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Biomaterials 31 (2010) 6249e6268

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Biomaterials

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Review

Iodinated blood pool contrast media for preclinical X-ray imagingapplications e A review

François Hallouard a, Nicolas Anton a,*, Philippe Choquet b,c, André Constantinesco b,c,Thierry Vandamme a

aUniversity of Strasbourg, Faculty of Pharmacy, CNRS 7199, Laboratoire de Conception et Application de Molécules Bioactives, équipe de Pharmacie Biogalénique,74 route du Rhin, BP 60024, F-67401 Illkirch Cedex, Franceb Institut de Mécanique des Fluides et Solides, University of Strasbourg, CNRS, F-67081 Strasbourg, FrancecCHRU de Strasbourg, Hôpital de Hautepierre, service de biophysique et de médecine nucléaire, 1 avenue Molière F-67098 Strasbourg, France

a r t i c l e i n f o

Article history:Received 25 March 2010Accepted 29 April 2010Available online 26 May 2010

Keywords:Molecular imagingNanoparticleLiposomeMicelleDendrimer

* Corresponding author. Tel.: þ33 3 68 85 42 13.E-mail address: nanton@unistra.fr (N. Anton).

0142-9612/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.biomaterials.2010.04.066

a b s t r a c t

The in vivo X-ray micro-computed tomography (micro-CT) is a very powerful and non-invasive tool usedto establish high-resolution images with isotropic voxels in typical scan times ranging from minutes totenths of minutes. This preclinical imaging technology is primarily adapted to visualize bones. X-rayimaging of soft tissues is made possible by using opaque compounds, providing contrast through tissuevascularization. Thus, using control agents with a long-lasting time in the blood, active or passive tar-geting of soft tissue is made possible in small animals. In this respect, the use of hydrophilic iodinatedX-ray contrast media remains limited due to their rapid blood clearance, albeit at a slightly slower pace inhumans as compared with rodents. The development of an iodinated contrast medium with increasedvascular residence time is thus necessary. This is precisely the scope of the present paper, which willreview and compare in detail the different vectors used as long-circulating iodinated contrast agents formicro-CT, i.e. liposomes, nano-emulsions, micelles, dendrimers and other polymeric particles. Thediscussion is focused, for each of these nanoparticulate systems, on their method of formulation andproduction, their stability properties, encapsulation properties, release properties, pharmacokinetics, andtoxicology. The different aspects relative to the adaptation of these properties and physico-chemicalcharacteristics for blood pool contrast agents aimed at angiographic micro-CT applications are alsodiscussed. The aim of this review is to propose an overview into the formulation and properties ofiodinated micro-CT contrast agents for preclinical applications.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

This review deals specifically with advances in the formulationof contrast agents with a long residence time in the blood pool forpreclinical X-ray micro-computed imaging. The main challengesare (i) to provide the highest concentration of contrast agent in theblood (i.e. iodine), (ii) to obtain a stable concentration of thiscontrast medium in the blood for several hours and (iii) to be ableto use passive or active targeting of these contrast agents for tissueor tumor characterization. In this paper wewill limit the discussionto iodinated contrast agents, these being the most developed for X-ray CT as they provide a good compromise between contrastingpower, safety (reduced toxicity) and cost.

All rights reserved.

2. Computed tomography

X-ray computed tomography (CT) is a non-invasive imagingmethod for which G. Hounsfield and A. Mc Cormack were awardedthe Nobel prize in 1979 [1]. It consists in reconstructing tomo-graphic images from the acquisition of 1D (fan beam geometry) or2D (cone-beam geometry) projections corresponding to theattenuation of X-rays through tissues. Both the X-ray source andopposite detector rotate simultaneously around the subject. Thereconstruction of tomographic slices is then achieved by processingthe projections taken from different angles. The X-ray attenuationof each voxel is measured in Hounsfield units (HU) with a scaledefined from values of air and water, respectively fixed at 1000 and0 HU. A particularity of the micro-CT equipment used in preclinicalimaging is its ability to give high-resolution isotropic voxels (typi-cally 0.1 � 0.1 � 0.1 mm3) due to cone-beam enlarged projectionson flat panel pixellated detectors.

F. Hallouard et al. / Biomaterials 31 (2010) 6249e62686250

Soft living tissues present very close densities, making it difficultto distinguish clearly between them. It is therefore essential to usecontrast media based on heavy elements, like iodine or barium,which allow significant enhancement of the differences in contrastbetween anatomic compartments [2,3]. However the main draw-back of the micro-CT lies in its projection image acquisition time,which may take around 10 min. Usual clinical X-ray contrast agents(hydrosoluble molecules) are very rapidly cleared from the bloodstream by the kidneys [4] and in experiments on rodents, their highheart rates (up to 600 bpm for mice) can induce even faster elim-ination of such agents. Furthermore, the small blood volume,particularly in mice, limits the volume that can be administered,thus requiring a high concentration, and consequently, high-density contrasting agents. Therefore, for the purposes of preclin-ical micro-CT imaging, it is essential to develop specific contrastagents which circulate for a longer time in the blood.

3. Blood pool contrast media

3.1. Definition

Blood pool contrast agents used in X-ray CT consist of a nano-metric-scale range of lipid or polymeric particulate systems con-taining a significant amount of heavy elements like iodine. Theseheavy atoms confer their contrasting power to the nano-objects.Lipid blood pool contrast agents are basically liposomes and nano-emulsions, while polymeric agents include block-copolymermicelles, dendrimers and classic polymer nanospheres and nano-capsules. Some publications even report nanoparticles composedonly of heavy metals like gold nanoparticles [5].

In order to increase their residence time in blood, most of thesenano-objects undergo controlled surface, form and sizemodifications,in order toprevent theirdiffusion throughvascular endotheliumor theactivation of the reticulo-endothelial system (RES) [6]. The massattenuation coefficient of heavy elements, related to the contrastingpower, increases with the atomic mass. Thus, for the formulation ofcontrast agents, research efforts were focused on the use of heavierelements (i.e. more contrasting) as well as on the number of theseelements per molecule. The aim was to improve the contrast and yetsubject patients to lower radiation exposure. However, these heavyelements present some drawbacks such as toxicity and/or potentialaccumulation in the organism [7]. In this respect, iodine appears to beanappropriateagent,offeringagoodcompromisebetweencontrastingpower, safety (reduced toxicity) and cost. That is why we have limitedthe present review to iodinated contrast agents.

A major advantage of using nanometric vectors is their potentialfor simultaneously encapsulating both drugs and one (or more)contrast agents. It is thus possible to produce multifunctional bloodpool contrast media for various imaging methods associated withdrugs [8,9]. These nanoparticulate forms contribute to both a reduc-tion of heavy atom (or derivative) toxicity and to an increase in activeorpassive targeting. Inpractice, these contrastmedia allow the invivopreclinical study of (i) physiological and pathological models and (ii)the evaluation of the pharmacokinetics of drugs encapsulated in suchcarriers [10]. However, in the case of small animals with a very weakblood volume (measurable in mL), the possibility of administeringdifferent contrast agents at the same time is limited. This hindrancecan be overcome by formulating multifunctional contrast media, inorder to carry out preclinical studies associating various methods ofmedical imaging on the same animal.

3.2. Long circulation properties

X-ray CT contrast agents are injected intravenously withoutspecific surface modification, they are rapidly opsonized and

removed from the circulation prior to completing their objective,because they are recognized as foreign particles by the organism(see Ref. [11] for details about immunologic process). It followstherefore that one fundamental property of blood pool contrastmedia or targeted contrast agents, is their long-circulating (orstealth) properties. The most frequent way to increase the bloodresidence time of these particulate contrast agents, is to modifytheir own surface by including hydrophilic polymers like poly(ethylene glycol) (PEG) [12e19]. These polymers have a high degreeof flexibility [20,21] due to the rapidmotion of their chains inwater.Hence, by coating nanoparticulate systems, PEG chains createa hydration shell composed of water molecules oriented andstructured in a cluster surrounding the polymer chains [20,22,23].The ability of PEG to increase the circulation lifetime of suchvehicles has been found to depend on both the amount of PEGincorporated and the length (and therefore the molecular weight)of the polymer [11,24,25]. The presence of PEG on the nanoparticlesurface reduces the binding with the plasma proteins, by increasingthe hydrophilicity, changing the nature of components, and theapparent electrical charge at nanoparticle surface [11,26e33].However, Vert and Domurado suggested that specific proteins maystill adsorb to a PEG coated surface, thus creating a friendly inter-face presenting a so-called “chameleon effect” [26]. These proteinsfixed to the PEG shell cause a reduction in cell uptake [34,35] andresult in dysopsonization. The liver endothelial cells, for instance,recognized Poloxamine-908 coated particles prior to opsonizationbut not subsequently [34]. PEG also seems to restrain liposomeaggregation and thus their elimination by the RES [36]. Incorpo-rating such hydrophilic synthetic polymers onto the nanoparticlessurfaces can be performed in several ways, depending on thenature of the nanoparticles [12]. In the case of lipid nanoparticulatesystems, like liposomes or nano-emulsions, polymers have to beable to anchor onto the surface and generally exhibit an amphi-philic structure. Furthermore, the hydrophobic or lipophilicanchors of such polymers must be long enough to prevent polymerseparation from the particle surface. It follows that the length of thepolymer lipophilic anchor has a significant role in the protectivenature of the polymer activity, i.e. particle stealth properties.Indeed, this is as important as the molecular weight of the hydro-philic polymer part, or the polymer surface density. In the case ofpolymeric nanoparticles, the surface treatment is mainly per-formed either by the co-incorporation of the surface modifyingagent during particle synthesis, or by external surface adsorption[37]. Finally, in the case of mineral nanoparticles, surface modifi-cation is also achieved by the polymer chain physical adsorption, orby a chemical grafting onto the particle surface. The particle sizeentrapping the contrast agent is also an important parameterinfluencing the long-circulating properties. In order to reside in theblood pool for a long time, their size must be larger than 10 nmwhich corresponds to the width of fenestrated capillaries [38], butmust be small enough (generally below 200 nm) as the RES uptakerate increases according to their size.

