Particle Toxicology - Chapter 21 docx

21.2.1.1 Magnetic Particles This in particular applies to magnetic nanoparticles and luminescent species that are applied for thedetection of pathogenic cells or tissue Table 21.2.. 2004

21 Nanoparticles in Medicine

Paul J A Borm

Centre of Expertise in Life Sciences, Hogeschool Zuyd

Detlef Mu¨ller-Schulte

Magnamedics GmbH

CONTENTS

21.1 Introduction 387

21.2 Particles in Nanomedicine: Health Benefits and Perspectives 388

21.2.1 Imaging and Diagnostic Tools 388

21.2.1.1 Magnetic Particles 388

21.2.1.2 Fluorescent Nanoparticles 390

21.2.1.3 Quantum Dots 391

21.2.2 Nanomaterials and Nanodevices 392

21.2.3 Drug Delivery Tools 393

21.2.3.1 Liposomes 394

21.2.3.2 Magnetoliposomes 395

21.2.3.3 Dendrimers 397

21.2.3.4 Fullerenes 398

21.2.3.5 Polymer Carriers 398

21.2.3.6 Magnetic Nanocarriers 399

21.3 Hazards and Risks of Nanoparticles 401

21.3.1 General Concepts 401

21.3.2 Toxicological Effects of Nanoparticles 402

21.3.2.1 Effects on Blood and Cardiovascular System 402

21.3.2.2 Uptake and Effects of Nanoparticles in the Brain 402

21.3.3 Current Data on the Toxicology of Engineered Nanoparticles 403

21.3.3.1 Quantum Dots 403

21.3.3.2 Carbon Nanotubes 403

21.3.3.3 Fullerenes 404

21.3.3.4 Dendrimers 404

21.3.3.5 Wear Particles from Implants 404

21.3.4 Nanomaterials in Medicine: Future Toxicological Needs 405

References 406

21.1 INTRODUCTION

Recent years have witnessed unprecedented growth of research and applications in the area of nanoscience and nanotechnology There is increasing optimism that nanotechnology, as applied to medicine, will bring significant advances in the diagnosis and treatment of disease Anticipated

387

applications in medicine include drug delivery, diagnostics, nutraceuticals, and production ofbiocompatible materials (ESF 2005; Ferrari 2005; Vision Paper 2005) Engineered nanoparticles(!100 nm) or nanostructured materials (NSM) are important tools to realize these applications.The reason why these nanoparticles (NP) are attractive for such purposes is based on their importantand unique features, such as their surface to mass ratio that is much larger than that of otherparticles, their quantum properties, and their ability to adsorb and carry other compounds NPhave a large (functional) surface that is able to bind, adsorb, and carry other compounds such asdrugs, probes, and proteins However, many challenges must be overcome if the application ofnanotechnology is to realize the anticipated improved understanding of the patho-physiologicalbasis of disease, bring more sophisticated diagnostic opportunities, and yield improved therapies.One of the most challenging problems that nanotechnology is facing is posed by research data withcombustion-derived nanoparticles (CDNP), such as diesel exhaust particles (DEP) Research hasdemonstrated that exposure to CDNP is associated with a wide variety of effects (review:Donaldson et al 2005) including pulmonary inflammation, immune adjuvant effects (Granumand Lovik 2002), and systemic effects including blood coagulation and cardiovascular effects(review: Borm and Kreyling 2004; Oberdo¨rster, Oberdo¨rster, and Oberdo¨rster 2005a; Oberdo¨rster

et al 2005b) Since cut-off size for their definition (100 nm) is the same, now both terms are used asequivalent The meeting of the worlds of nanoscience and engineered nanoparticles along withParticle Toxicology and its know-how of ultrafines, has led to an impressive series of workshopsover the past years However, little exchange of methods and concepts has taken place and thereforethe aim of this chapter is to discuss applications of nanoscale materials in nanomedicine along withtheir toxicological properties

21.2 PARTICLES IN NANOMEDICINE: HEALTH BENEFITS AND PERSPECTIVESNanomedicine uses nano-sized tools for the diagnosis, prevention, and treatment of disease toincrease understanding of the underlying complex pathophysiological mechanisms The ultimategoal is to improve quality of life The aim of nanomedicine may be broadly defined as the compre-hensive monitoring, control, construction, repair, defense, and improvement of all humanbiological systems, working from the molecular level using engineered devices and nanostructures

to ultimately achieve medical benefit In this context, nanoscale should be taken to include activecomponents or objects in the size range from one nanometre to hundreds of nanometres They may

be included in a micro-device (that might have a macro-interface) or a biological environment Thefocus, however, is always on nano interactions within a framework of a larger device or biologi-cally, within a sub-cellular (or cellular) system These definitions originate from a working groupinitiated in early 2003 by the European Science Foundation (ESF 2005)

