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12 Polymeric Nanoparticles forTumor-Targeted Drug DeliveryTania Betancourt, Amber Doiron,and Lisa Brannon-Peppas

CONTENTS

12.1 Introduction ........................................................................................................................215

12.2 Targeting to Cancer............................................................................................................21612.3 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 Specific Cancer Types ............................................223

12.6.1 Prostate Cancer.................................................................................................... 224

12.7 Targeted Nanoparticles and Imaging of Cancer................................................................22512.8 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 significantly 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.

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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 nonspecific

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 deficient 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 specific 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 beneficial 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, liposomesor 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 specific 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 DrugDelivery 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 first review recent work on targeting to the two most promising targetsfor cancer: Angiogenesis and folate receptors. We will then describe other potential targets for

cancer imaging and therapy with nanoparticles, including antibodies strategies using biotin.

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12.3 PASSIVE TARGETING AND THE EPR EFFECT

Although active targeting may be achieved through targeting to folate receptors, angiogenesis, or

targets more specific 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 specific targeting

systems can be developed. Preparation of nanoparticle systems that can avoid uptake by thereticuloendothelial 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

modified 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 betargeted, whether passively or actively, to cancerous tissues.

Extended circulation time and enhanced tumor targeting were seen for poly(ethylene oxide)-

modified poly(epsilon-caprolactone) nanoparticles in mice with tumors of MDA-MB-231, a human

breast carcinoma.14 These particles were loaded with [3H]-tamoxifen. The amount of labeled

tamoxifen at the tumor site, 6 h after injection, for those particles with PEO modification was at

least twice that of particles without modification 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 unmodified 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 first 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 five 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

modifications; the in vivo distribution of99mTc 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 99mTc 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.

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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 modified 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-modified 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-modified gelatin nanoparticles found in the tumor 4 h after injection

and later when compared to the number of nonmodified 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

infiltration 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 flow 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 oftumor 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 TARGETINGUSINGVASCULARENDOTHELIAL GROWTHFACTORRECEPTORS

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 havebeen 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 90Y caused a significant 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-

2-90Y nanoparticles.

12.4.2 TARGETINGUSINGINTEGRINS

The integrins represent another important cell surface molecule group for angiogenesis targeting

because some integrins, such asavb3and avb5, are upregulated on the endothelial cell surface of

neovasculature.30,31 The avb3 integrin is expressed on numerous tumor cell types; it is highly

expressed on neovascular endothelial cells. Hood and collaborators used 40-nm diameter



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cationic-lipid-based nanoparticles coupled to an organicavb3ligand that was shown to be specific

for avb3in cell studies. These nanoparticles, which contained a luciferase reporter plasmid, were

injected into mice with the avb3-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 efficacy, 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 90Y using nanoparticles targeted to the integrin avb3 with a

small molecule integrin agonist.29 In mice with K1735-M2 tumors, avb3-targeted90Y-nanoparticles

significantly delayed tumor growth compared to untreated tumors. TUNEL staining of tumor

sections showed widespread apoptosis in tumors treated with these targeted nanoparticles. Theauthors 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

efficacy due to the high payload delivered by the carriers.Additionally, PEGylated polyethyleneimine (PEI) nanoplexes with a cyclic disulfide 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 avb3targeting 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

sufficient 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 significant shrinkage of tumor

mass, a shrinkage that lasted for 12 days. The authors postulate that the light activated release ofreactive 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

avb3/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 five days in female Balb/cJ mice with

metastatic mammary tumor clone-66. A bi-exponential fix 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-specific nanoparticle

degradation and release of drug.35



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12.4.2.1 Integrins as Targets for Imaging

In an effort to image angiogenesis, Mulder and collaborators have created MR-detectable and

fluorescent liposomes that are targeted to avb3 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 increasein signal onT1-weighted images. Injection of these lipidic nanoparticles in nude mice with subcu-

taneously implanted LS174 human colon adenocarcinoma led to the ability to specifically image the

vascular endothelial cells in the tumor. Ex vivo fluorescence confirmed that RGD liposomes

specifically interacted with tumor endothelium associated with neovasculature.

