Tumor-Targeted Drug Delivery Tania Betancourt, Amber Doiron, and Lisa Brannon-Peppas CONTENTS 12.1 Introduction ........................................................................................................................215 12.2 Targeting to Cancer............................................................................................................216 12.3 Passive Targeting and the EPR Effect ...............................................................................217 12.4 Targeting to Angiogenesis .................................................................................................218 12.4.1 Targeting Using Vascular Endothelial Growth Factor Receptors...................... 218 12.4.2 Targeting Using Integrins ................................................................................... 218 12.4.2.1 Integrins as Targets for Imaging.........................................................220 12.5 Targeting Using Folate Receptors .....................................................................................220 12.5.1 Antibodies and Folate Receptors ........................................................................ 221 12.5.2 Folate-Targeted Nanoparticles for Gene Delivery ............................................. 222 12.6 Approaches for Cancer Targeting to Specic Cancer Types ............................................223 12.6.1 Prostate Cancer.................................................................................................... 224 12.7 Targeted Nanoparticles and Imaging of Cancer ................................................................225 12.8 Other Targets for Cancer ...................................................................................................225 12.9 Avidin and Biotin Targeting ..............................................................................................226 12.10 Conclusions ........................................................................................................................226 References.....................................................................................................................................226 12.1 INTRODUCTION Cancer is a disease that affects millions of people across the globe every year. The World Health Organization estimated that more than 10 million people developed a malignant tumor and more than 6.5 million people died from this disease during the year 2000. 1 In the United States, cancer is the second cause of deaths from disease after heart disease, accounting for more than half a million deaths every year. According to the American Cancer Society (ACS) cancer statistics, the overall cost for cancer for the United States in 2004 was $189.8 billion: $69.4 billion for direct medical costs, $16.9 billion for indirect morbidity costs, and $103.5 billion for indirect mortality costs. 2 Furthermore, while mortality rates of other major chronic diseases, such as heart and cerebro- vascular disease, decreased signicantly in the past half-century, cancer mortality rates have remained approximately constant. 2 This is a troubling fact because it suggests that recent detection and treatment options have not been able to improve mortality rates substantially. 215 q 2006 by Taylor & Francis Group, LLC Research in the past decade has focused on using unique characteristics of cancer cells and the vasculature surrounding those cells to deliver imaging agents, chemotherapeutic drugs, gene therapy, and other active agents directly and selectively to cancerous tissues. Many of these new formulations (described elsewhere in this volume) are liposomes, prodrugs, polymer conjugates, micelles, and dendritic systems. This chapter will concentrate on polymeric nanoparticles that have been studied as targeted systems for treatment and detection of cancer. 12.2 TARGETING TO CANCER Nanoparticles may be targeted to the growing vasculature serving the growing cancer or to the cancer cells themselves. Targeted delivery utilizes unique phenotypic features of diseased tissues and cells in order to concentrate the drug at the location where it is needed. Targeted delivery can be divided into passive and active targeting. Passive targeting tries to minimize nonspecic interactions between the drug carrier and nontarget sites in the body by detailing the physiochem- ical properties of the aberrant tissue such as size, morphology, hydrophilicity, and surface charge. 3 When targeting tumor tissue, the enhanced permeability and retention effect (EPR) is an example of passive targeting approach; it allows passage of drug carriers ranging in size from 10 to 500 nm through the highly permeable blood vessels that supply growing tumors and leads to entrapment of large molecules as a result of decient lymphatic drainage. 35 In fact, it has been reported that the intra-cellular openings in vascular endothelium of tumor blood vessels can be up to 2 mm in diameter and that the vessel leakiness in tumor vasculature can be up to an order of magnitude higher than that of normal blood vessels. 5 Active targeting utilizes biologically specic interactions including antigenantibody and ligandreceptor binding and may seek drug uptake by receptor- mediated endocytosis through association of the drug or drug carrier with such antigen or ligand. 3 Receptor-mediated endocytosis commonly occurs through clathrin-coated vesicles and is carried out in mammalian cells continuously for the uptake of nutrients and for modulation of signal transduction through the up- or down-regulation of signaling receptors. 6 Targeted delivery avoids the need for high systemic drug levels for the drug to be effective and consequently offers a more economic alternative for treatment. Not only is it useful for therapeutic purposes; it is also benecial in diagnosis. Recent research has pointed to its ability to concentrate imaging or contrast agents for the detection of malignancies and for monitoring the effects of therapeutic agents. 