In the course of extending our knowledge in the field of nanoparticles, the medical area haswitnessed in the last few years increasing attempts to exploit the intriguing perspectives nanotech-nology offers in medical diagnostics and analytics as well as look for a substitution or assistantmodalities for conventional x-ray analysis It is realistic to say that nanotechnological tools will beroutinely used in diagnosis long before being approved for the treatment of diseases Some tools arealready on the market or available on short-term, but many others still need considerable develop-ment (Table 21.1)

21.2.1.1 Magnetic Particles

This in particular applies to magnetic nanoparticles and luminescent species that are applied for thedetection of pathogenic cells or tissue (Table 21.2) The principle is based on the specific targeting

of these nanovectors onto the target tissue or cell which can afterwards be monitored either bymagnetic resonance imaging (MRI), ultrasound, or optical screening procedures such fluorescencemolecular tomography (Graves et al 2005) Due to the enormous know how accumulated in thescience area in the last decades, a broad spectrum of various nanocarriers have been developedwhich are able to meet the medical needs and prerequisites This applies to (i) the magneticproperties, (ii) size adaptation, and (iii) specific tissue targeting.

One of the key aims in modern diagnostics is undoubtedly the improvement and increase ofdetection sensitivity, leading ultimately to a detection of neoplastic chances in cancer in the earliestpossible stage This aspect certainly constitutes the focus of present research and development Themagnetic properties of nanoparticles are primarily exploited in MRI—where the contrast of thetarget tissue results from the diverse signal intensities this tissue delivers in response to a specifi-cally applied radio frequency pulse This response is a function of the proton density and magneticrelaxation time, which on its part is determined by the biochemical structure, and properties of the

TABLE 21.1

Anticipated Time Lines for Nano Based Diagnostic Tools

2005–2010 Cell sorting, on site detection in packaging, cell

2010–2015 Encapsulation, coating of contrast enhancement

2015–2020 Transfection nanodevice, implantable devices,

Source: Modified from ESF, European Science Foundation Policy Briefing: ESF Scientific Forward Look on Nanomedicine

2005 IREG Strasbourg, France, ISBN, 2-912049-520, 2005.

TABLE 21.2

Overview of Particles and Their Medical or Pharmaceutical Applications

Polylactic acid Polysaccharides Poly(cyano)acrylates Polyethyleinemine Polymer magnetic

tissue The effect of the magnetic nanoparticles on the relaxation times is to create local fieldinhomogeneity, which consequently shortens the relaxation times resulting in an enhancement ofthe image contrast In practice and especially for in vivo applications, the magnetic nanoparticlesare coated with a functional polymer that fulfils two major prerequisites:

† The polymer serves as matrix for attaching disease relevant moieties such as peptides,antibodies, or other small molecules that will bind to the pathological target

† The coating should assist the overall blood compatibility and in particular the increase ofthe blood half-lives, which is a crucial point as such particles are cleared by the liver andspleen within a few minutes A review by Bulte and Brooks (1997) summarizes thediverse approaches with special focus on variety of coatings and type of nanocarrier onthe contrast behavior

The potential of superparamagnetic iron oxide particles (SPIO) in MR lymphography usingdifferent administration routes and the usage of differently modified magnetic liposomes for thespecific imaging of an adenocarcinoma in a rat model have been addressed by Kresse, Wagner, andTaupitz (1997) and Pa¨user et al (1997) Likewise, Guimaraes et al (1994) demonstrated theimaging of hyperplastic and tumorous lymph nodes in rodents using commercial SPIOs Parallel

to the preparation and optimization of the different nanocarriers and iron oxides, respectively,which was subject of diverse research (Tiefenauer, Kuhne, and Andres 1993; Grimm et al 2000;Lawaczeck and Menzel 2004), the mode of applicability of these particles is primarily determined

by the targeting possibility This topic is basically addressed by the coupling of such targetingmediating species that show a high specific affinity towards the according target tissue or cell Zhao