3.3. Targeting properties

Numerous experimental and clinical reports have confirmeda passive accumulation of intravenously injected long-circulatingparticles in solid tumors [39e48]. This is explained by a larger porecutoff size in tumor vessels, but also by the enhanced permeabilityand retention effect (EPR) [46e50]. Whereas normal vesselspresent a pore cutoff size of 6e7 nm [51], subcutaneously growntumors exhibit a characteristic pore cutoff size ranging from200 nm to 1.2 mm [42]. It is also interesting to note that in the caseof hormone-sensitive tumors, the pore cutoff is reduced inregressing tumors after hormone withdrawal. The same effect has

Fig. 1. Schematic representation of liposomes used as X-ray blood pool contrast agent,(1) entrapping iodinated polymers within the phospholipid bilayer, or (2) encapsu-lating hydrophilic iodinated compound in its aqueous core.

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been observed with tumors in the brain. This effect of hormonewithdrawal shows that anti-hormonal drugs interfere with thepharmaceutical efficiency of anti-neoplasic drugs encapsulated inlong-circulating liposomes [41]. The influence of the tumor envi-ronment is due to the balance between host and tumor-elaboratedfactors that leads to the observed smaller microvessel pore cutoffsize. Host-elaborated factors form and regulate the bloodebrainbarrier [52] while tumor-elaborated factors induce hyper-permeability [45,53]. Finally, the tumor vessel pore cutoff sizeresults from the nature of the tumor, from the tumor microenvi-ronment and from the hormonal state [54e56]. Another tumorspassive targeting consists in using cationic liposomes, since thenegatively charged phospholipid head groups are preferentiallyexpressed on tumor endothelial cells [44,57e60].

To achieve a better selective targeting by long-circulatingnanoparticles, several strategies are explored. They can be classifiedinto two categories, consisting in (i) grafting specific ligands on thenanoparticulate surface, and, (ii) directing the formulationstowards stimuli-sensitive nanoparticles.

The sorts of ligands grafted on the nanoparticulate surface canbe antibodies like anti-HER2 [61], or endogenous molecules forwhich receptors are over-expressed at the targeted cell surface (e.g. folate [62e65] or transferrine [66,67]). Although antibody tar-geting is regarded as a promising strategy, some studies havereported it does not increase the tumor localization, but rather thetumor cell uptake [48,68]. To get round this drawback, Saul et al.have even designed long-circulating liposomes with a dual ligandapproach. They show that such immunoliposomes specificallytarget tumor cells, while sparing off-target cells, due to the factthat tumor cells typically over-express multiple types of surfacereceptors [69]. The grafting of targeting ligands onto PEG, in orderto build up targeting contrast agents, is carried out by usingseveral types of functionalized lipopolymers. These moleculesgenerally present a structure that follows the pattern: phospho-lipid-PEG-X, for which “X” is a reactive functional group, likehydroxyl, carboxyl or amino groups [70,71]. Such reactive PEGderivatives are easily adsorbed onto hydrophobic nanoparticles orare incorporated into liposomes or micelles via their phospholipidresidue. These conjugates can be incorporated (i) during nano-particle formulation, or (ii) through a post-insertion process [72].Finally, ligands can be added onto conjugates either before or afterthis step. There have been some studies done on active targetedblood pool contrast media [61,73e77]. Targeting ligands are linkedto blood pool contrast media via the PEG spacer. Ligands are thuspresent on a specific layer outside that of the dense PEG. On theone hand, this configuration allows the ligand-receptors to bindfor the selective targeting of contrast agents [73]. Such a strategycan, however, involve some undesired effects, such as an increaseduptake of these targeted nanoparticles by the RES [74,75].Furthermore, these targeted objects can also cause the develop-ment of an additional immune response, depending on the natureof the ligand and the blood pool contrast agent composition[61,76,77]. Fv fragments and chimeric antibodies appear lessimmunogenic than complete imunnoglobuline or antibodiesderived from animals [78]. Finally, the formulation has to bea compromise between the amount of fixed ligands to ensuresuccessful binding with the target and the stealth properties ofthe blood pool contrast agent to be maintained.

The development of stimuli-sensitive nanoparticles is based onthe fact that in various tissues [79e82], pathological processesinvolve an increase in local temperature, or acidosis. For instance,thermo- or pH-sensitive micelles, made of poly(N-iso-propylacrylamide) and poly(D,L-lactide), will specifically disinte-grate in such areas, inducing a very targeted drug release [83,84].Another example is the use of engineered protease sensitive

hemolysin, which is linked to long-circulating nanocarriers in orderto be activated by proteases released from cancer cells [85,86].

The combination of these two active targeting strategies wasdescribed using liposomes andmicelles [87]. The stimuli sensitivityPEG coating allow the preparation of multifunctional drug deliverysystems, which under normal (circulating) conditions are “shiel-ded” by the PEG, and becomes exposed after it is released.

4. Liposomes

4.1. Definition

Liposomes consist of amphiphilic phospholipids self-assembledinto vesicular structures [88]. The scope offered by the liposomeaqueous core makes these nano-objects prime candidates forcarrying and protecting different hydrophilic compounds at thesame time, such as both drugs and/or contrast agents [8,9,89,90].Liposomes are well characterized and biocompatible, which is dueto the innocuous nature of their components.

Thus, liposomes have been used as contrast agent carriers sincethe 80s [91]. The initial interest of using liposomes was to specifi-cally target contrast agents in the liver and spleen, in order to studyand diagnose solid tumors in these organs [92,93]. After thediscovery of PEG, long-circulating liposome formulations emerged,which, along with the loading of contrast agents, led to significantadvances in the diagnosis of diseases [94e98], image-guidedtherapeutic interventions [99] and assessment of treatmentoutcome [9,98].

Even if long-circulating liposomes contain a lower amount ofiodine (from 25 to 110 mg/mL) than conventional contrast agentsfor CT, their stealth properties ensure a higher X-ray attenuationefficiency [4]. Blood pool contrast agents thus help visualize detailsto a width of 100e200 mm with an acceptable irradiation dose[96,100]. A schematic illustration of X-ray contrasting liposomes isprovided in Fig. 1.

CT contrasting liposomes are also used to study solid tumors orthe regulation of transport pathways in tumor vessels [42]. Hoisaket al. created liposomes loaded with contrast agents for both CT andMRI. These liposomes provided anatomic and functional images,thus increasing the understanding of the disease processes [101].Clinically speaking, this formulation strategy may prove to bea powerful tool in the nanotherapeutic approach. Karathanasis et al.suggest that multifunctional nanocarriers will allow the on-linemonitoring and prediction of the outcome of breast cancer therapy[9]. Thus, such multifunctional nanocarriers can potentially provide

F. Hallouard et al. / Biomaterials 31 (2010) 6249e62686252

an optimized, personalized therapeutic regimen and avoid non-responders. Contrasting liposomes are also used to increase thediagnostic accuracy of pulmonary embolism, by monitoring theefficiency of t-PA in rabbits for 4 h, resulting in an optimizedtreatment [98].

4.2. Production of liposomes as blood pool contrast agent

Themethods for producing liposomes comprise 3 steps. The firstone is the hydration stage where multilamelar large vesicles (MLV)are produced using various methods (see Ref. [88]). These MLV arealso loaded at this stage. The second step is extrusion, at lowor highpressure, to control the liposome size [102,103]. It consists inpassing MLV through polycarbonate membranes of a defined poresize. The external layer of MLV is removed at every passage until thedesired size is obtained. Extruded liposomes generally havea slightly higher diameter than the membrane pore [43]. Further-more, some studies show that the industrial production of lipo-somes may now be possible as continuous extrusion methods havebeen developed [104e106]. The last step consists in removing non-encapsulated material from the bulk phase, by dialysis, ultracen-trifugation, gel-permeation chromatography or ion-exchangeresins. The whole liposome producing process lasts several hours[90,100,107,108].

When using liposomes as long-circulating particulate systems,the sizemust be controlled (between 10 nm and about 200 nm) andliposomes should be stealthy to increase their stability in blood[109]. Without such modifications, “nude liposomes” accumulaterapidly in the liver and spleen after an intravenous injection.

It is well acknowledged that the incorporation of poly(ethyleneglycol)-phosphatidyl ethanolamine conjugate (PEGePE) of up to10% in molar concentration remarkably prolongs the circulation ofliposomes [110e114]. Takeuchi et al. demonstrate that other poly-mers like poly(vinyl alcohol) (PVA-R) grafted onto the liposomesurface can also increase the stealth properties [19]. Liposomescoated, for example, with 1.3 mol% PVA-R (molecular weight:20 000), show stealth properties comparable to those of liposomesprepared with 8 mol% of DSPEePEG (molecular weight of PEG:2000).