7,8 To date, most systems for targeted delivery have utilized drug conjugates, liposomes or micelles. 911 Targeting of particulate systems has focused more often on passive targeting based on size than on active targeting. But systems that combine both methods, starting with passive targeting through EPR and enhancing the targeting through specic interactions are beginning to show great promise. While it is challenging to deliver a drug or imaging agent-containing nanoparticle directly and selectively to a cancerous cell or tissue, the additional challenges of then having that particle and/or its contents being transported into the targeted cell have often been overlooked. Couvreur presented some of these challenges at the 11th International Symposium on Recent Advances in Drug Delivery Systems and also summarized that presentation in a recent publication. 12 The tumor resistance can be due to the deliverance of nanoparticles to the tumor as well as to resistance to the active agent being delivered. In this article, many different pathways are described and the enhancement of drug delivery due to the presence of nanoparticles, especially polycyanoacrylate nanoparticles, is evaluated and summarized. The enhancement of drug permeability into cells due to interactions with biodegradation byproducts as well as the effect of nanoparticle surface charge is discussed. In this chapter, we will rst review recent work on targeting to the two most promising targets for cancer: Angiogenesis and folate receptors. We will then describe other potential targets for cancer imaging and therapy with nanoparticles, including antibodies strategies using biotin. Nanotechnology for Cancer Therapy 216 q 2006 by Taylor & Francis Group, LLC 12.3 PASSIVE TARGETING AND THE EPR EFFECT Although active targeting may be achieved through targeting to folate receptors, angiogenesis, or targets more specic to the cancer being treated, some enhancement of treatment to cancers on account of the EPR effect cannot be ignored and should be utilized until more specic targeting systems can be developed. Preparation of nanoparticle systems that can avoid uptake by the reticuloendothelial system (RES) is essential, and such particles are often referred to as stealth nanoparticles. The most effective polymer used as a coating on nanoparticles to avoid detection by the RES is poly(ethylene glycol). 13 The latter can be achieved by adsorption of the PEG-containing polymer onto the surface of nanoparticles, direct conjugation of the PEG to the nanoparticles, or inclusion of PEG in the polymeric backbone that makes up the nanoparticles. The PEG itself may then also be modied to give targeting capabilities and to avoid uptake by the RES. The PEG works to mask the surface of the nanoparticles by reducing the plasma and protein adsorption to the particles, reducing the complement activation and hence the recognition of the PEG as a foreign substance in the blood stream. The longer circulation time afforded PEGylated nanoparticles allows them time to be targeted, whether passively or actively, to cancerous tissues. Extended circulation time and enhanced tumor targeting were seen for poly(ethylene oxide)- modied poly(epsilon-caprolactone) nanoparticles in mice with tumors of MDA-MB-231, a human breast carcinoma. 14 These particles were loaded with [ 3 H]-tamoxifen. The amount of labeled tamoxifen at the tumor site, 6 h after injection, for those particles with PEO modication was at least twice that of particles without modication and four times that of labeled tamoxifen injection. The amount of labeled tamoxifen found in the blood stream at 6 h after injection was also at least twice that of the injection or unmodied nanoparticle formulations. The stability and circulation of PLGAmPEG nanoparticles containing cisplatin was investi- gated. It was found that while the mPEG content affected the drug release rate, the drug loading level had no effect on the drug release rate for in vitro studies. 15 The release was more that 60% completed within the rst 12 h in all cases. Data for blood levels was only presented for 3 h, so it is hard to draw any valid conclusions from this information. Nanoparticles of PLA and PEGPPGPEG were prepared containing irinotecan, a prodrug of an analogue of camptothecin. 16 Although little characterization beyond the average particles size (231 nm) was presented here and no in vitro studies were described, the in vivo studies are quite interesting. In this study, there was a modest increase in survival time in mice with M5076 tumors (early liver metastatic stage) after a single injection and more pronounced increases in survival time after either two or three repeat injections. It is noteworthy that the greatest survival times (20% survival at 45 days at the end of the study) were seen with two injections at days three and ve after the implantation of the tumor. Polycyanoacrylate nanoparticles have long been studied by Couvreur and collaborators as biodegradable nanoparticles for a variety of applications and therapies which are not limited to treatment of cancer. 17 A recent work describes the effectiveness of these nanoparticles as delivery systems for brain tumor targeting. Here they studied uncoated and PEG-coated nanoparticles and found that both types of particles showed accumulation in a well-established 9L gliosarcoma in rat studies. The PEG-coated particles showed the highest accumulation, with a tumor-to-brain ratio of 11. Poly(butyl cyanoacrylate) nanoparticles containing doxorubicin were prepared with no surface modications; the in vivo distribution of 99m Tc labeled nanoparticles was evaluated in mice inocu- lated with Daltons lymphoma tumor cells. 