nanotechnology for cancer therapy, 2007, p.810

q 2006 by Taylor & Francis Group, LLC q 2006 by Taylor & Francis Group, LLC q 2006 by Taylor & Francis Group, LLC Dedication To the loving memory of my dad, Mustafa Amiji, and to my mom, Shirin Amiji q 2006 by Taylor & Francis Group, LLC Foreword We are at the threshold of a new era in cancer treatment and diagnosis, brought about by the convergence of two disciplines—materials engineering and life sciences—that 30 years ago might have been difficult to envision The product of this curious marriage, nanobiotechnology, is yielding many surprises and fostering many hopes in the drug-development space Nanoparticles, engineered to exquisite precision using polymers, metals, lipids, and carbon, have been combined with molecular targeting, molecular imaging, and therapeutic techniques to create a powerful set of tools in the fight against cancer The unique properties of nanomaterials enable selective drug delivery to tumors, novel treatment methods, intraoperative imaging guides to surgery, highly sensitive imaging agents for early tumor detection, and real-time monitoring of response to treatment Using nanotechnology, it may be possible to leap over many of the hurdles of cancer drug delivery that have confounded conventional drugs These hurdles include hindered access to the central nervous system through the blood–brain barrier, sequestration by the reticulo-endothelial system, inability to penetrate the interior of solid tumors, and overcoming multi-drug resistance mechanisms These obstacles can be mitigated by manipulating the size, surface charge, hydrophilicity, and attached targeting ligands of a therapeutic nanoparticle The novelty of using nanotechnology for medical applications presents its own challenges, similar in many ways to the challenges faced by the introduction of the first synthetic protein-based drugs In the early 1980s, protein-based drugs offered an entirely new approach to targeting and treating disease They could be “engineered” to be highly specific and even offered the capability of separating the targeting and therapeutic functions of a drug, such as when monoclonal antibodies are conjugated to cytotoxic agents But monoclonal antibodies and recombinant protein-based drugs necessitated a different approach to lead optimization, metabolism, and toxicity screening from that of small-molecule drugs Factors such as stability, immunogenicity, and species specificity had to be considered and tested in ways that were not familiar to the developers of traditional drugs Nanotechnology-based drugs will catalyze a reevaluation of optimization, metabolism, and toxicity-screening protocols Interactions between novel materials and biological pathways are largely unknown but are widely suspected to depend heavily on physical and chemical characteristics such as particle size, particle size distribution, surface area, surface chemistry (including charge and hydrophobicity), shape, and aggregation state—features not usually scrutinized for traditional drugs Standard criteria for physicochemical characterization will have to be established before safety testing can yield interpretable and reproducible results Would nanotechnology-based drugs be successful? All drugs have to run an obstacle course through physiological and biochemical barriers For monoclonal antibody drugs, only a minute portion of an intravenously administered drug reaches its target However, drugs based on nanomaterials, including nanoparticles, have unique properties that enable them to specifically bind to and penetrate solid tumors Size and surface chemistries can be manipulated to facilitate extravasation through tumor vasculature, or the therapeutic agent can be encapsulated in polymer micelles or liposomes to prevent degradation and increase circulation half-life to improve the odds of it reaching the target This level of functional engineering has not been available to either small-molecule or proteinbased drugs For these drugs, modification of one characteristic, such as solubility or charge, can have dramatic effects on other essential characteristics, such as potency or target specificity Nanoparticles, however, introduce a much higher degree of modularity and offer at least four advantages over the antibody conjugates: (1) the delivery of a larger therapeutic payload per target recognition event, enhancing potency; (2) the ability to carry multiple targeting agents, enhancing q 2006 by Taylor & Francis Group, LLC selectivity; (3) the ability to carry multiple therapeutic agents, enabling targeted combination therapies; and (4) the ability to bypass physiological and biological barriers We would all like to hasten the day when chemotherapy—the administration of non-specific, toxic anti-cancer agents—is relegated to medical history Improvements in diagnostic screening and the development of drugs that target specific biological pathways have begun to turn the tide and contribute to a slow, yet consistent decline in death rates While confirming that early diagnosis and targeted