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UBC Theses and Dissertations

Pharmacokinetics, biodistribution and intratumoral distribution of Celludo nanoparticles Zhao, Yucheng 2016

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  Pharmacokinetics, Biodistribution and Intratumoral Distribution of Celludo Nanoparticles   by Yucheng Zhao  B.Sc. The University of Toronto, 2014  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2016  © Yucheng Zhao, 2016   ii  Abstract  Anti-tubulin agents are the most potent and broadest spectrum drugs for cancer therapy, including taxanes and vinca alkaloids. However, there are two major limitations for their clinical use: multidrug resistance (MDR), and significant side effects such as neutropenia and neuropathy. The overexpression of P-glycoprotein (Pgp) is the most commonly found mechanism for MDR in cancer. Our lab has screened several anti-tubulin agents against different MDR tumor cells. The results show that podophyllotoxin (PPT) remained highly active against the resistant cell lines with an IC50 of ~10 nM. However, PPT is insoluble and exhibits significant side effects due to poor selectivity. A nanoparticle dosage form of PPT was developed by covalently conjugating PPT and polyethylene glycol (PEG) to acetylated carboxymethyl cellulose (CMC-Ac) via ester linkages. The optimized polymer conjugates self-assembled into 20 nm particles (named Celludo) and displayed significantly improved efficacy against MDR tumors in mice compared to free PPT and the standard taxane chemotherapies. My thesis focused on developing a robust and reproducible HPLC method to measure PPT concentrations in biological samples in order to compare the pharmacokinetics (PK) and biodistribution (BD) of Celludo and free PPT. The kinetics of intratumoral distribution of the Celludo nanoparticles was also examined. Compared to free PPT, Celludo displayed extended blood circulation with 18-fold prolonged half-life, 9,000- fold higher area under the curve (AUC), and 1,000-fold reduced clearance compared to free PPT. The tumor uptake of Celludo was 500-fold higher than that of free PPT. With Celludo, the overall delivery to the tumor was 4.5-, 3.8-. 3.4-and 1.2- fold higher than that delivered to the liver, lung, heart, and spleen respectively. At 6 h, Celludo nanoparticles accumulated equally in the hypervascular and  iii  hypovascular region within the tumor. One and two days post-injection, the amount of Celludo in the hypervascular region remained the same, while the penetration to the hypovascular area increased constantly over 48 h post-injection. The data suggest that Celludo was an effective system targeting PPT to the tumor with enhanced penetration to the tumor core.  iv  Preface  This thesis is composed of one manuscript that will be submitted for publication. Details of the specific nature of the experiments and the scope of the thesis work were formulated in discussions between Dr. Shyh-dar Li, Dr. Aniruddha Roy and myself. During the study, I received training in the preparation of nanoparticle formulation from Dr. Aniruddha Roy. I performed the pharmacokinetics and biodistribution experiments and analyzed the data. I wrote this thesis under the guidance of my supervisor Dr. Shyh-dar Li.  v  Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Tables ............................................................................................................................... vii List of Figures ............................................................................................................................. viii List of Abbreviations .....................................................................................................................x Acknowledgements ..................................................................................................................... xii  Introduction ................................................................................................................1 1.1 Overview of multidrug resistance ................................................................................... 1 1.1.1 Structure and function of p-glycoprotein (Pgp) .......................................................... 1 1.1.2 Strategies to overcome Pgp overexpression-mediated MDR ..................................... 3 1.2 Anti-tubulin drugs for cancer therapy ............................................................................. 4 1.2.1 Podophyllotoxin .......................................................................................................... 5 1.2.2 Approaches to deliver PPT ......................................................................................... 7 1.3 Preliminary data of Celludo ............................................................................................ 8 1.4 Research hypothesis and aims of thesis ........................................................................ 11  Material and Methods ..............................................................................................12 2.1 Materials and reagents .................................................................................................. 12 2.2 Acetylation of the CMC ................................................................................................ 13 2.3 Synthesis of Celludo ..................................................................................................... 14 2.4 Preparation of Celludo nanoparticles and analysis of nanoparticle size ....................... 14  vi  2.5 Fluorescently labeled Celludo formulation ................................................................... 15 2.6 Pharmacokinetic and biodistribution study ................................................................... 15 2.7 Development of an UHPLC method to quantify PPT in biological samples ............... 16 2.7.1 Preparation of stock solutions ................................................................................... 16 2.7.2 Preparation of calibration standards for the analysis of free PPT ............................. 17 2.7.3 Preparation of calibration standards for the analysis of Celludo .............................. 17 2.7.4 Sample preparation ................................................................................................... 18 2.7.5 Instrumentation and experimental conditions ........................................................... 19 2.8 Pharmacokinetic studies................................................................................................ 20 2.9 Histology and immunohistochemical analysis.............................................................. 21 2.10 Statistical analysis ......................................................................................................... 22  Results........................................................................................................................23 3.1 Synthesis and characterization of the Celludo polymer and Celludo nanoparticles ..... 23 3.2 Development of bioanalytical assay to quantitate PPT ................................................. 25 3.2.1 Mass spectrometry/Liquid chromatography ............................................................. 25 3.2.2 Optimization of sample preparation .......................................................................... 28 3.3 Pharmacokinetics of Celludo and free PPT .................................................................. 29 3.4 Biodistribution of Celludo and free PPT ...................................................................... 31 3.5 Kinetics of intra-tumoral micro-distribution of Celludo ............................................... 33  Discussion ..................................................................................................................35 4.1 Conclusion and future plans.......................................................................................... 41   vii  List of Tables  Table 3-1 One compartmental pharmacokinetic analysis of free PPT and Celludo in BALB/c mice. t½: Half-life. Vd: Volume of distribution. Cl: Clearance. AUC0–240: Area under the curve from 0–240 h. C0: initial concentration measured (total PPT). MRT: mean of residence time.... 30 Table 3-2 Comparison of Celludo uptake in the tumor versus other major tissues. ..................... 