18 The nanoparticles were administered by subcutaneous injection and the concentration in a number of organs was followed for 48 hours and compared with that for 99m Tc labeled doxorubicin alone. When compared with the amount of doxorubicin alone, the amount of radioactivity in the tumor was higher at all times tested, with a 13-fold increase seen at 48 h. Polymeric Nanoparticles for Tumor-Targeted Drug Delivery 217 q 2006 by Taylor & Francis Group, LLC While the majority of work on targeted nanoparticles has been carried out with polylactides, polyglycolides and polycyanoacrylates, those are not the only materials that can be used in nano- particles. Some recent work with radiolabeled gelatin nanoparticles modied with poly(ethylene glycol) showed that adding the PEG to the surface of these nanoparticles increased the circulation time in mice with double the amount of PEG-modied nanoparticles in the blood stream 3 h after injection compared to the amount of control nanoparticles. 19 In addition, there was a four-fold increase in the amount of PEG-modied gelatin nanoparticles found in the tumor 4 h after injection and later when compared to the number of nonmodied gelatin nanoparticles. 12.4 TARGETING TO ANGIOGENESIS A recently explored and potentially promising target of cancer drug and gene nanoparticle therapy is tumor angiogenesis. It is now well established that tumor growth is dependent on new capillary inltration from surrounding, preexisting vasculature. 20,21 This is an important control point in cancer as much research has proven that tumors cannot effectively grow past a small size or metastasize without blood supply. 2224 Except for the cases of menstruation, wound healing, and tissue regeneration, capillaries do not increase in size or number under normal physiological conditions. Tumor growth is an exception to this physiological rule. Tumors are typically unable to affect angiogenesis when they are small and surrounded by healthy tissue. However, at the point in growth where nutrients, oxygen, and growth factors can no longer reach the cancer cells, blood ow is required to allow further growth of the tumor. After what is occasionally a substantial time period, the tumor may abruptly induce angiogenesis into the tissue. 23 Because understanding this step in the progression of cancer is thought to be of great importance, much research has been and continues to be focused on pinpointing the progression of cancer and on targeting the event for therapy. Much research has focused on targeted therapy of either chemotherapeutic agents to sites of tumor angiogenesis or of angiogenesis-inhibiting drugs to tumors with the goal of directly combat- ting the proliferation of newly forming capillaries in the tumor. Angiogenesis is a complex, multi- component process that involves many cell types, cytokines, growth factors and receptors, proteases, and adhesion molecules. 25 As a result, there are many potential targets for anti-angiogenic or chemotherapeutic therapy. Some recent advances in targeting approaches for nanoparticle drug delivery are discussed below. 12.4.1 TARGETING USING VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTORS Vascular endothelial growth factor (VEGF) is particularly important in the process of angiogenesis and has been shown to greatly affect tumor growth in animal models. 2628 The VEGF receptors have been used as a means to target the vascular bed in many instances. VEGF receptor-2 (VEGFR-2) has recently been used to target nanoparticles to tumor vascular beds by Li et al in mice with K1735-M2 tumors. 29 A succinyl-dextran-polymerized nanoparticle conjugated to rat anti-mouse VEGFR-2 antibody and radioisotope 90 Y caused a signicant tumor growth delay compared to conventional radiolabeled antibody and other controls. Additionally, anti-CD31 staining showed a decreased vessel density and damage to tumor vessels after treatment with the anti-VEGFR- 2- 90 Y nanoparticles. 12.4.2 TARGETING USING INTEGRINS The integrins represent another important cell surface molecule group for angiogenesis targeting because some integrins, such as a v b 3 and a v b 5 , are upregulated on the endothelial cell surface of neovasculature. 30,31 The a v b 3 integrin is expressed on numerous tumor cell types; it is highly expressed on neovascular endothelial cells. Hood and collaborators used 40-nm diameter Nanotechnology for Cancer Therapy 218 q 2006 by Taylor & Francis Group, LLC cationic-lipid-based nanoparticles coupled to an organic a v b 3 ligand that was shown to be specic for a v b 3 in cell studies. These nanoparticles, which contained a luciferase reporter plasmid, were injected into mice with the a v b 3 -negative cell line M21-L. The nanoparticles targeted to the neovasculature within the tumor (but not to the tumor cells themselves) with no expression else- where in the mice as detected by luciferase expression. In order to test therapeutic efcacy, NPs were conjugated to a mutant Raf gene that blocks angiogenesis. Systemic injection in the M21-L tumor-expressing mice rapidly induced apoptosis of endothelial cells within the tumor. Tumor regression was seen within 10 days. 32 In the same study with VEGFR-2 targeting discussed above, Li and collaborators also demon- strated targeting of the radioisotope 90 Y using nanoparticles targeted to the integrin a v b 3 with a small molecule integrin agonist. 29 In mice with K1735-M2 tumors, a v b 3 -targeted 90 Y-nanoparticles signicantly delayed tumor growth compared to untreated tumors. TUNEL staining of tumor sections showed widespread apoptosis in tumors treated with these targeted nanoparticles. The authors have postulated that this targeted nanoparticle radiotherapy has the potential to be used to treat a variety of solid tumors. They have also postulated that the use of nanoparticles increases efcacy due to the high payload delivered by the carriers. Additionally, PEGylated polyethyleneimine (PEI) nanoplexes with a cyclic disulde bond constrained ArgGlyAsp (RGD) peptide ligand at the distal end have been used to target integrin-expressing tumor neovasculature to deliver siRNA. Integrins are receptors for extracellular matrix components that contain a tripeptide RGD sequence. Therefore, RGD containing peptide sequences can target to cell surface integrins that are upregulated on neovasculature. The siRNA used inhibited angiogenesis by inhibiting VEGFR-2 expression. Intravenous injection of nanopar- ticles into nude mice with N2A tumors showed tumor uptake of the siRNA, inhibition of protein synthesis in the tumor, and inhibition of angiogenesis and tumor growth. 