therapies are pointing us in the right direction, the progress is still incremental Nanotechnology may be our best hope for overcoming many of the barriers faced by today’s drugs in the battle against cancer The combined creative forces of engineering, chemistry, physics, and biology will beget new and hopefully transformative options to old, intractable problems Piotr Grodzinski, Ph.D National Cancer Institute q 2006 by Taylor & Francis Group, LLC Preface With parallel breakthroughs occurring in molecular biology and nanoscience/technology, the newly recognized research thrust on “nanomedicine” is expected to have a revolutionary impact on the future of healthcare To advance nanotechnology research for cancer prevention, diagnosis, and treatment, the United States National Cancer Institute (NCI) established the Alliance for Nanotechnology in Cancer in September 2004 and has pledged $144.3 million in the next five years (for details, visit http://nano.cancer.gov) Among the approaches for exploiting developments in nanotechnology for cancer molecular medicine, nanoparticles offer some unique advantages as sensing, delivery, and image enhancement agents Several varieties of nanoparticles are available, including polymeric conjugates and nanoparticles, micelles, dendrimers, liposomes, and nanoassemblies This book focuses specifically on nanoscientific and nanotechnological strategies that are effective and promising for imaging and treatment of cancer Among the various approaches considered, nanotechnology offers the best promise for targeted delivery of drugs and genes to the tumor site and alleviation of the side effects of chemotherapeutic agents Multifunctional nanosystems offer tremendous opportunity for combining more than one drug or using drug and imaging agents The expertise of world-renowned academic and industrial researchers is brought together here to provide a comprehensive treatise on this subject The book is composed of thirty-eight chapters divided into seven sections that address the specific nanoplatforms used for imaging and delivery of therapeutic molecules Section focuses on the rationale and fundamental understanding of targeting strategies, including pharmacokinetic considerations for delivery to tumors in vivo, multifunctional nanotherapeutics, boron neutron capture therapy, and the discussion on nanotechnology characterization for cancer therapy, as well as guidance from the U.S Food and Drug Administration on approval of nanotechnology products Section focuses on polymeric conjugates used for tumor-targeted imaging and delivery, including special consideration on the use of imaging to evaluate therapeutic efficacy In Section 3, polymeric nanoparticle systems are discussed with emphasis on biodegradable, long-circulating nanoparticles for passive and active targeting Section focuses on polymeric micellar assemblies, where sophisticated chemistry is applied for the development of novel nanosystems that can provide efficient delivery to tumors Many of the micellar delivery systems are undergoing clinical trials in Japan and other countries across the globe Dendritic nanostructures used for cancer imaging and therapy are discussed in Section Section focuses on the oldest nanotechnology for cancer therapy—liposome-based delivery systems—with emphasis on surface modification to enhance target efficiency and temperature-responsive liposomes Lastly, Section focuses on other lipid nanosystems used for targeted delivery of cancer therapy, including nanoemulsions that can cross biological barriers, solid-lipid nanoparticles, lipoprotein nanoparticles, and DQAsomes for mitochondria-specific delivery Words cannot adequately express my admiration and gratitude to all of the contributing authors Each chapter is written by a world-renowned authority on the subject, and I am deeply grateful for their willingness to participate in this project I am also extremely grateful to Dr Piotr Grodzinski for providing the Foreword Drs Fredika Robertson and Mauro Ferrari have done a superb job in laying the foundation by providing a chapter entitled “Introduction and Rationale for Nanotechnology in Cancer.” I am grateful to Professor Kinam Park at Purdue University, Professor Robert Langer at MIT, and Professor Vladimir Torchilin at Northeastern University, who have been my mentors and collaborators, as well as many other researchers from academia and industry Special thanks are due to the postdoctoral associates and graduate students in my laboratory at Northeastern University who have been the “soldiers in the trenches” in our quest to use nanotechnology for the targeted delivery of drugs and genes to solid tumors Lastly, I am deeply q 2006 by Taylor & Francis Group, LLC grateful to the wonderful people at Taylor & Francis-CRC Press, including Stephen Zollo, Patricia Roberson, and many others, who have made the concept of this book into reality Any comments and constructive criticisms of the book can be sent to the editor at m.amiji@neu.edu q 2006 by Taylor & Francis Group, LLC 788 Nanotechnology for