33   viii  List of Figures  Figure 1-1 Structure of P-glycoprotein (A) and its mechanism to efflux an anticancer agent out of a cell. These diagrams are adapted from Jin MS 2012 and Sharom Oncol 2014 ........................... 3 Figure 1-2 Chemical structure of (A) Podophyllotoxin (B) Etoposide........................................... 7 Figure 1-3 TEM image of Celludo (A) and drug release kinetics of Celludo in serum (B). These diagrams were adapted from Roy BM 2015. ................................................................................ 10 Figure 1-4 In vivo efficacy of the Celludo against EMT6/AR1. Data = mean ± SEM (n=10). This diagram is adapted from Roy BM 2015. ....................................................................................... 10 Figure 2-1 H-NMR spectra of the PPT-CMC-Ac-PEG, CMC-Ac and PPT. ............................... 19 Figure 3-1 H-NMR spectra of the PPT-CMC-Ac-PEG, CMC-Ac and PPT. ............................... 24 Figure 3-2 Chemistry and preparation of the CMC-Ac nanoparticle system for drug delivery. Nanoparticle that contains podophyllotoxin (PPT) is named Celludo.......................................... 25 Figure 3-3 Representative product ion mass spectra of PPT (top) and CBZ (bottom) obtained with Turbo Spray in the positive ionization mode. ....................................................................... 27 Figure 3-4 Representative UHPLC-MS/MS chromatogram of blank plasma sample (top) and plasma samples containing 250 ng/mL PPT (middle) or 250 ng/mL CBZ (bottom). .................. 28 Figure 3-5 Pharmacokinetic profiles of free PPT and Celludo in BALB/c mice. Data = mean ± SD, n=3 ......................................................................................................................................... 30 Figure 3-6 Biodistribution of free PPT (A) and Celludo (B) in EMT6-AR1 tumor bearing mice. Data = mean ± SD, n=3 ................................................................................................................ 32 Figure 3-7 Kinetics of intratumoral distribution of DiI-Celludo. Tumor sections were stained with FITC-anti CD31 Ab and DAPI and scanned with TISSUEscope. Differential accumulation  ix  was found between hypovascular and hypervascular region. (A) Tumor sections at three different times points (6, 24 and 48 h). Blue: nuclei; green: blood vessel; red: DiI-Celludo. (B) Overall Dil fluorescence intensity in tumors (C): The tumor section image at 48 h. ...................................... 34 Figure 4-1 The glomerular filtration apparatus, taken in its entirety, possesses an effective size cut-off of 6-10 nm. The graph was adapted from Jochen, Nature, 2013. ..................................... 39     x  List of Abbreviations  ACN Acetonitrile AF Ammonium formate BD Biodistribution CAD Collision gas CBZ Cabazitaxel CMC  Carboxymethyl cellulose CMC-Ac Acetylated carboxymethyl cellulose Dil 1,1-dioctadecyl-3,3,3’3’-tetramethylindocarbocyanine perchlorate DTX Docetaxel  EDC  1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide EPR Enhanced permeability and retention  HPMA N-(2-Hydroxypropyl)methacrylamide HPV Human papilloma virus FA Formic acid GI Gastrointestinal  IS Internal standard MTBE Methyl tert-butyl ether MDR Multidrug resistance MRPs Multidrug resistance-associated proteins MRT Mean residence time  xi  NBD Nonbinding domain PEG Polyethylene glycol Pgp P-glycoprotein PK Pharmacokinetics PPT Podophyllotoxin PTX Paclitaxel RES Reticuloendothelial system TEM Transmission electron microscope TIC Total ion current TM Transmembrane domains UHPLC/MS/MS Ultra high performance liquid chromatography-tandem mass spectrometry Vd Volume of distribution    xii  Acknowledgements  I offer my enduring gratitude to my supervisor, Dr. Shyh-dar Li who has inspired me to pursue my work in this field and supported me each step of the way. I would like to thank my committee members: Drs. Urs Häfeli, Christian Kapsture, Artem Cherkasov for their guidance and direction on my research project. A big Thank you to Dr. Abby Collier as well for serving as my external examiner, and to Dr. Judy Wong for chairing my committee meetings.  I am grateful to Dr. Tara Klassen for collaborating with us for the bioanalytical method development using UHPLC/MS/MS in her lab and Dr. Andras Szeitz for invaluable training and advice during the LC method development. Thank you to Dr. Harvey Wong for analyzing the PK parameters using SAAM II software. Thank you to Drs. Aniruddha Roy and Jonathan P. May for advices and directions on my work. I would like to thank my colleagues in the Li lab for their help and encouragement over the past two years: Dr. Yang Yang, Dr. Mehrdad Bokharaei, Dr. Hamano Nobuhito, Wei-lun Tang, Wunan Zhang, and Jessie Zhang.  Finally, I would like to thank my parents Chonghao Zhao and Wen Jiang for their continuous encouragement and support throughout this period.  1   Introduction  1.1 Overview of multidrug resistance Cancer is a leading cause of deaths across the world. In 2016, an estimated 1.6 million new cases of cancer will be diagnosed, and it will account for 0.6 million deaths in the United States. Many novel chemotherapeutic agents have been discovered for cancer treatment during the past few decades. Although chemotherapy is regarded as the first line approach of treatment for advanced diseases, there is no significant decrease in the overall mortality rate of significant cancers. In many cases, cancer relapses because of the development of drug resistance (1). The ability of cancer cells become simultaneously resistant to different drugs is known as ‘multidrug resistance’ (MDR) (2). MDR has been detected in many types of cancers including breast, ovarian, lung, and lower gastrointestinal tract cancers (3). Only a few approaches have been developed to address MDR. Previous studies have proposed several mechanisms of resistance developed in the cell including reduced drug influx and activation of DNA repair and detoxifying systems (cytochrome P450) (4, 5). The most prevalent one remains the over-expression of the membrane efflux pump p-glycoprotein (Pgp). The details for Pgp are discussed in next session.   1.1.1 Structure and function of p-glycoprotein (Pgp) Majority agents used in chemotherapy are substrates of Pgp, hence are ineffective against tumors with Pgp over-expression. Pgp is a member of ATP-binding cassette sub-family B, also known as a multidrug resistance protein receptor (6, 7). The human ABC super family of membrane proteins contains 48 transporters, and is further divided into seven subfamilies (A-G). An ABC  2  transporter generally includes two transmembrane domains (TM), with six transmembrane segments each, and two nucleotide binding domains (NBD). Pgp contains 170-kDa membrane glycoprotein with 12 transmembrane spinning domains (8). The crystallographic structure of mouse Pgp has been found to consist of two homologous halves, and each includes a transmembrane domain and a cytoplasmic NBD (Fig 1-1A) (9). As an efflux transporter, Pgp exports the intracellular substrates through an ATP-dependent efflux, reducing their cytoplasmic levels (10). Pgp is also reported to function as a “flippase” that facilitates the transfer of substrates between the inner and outer membranes of the phospholipid bilayer (Fig 1-1B) (11). Another suggested model was named vacuum cleaner model that Pgp interacts with its substrate directly within the membrane and subsequently effluxes them to the extracellular medium (Fig 1-1B)(8). Pgp has a high binding affinity for a broad range of substrates, including many anticancer agents, immune-suppressants, anti-arrhythmics, protease inhibitors, and antifungals in the azole class. Therefore, overexpression of Pgp has been determined in clinical samples of refractory tumors.   