33 This study demonstrates tumor selective delivery through both the targeting ligand and gene pathway by using siRNA. In another a v b 3 targeting approach using an RGD peptide, Kopelman has created multifunc- tional nanoparticles of 3060 nm for the treatment of gliomas. 34 The nanoparticles are able to kill cancer cells by bombarding them with externally released reactive oxygen species created by photodynamic agents activated by laser light. The particles also contain superparamagnetic iron oxide and enhance imaging by magnetic resonance. The photodynamic sensitizer and MRI contrast agents are entrapped within a polyacrylamide core, the surface of which is coated with PEG chains and targeting RGD moieties. The particles containing photodynamic agents were shown to produce sufcient singlet oxygen to kill cells in vitro. Additionally, these nanoplatforms were injected into an in vivo rat intracerebral 9L tumor model, and diffusion MRI was performed at various times to evaluate the tumor diffusion, tumor growth, and tumor load. The gliomas treated with the nano- particles and irradiated with laser light caused regional necrosis and signicant shrinkage of tumor mass, a shrinkage that lasted for 12 days. The authors postulate that the light activated release of reactive oxygen from photosensitizer-containing nanoparticles is a viable approach for brain tumor treatment. Also, the incorporation of MRI contrast agents allows for monitoring of treatment and tumor progression in vivo. Carbohydrate based nanoparticles have also been used to target drugs to neovasculature via an a v b 3 /cyclic RGD peptide interaction. Inulin multi-methacrylate formed the core of the nanoparti- cles and was attached to the RGD targeting moiety with a PEG linker. Doxorubicin was loaded in the nanoparticles via covalent and noncovalent linkages. The pharmacokinetics and biodistribution of the doxorubicin loaded nanoparticles were studied over ve days in female Balb/cJ mice with metastatic mammary tumor clone-66. A bi-exponential x with a terminal half-life of 5.99 h was observed; decreasing drug concentrations with time in the heart, lungs, kidney, and plasma was also observed. Conversely, increasing drug accumulation was observed in the liver, spleen, and in the tumor where there was also the presence of high levels of doxorubicin metabolite. The presence of the high metabolite levels in the tumorsuggests nothing more than tumor-specic nanoparticle degradation and release of drug. 35 Polymeric Nanoparticles for Tumor-Targeted Drug Delivery 219 q 2006 by Taylor & Francis Group, LLC 12.4.2.1 Integrins as Targets for Imaging In an effort to image angiogenesis, Mulder and collaborators have created MR-detectable and uorescent liposomes that are targeted to a v b 3 integrin on neovasculature. 36 The MR contrast agent Gd-DTPA-bis(stearylamide) was incorporated into PEGylated liposomes covalently coupled to RGD peptide. It was observed that the liposomes effectively brought about an increase in signal on T 1 -weighted images. Injection of these lipidic nanoparticles in nude mice with subcu- taneously implanted LS174 human colon adenocarcinoma led to the ability to specically image the vascular endothelial cells in the tumor. Ex vivo uorescence conrmed that RGD liposomes specically interacted with tumor endothelium associated with neovasculature. In a study reported by Lanza, paramagnetic molecular imaging of angiogenesis was accom- plished in vivo with a v b 3 -targeted lipid encapsulated peruorocarbon nanoparticles of about 250 nm. 37 A Vx-2 carcinoma tumor in New Zealand rabbits was imaged with a 1.5-T MRI system with either nontargeted or a v b 3 -targeted nanoparticles. An eight-fold greater contrast enhancement was achieved with the targeted nanoparticles. In the second part of the study, pacli- taxel loaded nanoparticles were targeted to tissue factor (TF) proteins on vascular smooth muscle cells with a specic TF antibody. The TF-targeted paclitaxel particles inhibited cell proliferation while delivery of nanoparticles to targeted cells was conrmed with uorine spectroscopy. 12.5 TARGETING USING FOLATE RECEPTORS During the past few decades, there has been great interest in the utilization of folate receptors for the targeted delivery of therapeutic and imaging agents. A number of delivery systems have been utilized for this purpose, including drug conjugates, 3840 liposomes, 41 micelles, 42 viral vectors, 43 and nanoparticles. Folate (vitamin B-9) is essential for the synthesis of nucleotides and amino acids. Two main groups of molecules are responsible for transport of folate molecules in vivo. Most cells in the body express a folate anion transporter with micro-molar afnities for folates that participates in the transport of coenzyme 5-methyltetrahydrofolate, the physiologic circulating reduced form of folate. Folate receptors (FR), by contrast, are members of the glycosylphosphatidylinositol (GPI)-linked membrane glycoprotein family and have high afnity for folic acid, an oxidized form of folate, and 5-methyltetrahydrofolate, with binding afnities being in the nanomolar range (K D !1!10 K9 M) for the a isoform of FR. 4446 It has been observed with few exceptions that only cells involved in pathologic conditions, including cancer cells, express the high afnity folate receptors. These receptors are able to transport folic acid, folate-bound molecules, and even particles through receptor-mediated endocytosis. 