In vivo, several studies show a correlation between the size andblood pool circulating time of liposomes. Accumulation in thespleen and liver increase progressively with the size of the lipo-some, due to a better uptake by the RES [115e117]. However, somepoints remain unclear, such as the reasons why small liposomesshow a more enhanced liver accumulation than larger sized ones.Some authors explain this phenomenon by the fact that these smallliposomes have access to the hepatocytes through the fenestratedhepatic endothelium [118].

4.3. Liposome stability

In comparison to other nanoparticulate carriers, stability is themajor drawback of liposomes. Overall, liposomes undergo chemicaland physical degradation, like autoxidation, hydrolysis, aggrega-tionefusion and leakage of encapsulated compounds [119]. Theirstability is closely related to (i) their bilayer rigidity, (ii) thedifference in osmolarity between the inner aqueous core of lipo-somes and the external bulk environment, (iii) the presence ofpolymers, such as PEG, on the particle surface (which prevents boththe penetration of serum opsonins on their surface [43] and theincrease of steric stabilization) and finally (iv) the storage condi-tions. In practice, all these points can be controlled experimentallyand are regulated by the very nature of phospholipids and thepresence of additives like cholesterol. For instance, at a giventemperature (e.g. 37 �C), the liquid-crystalline forming phase

within the phospholipid bilayer will play on its rigidity [43]. Inaddition, liposomes can be destabilized by HDL (high-densitylipoprotein) particles in the absence of cholesterol [120,121].Seltzer et al. explain that highly stable liposomes can be producedwhen the osmotic pressure of the entrapped contrast agent is equalto that of the plasma. This is attributed to the inhibition of osmoticstress on the liposome membrane [122].

Solutions for reducing liposome oxidation include (i) theselection of phospholipids composed of saturated acyl-chains (ii)oxygen free storage conditions (iii) the addition of antioxidantcompounds in the aqueous liposome suspension and (iv) theenvironmental bulk pH fixed at 6.5 and a low storage temperatureto reduce hydrolysis [88]. Some processes involving freeze-dryingfollowed by rehydrating liposomes have also been shown toincrease their stability [119]. This protective effect was shownwiththe encapsulation of iopromide (a CT contrast agent).

4.4. Liposome encapsulation properties

The encapsulation properties of liposomes such as iopromidehave been shown by Schneider et al. in reference [104] to dependclosely on experimental conditions. Firstly, the concentration inencapsulated contrast agents does not actually promote theencapsulation rate, and can indeed induce the opposite effect.Secondly, performing freezeethaw cycles greatly improves theencapsulation efficiency of iopromide. Thirdly, the composition ofthe liposome bilayer itself may also influence the encapsulationrate. This is illustrated by the difference in iopromide encapsulationobserved when negatively charged phospholipids (like soy phos-phatidylglycerol (SPG)) are either added or not to the formulation[123]. Fourthly, the lipid concentration in the preparation can alsoincrease the liposome encapsulation features. Finally, the formu-lating process can influence encapsulation. For instance, in the caseof the extrusion step of MLV, part of the contrast agent will irre-mediably be released. There are recurrent problems when theencapsulated molecules are polar compounds and/or are not elec-trically repulsive to the bilayer, resulting in poor encapsulation afterlipid hydration [88]. Nevertheless, extensive research has beendone in this domain, and for example, Haran et al. discovered thatamphipathic weak bases like doxorubicin can be efficientlyencapsulated by using a pH gradient (generated by a gradient ofammonium sulfate) [124].

4.5. Liposome compounds leakage

The encapsulation of active ingredients within the aqueous coreof liposomes causes a gradual leakage of the encapsulated mole-cules, even when the osmotic pressures are well equilibrated. Thisis due to the compositional diffusion of the solutes through themembrane, tending to balance the chemical potentials. Thisleakage is also due to the influence of the bilayer morphology,rigidity, composition in phospholipids and the presence ofcholesterol. A study by Seltzer et al. emphasizes the links betweenthe nature of phospholipids and bilayer permeability. For example,the permeability measured through DSPCmembranes is lower thanthat using egg phosphotidylcholine [122]. Carruthers and Melchior[125] explain this difference by the correlation between perme-ability and the crystalline state of the bilayer (i.e. a transition fromlow to high permeability following a liquid-crystalline to fluidphase transition).

The presence and concentration of cholesterol have an effect onbilayer crystallinity, and thus on permeability [126]. For example,cholesterol added at 5 mol% to a dimyristoylphosphatidylcholine(DML) bilayer, decreases the membrane permeability at a temper-ature above the phase transition. At a lower temperature,

F. Hallouard et al. / Biomaterials 31 (2010) 6249e6268 6253

cholesterol increases membrane permeability. Furthermore, ata higher concentration of cholesterol (over 10 mol%), the perme-ability of the DML bilayer decreases regardless of the temperature[125].

Encapsulating contrast agents in liposomes nonethelessinvolves their non-encapsulated presence in the bulk phase. In vivo,this results in a contrast agent burst release in the kidney just afterintravenous injection [90]. The presence of this free contrast agentpoints to its leakage from liposomes. Mukundan et al. worked onreducing the leakage of an iodinated contrast agent beforeadministration, from about 10 to 3% [90,94,107]. Leakage of iodin-ated molecules from the blood pool contrast agent was avoided byusing iodinated phospholipids to form liposomes [127]. Elrod et al.thus provided liposomes with an iodine concentration of up to40 wt%, ranging from 50 to 150 nm in size. Furthermore, thissolution prevents the interaction of an iodinated contrast agent(present in the liposome membrane) with other loaded drugs(present in the liposome core).

4.6. Long-circulating liposomes pharmacokinetics

Research efforts have led to a reasonably good characterizion ofliposome pharmacokinetics [43], working on models like rodents,rabbits or primates [9,89,94,95,107,108,128]. The metabolism ofliposomes has been shown in the literature to carried out by theRES [96]. Jacobsen et al. highlighted an example of the biotrans-formation of liposomes loaded with iodixanol (a contrast agent) ina monkey liver [129]. Two new metabolites were generated, butonly when the contrasting molecule was encapsulated in lipo-somes. This case shows that even if liposomes are generallyconsidered to be innocuous, here they induced a metabolicresponse and thus potential toxicity. High doses of injected lipo-somes are able to induce RES saturation, with a consequentreduction in blood clearance [96,115,116,130,131]. Finally, the resi-dence time of stealth liposomes in the blood pool can vary from40 min to 3 days, depending on such factors as experimentalconditions, vesicle structure and composition.

However, the elimination route of long-circulating liposomescan differ considerably in function of the chemical composition ofthe phospholipids used in the formulation. For example, liposomesformed with DPPC or DSPEePEG are eliminated through the liver[8,108], with dose-independent clearance kinetics in the case ofDSPEePEG [39,110]. Liposomes formed with SPG or SPC are elimi-nated through the kidneys [106,128]. In addition, the RES uptakeincreases rapidly depending on the liposome size. This is why largeliposomes (e.g. 400 nm), allowing rapid visualization of the liverand smaller vesicles (e.g. 200 nm), may persist in the blood streamand temporarily evade phagocytosis [122,132]. As mentionedabove, the earlier burst-attenuation in the kidney observed afterintravenous injection is due to non-encapsulated contrastingmolecules. These free (i.e. non-encapsulated) molecules presenta t1/2 shorter than 15min not only for a CT iodinated contrast agent,but also for MRI contrast agents such as gadoteridol [4,8].

4.7. Liposomes toxicology

At high doses, liposomes are intrinsically toxic. Over 100 mg/kgbody weight in mice, liposomes induce hepatomegaly, granulomas,and splenomegaly [133e136]. PEG, used to prepare stealth lipo-somes with a molecular weight ranging from 1900 to 5000 Da, areconsidered to be non-toxic compounds [137]. However, Krauseet al. observed allergy-like side effects induced by liposome injec-tions in mice. These were strongly dependant on the size, surfacepotential and composition of the particles [138]. A human phase Itrial was performed with non-PEGylated liposomes of 340 nm,

loaded with an iodinated contrast agent. This showed dose-dependant effects on the leucocyte count, C-reactive protein (CRP)and adverse effects (AE). Leucocytes were depressed for the first 2 hbefore showing a rise, particularly pronounced for neutrophilicleucocytes [139]. A rise was also seen in CRP, once again indicatingthat cytokine-mediated reactions occurred. AE included chills(63e88%), and backache with a dose of over 30 mg I/kg and, in thecase of 100 mg I/kg, flu-like symptoms (13%) and nausea withvomiting (13e38%).

The mean lethal dose (LD50) of stealth iopromide-loaded lipo-somes in rats is 4.5 g of total iodine per kilogram [106], as opposedto that of nonliposomal iopromide (i.e. non-encapsulated) which is11 g I/kg [140]. Furthermore, LD50 of liposomes can also varyaccording to the surface potential of the liposomes. For instance,LD50 in mice generally ranges from 3 to 4 g lipid/kg for positively-charged liposomes, but can increase to up to 7 g lipid/kg whenliposomes are negatively charged [92]. The toxicity of iodinatedliposomes may in fact simply be explained by the high doses oflipids in the blood pool, due to the particular formulation. Theconsequences of the lipid toxicity of liposomes include milkyserum, affected glutamic-oxaloatic transaminase, gamma GT, bloodurea nitrogen and glutamic pyruvique transaminase [106]. More-over, toxicity is increased with the intracellular uptake of iopro-mide liposomes by the mononuclear phagocytic system [106].

5. Nano-emulsions

5.1. Definition

Nano-emulsions are nanometric sized emulsions, typicallyexhibiting diameters ranging from 20 to 200 nm [141]. Theseemulsions are also frequently known as miniemulsions, fine-dispersed emulsions or submicron emulsions. They are character-ized by a great stability in suspension due to their very small size.