3   Figure 1-1 Structure of P-glycoprotein (A) and its mechanism to efflux an anticancer agent out of a cell. These diagrams are adapted from Jin MS 2012 and Sharom Oncol 2014   1.1.2 Strategies to overcome Pgp overexpression-mediated MDR To overcome Pgp-induced MDR, compounds have been screened for their activity to inhibit Pgp. The first inhibitor identified was L-type calcium channel blocker verapamil, however, the potency to block Pgp was low and induced serious cardiac side effects (12). Up to now, there is no Pgp inhibitors approved in the clinic due to significant adverse effects. A Pgp inhibitor will also increase vascular permeability within the brain, leading to increased penetration of toxic agents to the brain (13). Alternatively, nanotechnology was employed to deliver anticancer drugs to reduce Pgp efflux. Nanoparticles have been shown to be internalized by tumor cells efficiently, and the drug dose carried by nanoparticles will be dumped inside the cells, overwhelming the Pgp efflux. In addition, some excipients in the nanoparticles, such as surfactants and polymers have been shown to inhibit Pgp. Conjugating a target ligand to the  4  nanoparticle surface will facilitate its recognition by a tumor surface antigen and cellular internalization (14). However, the intracellular released drug remains a substrate for Pgp and improvements in efficacy by nanoparticle delivery have been inconsistent.   1.2 Anti-tubulin drugs for cancer therapy Anticancer agents that can interfere with micro-tubulin function have been widely used for cancer treatment, and they are known as anti-tubulin agents. Microtubules play an important role in the survival and growth of cells, and this makes tubulin a good target for cancer therapy. Microtubules are composed of alpha- and beta- tubulins (15), and molecules bind to tubulin affect the mitosis, leading to apoptosis. Taxanes, including paclitaxel, docetaxel and cabazitaxel, are one of the most potent anti-tubulin agents. Taxanes bind to microtubules and stabilize them, increasing the microtubule polymer mass, inducing the formation of microtubule bundles in cells and leading to blockage at the metaphase-anaphase transition and eventually cellular apoptosis (16, 17). Another class of anti-tubulin agents destabilizes microtubules, including vinca alkaloids. Vinca alkaloids, such as vinblastine, vincristine and vinorelbine, bind to tubulin and prevent it from polymerizing into microtubules, which is a necessary component for cellular division. Both Taxanes and vinca alkaloids have shown significant potency against various cancers (18, 19).   Although the anti-tubulin agents are effective against a broad spectrum of tumors, the resistance of tumors to them is a substantial problem. In particularly, Pgp engages in the drug efflux. Both taxanes and vinca alkaloids are substrates for Pgp, thereby this membrane transporter will reduce  5  their intracellular concentration and cytotoxic activity. Roy et al reported that the activity of Docetaxel (DTX) decreased by 25-fold in Pgp overexpressing MDA-MB-231 human breast cancer cells compared to that in the parental cell line. They also showed that Pgp-overexpressing MDR humane prostate cancer cells (PC-3) and murine breast cancer cells (EMT6-AR1) were 500-fold and 5000-fold less sensitive to paclitaxel (PTX) than the parent PC-3 and EMT6 cell lines respectively. Among these anti-tubulin drugs, cabazitaxel (CBZ) displays decreased affinity with Pgp and is used for castration resistant prostate cancer after DTX failure. However, the activity of CBZ was still decreased by 100-fold in Pgp overexpressing cell lines.   In addition to the ineffectiveness of these agents in MDR tumors, these drugs suffer from low solubility and poor tissue selectivity. The low solubility of these compounds leads to the inclusion of a co-solvent and detergent in the formulation to increase the solubility. The presence of these solubilizing agents induces hypersensitive reactions. Besides, neutropenia and neurotoxicity are commonly reported in patients treated with these drugs due to poor tissue selectivity (20-22).    1.2.1 Podophyllotoxin Podophyllotoxin (PPT) has been used as folk medicine for centuries. PPT is the active constituent of Podophyllum, a member of the berberidaceae family genus. PPT was originally used to treat venereal warts by Kaplan in 1942, and its antineoplastic effect was demonstrated in 1947 (23). PPT is currently used for the topical treatment of external genital warts, as it inhibits the growth of epithelia cells infected by human papilloma virus (HPV) in the epidermis (24).  6  Unlike epi-podophyllotoxins (e.g. Etoposide) that are topoisomerase II inhibitors and substrates for Pgp and multidrug resistance-associated proteins (MRPs) (Fig 1-2B), PPT is an anti-tubulin agent and is not a substrate for Pgp and MRPs (25, 26). PPT performs similarly to vinca alkaloid, and these agents induce partial unfolding of tubulin at the carboxyl terminis of the secondary structure of ß-tubulin, preventing its polymerization (27-29). PPT binds to the colchicine-binding site in tubulin and inhibits the polymerization into microtubule, arresting mitosis and inducing apoptosis of cells (30). Anti-tubulin agents acting on this site often remain active against tumor cells that overexpress β-III tubulin (31), and do not cause neuropathy (32, 33). Meng et al showed that the average half-life of PPT in mice was 0.88±0.06 h and the average initial redistribution elimination half-life is 29.93±4.72 h, after intraperitoneal injection of 10 mg/kg PPT formulated in 20% ethanol. The plasma AUC was 63.58 ± 8.48 ug.min/mL with clearance of 0.16±0.02 mL/h. The data suggested PPT was eliminated from blood circulation rapidly, leading to poor antitumor efficacy.   Additionally, PPT induced severe toxicity after dermal application with degenerative changes in the liver, intestine, testis, and pancreas of animals after 72 h. Its clinical results in cancer patients were disappointing due to severe gastrointestinal (GI) side effects (30). These reports indicate that PPT exhibits low tissue selectivity, and suggest targeted delivery of PPT is required.   7   Figure 1-2 Chemical structure of (A) Podophyllotoxin (B) Etoposide  1.2.2 Approaches to deliver PPT Ugir et al conjugated PPT to a poly-(amidoamine) (PAMAM) dendrimer and developed a nanodevice (D-PODO) that released PPT in a sustained manner. The particle size of D-PODO was 8 -15 nm determined by transmission electron microscope (TEM). Only 10 % of drug was released from D-PODO in PBS in 6 days. No significant renal and hepatic toxicity was reported after intraperitoneal administration of 8 mg PPT/kg of D-PODO, whereas the same dose of PPT elevated serum levels of ALT, AST, creatinine and BUN. However, the antitumor efficacy of D-PODO was only mild, inhibiting 50-60% growth of skin tumors. Additionally, there is no in vitro drug release data reported in this study and further biodistribution and pharmacokinetic studies need to be performed to confirm its targeted delivery. Zhu developed a podophyllotoxin-loaded solid lipid nanoparticle formulation (PPT-SLNs). PPT was formulated with steric acid in the solid core stabilized by lecithin and Myrj59. The particle size was ~50 nm with entrapment efficiency of 71.6%, and the formulation released 87% of PPT in 36 h in Phosphate buffer. There was no in vivo efficacy reported.   8  1.3 Preliminary data of Celludo Our lab developed a polymer-based nanoparticle system by delivering water insoluble drugs. This proprietary system is produced by covalently conjugating polyethylene glycol (PEG: mPEG2000-OH) and a hydrophobic drug to acetylated carboxymethycellulose (CMC-Ac, MW 4-12 KDa) via ester linkages. Carboxymethylcellulose (CMC) is commonly used as a pharmaceutical excipient in many products and is approved for use in parenteral formulations (e.g., Vivitrol, Sandolog and Sandostatin). CMC is a good candidate for a polymer backbone in the drug delivery, because it has a higher carboxylate DS (0.8) than most other polysaccharides (0.2 - 0.5) (34). However, incompatibility with organic solvent still restrains its use in drug conjugation chemistry. In our lab, we modified CMC via the esterification of the hydroxyl groups of acetic anhydride. This modification allowed this acetylated CMC (CMC-Ac) dissolved in water-free organic solvents, such as ACN and DMSO. When a balance between hydrophobicity (insoluble drug) and hydrophilicity (PEG) is achieved, the resulting polymer can self-assemble into nanoparticles in an aqueous phase.   