47,48 FR are known to be overexpressed in various epithelial cancer cells, such as those of ovarian, mammary gland, colon, lung, prostate, and brain epithelial cancers, and in leukemic cells. 4961 Folate receptor overexpression has been correlated to poor prognosis. In addition, metastasized cancer cells have been found to overexpress the folate receptor to a larger degree than localized tumor cells. 62 This nding is of great importance. The only nonpathological tissues where FR is expressed are choroid plexus, placenta, lungs, thyroid, and kidney. 46,63 FR expression is limited to the apical (luminal) side of polarized epithelial cells, except for the cells of the proximal tubules in the kidney. As a consequence, FR is practically inaccessible to blood-borne folate-linked systems. 44,45 These characteristics make folate receptors very advantageous for targeted delivery of nanoparticles with high payloads of therapeutic agents, imaging agents, and even genes for the treatment, detection, and monitoring of cancer. What is more, the macromolecular size of nanoparticles will prevent gromerular ltration and the consequent exposure of kidney tissue to folate-targeted nanoparticles. Nanotechnology for Cancer Therapy 220 q 2006 by Taylor & Francis Group, LLC 12.5.1 ANTIBODIES AND FOLATE RECEPTORS The most common targeting moieties utilized for FR targeting include monoclonal antibodies to FR and folic acid. 46 To date, monoclonal antibodies to FR have not been utilized for targeted delivery of nanoparticles although successful experiences have been reported for radiopharmaceuticals. 64 The benets of targeting with folic acid are many: it is small in size, has high stability, lacks immunogenicity, and costs very little. 62 Folic acid conjugation at its g-carboxyl group is necessary for maintaining binding afnity to FR. The structure of folic acid is shown in Figure 12.1. Cellular uptake of drug carriers bound to folic acid is believed to be mediated by receptor mediated endocytosis although this process is not currently completely understood. A number of hypotheses have been proposed for this process, including clathrin and caveolar pathways. 63 It has been shown that folate-bound molecules are able to escape endosomes after receptor-mediated endocytosis because the process of endosomal acidication results in a conformational change in the receptor that facilitates folate ligand release. Consequently, folate-bound molecules provides a great oppor- tunity for the delivery of pH-sensitive biopharmaceuticals. 45,62 In most drug delivery systems investigated thus far, folic acid is incorporated to the drug delivery system through conjugation to a poly(ethylene glycol) (PEG) spacer utilizing well- known dicyclohexylcarbodiimide/N-hydroxysuccinimide (DCC/NHS) mediated chemistry. Such design aims to minimize steric hindrance for optimal folate recognition. This conjugation tech- nique, however, can activate both the g- and the a-carboxylic acids of folic acid. It should be said, though, that the g group is more reactive and is responsible for most of the linkages. Numerous attempts at nanoparticle targeting to the folate receptor have been reported. Many groups have formulated nanospheres of amphiphilic block copolymers including PEG. Park and collaborators reported in vitro results of the preparation and evaluation of methoxy poly(ethylene glycol) (PEG)-poly(3-caprolactone) (PCL) block copolymer nanospheres loaded with paclitaxel in which folic acid was conjugated to a modied amino-terminated PCL with a carbodiimide- mediated reaction. 65 Dialysis was used to create these nanospheres that ranged in diameter from 50 to 120 nm, depending on the ratio of the block copolymers. Paclitaxel loading efciencies of up to 55% were reported with this system, thus signicantly increasing the effective solubility of this agent in aqueous systems like the body. Because the folate moiety was conjugated to the hydro- phobic end of the block copolymer, it is expected that upon nanosphere formation it will be localized in the inner core of the particles. However, XPS characterization demonstrated the presence of nitrogen-containing molecules at the surface which could only be attributed to the folate linker. Although the targeting effectiveness of these particles was not determined, cyto- toxicity studies revealed that encapsulation of paclitaxel into the nanospheres reduced its HO N N N N NH 2 NH NH O O HO O OH FIGURE 12.1 Chemical structure of folic acid. Conjugation to folic acid for folate targeting is commonly done at the g-carboxyl group. Polymeric Nanoparticles for Tumor-Targeted Drug Delivery 221 q 2006 by Taylor & Francis Group, LLC cytotoxicity. Consequently, encapsulation offered a safe alternative to direct administration of this chemotherapeutic agent. Further studies should evaluate whether conjugation of folic acid to hydrophobic end of the block copolymer results in sufcient surface availability of this targeting agent. Another approach to folate targeting of nanoparticles has been the modication of surface properties of polymeric nanoparticles with polymer conjugates including the targeting agent. For example, Kim reported on the modication of anionic poly(lactic-co-glycolic acid) (PLGA) nano- particles prepared by emulsication and solvent evaporation with a cationic poly(L-lysine)- poly(ethylene glycol)-folate (PLLPEGFOL) copolymer. 