Blood pool contrast agents such as nano-emulsions target theliver in a similar way to chylomicrons and may therefore be used tostudy the liver metabolism [142]. Fenestra LC� is thus used todiagnose and study liver tumors [143e146] and optimum tumorvisualization in the liver is attained 4 h after injecting the nano-emulsions [145]. This has resulted in an enhanced resolution intumor detection, enabling the detection of tumors smaller than300 mm (in comparison, tumors up to 1 mm are routinely detectedwith a fair liver contrast). The visualization of liver tumors [144] isincreased by combining a non-stealth iodinated nano-emulsion(Fenestra LC�) which provides liver contrast with long-circulatingiodinated nano-emulsions (Fenestra VC�) contrasting the bloodpool. The combination provides a significant contrast to both theliver and the vessels, hence emphasizing the tumoral structures. Aschematic representation of the iodinated nano-emulsions isproposed with Fig. 2.

Badea et al. observed that, as with long-circulating liposomes,iodinated nano-emulsions at a dose of 0.4 mL induce a higher X-rayattenuation in the blood pool than a 1 h perfusion at 1 mL ofa contrasting molecule (Isovue-370�) used in clinical applications,despite a lower iodine concentration in nano-emulsions [147]. Thisphenomenon could be due to the pharmacokinetics of Isovue-370�

which depend on the flow rather than on the injected amount as isthe case for circulating blood pool contrast agents [148]. However,since the iodine content in Fenestra� is relatively low (limited bythe formulation at 5 wt%), a relatively large volume must beinjected compared to the total blood volume of the mouse. Thiscould result in modifications of the cardiac and pulmonary func-tions as well as in animal stress, which can interfere with the studyof these organs [142].

Fig. 2. Schematic representation of nano-emulsions used as X-ray contrast agent. The oily phase is generally composed of iodinated triglycerides (ITG). The nano-emulsions canpresent features of blood pool contrast agent (when surrounded by a PEG shell, right, see also Fig. 4) or not (left, in this case the liver will be constrasted, see also Fig. 3).

F. Hallouard et al. / Biomaterials 31 (2010) 6249e62686254

5.2. Nano-emulsions production

There are several methods and processes for producing nano-emulsions. These methods may be divided into two groups, “highenergy” and “low-energy” methods.

High energy emulsification processes follow two steps: First,droplets are created by deforming and disrupting the pre-emul-sified emulsion droplets. Second, the newly formed dropletsundergo steric stabilization by surfactant adsorption on theirinterface. In the literature, three sorts of devices can supplyenough energy to generate nano-emulsion droplets: ultrasoundgenerators, high pressure homogenizers, and rotor/stator devices.However, to date, only high pressure homogenizers have beenused in the formulation of nano-emulsions containing blood poolcontrast agents [143]. The mechanism of nano-emulsification isbased on the passage of macro-emulsion droplets through narrowgaps, by imposing high pressure. The resulting emulsion dropletsare very small, less than a micrometer, showing a narrow distri-bution [149].

There are, however, several drawbacks to the use of these highenergy methods. The first concerns the energetic yield of theprocess, since a huge amount of energy is wasted in heat dissipa-tion. The emulsion time and thus the energy involved in providinghomogeneous nano-emulsions increases in function of the volumeto nano-emulsify. Moreover, during the process of encapsulatingfragile or thermo-sensitive drugs in nano-emulsions, thesemethods can potentially induce degradation. Such drawbacks donot exist with low-energy methods, where nano-emulsions arespontaneously generated by diverting the intrinsic physico-chem-ical properties of the excipients in the formulation. Such methodsare easy to scale-up and in some cases do not need solvents duringprocessing [150].

5.3. Nano-emulsions stability

The great advantage of nano-emulsions is their stability. Sincethe size of nano-emulsion droplets is not affected by gravitation,they can be considered as Brownian particles and as such, preventsedimentation or creaming, flocculation and coalescence. For thesereasons, unlike micrometric scaled macro-emulsions, nano-emul-sions are stable for months [150], despite autoclave sterilization[151]. For example, Fenestra LC� and Fenestra LC� presenta stability (according to the manufacturer) of several months atroom temperature. The nanometric range of the nano-emulsiondroplets naturally prevents flocculation by steric stabilization.Steric repulsion, caused by the overlap of interfacial droplet layers[152], stems from the unfavorable mixing of the stabilizing chain ofthe adsorbed layer, depending on the interfacial density and layerthickness, as well as on the Flory-Huggins parameter (reflecting theinteraction between the interfacial layer and the solvent). Anotherparameter which plays a significant role in steric stabilization is thebending stress of the chains, occurring when the distance betweeninter-droplets is lower than the thickness of the interfacial layer.The predisposition for inducing droplet coagulation depends on theratio between the thickness of the interfacial layer and the particleradius [150]. The higher this ratio, the higher the stability. In thecase of nano-emulsions, this parameter is so high that it totallyprevents the suspension from droplet flocculation. Finally, thesenano-suspensions can be stable for months, destabilized only bythe phenomenon of Ostwald ripening [150,152].

5.4. Long-circulating nano-emulsions pharmacokinetics

After intravenous injection, the homogeneous biodistribution ofFenestra� in the blood pool is reached within 10 min [153]. In mice,

F. Hallouard et al. / Biomaterials 31 (2010) 6249e6268 6255

the contrasting effect in the blood pool (i.e. the concentration ofnano-emulsions in the blood), remaining after injection is ofaround 1 h for native nano-emulsions and can exceed 2 h forPEGylated ones [153,154]. In rabbits, PEGylated nano-emulsionsstill circulate in the blood pool for up to 3 h [144].

Iodinated nano-emulsions show a biphasic clearance curve withcalculated half-lives of 16.2 h and 30.6 min [154]. Uptake of thecontrast product by the spleen starts at 10 min after injection witha peak increase between 60 and 90 min [154]. In mice or rabbits,these nano-emulsions are totally cleared from the blood by the liverthrough the gall bladder 24 h after injection [153e155]. Thiselimination route is due to the nano-emulsion structure andcomposition, which is very close to that of chylomicrons. It thusfollows that nano-emulsion uptake is achieved by internalization inhepatocytes after specific binding to the apolipoprotein-E (apoE)receptors [153]. The nano-emulsions remain in the liver for a fewdays [156].

The pharmacokinetics of Fenestra VC� and Fenestra LC� aresimilar, the only difference being that Fenestra VC� presentsa prolonged residence time in the blood pool. This results in thespecific grafting of PEG onto the surface of the nano-emulsiondroplets of Fenestra VC�. The PEG appears to temporarily blockhepatocyte recognition by the apoE [154]. These nano-emulsionsin the liver will only provide a contrast in healthy tissues, due tothe fact that tumor tissues do not (or hardly) express apoEreceptors on their surface. For this reason micro-CT provideshypodense images of tumor tissues [145]. Figs. 3 and 4 presentillustration of contrasts obtained respectively with Fenestra VC�

and Fenestra LC�.The imaging apparatus-compatible iodine dose in the formula-

tions was shown by Suckow and Stout to be around 7.5 mL/kg,sufficient for 1 day of effective imaging. In mice, they also high-lighted the influence on pharmacokinetics of the strain [142],partially explained by the different efficiencies of the RES insequestering in the liver (and thus acting on the elimination rate ofthe contrast agent). Fenestra VC� at 10 mL/kg presents two imagingwindows [148]: The first window, appearing immediately after theintravenous injection allows the visualization of the vascularsystem and lasts 1 h. The second window highlights the spleen andliver up to 24 h after injection. In addition, the slight attenuationobserved in the kidneys seems to be due to the vascularity of theseorgans rather than to the elimination of the contrast medium.

Fig. 3. (A) Frontal (top left), transverse (top right) and sagittal (bottom) smoothed views oacquired in a healthy living mouse after Fenestra LC IV administration. Micro-CT: eXplore RSand the arrowhead the spleen. 3D isosurface rendering ((B) ventral view, (C) left lateral viewthe large hole in the liver marks the position of the gall bladder. A movie is available as su

5.5. Long-circulating nano-emulsions toxicology

High viscosity solutions administered by injection can inducea significant number of AE including pain, nausea, itching, burningand other sensations [157]. Iodinated nano-emulsions presenta viscosity 3e6-fold lower than clinical contrast media, whichpartially explains the greater tolerance when injected in mice andrabbits [148,153]. Emulsion cytotoxicity may be the result of eitherthe surfactant(s) used and/or of the oil [158]. The cell lysis inducedby the surfactants seems to result from an osmotic lysis or a solu-bilization. Low surfactant concentrations trigger the insertion ofthese amphiphiles within the membrane only, whereas at a highconcentration, membrane solubilization causes cell lysis [159].

Among theexcipients studiedbyAparicioet al. [158], somepresentahighhemolyticactivity (e.g.Labrasol� asanoilyphaseorbutyl lactateas a surfactant), and are therefore incompatible with the formulationof emulsions for intravenous administration routes.Other compoundshowever are known and recognized not to induce hemolysis below80mL/mL.These includeTween80�, CremophorEL�, CremophorELP�,isopropyl myristate and Miglyol-812� [158,160,161].