et al (2002) investigated the influence of the HIV-1 Tat peptide derivatized iron oxide ticles for the uptake in cells This peptide, which translocates exogenous molecules into cells,facilitates the cellular uptake of the nanoparticles in an exponential fashion and results in a100-fold increase in cell labeling efficiency Further approaches addressing this topic pertain tothe coating with galactosides (Weissleder et al 1990) for the targeting of hepatocytes via theasialoglycoprotein receptor, anti-carcinoembryonic antigen CEA coupled magnetic nanoparticles

nanopar-as models for tumor targeting (Tiefenauer, Kuhne, Andres 1993), and antibody coated nanoparticlesdirected against the HT-29 surface antigen showing their basic applicability as relaxation andtargeting agents (Cerdan et al 1989) Lanza et al (2004) concisely discuss the diverse aspects ofmolecular imaging including the physical magnetic parameters, the possibilities of active andpassive targeting into the diverse organ tissues, ligand coupling strategies with the emphasize onantibodies, avidin and aptamers, and the spectrum of diverse nanovectors ranging from gadoliniumloaded perfluorocarbon-lipid particles targeted towards fibrin, liposomes, and ab-integrin-targetednanoparticles specially designed for the detection of angiogenesis

Jaffer and Weissleder (2005) reviewed the different molecular imaging systems, includingMRI, nuclear, and optical imaging They also conducted a survey about the diverse imagingagents, their clinical applications with particular emphasis on cancer, atherosclerosis, apoptosis,and inflammatory enzyme activity imaging Apart from nanocarriers, Mikawa et al (2001) describe

a new approach in MRI contrast enhancement by using water-soluble gadolinium metallofullerenesused for both in vivo and in vitro tests They could show a 20-fold higher relaxivity than that of thecommercial MRI contrast agent Magnevistw In vivo MRI at lung, liver, spleen, and kidney of micecorroborated these findings

21.2.1.2 Fluorescent Nanoparticles

Other types of nanoparticles that have attracted much interest in the last few years in the area ofdiagnostics are the optical markers in the form of fluorescent nanoparticles Imaging of cell-surfacereceptors, antigens, and gene expressions using conventional fluorescent molecular probes or

fluorescent-tagged antibodies to trace tumor cells, apoptosis, and metastases have been described(Graves et al 2005) Despite their broad usage and successful application, which has resulted in thedevelopment of fluorescence molecular tomography, these probes have generally some disadvan-tages with regard to photo stability and show low in vivo biodegradability of tagged biomolecules,

or they show inappropriate bio distribution Hence, there is an ongoing tendency to use the potential

of these fluorescent markers in combination with a polymer carrier to exploit their full potential formolecular imaging in medical diagnostics

The number of fluorescent molecules potentially used as markers in bioscience is underlined bythe fact that there are a number of companies specializing in the commercialization of suchcompounds In this review, we will focus solely on two issues, namely, dye-doped silica carriersand quantum dots, as these appear to provide the most promising perspectives in this area Based onthe well-known Sto¨ber suspension process for the preparation of monodisperse silica nanoparticles,several researchers have used this intriguing technology to create fluorescent markers Because ofthe pronounced functionality of the silica matrix, isothiocyanate derivatized fluorophores in theform of rhodamine and fluorescein were directly coupled to the matrix This approach was inten-sively studied by van Blaaderen (2006), aiming to develop the coupling chemistry on derivatizednanoparticles, their fluorescence properties in different solvents, and the diverse physical character-ization using, e.g., light scattering and transmission electron microscopy An alternative route(Santra and Biomoleku¨le et al 2005) uses a W/O micro emulsion of a fluorescein-isothiocyanatederivatized silane precursors to obtain dye-doped nanocarriers To demonstrate the bio imagingpotential of these markers, the nanocarriers were modified with the Tat peptide and folic acid.Incubating these carriers with A-549 and lung adenocarcinoma cells revealed an extensive celllabeling A further basic method of preparing dye labeled particles was recently described (Graf

et al 1999) using a multi-step core-shell approach whose aim is to protect the encapsulatedfluorophores such as rhodamine B, coumarin, and pyrene with a silane layer