Cancer Therapy i.e., the molecular targets of anti-cancer drugs, is being recognized Transporting the cytotoxic drug to its intracellular target could potentially overcome MDR by bypassing the p-glycoprotein, i.e., the drug would literally be hidden from the p-glycoprotein inside the delivery system until it becomes selectively released at the particular intracellular site of action At the same time, a sub-cellular delivery system would significantly increase the subcellular bioavailability of any drug acting inside a cell 38.2 APOPTOSIS AND MITOCHONDRIA Apoptosis (programmed cell death) plays a central role in tissue homeostasis, and it is generally recognized that inhibition of apoptosis may contribute to cell transformation.1 Significant knowledge has been accumulated during the past several years about how the apoptotic machinery is controlled.2–20 As each new regulatory mechanism had been identified, dysfunction of that mechanism has been linked to one or another type of cancer.17 Dysregulation of the apoptotic machinery is now generally accepted as an almost universal component of the transformation process of normal cells into cancer cells A large body of experimental data demonstrates that mitochondria play a key role in the complex apoptotic mechanism.18–49 Mitochondria have been shown to trigger cell death via several mechanisms: by disrupting electron transport and energy metabolism, by releasing or activating proteins that mediate apoptosis, and by altering cellular redox potential A critical event leading to programmed cell death is the mitochondrial membrane permeabilization that is under the control of the permeability transition pore complex (mPTPC), a multiprotein complex formed at the contact site between the mitochondrial inner and outer membranes The mPTPC is widely accepted as being central to the process of cell death and has accordingly been recommended as a privileged pharmacological target for cytoprotective and for cytotoxic therapies in general.50 In particular, it has been suggested that targeting specific mPTPC components may overcome bcl-2 mediated apoptosis inhibition in cancer cells.1 Several studies have already demonstrated the feasibility of eliminating neoplastic cells by selectively inducing apoptosis (reviewed in reference 17) The design of mitochondria-targeted cytotoxic drugs has been formulated as a novel strategy for overcoming apoptosis resistance in tumor cells,1 which, intriguingly, opens up a whole new avenue for the therapy of cancer by “tricking cancer cells into committing suicide.”17 38.3 PROAPOPTOTIC DRUGS ACTING ON MITOCHONDRIA Several conventional anti-cancer drugs, such as doxorubicin, and cisplatin, have no direct effect on mitochondria.51 These conventional chemotherapeutic agents elicit mitochondrial permeabilization in an indirect fashion by induction of endogenous effectors that are involved in the physiologic control of apoptosis.1 However, a variety of clinically approved drugs such as paclitaxel,52–60 VP-16 (etoposide)61–64 and vinorelbine58 as well as an increasing number of experimental anticancer drugs such as betulinic acid, lonidamine, CD-437 (a synthetic retinoids) and ceramide (reviewed in1) have been found to act directly on mitochondria resulting in triggering apoptosis These agents may induce apoptosis in circumstances in which conventional drugs fail to act because endogenous apoptosis inducing pathways, e.g., such as those involving p53, death receptors or apical caspase activation, are disrupted, leading to the apoptosis-resistance of tumor cells For example, several in vitro and in vivo studies have shown that the synthetic retinoid CD437 is able to induce apoptosis in human lung, breast, cervical and ovarian carcinoma cells (reviewed in Kaufmann and Gores 2000) It could be demonstrated that in intact cells, CD437-dependent caspase activation is preceded by the release of cytochrome C from mitochondria.65 Moreover, it was shown that when added to isolated mitochondria, CD437 causes membrane permeabilization and that this effect is prevented by inhibitors of the mPTPC such as cyclosporine A CD437 constitutes q 2006 by Taylor & Francis Group, LLC DQAsomes as Mitochondria-Targeted Nanocarriers for Anti-Cancer Drugs 789 an experimental drug that exerts its cytotoxic effect via the mPTPC, i.e., by acting directly at the surface or inside of mitochondria The development of anti-cancer drugs whose cytotoxic effects depend on their direct interaction with mitochondria inside of living cells raises the issue of the intracellular distribution of these drugs after having been taken up by the cell, i.e., the question of their intracellular bioavailability Independent of their mode of cell entry that should mostly take place via passive diffusion through the cell membrane, drug molecules become randomly distributed among all cell organelles and, depending on the chemical nature of the drug, eventually metabolized Therefore, anti-cancer drugs that exert their cytotoxic activity by directly acting at or inside of mitochondria in living cells