In a study reported by Lanza, paramagnetic molecular imaging of angiogenesis was accom-

plished in vivo with avb3-targeted lipid encapsulated perfluorocarbon 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 avb3-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 musclecells with a specific TF antibody. The TF-targeted paclitaxel particles inhibited cell proliferation

while delivery of nanoparticles to targeted cells was confirmed with fluorine 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 affinities 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 affinity for folic acid, an oxidized form of folate, and

5-methyltetrahydrofolate, with binding affinities being in the nanomolar range (KD!1!10K9 M)

for thea isoform of FR.4446 It has been observed with few exceptions that only cells involved in

pathologic conditions, including cancer cells, express the high affinity 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 finding 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 forthe treatment, detection, and monitoring of cancer. What is more, the macromolecular size of

nanoparticles will prevent gromerular filtration and the consequent exposure of kidney tissue to

folate-targeted nanoparticles.



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12.5.1 ANTIBODIES ANDFOLATERECEPTORS

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 benefits of targeting with folic acid are many: it is small in size, has high stability, lacksimmunogenicity, and costs very little.62 Folic acid conjugation at its g-carboxyl group is necessary

for maintaining binding affinity 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 acidification 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 drugdelivery 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 theg- and thea-carboxylic acids of folic acid. It should be said,

though, that the ggroup 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 modified 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 efficiencies of up

to 55% were reported with this system, thus significantly 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

NH2

NH

NH

O

O

HO

O

OH

FIGURE 12.1 Chemical structure of folic acid. Conjugation to folic acid for folate targeting is commonlydone at the g-carboxyl group.



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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 sufficient surface availability of this

targeting agent.

Another approach to folate targeting of nanoparticles has been the modification of surface

properties of polymeric nanoparticles with polymer conjugates including the targeting agent. For

example, Kim reported on the modification of anionic poly(lactic-co-glycolic acid) (PLGA) nano-

particles prepared by emulsification 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 uponaddition 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 fluorescein 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 absentfrom cell media and increased cytotoxicity (anti-tumor efficacy) 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 efficacy 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 fromw50 nm at 378C tow135 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-TARGETEDNANOPARTICLES FORGENEDELIVERY

The use of folate-targeted nanoparticles for gene delivery has also been studied. In gene delivery,

tissue targeting is very important for the efficacy and safety of treatment. To date, the transfection



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efficiency 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 efficiencies 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-(N0,N0-dimethylaminoethane)-carbamoyl] cholesterol, and Tween 80 were

used to complex with DNA into cationic nanoparticles of 100200 nm in diameter with a modified

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 cationicnanoparticles 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 efficacy of nanoparticles complexed with a luci-ferase-encoding plasmid on FR(C) KB cells. The association and efficacy 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 onlybetween 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



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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. Specific targeting

systems that are in use as cancer therapeutics are shown in Table 12.1.8,72

12.6.1 PROSTATECANCER

Recently, aptamers have been used to target nanoparticulate systems to prostate-specific 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

modified with a prostate specific RNA aptamer (A10). Binding of the aptamer nanoparticles toLNCaP cells expressing prostate specific membrane antigen in vitro was significantly enhanced

when compared to a control of nontargeted particles. Additionally, very low binding was seen on

nonprostate specific 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 quantum dots (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-specificmembrane antigen. Specific 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 fluorescence imaging of cancer cells in vivo was

TABLE 12.1Targeting Systems Utilizing Antibodies Currently in Use to Treat Cancer

MechanismAntibody

Target Trade Name

Agonist activity CD40, CD137 Various

Antagonist activity CTLA4 MDX-010

Angiogenesis inhibition VEGF Avastine

Antibody-dependent cell-mediated cytotoxicity CD20 Rituxanw, HuMax-CD20, Zevalinw

Inhibition of binding of extracellular growth signals HER-2/neu Herceptin

Receptor blockage EGF receptor HuMax-EGFr

Toxin-mediated killing CD33 Mytotargw

Disruption signaling HER-2/neu Pertuzumab (2C4)

Complement-dependent cytotoxicity CD20 Rituxanw, HuMax-CD20

Blockage ligand binding EGF receptor Erbutixe

Antibody-dependent lysis of leukemic cells following

cell binding

CD52 Campathw

Inhibits phosphorilation of tyrosine kinases EGF receptor Iressa



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accomplished in this study. Nonspecific 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 PVA with 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 classified 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 T1 and T2. Gasco and collaboratorshave 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 waspossible to detect and image in vivo in rats. Incorporation of the Endorem in the SLN allowed 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 fluorescein 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 significantly 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 specifically 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



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H1299 cells was significantly 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 efficacy 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 modification 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 verified by targeting of the nanoparticles to biotinylatedagarose 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 fields 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. Until other

scientists determine how to stop cancer from occurring, those mentioned in this chapter and

many more besides them are doing their best to eliminate cancer.

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