Nanoparticles or polymers with a diameter > 10 nm or a MW >40 kDa display reduced renal clearance and prolonged blood circulation. These long circulating materials often show selective accumulated in tumors via the enhanced permeability and retention (EPR) effect. There are a number of polymer-drug conjugates advancing in clinical trials. NK105 and Genexol-PM (approved in South Korea) are Cremophor-free micellar nanoparticles displaying a high level of PTX loading and enhanced drug delivery to tumors compared to free PTX (35-37). Opaxil, a  9  polyglutamate-paclitaxel conjugates (Phase III), exhibited >12-fold higher accumulation in the tumor compared to PTX formulated with Cremophor EL (38)  We have employed the CMC-Ac system to deliver PPT and have optimized the polymer composition, which contains 15-20 wt% PPT and 40-45 wt% PEG. This optimized polymer self-assembled into stable particles with mean diameter of ~20 nm. This product is named Celludo.  Celludo released PPT at ~5%/day in serum (Fig 1-3). Celludo exhibited an IC50 of ~ 100 nM against various MDR tumor cell lines. The safety and antitumor efficacy of Celludo was examined in tumor bearing mice. Mice tolerated Celludo up to 180 mg PPT/kg (twice weekly for 3 i.v. doses) without showing significant body weight loss or signs of discomfort, while drug toxicity-induced animal euthanasia was recorded (2/10) with free PPT at 20 mg PPT/kg. Celludo displayed significantly improved efficacy against EMT6AR1 tumors in mice compared to free PPT, and 7/10 mice in the Celludo group were cured (Fig 1-4).  10   Figure 1-3 TEM image of Celludo (A) and drug release kinetics of Celludo in serum (B). These diagrams were adapted from Roy BM 2015.  Figure 1-4 In vivo efficacy of the Celludo against EMT6/AR1. Data = mean ± SEM (n=10). This diagram is adapted from Roy BM 2015.   11  1.4 Research hypothesis and aims of thesis The preliminary data suggested Celludo is promising new drug to treat multidrug resistant cancer. My thesis focused on comparing pharmacokinetics and biodistribution of free PPT and Celludo to confirm the drug targeting of Celludo. The intratumoral microdistribution of Celludo nanoparticles was also studied to examine the tumor penetration kinetics. My project contains three specific aims.  Aim 1: Develop a UHPLC method to quantify podophyllotoxin concentration in biological samples.  Aim 2: Compare pharmacokinetics and biodistribution of free PPT and Celludo Aim3: Study the intratumoral distribution of Celludo.   12   Material and methods  2.1 Materials and reagents Podophyllotoxin (> 98%) was purchased from Carbosynth Limited (Compton, Berkshire, UK). Cabazitaxel (> 99%) was purchased from Bolon Pharmachen Co, Ltd (Taizhou, Zhejiang, China), and ammonium formate (99.995% metals basis) was from Sigma-Aldrich (St. Louis, MO, USA). Methyl-tert-butyl ether, acetonitrile, and methanol (HPLC grade) were purchased from Fisher Scientific (Fair Lawn, NY, USA) and hydrochloric acid (1.0 M) from VWR (West Chester, PA, USA). Ultra- pure water was prepared in our laboratory using Milli-Q Synthesis system (Millipore, Billerrica, MA, USA). Sodium carboxymethylcellulose (CMC) (CEKOL 30000, degree of substitution = 0.82 was received from CPKelco (Atlanta, GA, USA). Slide-a-Lyzer dialysis cartridges were purchased from pierce Biotechnology (Rockford, IL, USA). Vivaspin 10 kDa MWCO ultracentrifugation filters were purchased from Fisher Scientific (Ottawa, ONT, Canada). Resistant EMT6/AR1 cells were a gift from Mr. Ian Tannock, Princess Margaret Hospital, Toronto. Formic acids (99.99%) and morpholine (99.99%) was purchased from Sigma-Aldrich (Oakville, ONT, Canada). BALB/c mouse plasma and liver homogenate were purchased from Bioreclamation IVT (Chestertown, NY, USA). Homogenate Navy RINO Lysis kit 50 for sample homogenizing was purchased from FroggaBio Inc (Toronto, ONT, Canada). All other general laboratory chemicals were purchased from Fisher Scientific Fisher Scientific (Ottawa, ONT, Canada) and VWR scientific (Mississauga, ON, Canada).   13  2.2  Acetylation of the CMC The method of acetylation of CMC was adapted from a method reported by Namikoshi (39). Sodium CMC (10 g) was weighed into a round-bottom flask, and was suspended in 20% sulfuric acid (200 mL) with vigorous stirring at room temperature for 2 h. The CMC was then centrifuged out of solution (4000 rpm, 5 min). The slurry of CMC-COOH was washed with deionized water 3 times until the water tested neutral. The white powder polymer product was dried by lyophylizer. The CMC-COOH was transferred to a round-bottom flask in an ice bath, and suspended in glacial acetic acid (50 mL). Acetic anhydride (30 mL) and sulfuric acid (1.2 mL) were added to slurry product. The flask was switched to silicon oil bath; the temperature was raised up to 50 °C. The reaction solution was concentrated by rotary evaporation (58 °C, 58 mbar) and was precipitated in deionized water. The water was extracted by centrifuging the suspension followed by the pellet re-suspended in water. The whole process was repeated until reaching a neutral pH. The polymer was transferred to a lyophylizer jar to be dried for overnight. The polymer was re-dissolved in 10 mL of acetone followed by centrifugation (4000 rpm, 5 min). The acetone supernatant was added to a beaker with 500 mL deionized water in. The Buchner funnel was set up with P8 filter paper, where the water solution was filtered off. The powder was scrapped off by spatula, and was added to 50 mL conical tube. The polymer was lyophilized again for overnight, and analyzed by 1H-NMR (Bruker, 500 MHz NMR spectrometer) in DMSO to confirm the presence of PPT and PEG, and to estimate molecular composition. Compounds were dissolved in DMSO-d6 for 1H-NMR analysis, and composition was estimated by integration of selected signals. A spectrum was analyzed by NMR eNotebook.    14  2.3 Synthesis of Celludo CMC-Ac (1000 mg, 3.90 mmol) was dissolved in 20 mL Acetonitrile (ACN) in a round bottom flask, followed by the addition of EDC HCl (1495 mg, 7.80 mmol), DMAP (1905 mg, 15.59 mmol), PEG-OH 2000 (3898, 1.95 mmol) and podophyllotoxin (1130 mg, 2.73 mmol). The solution was stirred over night at room temperature with protection from light. The solvent was removed by rotary evaporation (55 °C, 5 mbar), and the slurry product (5 mL) was transferred to a 50 L conical tube. Diethyl ether (45 mL) was added to precipitate the product, followed by centrifugation at 4000 for 5 min. The precipitated crude product was dissolved in 10 ml ACN and precipitated in 45 mL diethyl ether for two more times. The crude product was dissolved in ACN and then dialyzed against milliQ water. CMC-Ac was obtained after lyophylization. H-NMR (DMSO d6) analysis was conducted to confirm the chemical structure and composition of the polymer.  2.4 Preparation of Celludo nanoparticles and analysis of nanoparticle size Celludo nanoparticles were prepared in a controlled nanoprecipitation process using a two channel microfluidic system (NanoAssemlr, Precision Nanosystems International, Canada). The consistent size of nanoparticles was achieved by hydrodynamic flow system, with 1 mL Celludo polymer (30 mg) in ACN at a concentration of 30 mg/mL in one channel and 3 mL 0.9% NaCl in the adjacent channel. Total flow rate was maintained at 18 mL/min. The outlet stream was composed of the rapid mixing of aqueous and organic streams. The diluted nanoparticle solution was collected in conical tube, transferred to a dialysis cartridge (slide-A-Lyzer, 10000 MWCO), and dialyzed against 0.9% NaCl overnight. The buffer was replaced every 6hrs for three times. The particles were filtered through a 0.22 µm Millipore PVDF filter, transferred to a centrifugal  15  tube (10000 MWCO, Vivaspin), and concentrated for 1h to obtained final concentration at 18 mg/mL. PPT content was determined by H-NMR. 100 uL of Celludo solution was mixed with 900 uL of deuterated dimethysulphoxide containing 2-methyl-5-nitrobenzoic acid as an internal standard. Nanoparticle size and size distribution were determined by dynamic light scattering (DLS) using a particle analyzer (Zetasizer NanoZS, Malvern Instruments Ltd, Malvern, UK). The sample was diluted 10-fold in saline prior to analysis. The results were expressed as Z-Ave diameter. Free PPT formulation was prepared in a Tween80/ethanol/saline (20:13:67) solution, and was sterile filtered.  