerable lower Pt concentration in the tumor per mg of dry tissue. In fact, it has been observed that the mean fold increase (from day 0 to day 28) in tumor volume of the control (saline) treated group for the MDA-MB-231 xenograft (11.415 ± 1.906) was notably higher compared to the MCF-7 (3.486 ± 0.494) and BCM-4272 PDX (4.88 ± 0.319) xenografts. 86 The reason for the higher CDDP uptake in tumor for CDDP@US-tubes is likely due to their prolonged blood circulation time compare to free CDDP which facilitates substantial tumor targeting by the EPR effect. Consistent with previous reports, we observe short blood circulation time for free CDDP.117,118 The adsorption of free CDDP occurred very rapidly by various organs especially kidney, liver and spleen. However, the mice treated with W-CDDP@US-tubes and CDDP@US-tubes displayed a significantly different biodistribution behavior than mice treated with free CDDP, with much higher Pt accumulation in tumor, spleen, and kidney, while being similar in lung, stomach, heart, and liver. By also analyzing the Y concentration in tissue, we have determined the higher retention of US-tubes in spleen, liver, kidneys, and stomach, while the amount found in other organs was much lower after i.p. administration. 87 Chapter 6 Conclusion and Future Perspective Over the past decade, a number of nanoscale materials have been explored that exhibit many unique intrinsic physical and chemical properties and have much to offer in the field of medicine. Among the diverse classes of nanomaterials, CNTs, or more specifically US-tubes, hold great promise in the area of nanomedicine for the development of new nanoplatforms. In only a few short years, the US-tubes have been shown to be a popular, new platform for the delivery of various medical agents for both imaging and therapeutic purposes. In this work, for the first time, we have shown that US-tubes can be also utilized as a drug delivery platform to deliver the chemotherapeutic drug, CDDP. The therapeutic efficacy of a CDDP@US-tube material has been evaluated both in vitro and in vivo and found to exhibits superior efficacy when compared to free CDDP. The studies have revealed the ability of the US-tube platform to assist the delivery of encapsulated CDDP by increasing the accumulation of drug in resistance cells which suggests how the 88 CDDP@US-tube materials helps to overcome CDDP resistance. Although it has been demonstrated that passive tumor accumulation of CDDP@US-tubes in vivo occurs by the EPR effect. Tumor specific targeting of CDDP can be passively also achieved by functionalizing the US-tube platform with antibodies to further boost accumulation in tumor cells. We have also demonstrated that CDDP release from W-CDDP@US-tubes can be stimulated by radiofrequency (RF) fields to produce heat that disrupts the Pluronic coating and triggers CDDP releases in an RF-dependent manner. RFinduced release-dependent cytotoxicity of W-CDDP@US-tubes has been evaluated in vitro against two different liver cancer cell lines, Hep3B and HepG2, and found to exhibit superior cytotoxicity to W-CDDP@US-tubes not exposed to RF. While it has been demonstrated that a Pluronic coating significantly retards drug release from the CDDP@US-tubes, up to 30% of CDDP is passively released despite the Pluronic coating. Future studies will evaluate alternative temperature-sensitive coatings to allow stimulated drug release only in the presence of an RF field. From the above encouraging results, we believe that it might be possible to control CDDP release from CDDP@US-tubes by wrapping them with covalentlyattached activatable cancer-specific peptide sequences instead of surfactant molecules. Recently, a catalytic method to functionalized US-tubes with various amino acids and peptides was reported which improved solubility, biocompatibility, and biological targeting capabilities.60 In future work, using this functionalization protocol, a peptide chain, containing an enzyme-specific substrate sequence for a 89 cancer cell antigen, such as the well-known prostate specific antigen (PSA) and/or matrix metalloproteinase-2 (MMP-2) can be synthesized and covalently-attached to CDDP@US-tubes. 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