21.2.1.3 Quantum Dots

Compared to conventional organic fluorescent dyes, quantum dots—nanocrystalline tors for bioimaging purposes mainly composed of III–V materials, e.g., GaP, GaAs, and INAs orII–VI materials, e.g., CdSe, CdS, ZnSe—represent a novel class of marker systems with uniqueoptical properties (Figure 21.1) This is first of all reflected by the size- and composition-tunablefluorescence emission from visible to infrared light By reducing the size of the nanocrystal, weobserve a blue shift and vice versa in the emission (Murphy 2002; Parak et al 2003) Together withthe large absorption coefficient across a wide spectral range, the high quantum yield, longerfluorescent decay times in comparison to conventional dyes, and pronounced photo stability,these nanocrystals are an ideal marker for bioimaging However, to apply these nanocrystals for

semiconduc-in vivo imagsemiconduc-ing, two pre-requisites need to be fulfilled: the quantum dots must be water dispersible,and, due to the high toxicity of the basic chemical constituents, they have to be coated with abiocompatible matrix This prevents direct contact with the biological tissue thus preventingtoxicity (Chen and Yao 2004) This is achieved by coating the nanocarriers with a range ofdiverse polymers and surfactants Most promising approaches fulfilling the basic purposes aresiloxanes, polyethylene glycol, phospholipids, carboxymethyldextran, mercaptoacetic acid, dithio-threitol, glutathione, or synthetic polymers, e.g., in form of a block copolymers (Hirai, Okubo, andKomasawa 2001; Winter et al 2001; Dubertret et al 2002; Chen, Ji, and Rosensweig 2003; Parak

et al 2003; Gao et al 2004) Apart from the stabilization, the chemical functionality of the coatingsalso provides a basis to attach target-finding bioligands to the surface that paves a way in in vivoimaging Promising approaches in this field include the attachment of specific antibodies orpeptides for neuron targeting (Winter), DNA coupling for the detection of nucleotide poly-morphism (Parak et al 2003), and antibody and streptavidin for labeling breast cancer cells (Wu

et al 2003; Gao et al 2004) Apart from the bioimaging application, quantum dots are also very

suitable for optical coding in the scope of bioassays Han et al (2001) recently described multicolorpolystyrene CdSe/ZnS nanocrystals for multiplexed optical coding of biomolecules The extremelyhigh number of possible codes when using multiple wavelengths and multiple intensities areexemplarily demonstrated in a DNA hybridization assay We recently described a new approachfor highly fluorescent marker systems Using a novel inverse sol–gel suspension technique,quantum dots and fluorescence dyes can be easily encapsulated in a one-step procedure intosilica nano- and microbeads (Mu¨ller-Schulte 2004, Mu¨ller-Schulte et al 2005) The intriguingaspect of this novel technique is that it allows a synthesis of fluorescent carriers within a fewminutes thus providing a significant time saving in comparison to established methods, as well

as a simultaneous combinatorial encapsulation of diverse fluorescent compounds and magneticcolloids This opens up novel perspectives for cell screening and bioassays

Nanomaterials and nanodevices are critical in nanomedicine On the one hand, the principles ofmaterials science may be employed to identify biological mechanisms and develop medical thera-peutics On the other hand, the opportunities of nanomedicine depend on the appropriatenanomaterials and nanodevices to realize their potentials Nanomaterials and nanodevices fornanomedicine are produced largely based on nanoscale assemblies for targeting and ligand display.The field of medical implants is fast growing since new, light, durable, and biocompatibleimplants can be constructed on a tailor-made basis Medical implants are being used in every organ

of the human body Ideally, medical implants must have biomechanical properties comparable tothose of autogenous tissues without any adverse effects In each anatomic site, studies of the long-term effects of medical implants must be undertaken to determine accurately the safety and per-formance of the implants Today, implant surgery has become an interdisciplinary undertakinginvolving a number of skilled and gifted specialists Applications can be identified for each implantsite, from orthopaedics, dentistry, to cardiovascular surgery Artificial hips on one hand need to befixed steadily in the bone while on the other hand the knob needs to be flexible, biocompatible, andnot subject to wearing The success of total hip replacement depends in part on the materials,design, and processing of the materials used in the implant During surgery, the painful parts of the

Cdse

QD core-shell (e.g., CdSe/ZnS) (e.g., protein, peptide)Bioactive coating

damaged hip are replaced with artificial hip parts, which make up the prosthesis—a device thatsubstitutes or supplements a joint To duplicate the action of a ball-and-socket hip joint, theprosthesis has three parts (Figure 21.2):