2.5 Fluorescently labeled Celludo formulation Celludo polymer powder (10 mg) was dissolved in ACN (1 mL) containing 0.1 mg/mL Dil (1,1-dioctadecyl-3,3,3’3’-tetramethylindocarbocyanine perchlorate) and was precipitated into 3 mL 0.9% saline by rapid mixing to form nanoparticles using Nanoassemblr. The process for Celludo-Dil preparation was identical to Celludo nanoparticles. Dil content of Celludo nanoparticles was determined by dissolving Celludo in DMSO and was quantified by fluorescence (Excitation filter: 535 nm; Emission Filter 590 nm).   2.6  Pharmacokinetic and biodistribution study Female BALB/c mice (aged 6 weeks, 18-20 g) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The experimental protocols in this study were approved by the Animal Care Committee of the University of British Columbia (Vancouver, BC, Canada) in accordance with the policies established in the Guide to the Care and Use of Experimental Animals prepared by the Canadian Council of Animal Care. The PK and BD studies were performed in BALB/c  16  mice bearing s.c. EMT6AR1 tumors: EMT6AR1 cells (2×105 cells/100 ul media) were s.c. inoculated into the shaved right lateral flank of BALB/c mice. One week later, when tumors reached ~200 mm3, free PPT (20 mg/kg) or Celludo (180 mg PPT/kg) were administrated by an i.v. injection through the tail vein. At selected time-points (6-96 h), blood samples were collected in EDTA-containing tubes, and animals were euthanized, followed by tissue collection. Blood was centrifuged at 2500 rpm for 10 min, and plasma was collected and stored at -80 ºC until analysis. Organs (heart, lung, liver, spleen, kidney, brain, muscle, intestine) and tumor were rinsed with buffer, and frozen at -80 ºC until analysis.   2.7 Development of an UHPLC method to quantify PPT in biological samples  2.7.1 Preparation of stock solutions  A master stock solution of PPT (100 μg/mL) was prepared in methanol and diluted 2-fold to yield the working stock solution A (50 μg/mL). This solution was diluted 50-fold to yield the working stock solution B (1 μg/mL). The working stock solutions A and B were further diluted with methanol to yield a series of diluted working stock solutions, which were used to prepare the calibration standards for the analysis of free PPT. A stock solution of Celludo (4 mg/mL) was prepared in Milli-Q water. The working stock solution B was further diluted to yield the working solution C. The working solution C was further diluted with water to yield a series of diluted working stock solutions, which were used to prepare the calibration standards for the analysis of Celludo. A 100 μg/mL CBZ internal standard (IS) solution was also prepared in methanol and diluted to yield the 5 µg/mL IS working stock solution. Morpholine, base was used to liberate the  17  PPT from polymer, was diluted 50 times by water. The solutions were stored at 4 °C until analysis  2.7.2 Preparation of calibration standards for the analysis of free PPT Series of working stock solutions of PPT were prepared in methanol and used for the calibration standards in mouse plasma. The calibration standards were prepared by pipetting 10 µL of blank mouse plasma into tubes, adding 100 µL of water and spiking them with 10 μL aliquots of appropriately diluted working stock solutions of PPT. A volume of 20 µL of 20% formic acid (FA) solution was also added. The samples were further processed as described in the Sample preparation section. Calibration curves were prepared freshly on the day of a batch analysis in the concentrations of 5.0, 12.5, 25, 50, 125, 250, 500, 2,500, 5,000 ng/mL of PPT in mouse plasma. A blank mouse plasma sample was also prepared.  2.7.3 Preparation of calibration standards for the analysis of Celludo  Analysis of coupled PPT in Celludo required a chemical treatment of the nanoparticle with morpholine to hydrolyze and liberate PPT. Morpholine was chosen as a base to degrade the ester bonds and liberate PPT from the polymer conjugate, and then the reaction was quenched with. The standards for coupled PPT in Celludo were prepared by serial a dilution of the Celludo nanoparticle solution in the range of 500 ng/mL to 0.5 mg/mL PPT in mouse plasma. The calibration standards were prepared by pipetting 10 uL of blank mouse plasma into tubes and spiking 10 uL aliquots of appropriately diluted working stock solutions into the tubes, followed by the addition of 10 uL of 250 mM morpholine solution. A volume of 100 uL water was added.  18  The tubes were vortex-mixed and incubated in a water bath at 37 ºC for 30 h. Subsequently, 20 uL of 20% FA and 50 uL of the 5 µg/mL IS solution were added. 20 % FA improved PPT extraction from plasma with increased recovery. The samples were further processed as described in the section 2.7.4. Calibration curves were prepared freshly on the day of a batch analysis in the concentration of 500, 1000, 2500, 5000, 10,000, 25,000, 50,000, 100,000, 250,000 ng/mL of PPT in mouse plasma. A blank mouse sample was also prepared.  2.7.4  Sample preparation Tissues (~100 mg), 0.9 m-2.0 m stainless steel blends, and 0.5 mL water were combined in screw-up Navy bead lysis kit (FroggaBio, Toronto. ON). The Bullet Blender® Gold was employed to homogenize the samples by subjecting for 5 mins at speed 12. Plasma and tissue homogenates contained a mixture of covalently coupled and released PPT. A 10 uL aliquot was transferred to a glass vial and combined with 100 uL water, 10 uL blank plasma or liver homogenates and 10 uL of 250 mM morpholine. The dilutions were vortex-mixed and incubated in a water bath at 37 ºC for 2 h. Subsequently, 50 uL of 5 µg/mL IS solution and 2 mL methyl tert-butyl ether were added to both dilutions and calibration standards as described previously. The mixtures were vortex-mixed for 30 s and the tubes were stored at -80 ºC for 10 mins. The tubes were removed from –80°C and the top layers were transferred to a clean set of tubes. The organic layer was brought to dryness in a Zymark TurboVap LV sample evaporator (Zymark Corporation, Hopkinton, Mass, USA), under nitrogen, at 35°C, and the dried residues were reconstituted with 100 μL of water : methanol, 50 : 50 (vol/vol) mixture containing 2.5 mM AF.  19   Figure 2-1 H-NMR spectra of the PPT-CMC-Ac-PEG, CMC-Ac and PPT.  2.7.5 Instrumentation and experimental conditions Plasma and tissue concentrations of Celludo and free PPT were determined by an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS) method using cabazitaxel (CBZ) as an internal standard (IS). The UHPLC/MS/MS system consisted of an Agilent 1290 Infinity Binary Pump, a 1290 Infinity Sampler, a 1290 Infinity Thermostat, and a 1290 Infinity Thermostatted Column Compartment (Agilent, Mississauga, Ontario, Canada) connected to an AB Sciex QTrap® 5500 hybrid linear ion-trap triple quadrupole mass spectrometer equipped with a Turbo Spray source (AB Sciex, Concord, Ontario, Canada). The mass spectrometer was operated in positive ionization mode and data were acquired using the Analyst 1.5.2. software on a Microsoft Windows XP Professional operating platform. Chromatographic analyses were performed using a Waters Acquity UPLC BEH C18, 1.7 µm 2.1 x 50 mm column, which was protected by a Waters Acquity UPLC BEH C18 VanGuard (1.7  20  µm, 2.1 × 5 mm) guard column (Waters Corp., Milford, MA, USA). The columns were maintained at 30 °C and the auto sampler tray temperature was maintained at 10 °C. Solvent A was water with 2.5 mM AF (ammonium formate), solvent B was methanol with 2.5 mM AF. The mobile phase initial conditions were solvent A (50%) and solvent B (50%), which was ramped to solvent A (5%) by 1.3 min, held until 3.0 min and followed by an equilibration with solvent A (50%) and solvent B (50%) for 2 min. The flow rate was 0.2 mL/min, injection volume was 5 µL with a total run time of 5.0 min. The mobile phase flow was diverted to the waste before 1.4 min and after 3.4 min during the chromatographic run. Mass spectrometric conditions were as follows. Curtain gas 30 units, collision gas (CAD) high, ionspray 5500 V, temperature 450 °C, ion source gas 1, 40 units, ion source gas 2, 60 units. Nitrogen gas was used for curtain gas, collision gas, ion source gas 2 (vaporizing gas), and zero air was used for ion source gas 1 (nebulizing gas). Entrance potential 10 units, resolution Q1 unit, resolution Q3 Unit, and dwell time was 150 msec. PPT and CBZ were monitored using the total ion current (TIC) of the multiple reaction monitoring (MRM) transitions as follows. For PPT (declustering potential DP, 146, collision energy CE, 13, collision cell exit potential CXP, 16), m/z 415.3  397.1, (DP, 156. CE, 25, CXP, 12) m/z 415.3  313.0, (DP, 151, CE, 19, CXP, 10), m/z 415.3  247.1; for CBZ, (DP, 101, CE, 17, CXP, 12), m/z 836.5  730.4, (DP, 111, CE, 21, CXP, 14), m/z 836.5  433.2). Using the current experimental conditions, the chromatographic retention times were for PPT and CBZ 2.12 min and 2.66 min, respectively.   