6. Micelles

6.1. Definition

Micelles are self-assembled, mainly spherical shaped, molecularclusters in water [162]. These nanoparticles are formed when theconcentration of soap rises above the critical micelle concentration(CMC). When the bulk phase is aqueous, micelles have an innerhydrophobic core and an outer hydrophilic shell, a structure whichallows them to carry different hydrophobic compounds. Suchmicellar systems can be used as blood pool contrast media whentheir CMC is very low, which is the case for polymeric micelles(which can be 1000-folds lower than the one of surfactant micelles[163]). This explains our choice to focus only on polymeric micelles.Schematic representation of polymeric micelles used as blood poolcontrast agents is presented in Fig. 5 (modified from Ref. [164]).

6.2. Micelles production

Polymeric micelles are generally formed from copolymers,a conjugate of hydrophilic and hydrophobic polymers (blocks).

f a micro-CT whole body reconstructed volume (voxel size 93 mm 93 mm 93 mm)Locus, GE Healthcare, Waukesha, USA; parameters: 80 kV, 450A. The star show the liver) showing the skeleton (in white), the liver (in orange) and the spleen (in green). In (B),pplementary data (movie2.mov).

Fig. 4. Transverse (A), sagittal (B) and frontal (or four cavities, C) views of a micro-CT heart volume (voxel size 93 mm 93 mm 93 mm) acquired on a healthy living mouse, followingIV administration of Fenestra VC. Micro-CT: explore RS Locus, GE Healthcare, Waukesha, USA; parameters: 80 kV, 450A. (1) Right ventricle, (2) Left ventricle, (3) Papillary muscles,(4) Caudal vena cava, (5) Pulmonary veins, (6) Left cranial vena cava, (7) Aorta, (8) Left auricle, (9) Right auricle, (10) Pulmonary trunk. Lateral (D) and ventral (E) volume renderingviews of the thorax, enhancing (in red) the vascular bed. A movie is available as supplementary data (movie1.mov).

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These micelles are used as diagnosis agents or drug carriers byeither (i) covalent incorporation of the contrast agent or drugswithin the amphiphilic polymer structure or (ii) solubilization intothe hydrophobic core of the micelles (Fig. 6) [165], as detailedbelow. Some examples present the formulation of polymericmicelles by the chemical combination of hydrophilic polymers withhydrophobic pharmaceutical agents [166,167], resulting in amphi-philic macromolecule-forming micelles. In the formulation of long-

circulating micelles, the hydrophilic polymer used is often PEG, forits recognized and efficient steric protection in biological media[168].

To date, only there has been only one published formulation ofiodinated polymeric micelles, used particularly as a blood poolcontrast agent for micro-CT [169]. For this agent, iodine is cova-lently grafted onto a polymer molecule, which, in an aqueousmedium, induces rapid micelle formation. The nanoparticles

Fig. 5. Schematic representation of polymeric micelles formation and used as X-ray contrast agent. Contrasting species (stars) can be: (1) directly solubilized within the micellecore; (2) Linked to the hydrophilic part of the polymer; (3) incorporated linked onto a lipophilic molecule within the micelle core; (4) Linked to the hydrophilic part of the polymer.

Fig. 6. Example of an iodinated block-copolymer (N-triiodibenzoyl-PLL-PEG) used toform a micellar blood pool contrast medium, from Ref. [165].

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exhibit a size up to 100 nm, with an iodine content of around45 wt%.

Active targeting strategies are possiblewithmicelles, by graftingthe specific targeting function onto the micelle surface, i.e. onto theperipherical part of the hydrophilic polymer block [170]. Someadapted micellar systems are also compatible with a passive tar-geting mechanism (introduced above) which is useful in that manypathological processes are accompanied by a local temperatureincrease and/or a local acidosis [79,80]. Hence, micelles made ofthermo- or pH-sensitive components, such as poly(N-iso-propylacrylamide) with poly(D,L-lactide), can specifically disinte-grate in such diseased zones, resulting in a very targeted drugrelease [83,84].

6.3. Micelles stability

From a thermodynamic point of view, intravenously injectedmicelles are strongly diluted in the blood circulation. Should theamphiphile concentration decrease to below the CMC, this dilutioncan induce the dissociation of micelle clusters. However, evenwhenthe theoretical conditions leading to cluster dissociation arefavorable, a cluster may not occur instantaneously, taking froma fewminutes to a few days (if the dissociation process is kineticallytrapped) [162]. One of the particularities of amphiphilic polymers isa CMC in the micromolar or even nanomolar range [171,172],enabling the preparation of physiological stable micelles [173]. Thetwo main parameters that play a role in micelle stability are (i) theforce of Van derVaal’s bonding between the hydrophobic innerblocks and (ii) the molecular size of the hydrophilic external part ofthe macromolecule [169]. Furthermore, interactions between thepolymeric micelles and blood plasma components seem to berelatively low, enhancing the stability and the stealth properties.Torchilin et al. did not report any change in themicelle size after 1 hof incubation with rat plasma [169].

6.4. Micelles encapsulation properties

The process of the solubilization of hydrophobic compoundswithin the lipophilic core of micelles (i.e. a non covalent incorpo-ration), was investigated in detail [174]. This study, of a mathe-matical nature, emphasized the loading process of the micelles:first, the establishment of the micellar structures, then the gradualaccumulation of lipophilic compounds within their core. As inmicroemulsion systems, this hydrophobic loading process inducesan increase in the size of the micelles by enlarging their core. At thispoint, it is important to note that both the length of the parts of theamphiphilic polymer lipophilic forming core and the hydrophilic

forming shell strongly influence the drug loading efficiency [175].The bigger the hydrophobic polymer blocks, the higher the abilityto entrap lipophilic guest molecules. The length of the hydrophilicblocks can increase the CMC and decrease the aggregation number,thus resulting in a decrease in the micelle loading efficiency. Anincrease in temperature, or the presence of lipophilic functions (e.g.aromatic ring) on the block-copolymer, on the other hand,enhances this loading efficiency [176].

The literature provides examples inwhich drug solubilization inmixed micelles considerably increases the loading rate. InRef. [177], for instance, the encapsulation of paclitaxel is twicehigher when egg phosphatidylcholine (ePC) is added to a poly(ethylene glycol)-phosphatidyl ethanolamine conjugate (PEGePE)based micelle composition. Such a strategy can be useful when thepolymers do not exhibit a large hydrophobic domain [178], which isthe case when ePC is present.

6.5. Long-circulating micelles pharmacokinetics

Blood pharmacokinetics of radiolabeled polymeric micelles hasbeen largely reported in literature [179e184]. A micellar suspen-sion is a thermodynamically stable state involving the establish-ment of equilibrium between single polymer molecules (at CMC)and the micelles. This has a significant effect on the in vivo phar-macokinetics [162]. The clearance of polymeric micelles in bloodusually shows a biphasic profile with an initial fast distribution inthe first 1 or 2 h, followed by a slower elimination phase. Forexample, iodine-containingmicelles formed fromMPEG-iodolysinecan stay several hours in the blood pool in rats and rabbits[166,169]. These micelles gradually accumulate in the liver andspleen. The half-life observed for themicelle elimination profile canvary from 2 h to a much longer period, up to around 90 h. This largerange of possible kinetics, as well as the elimination route (mainly

F. Hallouard et al. / Biomaterials 31 (2010) 6249e62686258

urinary and/or hepatic), are in fact closely related to the nature ofthe polymer-forming micelles, depending on the chemical natureof the polymer, and also on the amphiphilic properties such as theratios between hydrophobic and hydrophilic chains or the micellesize. For instance, micelles formedwith poly(D,L-lactide)-block-poly(ethylene oxide) (PLA-b-PEO) (PLA/PEO ¼ 5000/5000 Da) mainlyinduce a hepatobiliary excretion [179] while micelles formed withPLA-b-PEO (PLA/PEO ¼ 2000/3000 Da) are eliminated by thekidney [180]. Finally, the limits of the micelle size are defined (i) forthe lowest by the width of fenestrated capillaries which is 10 nm,and (ii) for the highest, around 250 nm, given that bigger particlesare actively cleared by phagocytosis. Precise control of the micellesize is also useful to prevent their penetration across physiologicalbarriers [38].

6.6. Long-circulating micelles toxicology

As regards the toxicology of micelles, several studies haveshown that encapsulating drugs in polymeric micelles clearly bothreduces adverse effects and preserves drug activity. This has beenobserved, for instance, on micelles composed of block-copolymersPEGePLA or PEG-poly(aspartic acid), entrapping active guestmolecules like paclitaxel, cisplatin or doxorubicin [180,185e189].

The in vitro evaluation of PEGePLA micelles emphasizes a celllysis in L-929mouse fibroblast cells [180], but without hemolytic orgenotoxic effects. The in vivo evaluation of these micelles in micedid not disclose intracutaneous reactivity and experiments in ratsinvolving weight assessment, a complete blood cell count, serumchemistry and major organ histology did not highlight evidence ofsystemic toxicity. In vivo, formulations of cisplatin-containing PEG-poly(aspartic acid) micelles present a similar antitumor activity anda lowered nephrotoxicology when compared to cis-Diamminedi-chloroplatinum (II) (CDDP) against MKN 45 human gastric cancer(even if the in vitro activity of the former is significantly reducedcompared to CDDP, i.e. around 10e17%). This is due to a betteraccumulation of cisplatine in solid tumors when it is encapsulatedin micelles [186].