would dramatically benefit from a delivery system that can selectively transport these drugs to and into mitochondria One of the major roles of mitochondria in the metabolism of eukaryotic cells is the synthesis of Adenosine triphosphate (ATP) by oxidative phosphorylation via the respiratory chain According to Mitchell’s chemiosmotic hypothesis, electrons from the hydrogens on Nicotine amide adenine dinucleotide (NADH) and Flavin adenine dinucleotide (FADH2) are carried along the respiratory chain at the mitochondrial inner membrane, thereby releasing energy that is used to pump protons across the inner membrane from the mitochondrial matrix into the intermembrane space This process creates a transmembrane electrochemical gradient that includes contributions from both a membrane potential (negative inside) and a pH difference (acidic outside) The membrane potential of mitochondria in vitro is between 180 and 200 mV, the maximum a lipid bilayer can sustain while maintaining its integrity.66 Although this potential is reduced in living cells and organism to about 130–150 mV as a result of metabolic processes such as ATP synthesis and ion transport,67 it is by far the largest within cells Most interestingly, carcinoma cells posses a different mitochondrial membrane potential relative to normal cells It has been found that in many carcinoma cell lines, the mitochondrial membrane potential is higher than in normal epithelial cells (reviewed in reference 68) For example, the difference of the mitochondrial membrane potential between the colon carcinoma cell line CX-1 and the control green monkey kidney epithelial cell line CV-1 has been reported to be approximately 60 mV.68 Moreover, some carcinoma cells, in particular, human breast adenocarcinoma-derived cells, have in addition to the higher mitochondrial membrane potential also an elevated plasma membrane potential relative to their normal parent cell lines.68–75 The striking difference between normal cells and human adenocarcinoma cells regarding the electrical charge of both plasma and mitochondrial membranes has lead numerous investigators during the 1990s to explore fundamentally new strategies for the selective targeting of cancer cells Their attempts have been based on compounds with a delocalized charge that have long been known to accumulate in mitochondria of living cells in response to the mitochondrial membrane potential Many of these DLCs are toxic to mitochondria at high concentration For example, the rhodacyanine official name of this compound, unknown what the letters stand for (MKT-07771,76–80) was the first DLC to be approved by the FDA for clinical trials for the treatment of carcinoma.81 The trials were discontinued, however, because efficacy in tumor cell killing was not demonstrated at the particular approved dosage and drug regimen.68 From the phase I clinical trial, it was concluded that it is feasible to target carcinoma cell mitochondria with rhodacyanine analogues if drugs with higher therapeutic indices could be developed.81 The DQAsomal-based strategy involving the use of dequalinium chloride, a typical representative of DLCs, for tumor and mitochondria-specific targeting factually starts where these failed attempts of the 1990s have left off This approach combines the well-proven ability of DLCs to specifically target carcinoma cell mitochondria with the selective delivery of apoptotically active compounds known to trigger programmed cell death via directly acting at the mitochondrial surface The DQAsomal approach utilizes the mitochondria-specific affinity of DLCs for the delivery of pro-apoptotic drugs to mitochondria Therefore, the actual cytotoxic effect is caused q 2006 by Taylor & Francis Group, LLC 790 Nanotechnology for Cancer Therapy by apoptosis-triggering drugs Any inherent cytotoxicity of the carrier system, i.e., the DLCs used, might only add to the overall efficiency of killing carcinoma cells 38.4 MITOCHONDRIOTROPIC VESICLES (DQASOMES) Dequalinium chloride (DQA, Figure 38.1a) represents a typical mitochondriotropic delocalized cation that already almost 20 years ago was shown to selectively accumulate in carcinoma cell mitochondria.73 Most interestingly in the context of this chapter, it was demonstrated recently that dequalinium B, a new boron carrier for neutron capture therapy, also accumulates preferentially in carcinoma cells over non-transformed cells.82 Dequalinium is a dicationic compound resembling “bola”-form electrolytes, i.e., it is a symmetrical molecule with two charge centers separated at a relatively large distance Such symmetric bola-like structures are well known from archaeal lipids that usually consist of two glycerol backbones connected by two hydrophobic chains.85,86 The self-assembly behavior of bipolar lipid from Archaea has been extensively studied (reviewed in Gambacorta, Gliozi, and De Rosa 1995) Generally, it has been shown that these symmetric bipolar archaeal lipids can self-associate into mechanically, very stable monolayer membranes The most striking structural difference between