2.8 Pharmacokinetic studies PK parameters were calculated with SAAM II (Saam Institute, University of Washington, Seattle, WA) software, using one-compartmentalized data analysis.   21   2.9 Histology and immunohistochemical analysis Histology and immunohistochemistry slides were prepared at the Toronto General Hospital Pathology Research Program lab (Toronto, ONT, Canada). Harvested tumor tissues were flash frozen and embedded in OCT and stored at -80 °C until sectioned.  5µm serial tissue sections were cut, collected on super frost plus slides, air dried for 30 minutes prior to storing at -80 °C until starting the staining procedure.  Sections were thawed at room temperature for approximately 5 minutes and then fixed in 2% paraformaldehyde for 20 minutes. The sections were then washed in running tap water for 5 minutes.  Sections were Permeabilized in PBS-T + 0.5% Triton X-100 for 15 minutes.  Block non-specific antibody binding with Dako diluent containing 5% Normal Goat serum for 30 minutes or 1 hr.    The serial section was incubated with Rat Anti-mouse CD31 (BD Pharmingen, San Jose, CA, USA) at a dilution of 1:300 made up in Dako diluent for 1 hr at room temperature.  A secondary antibody incubation followed consisting of Goat anti-Rat Alexafluor 488 (Thermo Fisher Scientific/Life Technologies Inc, Burlington, Canada) diluted to 1:200 and incubated for 1 hour at room temperature. The immunofluorescent staining was digitized on a scanning laser confocal microscope (TISSUEscope 4000, Huron Technologies) to acquire positive staining for each primary antibody, DiI and Dapi.   22  2.10 Statistical analysis All data are expressed as mean ± SD. Statistical analysis was conducted with the two-tailed unpaired t test for two group comparison or one-way ANOVA, followed by the Turkey multiple comparison test by using GraphPad Prism (for three or more groups). A difference with p < 0.05 was considered to be statistically significantly.  23   Results  The first part of my project is to synthesize Celludo nanoparticles and characterize them to confirm the batch I have generated was consistent with the previously reported one.   3.1 Synthesis and characterization of the Celludo polymer and Celludo nanoparticles Sodium CMC was converted to its free acid form and then acetylated to produce acetylated CMC (CMC-Ac). The CMC-Ac polymer was in a white powder form, which was soluble in DMF, THF and ACN, without gelling. H NMR (DMSO) δ: 3.0-5.5 (m, H in CMC), 2.02 (m, acetyl CH3). PPT, PEG and CMC-Ac were reacted in the presence of EDC and DMAP in anhydrous ACN, and PPT and PEG were conjugated to CMC-Ac via ester linkages. The synthesis scheme and 1H NMR spectra of the PPT-CMC-Ac-PEG, CMC-Ac and PPT are shown in Fig 3-1. Based on the NMR analysis, Celludo polymer contained 15.6 ± 2.0 wt% PPT and 42.1 ± 3.4 wt% PEG. 2-methyl 5-nitro benzoic acid was selected as an internal standard to determine relative percentage of PPT and PEG by integration of the 1H NMR spectra. The Celludo nanoparticles were prepared by a microfluidic mixing device Nanoassemblr. The particle size was 21.0 ± 0.1 nm with a polydispersity index of 0.101 ± 0.023 and zeta potential of -5.14 ± 7.32 mV. Free PPT in the nanoparticles was found to be less than 1.5 % of the total drug content using HPLC.   24   Figure 3-1 H-NMR spectra of the PPT-CMC-Ac-PEG, CMC-Ac and PPT.  25   Figure 3-2 Chemistry and preparation of the CMC-Ac nanoparticle system for drug delivery. Nanoparticle that contains podophyllotoxin (PPT) is named Celludo.  3.2 Development of bioanalytical assay to quantitate PPT The first aim of my thesis is to develop a sensitive and reproducible UHPLC/MS/MS method for the quantification of free and conjugated PPT in mouse plasma and tissue samples. This method is critical for studying pharmacokinetics and tissue distribution of PPT formulations in mice.  3.2.1 Mass spectrometry/Liquid chromatography CBZ was served as an internal standard for measurement accuracy, and mass spectrometry was applied to measure PPT and CBZ. To avoid formation of undesirable adducts, 2.5 mM ammonium formate (AF) was included in the mobile phase. Turbo Spray and positive ionization mode allowed the formation of positively charged molecular ions of PPT (m/z 415.3) and CBZ (m/z 836.5) that can be detected by the MS detector as shown in Fig 3-2. The mass spectrometric parameters, such as declustering potential (DP), collision energy (CE), collision cell exit  26  potential (CXP) were optimized to achieve the highest sensitivity. PPT and CBZ were monitored using the total ion current (TIC) of the multiple reaction monitoring (MRM) transitions as follows. For PPT (DP, 146, CE, 13, CXP, 16), m/z 415.3  397.1, (DP, 156. CE, 25, CXP, 12) m/z 415.3  313.0, (DP, 151, CE, 19, CXP, 10), m/z 415.3  247.1; for CBZ, (DP, 101, CE, 17, CXP, 12), m/z 836.5  730.4, (DP, 111, CE, 21, CXP, 14), m/z 836.5  433.2).  PPT and CBZ were separated on a C18 column (1.7 µm, 2.1 x 50 mm) with a gradient mobile phase, and were detected by the MS/MS system described in section 2.7.5.  The total run time was 5 min, and the retention time for PPT and CBZ were 2.13 mins and 2.65 mins, respectively. A representative chromatogram obtained from a mouse plasma sample containing 250 ng/mL PPT is presented in Fig 3-3. Calibration curves were constructed for the free PPT and Celludo and the curves were weighted with a weighting factor of 1/x2. The correlation coefficient was r ≥ 0.98 after weighting. The linear range of the method for the free PPT was established as 5 ng/mL to 5000 ng/mL, and that for Celludo was 500 -20,000 ng/mL. The lower limit of quantification was 5 ng/mL for free PPT and 500 ng/mL for Celludo.  27   Figure 3-3 Representative product ion mass spectra of PPT (top) and CBZ (bottom) obtained with Turbo Spray in the positive ionization mode.  28   Figure 3-4 Representative UHPLC-MS/MS chromatogram of blank plasma sample (top) and plasma samples containing 250 ng/mL PPT (middle) or 250 ng/mL CBZ (bottom).   3.2.2 Optimization of sample preparation NAVY kit tubes with a size of 2 mL were used to homogenate tissue samples. Blank mouse plasma was used for the preparation of the calibration curves for the plasma samples, and blank mouse liver homogenates purchased from Bioreclamation IVT were used for the preparation of the calibration curves for the rest of the mouse tissue samples. A volume of 10 uL of plasma or tissue homogenate was pipetted into disposable borosilicate glass tubes and 100 µL of water was added.  The incubation conditions for morpholine were optimised for the highest release of PPT from the polymer, in which 250 mM morpholine incubated for 30 mins achieved the highest drug recovery. Formic acid was selected to quench the reaction of nanoparticle cleavage process with  29  morpholine. Methyl-tert butyl ether (MTBE) was used as the solvent for extraction PPT and CBZ, which is also safe and easy to handle with.  3.3 Pharmacokinetics of Celludo and free PPT Free PPT and Celludo were administrated intravenously to BALB/c mice bearing s.c. EMT6AR1 tumors at the MTD (20 mg PPT/kg) for free PPT and at the maximum deliverable dose (180 mg PPT/kg) for Celludo. Blood samples were collected at selected time points, and PPT was extracted and analyzed by UHPLC-MS. As shown in Fig 3-4, Celludo displayed significantly extended blood circulation compared to free PPT. Cmax in the free PPT group was only 1.8 µg/mL after 1 h administration, and the plasma concentration decreased rapidly to 26.6 ng/mL in 8 h. The plasma concentration of PPT measured in the Celludo group at 6 h post injection was 1,730 µg /mL, and the concentration gradually decreased to 8.4 µg /mL in 96 h. During the examined time period, only 0.1% of PPT was released from Celludo into the plasma. The PK parameters for free PPT and Celludo were calculated using SAAM II (Saam Institute, University of Washington, Seattle, WA) software, using one-compartmentalized data analysis (Table 3.1). Compared to free PPT, Celludo showed an 18-fold prolonged half-life (t1/2), a 9,000-fold higher area under the curve (AUC), a 60-fold lower volume of distribution (Vd) 1,000-fold reduced clearance and 18-fold mean residence time (MRT).   