† The stem, usually made from metal which has to be fixed in the bone

† The ball or head, made of ceramic or metal

† The shell and accompanying liner, with the shell made of metal and the liner made ofcross-linked polyethylene The liner may also be made of ceramic or metal

Drug delivery and related pharmaceutical development in the context of nanomedicine should beviewed as science and technology of nanometre size scale complex systems (10–1000 nm),consisting of at least two components, one of which is an active ingredient (Duncan 2003;Ferrari 2005) The whole system leads to a special function related to treating, preventing, ordiagnosing diseases, sometimes called smart-drugs or theragnostics (LaVan, McGuire, andLanger 2003) Depending on the origin, the materials employed include synthetic or semi-syntheticpolymers and natural materials such as lipids, polymers, and proteins (Aston 2005) The primarygoals for research of nanobiotechnologies in drug delivery include:

– Faster development of new safe medicines,

– More specific drug delivery and targeting, and

– Greater safety and biocompatibility

Currently, the development of a new drug is estimated at over 700 billion euro, and the number

of FDA approvals has been gradually decreasing over the past decade It is anticipated that with the

Acetabular shell

Polyethylene liner Femoral head

Longevity highly crosslinked polyethylene surface

Neck Stem

FIGURE 21.2 Schematical depiction of the building parts of an artificial hip, which is probably the mostsuccessful medical implant over the past 20 years Dependent on the material and the wear forces, every stepcauses the release of a significant amount of particles (wear debris) into the surrounding tissue

help of nanoscience, so-called orphan drugs may be further developed and drugs that were nated in the development of the process may be reactivated However, the pharmaceutical industry

elimi-is currently showing little interest and major developments occur in academia It elimi-is therefore quiteobvious that the nanomaterials segment, which includes several long-established markets such ascarbon black rubber filler, catalytic converter materials, and silver nanoparticles used in photo-graphic films and papers, presently accounts for over 97.5% of global nanotechnology sales By

2008, the nanomaterials share of the market will have shrunk to 74.7% of total sales (BCC 2004).Nanotools will have increased their share to 4.3% ($1.2 billion), and nanodevices will have estab-lished a major presence in the market with a 21% share ($6.0 billion) The Life Science applicationsare thought to induce the latter increase

The main issues in the search for appropriate carriers as drug delivery systems pertain to thefollowing topics that are basic prerequisites for design of new materials They comprise (i) biocom-patibility, (ii) biodistribution, (iii) functionality, (vi) targeting and (v) drug incorporation andrelease ability Certainly none of the so far developed carriers fulfill all of these parameters tothe full extent; the progress made in nanotechnology, inter alia, emerging from the progress in thepolymer-chemistry, however, can provide an intriguing basis to tackle this issue in a promisingway The following paragraphs briefly discuss different particle types currently used in nanome-dicine for drug delivery

21.2.3.1 Liposomes

One of the earliest approaches in the field of drug delivery concerns liposomes that are nanovesiclescomposed of phospholipids Starting with the pioneering work by Gregoriadis (1973) and later byFendler (1977), the cell-like structure of these vectors and their enormous chemical and physicalversatility in terms of size, lipid composition, surface charge, fluidity, surface functionality, anddrug incorporation ability make these carriers the material of choice when approaching thesesubjects Due to the potential variety of the liposomes, researchers have pursued diverse routes

in the development in order to fulfill the above-defined parameters Bio- and blood compatibilityrepresent the starting prerequisites for a carrier design because they directly determine the bloodhalf-life and hence the pathway and biodistribution of a particle Several attempts have beendescribed to design liposomes avoiding the uptake by the immune system—particularly thereticuloendothelial system (RES), to clear foreign bodies—and to enhance blood half-life fromgenerally less than one hour to several hours This objective has been addressed by modifying theliposome surface Iga et al (1994) tested various lipid compositions and found that the incorpor-ation of negatively charged polyoxyethylene-stearyl-derivatives revealed the greatest reduction inRES uptake This is caused by the negative charge and the specific chain length of the poly-oxyethylenes The most promising approach in enhancing liposome bloodhalf-life comes frompreparations, which incorporated either monosialogangliosides or polyethyleneglycols into thelipid membrane Several authors (Gabizon et al 1990; Allen and Hansen 1991; Torchilin et al.1994) have comprehensively addressed this issue leading to the concept of so-called stealth lipo-somes Sphingomyelin–egg phosphatidylcholine–cholesterol-composed liposomes with a half-life