dequalinium and archaeal lipids lies in the number of bridging hydrophobic chains between the polar head groups Contrary to the common arachaeal lipids, in dequalinium there is only one carbohydrate chain that connects the two cationic hydrophilic head groups Therefore, this type of bola lipids has been named single-chain bola-amphiphile.87,88 The self-association behavior of this single-chain cationic bola amphiphile was investigated using Monte Carlo computer simulations (Figure 38.1c, left panel),83 several electron microscopic (EM) techniques (Figure 38.1c, right panel) as well as dynamic laser light scattering.84 It was found that, upon sonication, dequalinium forms spherical aggregates with diameters between about 70 and 700 nm that were termed DQAsomes 84 Freeze fracture images (Figure 38.1) show both convex and concave fracture faces These images strongly indicate the CH3 CH3 NH2 N CH CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 N NH2 (a) OR (b) A B C 0.2μm (c) FIGURE 38.1 (a) Chemical structure of dequalinium chloride with overlaid colors indicating, in blue, the hydrophilic part and in yellow the hydrophobic part of the molecule (b) Theoretical possible conformations of dequalinium chloride, i.e., stretched versus horseshoe conformation, leading to either a monolayer or a bilayer membranous structure following the process of self-assembly (c) Left panel: Monte Carlo Computer Simulations demonstrate the possible self-assembly of dequalinium chloride into vesicles (From Weissig, V., Mogel, H J., Wahab, M., and Lasch, J., Proceed Intl Symp Control Rel Bioact Mater., 25, 312, 1998.) Right panel: Electron microscopic images of vesicles (DQAsomes) made from dequalinium chloride, from left to right: Negatively stained, rotary shadowed, freeze fractured (From Weissig, V., Lasch, L., Erdos, G., Meyer, H W., Rowe, T C., and Hughes, J., Pharm Res., 15(2), 334–337, 1998 With permission.) q 2006 by Taylor & Francis Group, LLC DQAsomes as Mitochondria-Targeted Nanocarriers for Anti-Cancer Drugs 791 FIGURE 38.2 Top panel: Structure of the cyclohexyl derivative of dequalinium Bottom panel: Schematic illustration of the stabilizing effect of the cyclohexyl ring system (black circles) liposome-like aggregation of dequalinium Negatively stained samples (Figure 38.1) demonstrate that the vesicle is impervious to the stain and appears as a clear area surrounded by stain with no substructure visible Particle size measurements of DQAsomes stored at room temperature for 24 and 96 h (not shown) not show any significant changes in their size distribution in comparison to freshly made vesicles measured after one hour.84 This indicates that DQAsomes not seem to precipitate, to fuse with each other, or to aggregate in solution over a period of several days The use of dequalinium derivatives for the preparation of DQAsome-like vesicles leads to vesicles with different size distributions.89 For example, substituting the methyl group by an aliphatic ring system (Figure 38.2) confers superior vesicle forming properties to this bolaamphiphile These DQAsome-like vesicles, i.e., vesicles prepared from the cyclohexyl-derivative of DQA shown in Figure 38.2, have a very narrow size distribution of 169G50 nm and can be stored at room temperature for at least five months In contrast to vesicles made from dequalinium, bolasomes made from the cyclohexyl derivative are also stable upon dilution of the original vesicle preparation Whereas dequalinium-based bolasomes upon dilution slowly disintegrate over a period of several hours, bolasomes made from the cyclohexyl compound not show any change in size distribution following dilution It appears that bulky aliphatic residues attached to the quinolinium heterocycle favor self-association of the planar ring system It has, therefore, been speculated that the bulky group sterically prevents the free rotation of the hydrophilic head of the amphiphile around the CH2-axis (Figure 38.2, bottom panel), contributing to improved intermolecular interactions between the amphiphilic monomers 38.5 DQASOME-MEDIATED DELIVERY OF pDNA TO MITOCHONDRIA IN LIVING MAMMALIAN CELLS During efforts in developing a mitochondria-specific DNA delivery vector, it could be demonstrated that DQAsomes are able to selectively deliver plasmid DNA (pDNA) to mitochondria within living mammalian cells It has been shown, in particular, that DQAsomes stably incorporate pDNA,84 protect the pDNA from nuclease digestion, and mediate its cellular uptake most likely via non-specific endocytosis.90 Using membrane-mimicking liposomal membranes and isolated rat q 2006 by Taylor & Francis Group, LLC 792 Nanotechnology for Cancer Therapy liver mitochondria, it was shown that DQAsome/pDNA complexes become destabilized upon contact with mitochondrial membranes, but not at cell plasma membranes.91,92 The selective destabilization of DQAsomes at mitochondrial membranes may either be caused by the difference in the lipid composition between cytoplasmic and mitochondrial membranes91 or by the membrane potential-driven diffusion of