30   Figure 3-5 Pharmacokinetic profiles of free PPT and Celludo in BALB/c mice. Data = mean ± SD, n=3  Table 3-1 One compartmental pharmacokinetic analysis of free PPT and Celludo in BALB/c mice. t½: Half-life. Vd: Volume of distribution. Cl: Clearance. AUC0–240: Area under the curve from 0–240 h. C0: initial concentration measured (total PPT). MRT: mean of residence time.    31  3.4 Biodistribution of Celludo and free PPT BALB/c mice bearing EMT6AR1 tumors were treated with either 180 mg PPT/kg of Celludo or 20 mg PPT/kg of free PPT. Mice were sacrificed at selected time points, and the liver, spleen, kidney, tumor, lung, heart, intestine, brain and muscle were collected for drug concentration analysis by UHPLC-MS/MS. As shown in Fig 3-5A, free PPT uptake by tissues was low (< 5 µg /g) and the drug retention was short (<6-9 h), including the liver, spleen, tumor, kidney, heart and lung. No drug uptake was measured in the intestine, brain and muscle. On the other hand, the tissue uptake of Celludo was significant, especially in the liver, spleen and kidney. In the liver, the uptake peaked at 24 h at 270 µg/g, but declined rapidly to 14.5 µg/g at 48-96 h. The spleen uptake of Celludo increased over time and peaked at 48 h at 187 µg/g. Celludo accumulated in the tumor as fast as 6 h post injection and plateaued for 96 h at 123 µg/g to 170 µg/g. Interestingly, the uptake of Celludo by the kidney was high 6 h after injection and the level remained similar for 96 h (157-275 µg/g). A relatively low level of Celludo was detected in the hearts and lungs at all time points (< 65 µg/g), and the drug content is under the limit of detection in the intestines, brains and muscles. In the tumor, Celludo displayed 500-fold increased delivery compared to free PPT. In the Celludo group, overall delivery to the tumor was 4.5-, 3.8-, 3.4-, and 1.2-fold higher compared to the liver, lung, heart and spleen, respectively (Table 3.2). Celludo uptake by the kidney was the highest among the examined tissues, and was 1.4-fold higher than that by the tumor.  32    Figure 3-6 Biodistribution of free PPT (A) and Celludo (B) in EMT6-AR1 tumor bearing mice. Data = mean ± SD, n=3  33   Table 3-2 Comparison of Celludo uptake in the tumor versus other major tissues.  3.5 Kinetics of intra-tumoral micro-distribution of Celludo To examine the intratumoral micro-distribution of Celludo nanoparticles, DiI-labeled Celludo was intravenously injected into BALB/c mice bearing s.c. EMT6AR1 tumor. At 6-48 h post injection, tumors were isolated, sectioned, labeled with FITC-anti-CD31 antibody (blood vessels) and DAPI (nuclei), and scanned with TISSUEscope for analysis of Celludo penetration within the tumor. Areas that are highly positive with FITC-CD31 are defined as hypervascular regions, while the opposite is determined as hypovascular. As shown in Fig 3-6A, Celludo nanoparticles accumulated in the tumors as early as 6 h after injection with low penetration to the hypovascular region. Over time, Celludo nanoparticles displayed increased accumulation in the hypovascular areas, whereas the levels of Celludo in the hypervascular region remained unchanged for 48 h (Fig 3-6B). The data indicated that Celludo nanoparticles extravasated into the hypervascular areas in the tumors in the beginning, and then continuously migrated from the hypervascular areas to the hypovascular regions with significant accumulation in the hypovascular part of the tumor at later time points. Figure 3-6C shows that the DiI-Celludo nanoparticles preferentially accumulated in the hypovascular tumor core at 48 h post injection.   34   Figure 3-7 Kinetics of intratumoral distribution of DiI-Celludo. Tumor sections were stained with FITC-anti CD31 Ab and DAPI and scanned with TISSUEscope. Differential accumulation was found between hypovascular and hypervascular region. (A) Tumor sections at three different times points (6, 24 and 48 h). Blue: nuclei; green: blood vessel; red: DiI-Celludo. (B) Overall Dil fluorescence intensity in tumors (C): The tumor section image at 48 h.   35   Discussion  PPT shows great potential in inhibiting MDR tumor cells, but suffers from poor solubility and tissue selectivity, inducing significant side effects. Little research has been performed to improve the delivery of PPT. A broad panels of natural and synthetic polymers, including hydroxypropylmethacrylate (HPMA), polysaccharides, PEGs, and polyglutamatic acid, have been explored as delivery vehicles for anticancer drugs via polymer-drug conjugates (40, 41). Among these polymers, polysaccharides conferred advantages including low cost, great availability, and stable physicochemical and biological characteristics (42, 43). However, the use of polysaccharides is limited due to their low degree of substitution (DS, <20%) for drug conjugation, leading to rapid release (>20-30 % per day) (43).   Carboxymethylcellulose (CMC) was selected as the polymer backbone to synthesize a polymer conjugate with PPT, as CMC is safe and biocompatible, and contains a high density of carboxylate groups (DS = 0.82) for the efficient drug coupling. However, CMC is insoluble in organic solvent and gels in aqueous phase, posing significant challenges in reacting with hydrophobic compounds in a homogenous phase. Therefore, the hydroxyl groups on CMC were first acetylated to synthesize CMC-Ac, which displayed increased solubility in organic solvents such as ACN and DMSO without gelling. The CMC-Ac could thus be reacted with a hydrophobic drug and polyethylene glycol (PEG) in water-free conditions. Acetylating hydroxyl groups on the CMC could also reduce polymer cross-linking during the EDC coupling reactions that linked the hydrophobic drug and PEG to the polymer via ester linkages.  Compared with other polysaccharide-drug conjugates, our CMC-Ac system showed increased drug loading (~40  36  % content of the polymer conjugate) (34). PEG was included in this system to maintain the hydrophilicity and hydrophobicity balance to control the self-assembly of the polymers into nanoparticles in an aqueous phase. In addition to prevent the formation of large aggregates, PEG introduces steric hindrance around the nanoparticles, reducing serum protein binding and the subsequent clearance by macrophages to allow prolonged blood circulation and increased tumor delivery (44, 45).  In this study, a UHPLC/MS/MS method was developed to quantify PPT in biological samples to study the PK and BD of Celludo and free PPT. CBZ was chosen as the internal standard for PPT because of their similar MS ionization characteristics and chromatographic behaviors. 4-deoxypodophyllotoxin (4-DPPT) and etoposide (ETP) were also investigated as IS candidates for their structural similarity to PPT. Under the same chromatographic conditions, 4-DPPT was found to co-elute with PPT, and PPT fragmented to 4-DPPT via in-source fragmentation. Under the same conditions, ETP fragmented to PPT. Additionally, ETP is a relatively polar compound and could not be efficiently extracted from the biological samples with PPT. Therefore, 4-DPPT and ETP could not be used as an IS to quantify PPT. For the optimization of sample preparation, liver homogenates were considered as an acceptable representative matrix for the analysis of PPT than other tissue homogenates due to its high lipid content. Dilution of the tissue samples with 100 µL of water was required to reduce sample denaturation after the addition of the drug and IS dissolved in methanol. Morpholine was shown to effectively cleave the ester bonds to release PPT from the polymer conjugate. Formic acid was added to quench the reaction to improve the drug extraction. MTBE was selected as an extraction solvent owing to its wide-spread use for extractions of pharmaceuticals from a variety of biological matrices (46).   