7. Dendrimers

7.1. Definition

Dendrimers are polymeric complexes composed of a series ofpolymeric branches linked together by an inner core (Fig. 7)[190,191]. Dendrimer structures are well-controlled and well-defined, potentially exhibiting a high functionality and a lowpolydispersity (in spite of their large molecular mass). A typicaldendrimer is composed of three different topological parts: (i) Afocal core which can encapsulate various chemical species. (ii)Several internal layers (called generations) composed of repeatingunits surrounding this core, which can also solubilize various smallguest molecules (like clinical contrast agent). (iii) A multivalentsurface forming the external part which can potentially be func-tionalized to create specific interactions with the surroundingenvironment. This surface defines the dendrimer’s macrocospicproperties [192,193].

In literature, two examples of blood pool contrast media areformulated with dendrimers. The first one is a polyamidoamine(PANAM) molecule exhibiting 4 generations and a water-solublepart (triiodo amino acid) grafted onto the surface [194]. The secondone is a dendrimer composed of iodinated poly-L-lysine [191].

Compared to traditional linear, branched or cross-linked poly-mers, dendrimers exhibit unique chemical properties and a predi-cable three-dimensional architecture [195]. When their structurescomprise several generations, the external surface becomes so

dense that the focal core is totally isolated from the outer envi-ronment. Thus, the morphology of dendrimers with 4 or moregenerations presents a core/shell structure, with a highly congestedsurface forming a polymeric capsule, and a free-space core. Den-drimer synthesis is a step-by-step process (see below), whichallows site-selective functionalization and therefore total control ofthe dendrimer solubility. In fact, the solubility depends essentiallyon the peripheral functionalities. A final word to mention thatdendrimers composed of an apolar core and a polar shell are calledunimolecularmicelles and that, unlikemicelles, their stability is notdependent on their concentration [196].

7.2. Dendrimers production

There are two strategies for dendrimer synthesis, namely the“divergent approach” and the “convergent approach”.

The “divergent approach”was introduced in the 1980s [197,198]and consists of a stepwise, layer-by-layer synthesis from the focalcore towards the molecule periphery. It involves two kinds of basicoperations: (i) grafting the building blocks and (ii) deprotecting theend-functionalities of the periphery to create a new reactivesurface. This process is repeated until the desired number ofgenerations is obtained. However, in the fabrication of large den-drimers, the “divergent approach” proves to be limited, intrinsicallydue to the own synthesis process. In fact, increasing the number ofgenerations causes an exponential increase in surface functional-ities. As a result, the newly added homogeneous generationsbecome difficult with high generation dendrimers. Consequently,separating the molecules for final use from the other byproducts(i.e. thosewithout the required number of generations) proves to bequite difficult since their molecular weights are very similar.

The “convergent approach” consists in building the macromol-ecule from the part which will ultimately become its external shell.This time, the number of reactions decreases exponentially, as dothe reaction steps [199]. Compared to the “divergent approach” thepurification step in this process appears easier due to (i)the reduced number of resulting byproducts and (ii) the fact thatthe difference in molecular weight between the final dendrimersand the byproducts is now larger. However the reactive grouplocated at the focal point can be covered as the number of gener-ations increases, thus also raising steric congestion. A final remark:the reaction time for generating dendrimers using the divergentapproach is significantly longer than for the convergent approach.

Alternative preparation methods have also been developed,such as the so-called “double-stage convergent growth approach”[192] or the “double-exponential dendrimer growth approach”[200], with the aim of reducing the number of syntheses andpurification steps, thus increasing yields. One of these alternativeapproaches, known as “orthogonal-coupling”, even proposesa means of synthesis which does not require activation or depro-tection steps to add each new generation [201].

As regards the synthesis of dendrimers for active targeting, theliterature provides examples of targeting moieties grafted orintroduced onto the dendrimer periphery [202e205]. Activecompounds (e.g. contrast agents or drugs) are incorporated intodendrimers using the 3 following techniques [190]: (i) encapsu-lating guest molecules in the “empty” core of dendrimers usinga simple diffusion process (incubation) [206e210]; (ii) forminga network between dendrimers and active molecules [211]; (iii)linking the active ingredients directly onto the dendrimer surface[191,212,213].

Iodinated dendrimers for CT imaging are synthesized using the“divergent approach” [191]. The dendrimers are built with a PEG-core (which gives them an elongated shape), and with nonioniccontrast agents (iobitridol) covalently fixed onto the surface.

Fig. 7. Structure of an iodinated dendrimer used as a blood pool contrast agent, from [191].

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However, when compared to the other blood pool contrast agents,a huge drawback of such a system is the production time. Thecoupling reactions, for instance, can take from 72 up to 120 haccording to the generation.

7.3. Dendrimers stability

Dendrimer stability depends on (i) the nature of the dendrimersand (ii) the presence of a coating layer on the dendrimer surface.Poly(propyleneimine) (PPI) dendrimers are resistant to prolongedheating, solvent extraction or sonication [211], but seem unstable inthe presence of light [210], whereas iodinated poly-L-lysine den-drimers are stable enough to be sterilized in an autoclave with nosign of degradation [191]. In fact, in experimental and thermody-namic conditions, each individual system has its own particularstability. Formulations of coated dendrimers have a higher stabilitythan non-coated ones [214]. However, dendrimer stability (coatedor not) was enhanced in storage temperatures of around 2e8 �C, asshown by Bhadra et al.

7.4. Dendrimers encapsulation properties

Dendrimer encapsulation properties depend on (i) the nature ofthe polymer (ii) the shape of the dendrimer (iii) the number ofdendrimeric generations (iv) the presence and length of thePEG-core, (v) the coating of the dendrimers and (vi) the experi-mental conditions.

Dendrimers with a limited amount of generations present“open” and amorphous structures, whereas the ones withnumerous generations adopt a spherical conformation, capable ofincorporating drugmolecules [196]. In this case the domains able tosolubilize or induce complexation of guest molecules are enlargedinside the particle. Thus, the encapsulation efficiency is improvedand we can observe an increase in the dendrimeric generations[214]. An illustration is provided by the dissolution of pyrene indendrimers made of 4,4-bis(40-hydroxyphenyl)pentanol buildingblocks, which increases with the number of generations [215].Release rates are also influenced by the number of dendrimericgenerations. An example is provided with the solubilization ofpaclitaxel in poly[oligo(PEG)] dendrimers [216]. However, thisphenomenon also depends on the chemical nature of the blockscomposing the macromolecule, since the opposite effect, accom-panied by a decrease of the initial burst release has been observedfor poly-L-lysine peptide dendrimers with a PEG amine core [214].

The nature and molecular weight of the core also play a signifi-cant role in the encapsulation and release properties. PEG-coredendrimers exhibit a structural fluidity which facilitates thespreading and loading of drug molecules in their core (in compar-ison with other polymers) [214]. The encapsulation rate is alsoprolonged as the PEG-core molecular weight is increased due to thehigh availability of polymer groups for complexation, increasing thehydrotropic solubilizing features of dendrimers.

Finally, as regards the specific role of dendrimer coating, itappears that the nature and the molecular weight of the polymer

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blocks used significantly influence the release properties. Theencapsulation rates and release kinetics are respectively improvedand slowed down in PEG or chondroitin sulfate A (CSA) coateddendrimers as opposed to non-coated dendrimers [206,214,217].This result is attributed to the increase of steric hindrance andviscosity at the dendrimer surface [214], acting on the moleculartransfer at the dendrimer periphery. This phenomemon is nothowever a rule, since dendrimers coated with PEG-2000 areeffectively more efficient than similar dendrimers coated with PEG750, but dendrimers coated with longer PEG, like PEG-5000 appearless efficient than PEG-2000 dendrimers [218]. This effect increaseswith the molecular weight of the PEG, and has been attributed tothe PEG shell disruption due to the interpenetration of PEG chainsbetween adjacent dendrimers.

Other physico-chemical parameters can actively play on therelease kinetics, such as for instance the temperature on PPI den-drimers [210]. The mechanism proposed by Gajbhiye et al. isexplained by relating the premature release induced by tempera-ture with conformational changes, which creates openedstructures.

Dendrimers exhibiting particular structures, such as “coreddendrimers” resembling hollow nanospheres, are derived fromnative dendrimers by a post-synthetic removal of the core from thedendritic architecture [219,220]. The structural integrity of thedendrimers is maintained by cross-linking the peripheral surfacegroups. However, the loading process of active guest moleculeswithin these structures remains unclear, though it may be achievedduring the synthesis stage [211,221]. As a result, the very dense(cross-linked) surface shell prevents drug leakage and in fact therelease begins with the degradation of the dendrimer itself.

Finally, it is worth noting that unimolecular micelles havea limited encapsulation rate, because of their solubilizationcapacity, reduced due to the conformational collapse of thehydrophobic core in water [196]. This phenomenon can be pre-vented by reinforcing the structures with rigid linkages, whichfinally maintain the hydrophobic cavities within the dendrimers, inturn dispersed in an aqueous medium.

7.5. Long-circulating dendrimers pharmacokinetics

Dendrimer pharmacokinetics depend not only on their stabilityand encapsulation properties, but also considerably on their nature,structure, the number of generations and the peripherical layer. Forinstance, the pharmacokinetics of polyamidoamine (PANAM) den-drimers depend on (i) the surface potential of the dendrimersurface. Hence, the blood clearance of cationic PANAM dendrimersadministered intravenously to Wistar rats is significantly shorterthan that of anionic PANAM dendrimers. (ii) the number of den-drimeric generations [222], which also plays a role in stealthproperties: the fewer the number of generations, the better thestealth properties. The pharmacokinetics can also be improved bya specific grafting of folic acid on the dendrimer surface [204].Finally, these PANAM dendrimers are eliminated by different routesdepending on the number of generations. From 3 to 5.5 genera-tions, there is an accumulation in the liver. For 7 generations andmore, elimination is by urinary excretion [223]. As a result, the half-time of 5-generation PANAM dendrimers is around 1 h [222],whereas similar 5-generation PPI PEGylated dendrimers can takeup to 8 hours [210], and accumulate in the liver, kidneys, lung andspleen.