individual dequalinium molecules from the DQAsome/pDNA complex into the mitochondrial matrix leading to the disintegration of the complex However, the fact that DQAsomes not lose their cargo (pDNA) during their contact with the plasma cell membrane (i.e., during their cellular uptake) but release the entrapped pDNA upon contact with mitochondria appears as a very attractive feature of DQAsomes as a mitochondria-specific drug delivery system Any encapsulated drug would potentially be released upon contact with mitochondrial membranes leading to the desired high local drug concentration in the immediate proximity to the mPTPC that is recognized as a major target for apoptotically active experimental drugs.1,93,94 Before translocating to mitochondria in response to the mitochondrial membrane potential, however, endocytosed DQAsomes have to be released from endosomes into the cytosol From studies about the intracellular fate of cationic liposome/DNA complexes (lipoplexes), it is known that cationic lipids exert a destabilizing effect on endosomal membranes, leading to the release of at least a fraction of the lipoplex from early endosomes.95–98 In agreement with these data, it was found that dequalinium-based DQAsomes also display endosomolytic activity.99 It was shown that adding DQAsomes to liposomes mimicking the lipid composition of endosomal membranes reproducibly lead to the release of liposomal encapsulated fluorescence marker.99 Direct evidence for the ability of DQAsomes to transport pDNA selectively to the site of mitochondria was provided by studying the intracellular distribution of mitochondrial leader sequence peptide -pDNA conjugates in cultured BT20 cells using confocal fluorescence microscopy.100 Figure 38.3 shows images representative of the confocal fluorescence micrographs obtained in this study The green and red channels used to generate the overlaid images in the far right column are shown separately in the preceding columns to facilitate a careful comparison of the observed staining patterns The characteristic mitochondrial staining pattern seen with the red channel is a strong indicator of mitochondrial viability in the imaged cells From the composite image obtained by overlaying the green and red channels, it can be seen that a sizeable fraction of the intracellular green fluorescence co-localized with the red mitochondrial fluorescence (depicted as white areas in Figure 38.3c) These observations indicate that, in addition to mediating the cellular uptake of the pDNA conjugate, the use of DQAsomes resulted in a definite association of an appreciable fraction of the internalized conjugate with mitochondria FIGURE 38.3 (See color insert following page 522.) Representative confocal fluorescence micrographs of BT20 cells stained with Mitotrackerw Red CMXRos (red) after exposure to fluorescein labeled linearized MLS-pDNA conjugate (green) complexed with DQAsomes; (a) red channel, (b) green channel, (c) overlay of red and green channels with white, indicating co-localization of red and green fluorescence.(From D’Souza, G G., Boddapati, S V., and Weissig, V., Mitochondrion, 5(5), 352–358, 2005 With permission.) q 2006 by Taylor & Francis Group, LLC DQAsomes as Mitochondria-Targeted Nanocarriers for Anti-Cancer Drugs 793 38.6 ENCAPSULATION OF PACLITAXEL INTO DQASOMES Paclitaxel, generally known as an anti-microtubule agent, has recently been demonstrated to trigger apoptosis by directly acting on mitochondria.54,55,58 It has been shown that clinically relevant concentrations of paclitaxel directly target mitochondria and trigger apoptosis by inducing cytochrome c (cyt c) release in a permeability transition pore (PTP)-dependent manner.54 This mechanism of action is known from other pro-apoptotic, directly on mitochondria acting agents.51 A 24-hour delay between the treatment with paclitaxel or with other PTP inducers and the release of cyt c in cell-free systems compared to intact cells has been explained by the existence of several drug targets inside the cell Making only a subset of the drug available for mitochondria.54 Likewise, other anti-tubulin agents such as vinorelbine or nocodazole could also been shown to trigger the release of cyt c via the direct interaction with mitochondria101 that subsequently resulted in apoptotic cell death The detection of tubulin as an inherent component of mitochondrial membranes able to interact with the voltage-dependent anion channel102 and thereby able to influence the mPTPC seems to make the rather surprising identification of mitochondria as a target for well-established anti-tubulin agents plausible For encapsulation of paclitaxel into DQAsomes, dequalinium chloride and paclitaxel were dissolved in methanol followed by removing the organic solvent.103 After adding buffer, the suspension was sonicated with a probe sonicator until a clear opaque solution was formed To remove any undissolved material, the sample was centrifuged for 10 at 3000 rpm The solubility of paclitaxel in water at 258C at pH 7.4 is with 0.172 mg/L (0.2 mM) extremely low, making