37   Celludo demonstrated a significantly improved PK profile compared to free PPT with 18-fold prolonged half-life. Celludo was also characterized with increased Cmax and AUClast and decreased Vobs and Clobs over free PPT, indicating that Celludo exhibited increased retention in the plasma. The extended plasma circulation of Celludo would potentially lead to increased tumor accumulation. During the examined time period, only 1.5 % of PPT was released from Celludo into the plasma, suggesting that the systemic toxicity induced by free PPT could be reduced.   The prolonged blood circulation of Celludo led to increased accumulation in tissues, including the liver, spleen, tumor, kidney, heart and lung. In particular, the tumor uptake of Celludo was 500-fold higher than that of free PPT. The drug level in the tumor treated with Celludo persisted for >96 h. This result supports the efficacy data: while free PPT only exhibited moderate activity against EMT6AR1 tumor in mice, Celludo effectively regressed the tumor and cured 7/10 mice. The drug levels in tissues in the free PPT treatment group were significantly lower than that in the Celludo group, and this could be attributed to the short circulation half-life (0.66 h) and low AUC of free PPT. The highest tissue accumulation of free PPT was 1 h post injection in all the major tissues, and the drug levels declined rapidly to less < 0.3 µg/g at 9 h post injection. The BD data are consistent with the PK data.   High levels of PPT were detected in the liver and spleen after treatment of Celludo. Various types of nanoparticles haven been shown to exhibit increased uptake by macrophages in the liver and spleen (47, 48) . Although PEGylation reduces serum protein binding to nanoparticles,  38  PEGylated nanoparticles are still effectively recognized and cleared by the macrophages after opsonization. As the target cell population in the liver for nanoparticles is macrophage that exhibits a fast turn-over rate, hepatic toxicity is not always reported with nanoparticle formulations, such as Doxil (PEGylated liposomal doxorubicin) and Abraxane (Nab-paclitaxel). However, if the nanoparticles are digested in the liver macrophages and the drug is released to the neighboring hepatic cells, liver toxicity may occur. Our data show that the liver levels of PPT in the Celludo group peaked at 24 h post injection and rapidly decreased afterwards; suggesting Celludo nanoparticles and PPT were rapidly metabolized in the liver. Vasilev al et characterized the metabolism of deoxypodophyllotoxin (DPT, a podophyllotoxin derivative lignin), identifying 4 different metabolites from the incubation of DPT with rat liver microsomes using the LC-ESI/MS techniques (49). It was found that CYP3A4 and CYP2C19 were the major CYP isozymes in the metabolism of DPT. The metabolism of PPT and Celludo is yet to be studied, and the impact on liver toxicity must be carefully monitored. One of the most advanced polymer-drug conjugates, Opaxio™, the most advanced polymer-drug conjugate (Phase III), exhibited 5- to 8-fold increased uptake by the spleen and liver compared to the tumor(50-53). On the other hand, the overall delivery of Celludo to the tumor was 4.5- fold and 1.2-fold higher than that in the liver and spleen, respectively, suggesting improved tissue selectivity of Celludo towards tumors.   39   Figure 4-1 The glomerular filtration apparatus, taken in its entirety, possesses an effective size cut-off of 6-10 nm. The graph was adapted from Jochen, Nature, 2013. The overall uptake of Celludo by the kidney was 1.4-fold higher compared to the tumor. This result is somewhat unexpected, as the cut-off size for renal clearance of nanoparticles is 6-10 nm and the diameter of Celludo is ~20 nm (54). The prolonged blood circulation of Celludo (~12 h) indicates that Celludo was not cleared by the renal filtration; and therefore, the increased kidney uptake of Celludo is likely mediated by accumulation of Celludo in the renal tissue. The renal glomerular filtration barrier is a three-layer structure composed of fenestrated glomerular endothelial cells with pores diameters in the range of 60-80 nm, glomerular basement membrane between the two cellular layers (rich in heparin sulfate and charged proteoglycans which provides size and charge selectivity), and podocytes (with interdigitating foot processes that form filtration slits of 32 nm) (Fig 4-1) (55-57). It was suggested that Celludo nanoparticles would be able to penetrate through the fenestrated glomerular endothelial layer and interact with the glomerular basement. The high uptake of Celludo by the kidney poses a significant concern in  40  the nephrotoxicity. However, in our preliminary test, no elevation of renal toxicity markers (creatinine, BUN) was measured in serum from the treated mice. The safety profile of Celludo will be examined thoroughly in the future, especially in the liver and kidney. Nevertheless, this interesting result of enhanced drug delivery to the kidney suggests that the CMC-Ac system can be further developed as a platform technology for targeting drugs to the kidney, which remains an unmet challenge.    Naoparticles have been shown to exhibit limited penetration within tumors: they often accumulate around the blood vessels. This uneven intratumoral distribution of nanoparticles poses a significant concern, as some parts of the tumor could be left untreated after nanoparticle therapy, resulting in disease recurrence. The 20 nm Celludo nanoparticles showed visible but low accumulation in the tumor at 6 h, but the uptake by the tumor increased over time. The results are consistent with the BD data measure by UHPLC. At 6 h, slightly more Celludo nanoparticles were detected in the hypervasular area compared to the hypovascular region. As the time increased, the amount of Celludo in the hypervascular area remained the same, while that in the hypovascular region increased by 5-fold. At 48 h post-injection, most of the Celludo nanoparticles were detected in the hypovascular core of the tumor. The data suggest Celludo nanoparticles extravasated and accumulated around the blood vessels at the early time point after injection. While the Celludo nanoparticles continued to extravasate into the hypovascular region in the tumor, they also effectively migrated from the hypervascular area to the hypovascular core, resulting in significant accumulation in the tumor core after 48 h. The hypoxic tumor core harbors aggressive tumor cells and effective drug delivery to this area will greatly enhance the  41  therapy. Indeed, our efficacy data show that Celludo effectively regressed EMT6AR1 tumors in mice and cured 7/10. In previous studies, Cabral et al. and Lee et al. demonstrated that smaller particles (~30 nm) exhibited increased tumor penetration compared to the larger counterparts (58-60).   4.1 Conclusion and future plans A robust UHPLC method has been developed to measure PPT concentration in biological samples, allowing the comparison of the PK and BD of free PPT and Celludo. Celludo displayed 18-fold prolonged blood circulation and 9,000-fold increased AUC compared to free PPT. The extended PK of Celludo led to increased tissue uptake in the tumor and major tissues, including the liver, spleen, and kidney. Among these major tissues, Celludo displayed 4.5-fold increased uptake by the tumor compared to the liver. Celludo increased the tumor delivery by 500-fold compared to free PPT. Within the tumor, Celludo nanoparticles preferentially accumulated in the hypovascular core. The results support that Celludo was a long circulating (t1/2 ~ 12 h) nanoparticle formulation that selectively targeted tumor and exhibited enhance tumor penetration.   While the results are encouraging, Celludo also exhibited increased uptake in the liver and kidney, and the hepatic toxicity and nephrotoxicity must be carefully examined. Renal and hepatic toxicity markers should be carefully monitored during the therapy, including serum levels of ASL, ALT, creatinine and BUN. Tissues should be collected after the treatment, and the histology must be examined by a certified pathologist. It remains unclear why Celludo exhibited increased uptake by the kidney. Nevertheless, this interesting result opens a new opportunity for  42  the CMC-Ac delivery system for targeting drugs to treat renal diseases. Finally, the underlying mechanism for Celludo nanoparticles to preferentially accumulate in the hypovascular tumor core needs to be elucidated. 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