of over 20 h show a dosage-independence of blood clearance (Allen and Hansen 1991) The reasonfor this effect is mainly attributed to the sterical hindrance of opsonins Apart from directlyinfluencing the blood-half lives, the composition and derivatization of the lipids also exert animpact on the tissue distribution of the vesicles This and the aspects concerning, amongstothers, the stability, RES uptake, pharmacokinetics, lipid composition, particles size, surfacemodification, and surface charge have been concisely reviewed by Woodle and Lasic (1992).Following the development of providing appropriate routes for fundamentally improving thebiocompatibility and biodistribution, the application of these nanovectors notably in cancer thera-pies also require functionality, i.e., the possibility to attach targeting finding moieties to theliposome surface thus enabling tissue targeting Several research groups, whereby mainly tumor

antibodies were attached via the functional lipid phosphatidylethanolamine, have addressed thisobjective Jones and Hudson describe anti-placental alkaline phosphatase antibodies linked toimmunoliposomes that could be effectively targeted to tumor cells Similarly, specific antibody-linked polyethyleneglycol liposomes containing entrapped doxorubicin, targeted to KLN-205 lungcarcinoma cells, were capable of reducing the tumor in a mice model significantly (Ahmad et al.1993) The almost unlimited application potential of the liposome technology is furthermore under-lined by research done by Flasher, Konopka, and Chamow (1994), who targeted CD4 coatedliposomes to immunodeficiency virus type 1-infected cells opening up novel perspectives inanother highly explosive medical area.

The final parameter to be addressed and which determines the efficacy and applicability of adrug carrier is the incorporation capacity for a specific drug and its release once the target tissue hasbeen reached (Figure 21.3) Several authors (Kim 1993; Allen 2002; Park 2002) have reviewed thisissue and describe a multitude of well-known anti-cancer drugs in liposome formulations, thetargeting aspect, and the medical therapeutic background Based on the amount of publications,liposome based papers represent by far the biggest portion in this list followed by polymer nano-particles Despite some drawbacks of the liposome concept regarding mainly lack of chemical,biological, and physical stability, the commercialization of liposome tumor therapeutics amounting

to more than $200 million US in the year 2003 underlines the promising basis of this technology(Wagner and Wechsler 2004)

Tumour cell Cytotoxic payload

released into targeted cancer cell, leading to cell death

Irradation activates nanoparticles

Normal cell

Blood vessel

FIGURE 21.3 (Seecolor insert) Multi-component targeting strategies using nanoparticles in treating cancer.Nanoparticles extravasate into the tumor stroma through the fenestrations of the angiogenic vasculature, demon-strating targeting by enhanced permeation and retention The particles carry multiple antibodies, which furthertarget them to epitopes on cancer cells, and direct antitumour action Nanoparticles are activated and release theircytotoxic action when irradiated by external energy Not shown: nanoparticles might preferentially adhere tocancer neovasculature and cause it to collapse, providing anti-angiogenic therapy The red blood cells are notshown to scale; the volume occupied by a red blood cell would suffice to host 1–10 million nanoparticles of

10 nm diameter (Reproduced from Ferrari, M., Nat Rev., 5, 161–171, 2005 With permission.)

of molecule and cell separation, this concept is just beginning to penetrate the medical area.The intriguing perspectives are: (i) to maneuver or target these carriers into special locations

or tissues using a hand or a electromagnet and (ii) to inductively heat up these magneticparticles in an high frequency magnetic field (induction coil) within the scope oftumor hyperthermia

De Cuyper and his group conducted pioneering work for the synthesis and modification ofmagnetoliposomes This technique comprises a dialysis procedure of preformed lipid vesicles in thepresence of lauric acid-coated magnetite nanoparticles (De Cuyper and Joniau 1988; De Cuyper1996) Hodenius et al (2002) described the modification of the magnetoliposomes with biotinylatedlipids for streptavidin targeting Viroonchatapan et al (1995) presented thermosensitive dextranmagnetite-incorporated liposomes with varying magnetite contents for application in hyperthermiaand for potential cancer treatments Ito et al (2004) described real-life applications of magneticimmunoliposomes for tumor treatment Anti-HER2 antibodies were attached to the vesicles andshowed 60% incorporation into SKBr3 cells Subsequent exposure of the cells to 42.58C using analternating magnetic field resulted in a strong cytotoxic effect Shinkai et al (1994) preparedmagnetoliposomes for hyperthermia treatment of cancer by coating a special lipid compositiononto magnetite particles The nanovectors carrying antibodies directed to the surface antigens ofhuman colonic cancer cells BM314 and glioma cells U251-SP showed a 12 times more efficient celluptake than control cells Zhang et al (2005) investigated the potential of negatively chargedpaclitaxel magnetic liposomes as carriers for breast carcinoma via parenteral administration.Their studies demonstrated that these magnetoliposomes could be delivered to tumors more effec-tively than conventional vesicles, resulting in a higher potency on the therapy of breast cancer thanother formulations