any separation procedure of non-encapsulated paclitaxel unnecessary However, for control, a paclitaxel suspension was probe sonicated under identical conditions used for the incorporation of paclitaxel into DQAsomes but in the complete absence of dequalinium As expected, upon centrifugation, no paclitaxel was detectable in the supernatant using UV spectroscopy at 230 nm.103 Following this procedure, paclitaxel can be incorporated into DQAsomes at a molar ratio paclitaxel to dequalinium of about 0.6 In comparison to the free drug, encapsulation of paclitaxel into DQAsomes increases the drug’s solubility by a factor of about 3000 Weight distribution analysis 90% Dust 0.00% 50% 10% 200 600 1000 Size [nm] FIGURE 38.4 Paclitaxel encapsulated into DQAsomes Left panel: transmission electron microscopic image (uranyl acetate staining); middle panel: Size distribution; right panel: Cryo-electron microscopic image (From Cheng, S M., Pabba, S., Torchilin, V P., Fowle, W., Kimpfler, A., Schubert, R and Weissig, V J Drug Deliv Sci Technol., 15(1), 81–86, 2005 With permission.) q 2006 by Taylor & Francis Group, LLC 794 Nanotechnology for Cancer Therapy 0.9 Paclitaxel / DQA (molar) 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Day Day Day Day Day 10 FIGURE 38.5 Paclitaxel remains stably incorporated in DQAsomes: Molar ratio of paclitaxel to DQA in DQAsomes upon storage at 4C (From Cheng, S M., Pabba, S., Torchilin, V P., Fowle, W Kimpfler, A Schubert, R., and Weissig, V., J Drug Deliv Sci Technol., 15(1), 81–86, 2005 With permission.) Considering the known spherical character of DQAsomes, the results of an EM analysis of paclitaxel-loaded DQAsomes seem rather surprising The transmission EM image (Figure 38.4, left panel) and the cryo-EM image (Figure 38.4, right panel) of an identical sample show with a remarkable conformity worm- or rod-like structures roughly around 400 nm in length, the size of which could also be confirmed by size distribution analysis shown in Figure 38.4 (middle panel) These complexes may represent the formation of worm-like micelles as recently described for selfassembling amphiphilic block co-polymers.104 Figure 38.5 shows data about the stability of paclitaxel-containing DQAsomes upon storage Over a period of ten days, the ratio of paclitaxel to dequalinium in the DQAsome preparation remains almost constant, indicating that paclitaxel remains stably incorporated in DQAsomes However, from a gradual decrease of the total concentration of paclitaxel and of dequalinium over several days and to the same extent (by approximately 20–30% after four days, data not shown) can be concluded that paclitaxel-loaded DQAsomes tend to form larger aggregates that are removable by centrifugation This tendency to form aggregates is well known from artificial phospholipids vesicles (liposomes) and is generally reversible by shaking or slightly vortexing the preparation The stability of paclitaxel-loaded DQAsomes is under physiological conditions in comparison to their shelf-life stability significantly reduced In general, upon transferring paclitaxel-loaded DQAsomes either into serum-free cell medium (DMEM) at 378C or into complete serum, they form large aggregates leading to precipitation However, this process of aggregation seems to be kinetically controlled, giving at least a portion of monomeric paclitaxel-loaded DQAsomes sufficient time to reach their target The tendency to form aggregates under physiological conditions is common to all cationic carrier systems Nevertheless, it should be mentioned that cationic lipoplexes and polyplexes are being tested in numerous gene therapeutic clinical trials Also, cationic colloidal carriers (Catioms) are currently being successfully developed for systemic drug administrations.105–107 38.7 DQASOMAL-ENCAPSULATED PACLITAXEL TRIGGERS APOPTOSIS IN VITRO To test if DQAsomal-encapsulated paclitaxel triggers apoptosis at paclitaxel concentrations where the free drug does not have a significant cytotoxic effect, human colo 205 colon cancer cells were incubated with the free drug, with empty DQAsomes, with a mixture of empty DQAsomes and the free drug, and with the DQAsomal-encapsulated drug Following the staining of the treated cells q 2006 by Taylor & Francis Group, LLC DQAsomes as Mitochondria-Targeted Nanocarriers for Anti-Cancer Drugs 795 25 % of apoptosis cells 20 15 10 Empty DQAsomes mixture DQAsomes + free paclitaxel DQAsomal encapsulated paclitaxel FIGURE 38.6 Human colo 25 colon cancer cells were treated in triplicates with empty DQAsomes (20 nM DQA) with a mixture of empty DQAsomes (20 nM) and free paclitaxel (10 nM) and DQAsomal-encapsulated paclitaxel (20 nM DQA/10 nM paclitaxel) In each case, approximately 400 cells were counted (From Chen, S M and Weissig, V., 2006, manuscript in preparation) with the DNA-binding fluorophore Hoechst 33258, apoptotic nuclei showing the typical apoptotic condensation and fragmentation of chromatin were counted and expressed as percent of the total number of nuclei Figure 38.6 shows that under identical incubation