66 In this design, the polycation PLL block attaches to the PLGA nanoparticle through ionic interactions. Surface coating is achieved by simple incubation in an aqueous solution containing the PLLPEGFOL copolymer. The PEG- folate end of the copolymer is oriented toward the outer aqueous phase for better interaction between folate and the targeted cell membrane receptor. XPS characterization demonstrated the presence of nitrogen from folic acid on the surface of the coated nanoparticles. In vitro cellular studies with FITC-labeled nanoparticles revealed an increase in uptake of coated nanoparticles with increased conjugate-to-nanoparticle ratio in KB cells. Since a decrease in uptake was seen upon addition of free folic acid in the medium, the transport of nanoparticles into the cells was attributed to endocytosis mediated by the folate receptor. Dendritic polymer systems of polyamidoamine (PAMAM) with folic acid as the targeting agents and drug and imaging agent (methotrexate or tritium and uorescein or 6-carboxytetramethylrho- damine) were prepared and tested in mouse models. 67 It was found that targeted systems, as opposed to nontargeted systems, slowed the rate of tumor growth and even showed a complete cure in one mouse. This study was conducted with twice-weekly tail vein injections. The biodistribution studies showed that, for a single targeted injection of nanoparticles, a very high amount of the nanoparticles accumulated within the tumor by day 1; this level remained high through at least day 4. Nanometric particles prepared from drug-polymer conjugates have also been reported. In one notable study, Yoo and collaborators reported on the formulation of doxorubicin-PEG-folate nano- aggregates encapsulating additional doxorubicin in their core. 11 These aggregates are formed spontaneously when an organic phase containing the copolymer and solubilized doxorubicin is dispersed into an aqueous phase containing triethylamine. The basic aqueous environment results in deprotonation of doxorubicin, causing it to form aggregates with the hydrophobic copolymer. The average aggregate diameter was approximately 200 nm. In vitro cellular studies showed increased uptake of the nano aggregates in cells expressing the folate receptor when folic acid was absent from cell media and increased cytotoxicity (anti-tumor efcacy) of the aggregates compared to the free drug in cells expressing the FR. In vivo studies in mouse xenografts (KB cells) showed that the nanoaggregates had superior therapeutic efcacy than both doxorubicin aggregates without the folic acid ligand and free doxorubicin in solution. Nanoparticles of temperature-responsive hydrogels conjugated to folic acid have also been studied with the purpose of delivering chemotherapeutic agents. Nayak reported on poly (N-isopropylacrylamide) (pNIPAM) nanoparticles that exhibit lower critical solution temperature (LCST) behavior. 68 Fluorescent agents were included in the core of these nanoparticles to facilitate with tracking; amine comonomers were incorporated into the other pNIPAM shell for conjugation to folic acid. These nanoparticles swell when their temperature falls under their LCST. Indeed, the nanoparticle size increased from w50 nm at 378C to w135 nm at 258C. In vitro cell uptake studies showed a 10-fold increase in the intake of nanoparticles conjugated to folic acid compared to those without the targeting agent in KB cells (FRC). 12.5.2 FOLATE-TARGETED NANOPARTICLES FOR GENE DELIVERY The use of folate-targeted nanoparticles for gene delivery has also been studied. In gene delivery, tissue targeting is very important for the efcacy and safety of treatment. To date, the transfection Nanotechnology for Cancer Therapy 222 q 2006 by Taylor & Francis Group, LLC efciency offered by synthetic gene delivery systems is very low compared to that of viral vectors. This is also true for nanoparticle-based systems. Extensive research is being done in the area to improve transfection efciencies by designing better delivery systems with improved targeting ability, DNA stabilization, and cellular interaction. Mansouri reported on the formulation of nanoparticles through the complex coacervation of chitosan-folic acid conjugates with DNA. 69 Chitosan is a biocompatible polycation that joins to form a complex with DNA through electrostatic interactions and provides protection against nuclease degradation. Complex coacervation is an optimal preparation technique for the encapsu- lation of DNA because it avoids the use of organic solvents and high-energy ultrasonication. Results revealed that the integrity of plasmid DNA in the particles was maintained and that conjugation of chitosan to folic acid did not interfere with the electrostatic interaction between chitosan and DNA. As expected, the ratio of chitosan to DNA, and consequent charge ratio, had an effect on particle size and zeta potential. Nanoparticles smaller than 200 nm were obtained with a chitosan amino group to DNA phosphate group ratio of more than 2. The use of these nanoparticles consequently offers a promising alternative for nonviral gene therapy for the treatment of cancer and other diseases in which folate receptors are overexpressed. Nanoparticles with a poly(L-lactic acid) (PLL) core and a polyethyleneimide (PEI) surface conjugated to folate were utilized for delivery of plasmid DNA. 70 Folic acid was conjugated to the N-terminal amino group of PEI. PEI is a polycation that has been used in the past for DNA condensation and delivery because it protects DNA from degradation through an endosomal escape mechanism. Here nanoparticles were prepared through the self-assembly of the amphiphilic folate- PEIPLL copolymer with DNA in an aqueous medium. Nanoparticles of approximately 100 150 nm in diameter and spherical shape were produced. In vitro luciferase transfection studies revealed that this system actually resulted in lower luciferase expression than PEIDNA complexes. In a separate report, folate-polyethyleneglycoldistearoylphophatidylethanolamine conjugate (f-PEGDSPE), 3([N-(N 0 ,N 0 -dimethylaminoethane)-carbamoyl] cholesterol, and Tween 80 were used to complex with DNA into cationic nanoparticles of 100200 nm in diameter with a modied ethanol injection method. 