A new potential approach using surface modified magnetoliposomes (MP) in HIV-infectionwas described by Mu¨ller-Schulte et al (1997), as shown in Figure 21.4 Due to the dramaticdevelopment of this infection worldwide, the lack of effective drugs, as well as a disappointingperspective for vaccination treatments, new therapeutic measures are highly desirable Theapproach described exploits the heat sensitivity of HIV-viruses that are known to be irreversiblyinactivated at 508C–608C Thus heat treatment is applied in this method: magnetoliposomes with

a size of 50–100 nm are injected into the patient To direct the MP to the site of the infection,one can exploit the same biochemical mechanism that HIV uses to infect the target cells (mainlyT4 helper/inducer cells and macrophages) This initial infection is brought about by theinteraction of the HIV envelope protein gp120 with the CD4 receptor of the T4 helper cell.This leads to the incorporation of the virus in the target cell, which then triggers all the requiredsteps for virus proliferation To target the liposomes, these CD4 receptors are chemically bound

to the vesicles With this receptor, the MP imitate the target cells and can hence bind to thegp120 envelope protein of HIV, as well as the HIV infected cells which also contain the gp120ligand The concomitant presence of the gp120 envelope protein on the infected cells is a result

of the infection and proliferation process respectively (budding process) This opens up theexiting perspective to simultaneously target the MP to the HIV infected cells After MP areadministered and attached to the target organs (HIV and infected cells), the magnetic liposomesare inductively heated up to the appropriate temperatures of 508C–608C using an external highfrequent alternating magnetic field (induction heating) Such induction devices are state-of-the-art The special technique makes it possible for only the viruses and infected cells to be heated

up, not the residual tissue This is achieved by selecting specific frequencies that abrogatecoupling of the tissue so that all the energy is adsorbed by the liposomes This selectiveheating also allows the application of higher temperatures (O508C), thus shortening theoverall duration of treatment to a few minutes The course of the disease can be monitoredafter each treatment by using, e.g., the T4 count Depending on infection status, an additionaltreatment can be applied any time

21.2.3.3 Dendrimers

The class of dendrimers constitutes another type of promising carriers for drug delivery purposes.Although the preparation of these highly branched polymer particles has been investigated for thelast two decades and their intriguing chemical and physical properties concerning shape, size, andfunctionality reviewed (Tomalioa et al 1990), their potential application in biosciences has justbegun to conquer this area The benefits emerging from these nanodevices are (i) their exactadaptable size in the range of 5–50 nm, (ii) their chemical variability and (iii) the high loadingcapacity with active agents which is 25% higher than with conventional polymer–drug-conjugateswhich lie in the range of not more than 10% (Malik, Evagorou, and Duncan 1999) Notably, theadaptable small size of the dendrimers in combination with a biocompatible polymer is a pre-condition for an appropriate tissue distribution and for targeting and lowering the toxicity The class

of dendrimers best examined so far is poly(amidoamines), which exhibit pronounced ibility, water solubility, non-immunogenicity, and high chemical functionality All of theseproperties distinguish the vectors and make them an ideal platform for the development of drugcarrier systems (Patri, Majoros, and Baker 2002) Methotrexate and folic acid conjugated dendri-mers have been designed as a carrier for the targeted delivery of chemotherapeutic and imagingagents to specific cancer cells (Majoros et al 2005), underlining the potential of this kind ofnanodevices in medical therapy

biocompat-AIDS virus CD4

Sensitherm A Nanoparticles

Magnetic field

FIGURE 21.4 Graph demonstrating a strategy to bind and destroy HIV infected cells by magnetoliposomes(MP) To direct the MP to the site of the infection, one can exploit the same biochemical mechanism that HIVuses to infect the target cells (mainly T4 helper/inducer cells and macrophages) This initial infection isbrought about by the interaction of the HIV envelope protein gp120 with the CD4 receptor of the T4 helpercell This leads to the incorporation of the virus in the target cell, which then triggers all the required steps forvirus proliferation To target the liposomes, these CD4 receptors are chemically bound to the vesicles Withthis receptor, the MP imitate the target cells and can hence bind to the gp120 envelope protein of HIV as well asthe HIV infected cells which also contain the gp120 ligand