When iodinated dendrimers are injected in rats, an intravas-cular CT enhancement occurs, lasting for at least 32 min [191]. Thedendrimer concentration follows a monoexponential kineticpatternwith a half-time in the blood pool of approximately 35min.The estimated volume of distribution is 13%, an intermediate value

between a normal extracellular fluid volume (about 2) and theaccepted normal intravascular volume (about 5%). However, theelimination route remains unclear.

7.6. Long-circulating dendrimers toxicology

In vitro experiments repeatedly reveal that the short-termtoxicology of large dendrimers depends mainly on (i) the nature oftheir surface functionalities and (ii) the number of dendrimergenerations [210,223,224].

The first factor to consider is the dendrimer surface potential,which influences the cytotoxicity and thus the haematoxicity [222].Hence, in the case of PANAM dendrimers, a positively-chargedsurface will cause significantly higher cytotoxicity compared toa negatively charged one [225]. This can be explained by thedisruption of the cell membrane inducing cell lysis caused by thespecific interaction of cationic dendrimers with the phospholipidicbilayer. PANAM dendrimers also exhibit lower toxicity than themore flexible amino-functionalized linear polymers due to a loweradherence of the rigid globular dendrimers to globular surfaces[226]. The type of amine functionality is also important. Thus,primary amines are more toxic than secondary or tertiary amines[195].

Finally, the water-soluble PEG-functionalized dendrimersgenerally have a prolonged circulation time in the body, mainly dueto a lower sequestration or RES uptake. This reduced interactionwith the organism, in addition to reduced surface charges (due tothe nonionic polymers), also results in generally reduced cytotox-icity [225].

8. Others polymeric nanoparticles

8.1. Definition

Nanospheres and nanocapsules are the most common nano-metric formulations of polymeric molecules. Nanospheres are solidmatrix particles and nanocapsules present a core/shell structure, inwhich the surrounding shell has a solid polymeric structure and thereservoir core can be a liquid or semisolid core at room temperature[227]. Depending on the hydrophobicity of their core, nanocapsulescan solubilize both hydrophilic and lipophilic compounds.

To date, the literature provides two formulations of nano-particles, i.e. nanocapsules [228] and nanospheres [229], whichwere used as blood pool contrast agents (see below). The nano-capsules encapsulate an iodinated oil (Lipiodol�) in a PEG-Pluronic� membrane, and the nanospheres are directly composedof an iodinated polymer having presenting 58 wt% of iodine.Schematic representation of polymeric nanoparticles used as bloodpool contrast agents is presented in Fig. 8.

8.2. Polymeric nanoparticles production

A wide variety of polymeric nanoparticle-generating processesexist and can be divided into two groups: (i) the two-step methodsand (ii) the one-step methods. As this technology is already widelyand comprehensively described in literature [150,230e232]. Wehave restricted ourselves here to providing a simple, global over-view of the formulation processes.

8.2.1. The two-steps methodsTwo-step methods consist of a nano-emulsification step fol-

lowed by polymeric nanoparticle formation. The nano-emulsionformulation processes are similar to those described above (cf:Nano-emulsion production). The second step of polymeric particlefabrication is achieved using these means: (i) polymer nano-

Fig. 8. Schematic representation of polymeric nanoparticles used as X-ray contrastagent. Contrasting compounds (stars) can be either adsorbed onto the nanoparticlesurface, or encapsulated in its core.

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precipitation (ii) polymer gelation or even (iii) in situ polymersynthesis within the nanodroplets.

The polymer precipitation. Polymer precipitation is induced byremoving the polymer solvent from discrete nano-emulsiondroplets, obtained by (i) solvent evaporation, (ii) solvent diffusiontowards the bulk phase or (iii) reverse salting-out. The firstexperimental condition required in the solvent evaporationprocess is, of course, the use of a volatile solvent like dichloro-methane or chloroform as a polymer solubilizing medium [227].Ethyl acetate is also used as it has a better toxicological profile[227], even though its evaporation is slower. For instance, it takesabout 120 min to evaporate 10 mL of ethyl acetate in an emulsionof 50 mL [233]. This emulsification-solvent evaporation methodhas been largely used in literature to prepare nanoparticles madeof poly(lactide) (PLA), poly(lactide-co-glycolide) (PLGA) and poly(caprolactone) (PCL) [227,234], and also in the formulation ofnanoparticles built with amphiphilic copolymers like PEGePLA,PEGePLGA, PEGePCL, PEGePACA and polysaccharideePCL[235e238]. In the latter case, surfactants can be used to replaceamphiphilic copolymers.

Regarding the fabrication of nanoparticles by the emulsifica-tion-solvent diffusion method, the fundamental property ofa solvent is its partial miscibility with the bulk aqueous phase aswell as the ability to solubilize polymers [239]. The aqueous phaseused in the preparation of the o/w emulsion template is firstsaturated with the solvent (identical to that present in the organicphase solubilizing polymer) and the solvent present in the organicphase is in turn saturated with water. The nano-emulsion thusformed is then diluted with an extensive amount of pure water,which results in solvent displacement (from the dispersed towardsthe continuous phase), leading to discrete polymer nano-precipi-tation within the nano-emulsion droplets. Several solvents meetthese specifications, such as ethyl acetate, benzyl alcohol or iso-propyl acetate. The polymers generally used are PLA, PLGA and PCL[239e241]. In the nanocapsule formulation process, a small amountof oil is mixed in the organic solvent/polymer phase [242,243]. Themean diameter and polydispersity of nanoparticles and nano-capsules produced by this method are reduced by the stirring rateand the concentration of stabilizing agents [227]. Increasing thepolymer concentration in the emulsion droplets on the other hand,will increase the nanoparticle size [244]. The viscosity, pH of thebulk and volumic fraction between the dispersed and bulk phasesonly have a limited influence on the size and polydispersity.

Finally, as regards the emulsification-reverse salting-outprocess, the solvent needs to be totally miscible in water. The

solvent generally used is acetone [245,246]. The method consists inreducing the solvent miscibility with the aqueous bulk phase, bydissolving (in the water bulk) small molecules at a high concen-tration (several mol/L). Compounds used may be sucrose or salts,such as magnesium chloride, calcium chloride or magnesiumacetate. They induce an increase of the chemical potential ofacetone in water (thereby reducing its solubility). The nano-emulsion is then formulated, that is, (solventþ polymer) dispersedin (water þ sucrose or salts). Polymer nano-precipitation is ach-ieved by the dilution of the emulsion with a large excess of water.This phenomenon is called the “reverse salting-out effect”, as waterdilution inhibits the salting-out effect and allows the solventdiffusion towards the bulk phase. The last step of the process is thepurification of the nanoparticle suspension by eliminating thesolvent and salting-out agent. This method is used to encapsulatelipophilic drugs pre-incorporated in the polymer solution[247,248].

The polymer gelation. The gelation of polymers dispersed inemulsion droplets is a method which provides nanoparticulate gelsuspensions. The method is in fact strongly related to the nature ofthe polymer, i.e. the mechanism of gelation. With polymers likeagarose, for instance, dropping the temperature induces gelation.The agarose suspension is nano-emulsified at a high temperatureand nanoparticles are obtained by cooling down the system [249].Other gelation mechanisms are also widely used. Polymers likealginate or pectin, for instance, are made into a gelled structure bycreating a complex chelation using salt or by modifying the pH ofthe solution. Nanoparticles are obtained by mixing two differentemulsions, one containing droplets loaded with polymers and theother the gelling or the pH controlling agent [227].

The polymer in situ synthesis. A general experimental procedureis often used for the formulation of polymeric nanospheres by insitu polymerization (like in the case of polymeric nanospheres[229]). It consists in formulating the nano-emulsions (exclusivelyby high energy methods) made of monomer droplets taken ashydrophobes, stabilized with surfactants and dispersed in theaqueous phase [150]. This emulsification step is followed by poly-mer synthesis, principally described as the radical polymerizationof droplets. An initiator can be added in the aqueous phase, chosenfor its partial solubility in both the hydrophobic phase as well as thehydrophobe droplets and activated by raising the temperature,using UVs, ultrasounds, or even enzymes [150]. Other polymeri-zation mechanisms are sometimes used in the fabrication of poly-meric nanospheres, such as non-radical polymerization, includingpolyaddition, anionic polymerization and metal catalyzed poly-merization reactions [150].

Regarding the formulation of nanocapsules by in situ polymer-ization, it is worth noting the adaptability of the process of nano-sphere formulation, given that the polymerization reaction is notdependent on the nano-emulsification procedure chosen. Threemethods of formulation are adopted, whereby the monomer isintroduced (i) in the continuous phase, reacting with the materialconstituting the droplets, (ii) in the droplets, either reacting withthe continuous phase or with an initiator added in the continuousphase and (iii) in both phases reacting together, creating an inter-facial polymer film. Comprehensive reviews can be found in theliterature, describing these processes in great detail [150,230].