71 The formulation was carried out by dissolving the lipids in ethanol and then removing the solvent through evaporation in the presence of water. The folate moiety, which is conjugated to the PEG end of one of the lipid conjugates, naturally localizes at the surface of the nanoparticles because of PEG migration toward the water phase. Tween 80, a nonionic surfactant, and PEG were incorporated with the purpose of improving the in vivo stability of the cationic nanoparticles through steric hindrance. The size of the nanoparticles with higher PEG content was maintained in the presence of serum. This suggests that these nanoparticles are better able to maintain their structural integrity in the presence of anionic competitors present in blood. Folate targeting enhanced association and transfection efcacy of nanoparticles complexed with a luci- ferase-encoding plasmid on FR(C) KB cells. The association and efcacy were reduced when folic acid was present in the medium, thus revealing the involvement of the folate receptor in the transport of the plasmid DNA into the cells. A possible limitation of folate targeting is the noted variability of FR expression levels not only between patients, but also within a single tumor. 44 In addition, it has been reported that expression of FR in cancerous cell lines is not representative of those one sees in vivo. 44 Consequently, screening protocols for FR expression will need to be utilized clinically in order to determine if folate-targeted therapies are appropriate. 12.6 APPROACHES FOR CANCER TARGETING TO SPECIFIC CANCER TYPES In a recent review, Kim and Nie describe passive and active targeting methods and then go into detail on a number of active targeting techniques. 72 The active target combinations they mention Polymeric Nanoparticles for Tumor-Targeted Drug Delivery 223 q 2006 by Taylor & Francis Group, LLC are lectincarbohydrate, ligandreceptor and antibodyantigen. One limitation of using these targeting strategies is that the lectincarbohydrate targeting systems are usually targeted to whole organs, making them inappropriate for targeting a cancerous part of a particular organ or tissue. New antibody systems show a great deal of promise. Unfortunately, they also have poten- tially harmful side effects such as advanced gastric adenocarcinoma. The latter arises when one attempts to target breast cancer on account of the fact that antigen-positive normal cells to the antibody BR96 in gastric mucosa, small intestine, and pancreas. Some of the aspects of angiogenic targeting mentioned here have already been covered elsewhere in this chapter. Specic targeting systems that are in use as cancer therapeutics are shown in Table 12.1. 8,72 12.6.1 PROSTATE CANCER Recently, aptamers have been used to target nanoparticulate systems to prostate-specic membrane antigen, a known prostate cancer tumor marker. A model drug, rhodamine-labeled dextran, was encapsulated in PEGylated poly(lactic acid) nanoparticles, which were subsequently surface modied with a prostate specic RNA aptamer (A10). Binding of the aptamer nanoparticles to LNCaP cells expressing prostate specic membrane antigen in vitro was signicantly enhanced when compared to a control of nontargeted particles. Additionally, very low binding was seen on nonprostate specic membrane antigen expressing cells (PC3). The nanoparticles were shown to both target and be taken up by the prostate cancer epithelial cells. This evidence points to the conclusion that this novel aptamer-based, targeted nanoparticle delivery approach can be effective. 73 Gao and collaborators report the use of quantumdots (QD) for in vivo targeting of prostate cancer and imaging of tumors. 7 The core-shell CdSeZnS quantum dots contain tri-n-octylphosphine oxide (TOPO) that binds to a covering of high molecular weight ABC triblock copolymer of polybutylacrylate, polyethylacrylate, polymethacrylic acid, and an 8-carbon alkyl side chain. The complex is functionalized with PEG molecules and monoclonal antibodies to prostate-specic membrane antigen. Specic binding was shown for prostate cancer lines whereas low binding was seen to normal cells. The QD-antibody formulations were studied in vivo in a mouse model human prostate cancer. The nanoparticles were shown to target to the tumor both by passive and active antibody targeting. Sensitive and multicolor uorescence imaging of cancer cells in vivo was TABLE 12.1 Targeting Systems Utilizing Antibodies Currently in Use to Treat Cancer Mechanism Antibody Target Trade Name Agonist activity CD40, CD137 Various Antagonist activity CTLA4 MDX-010 Angiogenesis inhibition VEGF Avastine Antibody-dependent cell-mediated cytotoxicity CD20 Rituxan w , HuMax-CD20, Zevalin w Inhibition of binding of extracellular growth signals HER-2/neu Herceptin Receptor blockage EGF receptor HuMax-EGFr Toxin-mediated killing CD33 Mytotarg w Disruption signaling HER-2/neu Pertuzumab (2C4) Complement-dependent cytotoxicity CD20 Rituxan w , HuMax-CD20 Blockage ligand binding EGF receptor Erbutixe Antibody-dependent lysis of leukemic cells following cell binding CD52 Campath w Inhibits phosphorilation of tyrosine kinases EGF receptor Iressa Nanotechnology for Cancer Therapy 224 q 2006 by Taylor & Francis Group, LLC accomplished in this study. Nonspecic uptake was seen in the liver and spleen with little or no uptake in other organs. 12.7 TARGETED NANOPARTICLES AND IMAGING OF CANCER Hofmann and collaborators have evaluated the ability of super paramagnetic iron oxide nanoparti- cles (SPION) to interact with human melanoma cells in such a way that these particles could be selectively targeted to tumor cells ands then imaged using MRI. 74 They varied the coating placed on these particles and found that when comparing particles coated with poly(vinyl alcohol) (PVA), a vinyl alcohol/vinyl amine copolymer (amine-SPION), PVA with randomly distributed carboxylic groups or PVAwith randomly distributed thiol groups, human cells in culture would interact strongly only with the amine-SPION particles. Furthermore, these particles showed the lowest cytotoxicity. This human study involved intravenous administration of Combidex (Advanced Magnetics, Inc., ferumoxtran-10, a molecular imaging agent of iron oxide nanoparticles and a dense packing of dextran derivatives) to 18 men ages 2146 with diagnosed testicular cancer. 