21.2.3.4 Fullerenes

Fullerenes are a class of compounds that are in a comparable stage of development as the mers The exact spherical carbon structure of fullerenes consists of regularly arranged five and sixcarbon rings that provide an interesting basis for the development of well defined devices formedical therapy However, although discovered about 20 years ago and despite the enormousquantity of papers dealing with the issues such as synthesis, derivatization, investigation of thephysical structure and various properties, applications in the medical therapeutic area are ratherscarce Karam, Mitch, and Coursey (1997) describe fullerenes containing gamma ray emittingTc-99m, the first direct encapsulation of a radionuclide during fullerene formation These appro-priately derivatized “radioendofullerenes” could potentially be used as a radiopharmaceutical formedical imaging Later Wang et al (2004) reviewed the structure and properties of fullerenes andderivatives, their ability to function as carriers for singlet oxygen photosensitizers and theirpotential applications in photodynamic therapy The perspectives of fullerenes in the field ofpharmaceutics were recently reviewed by Gorman (2002)

dendri-21.2.3.5 Polymer Carriers

Polymers as basic material for the design of drug carriers certainly surpass the liposomes with regards

to variability This has resulted in an enormous amount of diverse synthetic, semi-synthetic, andnatural polymers Above all, a copolymerization or combination of these three basic polymersleads to an almost unlimited number of various materials, bringing science a step closer

to fulfilling the ultimate goal of achieving optimally designed carrier systems In view of this widefield, we will focus on those approaches that provide the most promising therapeutic perspectives

As basic synthetic material, polylactic acid, polyacrylates, polycyanoacrylates, poly(ethyleneimine),and silica gels were in the focus of recent research Semisynthetic materials include modified poly-saccharides such as agarose, or poly(amino acids) such as poly(L-lysine) and poly(glutamic acid),whereas natural polymers comprise hyaluronic acid, polysaccharides, dextrin, chitosan, gelatin,peptides, and proteins The main criteria for selecting theses polymers are basic biocompatibilityand functionality Brannon-Peppas and Blanchette (2004) have reviewed the aspect of biocompati-bility by resuming that small sizes and hydrophilicity of the nanocarriers are one of the majorparameters to circumvent elimination by the RES system Targeting of the nanodevices to tumortissue, addressing important physiological aspects such as tissue permeability, angiogenesis, andtumor vasculature, particularly specifying a number of concrete anti-angiogenic drugs, are alsodiscussed

The topic of biocompatibility is furthermore addressed by using either natural hydrophilicpolymers such as polysaccharides, serum albumin, gelatin, or chitosan, or applying biodegrad-able polymers whose most prominent representative are poly(lactid) or poly(lactid-co-glycolic)acid Nanocarriers made of these polymers loaded with paclitaxel showed an in vitro payloadrelease of over 50% within the first 24 h (Brannon-Peppas and Blanchette 2004) Protein encap-sulated polylactid acid nanocarriers were also used to demonstrate the positive influence ofPEGylation on biocompatibility (Quellec et al 1998) thus constituting the basis for a novelstealth concept among synthetic polymers Two hundred nanometers nanoparticles made fromchitosan and carboxymethyl cellulose might be further candidates to fulfill the basic require-ments of biocompatibility Enzymatic degradation of these carriers revealed an over 80% release

of DNA fragment within 6 h (Watanabe and Iwamoto 2005) thus supplying a basis for priate drug delivery systems Chitosan nanoparticles were the subject of recent research testingdiverse formulations that demonstrated their potential for nasal insulin delivery devices (Dyer

appro-et al 2002) Complappro-eting the spectrum of diverse carrier systems, Lemarchand and Gref (2004)highlight the background and aspects of synthesis, characterization, pharmacokinetics, targeting,and application of polysaccharide derivatives in cancer therapy and diagnostics It was shown