conditions, 10 nM paclitaxelencapsulated in DQAsomes more than doubles the number of apoptotic nuclei in comparison to the control whee cells were treated with a mixture of empty DQAsomes and 10 nM free paclitaxel Likewise, a DNA ladder caused by DNA fragmentation typical for apoptosis could be detected upon incubation of colon cancer cells with 10 nM DQAsomal-encapsulated paclitaxel but not upon incubation with the free drug either alone or in mixture with empty DQAsomes (Figure 38.7) Incubating the cells for the same period of time, the amount of free paclitaxel had to be increased at least 5-fold over the amount of DQAsomal-encapsulated drug in order to generate a DNA ladder (not shown) FIGURE 38.7 Human colo 25 colon cancer cells were incubated for 30 h with buffer (lane 2), 20 nM empty DQAsomes (lane 3), 10 nM free paclitaxel (lane 4), a mixture of 20 nM empty DQAsomes and 10 nM free paclitaxel (lane 5), and 20 nM DQAsomes with 10 nM encapsulated paclitaxel (lane 6) White arrow heads indicate the apoptotic DNA ladder (Chen, S M and Weissig, V., 2006, manuscript in preparation) q 2006 by Taylor & Francis Group, LLC 796 Nanotechnology for Cancer Therapy Considering that paclitaxel, generally known as an anti-microtubule agent, has recently been demonstrated to trigger apoptosis by directly acting on mitochondria,54,55,58 it can be concluded from the data shown in Figure 38.6 and Figure 38.7 that encapsulating paclitaxel into a mitochondria-specific drug delivery system appears to increase the sub-cellular, i.e., mitochondrial, bioavailability of the drug 38.8 TUMOR GROWTH INHIBITION STUDY WITH PACLITAXEL-LOADED DQASOMES IN VIVO Paclitaxel-loaded DQAsomes were tested for their ability to inhibit the growth of human colon cancer cells in nude mice.103 COLO-205 cells were inoculated s.c into the left flank of nude mice that all formed palpable tumors within seven days after cell injection For controls with free paclitaxel, the drug was suspended in 100% DMSO at 20 mM, stored at 48C, and immediately before use, diluted in warm medium In all controls, the dose of free paclitaxel and empty DQAsomes, respectively, was adjusted according to the dose of paclitaxel and dequalinium given in the paclitaxel-loaded DQAsome sample Because of the lack of any inhibitory effect on tumor growth, the dose was tripled after 1.5 weeks Figure 38.8 shows that at concentrations where free paclitaxel and empty DQAsomes not show any impact on tumor growth, paclitaxel-loaded DQAsomes (with paclitaxel and dequalinium concentrations identical to controls) seem to inhibit the tumor growth by about 50% Correspondingly, the average tumor weight in the treatment group after sacrificing the animals after 26 days is approximately half of that in all controls (Figure 38.9) Although this result seems to suggest that DQAsomes might be able to increase the therapeutic potential of paclitaxel, the preliminary character of this first in vivo study should be emphasized 1400 Average tumor volume [mm3] 1200 Hepes buffer 1000 Free paclitaxel 800 Empty DQAsomes 600 Paclitaxel-loaded DQAsomes 400 200 0 10 20 Day after tumor implantation 30 FIGURE 38.8 Tumor growth inhibition study in nude mice implanted with human colon cancer cells The mean tumor volume from each group was blotted against the number of days Each group involved eight animals For clarity, error bars were omitted Note that after 1.5 weeks, the dose, normalized for paclitaxel, was tripled in all treatment groups (From Cheng, S M., Pabba, S., Torchilin, V P., Fowle, W., Kimpfler, A., Schubert, R., and Weissig, V., J Drug Deliv Sci Technol., 15(1), 81–86, 2005 With permission.) q 2006 by Taylor & Francis Group, LLC DQAsomes as Mitochondria-Targeted Nanocarriers for Anti-Cancer Drugs 797 2.000 Average tumor weight [g] 1.600 1.200 0.800 0.400 0.000 Hepes buffer Free paclitaxel Empty Paclitaxel-Loaded DQAsomes DQAsomes FIGURE 38.9 Average tumor weight (n Z 8) at time of sacrifice of nude mice implanted with human colon cancer cells For treatment group (paclitaxel-loaded DQAsomes) versus all three control groups combined PZ 0.054 (parametric Student t-test) (From Cheng, S M., Pabba, S., Torchilin, V P., Fowle, W., Kimpfler, A., Schubert, R., and Weissig, V., J Drug Deliv Sci Technol., 15(1), 81–86, 2005 With permission.) 38.9 CONCLUDING REMARKS DQAsomes and DQAsome-like vesicles have been established as the first mitochondria-specific cationic drug 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To advance nanotechnology research for cancer prevention, diagnosis, and treatment, the United States National Cancer Institute (NCI) established the Alliance for Nanotechnology in Cancer in September... $144.3 million in the next five years (for details, visit http://nano .cancer. gov) Among the approaches for exploiting developments in nanotechnology for cancer molecular medicine, nanoparticles... neutron capture therapy, and the discussion on nanotechnology characterization for cancer therapy, as well as guidance from the U.S Food and Drug Administration on approval of nanotechnology products