75 The Combidex was imaged using MRI. From this study, it seems evident that those lymph nodes with a higher signal were classied as being malignant. However, based on the information from Advanced Magnetics, these particles should accumulate selectively in noncancerous lymph node tissue. The particles are still experimental and rightly so. Although treatment of cancer with targeted nanoparticles is an important goal, more accurate imaging of cancer is needed to allow for the optimal treatment for each patient. Towards that end, a considerable amount of research is underway with various imaging techniques to establish more accurate determination of the presence and extent of cancer growth and metastases. Because magnetic resonance imaging (MRI) is a widely used imaging technique, much work is currently being done to develop targeted imaging agents for MRI. Some of these involve paramagnetic and superparamag- netic iron oxides due to their ability to affect water relaxation times T 1 and T 2 . Gasco and collaborators have prepared solid lipid nanoparticles containing Endorem, superparamagnetic iron oxide nanopar- ticles (Guebert and Advanced Magnetics), using either a multiple emulsion technique or an oil in water emulsion technique. 76 Although the loading rates achieved were less than 1 wt% iron, it was possible to detect and image in vivo in rats. Incorporation of the Endorem in the SLNallowed passage across the blood brain barrier, passage which was not possible with Endorem alone. Often development of nanoparticle systems is a two-pronged approach involving both drug and imaging agent. If a targeting system is successful, one should be able to enhance the imaging of a cancer and then kill it with the same system. One such study involved the preparation of glycol chitosan nanoaggregates to which either uorescein isothiocyanate (FITC) or doxorubicin (Dox) was conjugated. 12 Based on a single tail-vein injection of FITC-conjugates, levels remained high for eight days and gradually increased in the tumors of rats with II45 mesothelioma cells, in the kidney, and to a lesser extent in the spleen. Meanwhile, levels decreased in other organs. The liver showed some accumulation at day 3 but was signicantly lower at day 8 relative to days 1 and 3. The performance of these systems, as evidenced by a decrease in tumor volume, was excellent with a consistent decrease in tumor volume after day 13 when a tail-vein injection of Dox-nanoaggregates is given at days 13 and 19. 12.8 OTHER TARGETS FOR CANCER A new approach to targeting is the use of lectins, which are plant proteins that specically recognize cell surface carbohydrates. The latter function as selective cancer-cell-targeting agents. PLGA nanoparticles of mean diameter 331 nm incorporating isopropyl myristate were used to deliver paclitaxel to malignant A549 and H1299 and normal CCL-186 pulmonary cells in vitro by means of wheat germ agglutinin lectin as the targeting molecule. The in vitro cytotoxicity against A549 and Polymeric Nanoparticles for Tumor-Targeted Drug Delivery 225 q 2006 by Taylor & Francis Group, LLC H1299 cells was signicantly increased with wheat germ agglutinin-conjugated PLGA nanoparti- cles containing IPM and paclitaxel compared to controls. 77 In a subsequent study, Mo and Lim evaluated in vivo efcacy of the same nanoparticles in a SCID mouse model injected with an A549 tumor nodule. 78 One injection of wheat germ agglutinin-conjugated PLGA nanoparticles containing IPM and a paclitaxel dose of 10 mg/kg inhibited tumor growth without appreciable weight loss. Tumor doubling was increased to 25 days compared to 11 days for conventionally formulated paclitaxel. 12.9 AVIDIN AND BIOTIN TARGETING Although it is by far the most widely utilized polymer for surface modication of nanoparticles, PEG is not the only compound that can be included at the surface of nanoparticles. Nor is it necessary to achieve active targeting. Saltzman has recently reported a method for incorporating avidin-fatty acid conjugates into the surface of PLGA nanoparticles. 79 This method resulted in avidin at the surface of the nanoparticles that remained active for weeks. The ability of these nanoparticles to target to biotin was veried by targeting of the nanoparticles to biotinylated agarose beads. Not only could this system be used for targeted delivery; it can also be utilized to selectively modify surfaces for tissue engineering. In another such example, Hunziker has prepared biotin-functionalized (poly(2-methyl- oxazoline)-b-poly(dimethylsiloxane)-b-poly(2-methyl-oxazoline) triblock copolymers. 80 The biotinylated targeting agents were added using streptavidin as a coupling agent. Uptake of these nanocontainers was seen in the presence of the target receptor, the macrophage scavenger receptor SRA1, but not in the absence of this target. 12.10 CONCLUSIONS The amount of research in targeted, polymeric nanoparticles for cancer imaging and therapy has increased dramatically in the past 510 years. Seeing actual products using targeted therapies has no doubt fueled that work. In the next decade, we will certainly see products, whether with polymeric nanoparticles or some other type of delivery system, using folate receptors and carrying imaging agents. All of these technologies, driven by the elds of fundamental immunology, biochemistry, polymer chemistry, and biomedical engineering, are bringing us closer to the time when cancer may be treated on an individual basis. One patients diagnosis and treatment will be unique to her condition and will be the most effective treatment possible for her. 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