8.2.2. The one-step methodsThe nano-precipitation of a polymer. The method consists in

inducing the precipitation of preformed polymers in the form ofnanoparticles. The polymers are homogeneously solubilized ina solvent beforehand. In principle, the polymer solvent has to befully miscible in the polymer non-solvent (generally acetone andwater respectively). Therefore, when the organic phase solubilizing

F. Hallouard et al. / Biomaterials 31 (2010) 6249e62686262

polymer is poured into the aqueous phase, the resulting solventmixture is unable to solubilize the polymer and nano-precipitationoccurs. The solvent is then removed by evaporation [250e252] andthe resulting nanoparticles have a well-defined size with lowpolydispersity. Critical factors of this formulation process are themiscibility of the organic solvent with the non-solvent and also thenature of the polymer/solvent interactions [252]. The surfactantnature of polymers plays a significant role in the process, increasingits yield. Thus, this method is well adapted for the formulation ofparticulate drug nanocarriers with amphiphilic copolymers, thedrug being added in the polymer organic phase before the forma-tion of nanoparticles [253e255].

The methods based on ionic gelation. This method consists of thegelation of a charged polysaccharide (e.g. chitosan) in the presenceof oppositely charged small molecules (e.g. tripolyphophates). Thepolymer gradually forms nanoparticulate complex clusters. Inaddition, the presence during this process of PEO or polyethyleneoxide polypropylene oxide (PEOePPO) confers to these objectsa specific PEO coating, which increases particle stability and theirstealth properties in the blood pool [256,257]. The resultingnanoparticle properties, i.e. size, polydispersity and stability aredirectly linked to the molecular weights of the differentcomponents.

8.3. Polymeric nanoparticles stability

The stability of polymeric nanoparticles depends on (i) thenature of the polymer and the formulation method (ii) the nano-particle morphology, such as the presence of surface coating, and(iii) the sterilization method used.

In nano-precipitation methods, surfactants are generally used topreserve nanoparticle suspensions from aggregation over longstorage periods [250]. The stability of nanoparticles formed by ionicgelation, e.g. chitosan nanoparticles, can be sensitive to composi-tion factors such as the concentration of KCl or pH which, near theisoelectric point, leads to particle disintegration [258].

The effect of the presence of PEG on the nanoparticle surfaceseems also to depend on the nature of the polymer. The degrad-ability in serum of poly(hexadecylcyanoacrylate) particlesincreases when they are PEGylated [253] whereas PEG-poly(hydroxybutyrate) (PEGePHB) copolymers have a better thermalstability than PHB polymers [259].

If the nature of the polymer and the formulation process areimportant, the same is true for the purification and sterilizationsteps, which play a significant role in particle destabilization. Forinstance, purification by high-speed centrifugation can increase theparticle size due to aggregation [75]. The autoclave can also impacton the size of nanoparticles and/or catalyze chemical reactionsbetween polymers and the other constituents [260]. An example isprovided by poly(isobutylcyanoacrylate) (PBCA) nanocapsules forwhich the size increases from 200 to 500 nm after the sterilizationstep, attributed to the swelling of the polymeric membrane or tothe expansion of the oily phase [261]. Sterilization by gammairradiation provides the nanoparticles with a large amount ofenergy which helps induce the fragmentation of covalent bondsand as a result produces free radicals, thus modifying the nano-particle properties and/or forming toxic compounds [258,262,263].However, this phenomenon depends on the nature of the polymer.Gamma irradiation induces cross-linking reactions between PCLchains [227] as well as PLA chain scissions [264], but does not alterPBCA nanoparticles. On the other hand, sterile filtration and highhydrostatic pressure treatment do not induce physical damage onpolymeric nanoparticles [265,266].

Nanoparticle storage is optimal in a dried form, in order toprevent microbiological contamination, polymer degradation by

hydrolysis, aggregation and sedimentation [267]. For instance, it isthe case with the iodinated nanocapsules formulated by Ho Konget al. presented above [228] which are lyophilized before storage.Suspension drying is performed by lyophilization or spray-drying.For lyophilization techniques yielding a high concentration ofnanoparticles in the final dried product, the presence of cryopro-tectants like trehalose or mannose helps reduce destabilization dueto freezing, drying stress and aggregation [268]. The spray-dryingprocess is better adapted to heat-sensitive molecules, the maindrawback here being the redispersion of the powder [269]. Afterlyophilization or spray-drying, nanoparticles should be stored ata temperature below the polymer glass transition temperature, toprevent nanoparticle aggregation [227].

8.4. Polymeric nanoparticles encapsulation properties

Here again, the potential encapsulation properties of polymericnanoparticles depend on (i) the nature and shape of the polymer(ii) the formulation method and (iii) the stability of the particles.

Active compounds can be entrapped within the nanoparticles oradsorbed onto their surface. Accordingly, they help encapsulateeither lipophilic or hydrophilic molecules, covalently associatedwith the core or not. For example, Lipiodol, an iodinated oil, wasencapsulated in PEG-Pluronic� nanocapsules at a rate of 82% [228].Lipiodol was not released from these nanocapsules for at least 3days. However, in the case of aqueous nanocapsules, small mole-cules escape very rapidly. The strategy used to counter this draw-back is the association within the nanocapsule core of these smallmolecules, with an oppositely charged polyionic compound [270].

Others factors can influence encapsulation properties and areclosely related to the formulation methods. In the ionic gelationmethod, the encapsulation properties mainly depend on thestrength of the association between the compounds and the poly-saccharides [271]. In interfacial polycondensation reactions, thethickness and porosity of the nanocapsule wall are correlated withthe monomer concentrations and molecular weight, thus influ-encing drug loading and release [272e275].

8.5. Long-circulating polymeric nanoparticles pharmacokinetics

In order to increase the blood circulation of polymeric nano-particles, the surface treatment of these particles with the co-incorporation of PEG is more efficient than the external surfaceadsorption method [37].

The PEG content in the copolymers greatly influences theamount of phagocytosis in vitro of nanoparticles [276]. An illus-tration is provided by the intravenous administration of PLAePEGand PLGAePEG nanoparticles, which remain in the systemiccirculation for hours, whereas PLA and PLGA nanoparticles areremoved from the bloodwithin a fewminutes [277]. In cases wherenanoparticles are formed from a PLGA/PLGAePEGmixture, the rateof nanoparticles remaining in the blood circulation rises with theincrease of the PLGAePEG content [278,279]. After incubation withserum, PEGePLGA block-copolymer nanoparticles stay longer inthe blood pool than identical non-incubated ones [280].

Chitosan nanoparticles are a further illustration of poly-saccharide nanoparticles, for which the main polymer used isinsoluble inwater at a neutral pH (7). Some chitosan derivatives areformulated to obtain stable water-soluble chitosan nanoaggregates.Glycol-chitosan nanoaggregates, for instance, are maintained in theblood pool at a high level for 3 days in spite of their 250 nm size[281]. In tumor-bearing rats, these aggregates are distributedmainly in the kidney, liver and tumors.

There are further examples of nanocapsules with a PEG-Pluronicshell encapsulating an iodinated oil, that remain in the blood

F. Hallouard et al. / Biomaterials 31 (2010) 6249e6268 6263

stream 4 h after intravenous administration. The nanocapsulesaccumulate in the spleen and liver, due to the RES uptake, probablyas the result of protein adsorption onto the polymer capsule surfaceleading to an increase in size. These polymeric nanoparticles arecompletely eliminated 72 h after administration [228].

8.6. Polymeric nanoparticles toxicology

The toxicology of polymeric nanoparticles is closely linked tothat of the polymer used and thus to its biocompatibility andbiodegradability. Biocompatible polymers (i) should have anacceptable shelf life (ii) can be cleared from the body and shouldhave non-toxic degradation products if metabolized. These twopoints are fundamental and justify the fact that, in spite of themultitude of polymers available today, only a limited number canbe used as blood pool contrast agents: these mainly being PLA,PLGA PCL or PHB.

The cytotoxicity of polymeric nanoparticles depends on severalfactors. Polymers are potentially toxic, but the presence of PEG onthe surface of PLGA or PHB nanoparticles for instance, diminishestheir hematotoxicity [282,283]. Furthermore, the proportion of PEGin PEGePLGA or PHBePEGePHB nanoparticle copolymers can havea significant effect on hemolysis. Hence, polymer biocompatibilityis enhanced with both the amount of PEG (50e85 wt%) inPEGePLGA copolymers and the length of PHB segments in thePHBePEG-PHB blocks. Note also that the concentration of nano-metric particles can affect the hemolysis rate and hemostasis asobserved with PHBePEGePHB and PEGePLGA nanoparticles. Thus,during hemostasis, the increase in concentration of PEGePLGAnanoparticles reduces the clotting time and the strength of thefibrin clot. The prothrombin activity is also decreased witha concentration of 560 mg/mL of uncoated PLGA nanoparticles[282,283].

As for iodinated nanocapsules used as CT contrast agents(composed, as explained earlier, of a polymer shell of PEG cross-linked with Pluronic�), their in vitro toxicology is very similar tothat of iopromide, a clinical contrast agent [228].

9. Conclusion

Over the past few years, major progress in the formulation ofnano-delivery systems hasmade it possible to develop new types ofcontrast agents. Because of their long residence time in the bloodpool, these contrast agents can be used to study or monitor (i)vascular diseases such as pulmonary embolism or atherosclerosisor (ii) pathologies in which the blood flow plays a crucial role: solidtumors, for instance. The main advantages of using CT for thesediseases are its high-resolution and short waiting time. However,these contrast media are still too new to provide a complete eval-uation of their toxicity.

Appendix. Supplementary information

Supplementary material associated with this article can befound in the on-line version at doi:10.1016/j.biomaterials.2010.04.066.

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