Pathogen-mimetic stealth nanocarriers for drug delivery: a future possibility

BASIC SCIENCE

Nanomedicine: Nanotechnology, Biology, and Medicine7 (2011) 730–743

Review Article

Pathogen-mimetic stealth nanocarriers for drug delivery:a future possibility

Miguel Cavadas, MSca, África González-Fernández, MD, PhDb, Ricardo Franco, PhDa,⁎aREQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal

bImmunology, Biomedical Research Center (CINBIO) Universidade de Vigo, Campus Lagoas Marcosende, 36310, Vigo, Pontevedra, Spain

Received 29 September 2010; accepted 18 April 2011

nanomedjournal.com

Abstract

The Mononuclear Phagocyte System (MPS) is a major constraint to nanocarrier-based drug-delivery systems (DDS) by exerting anegative impact on blood circulation times and biodistribution. Current approaches rely on the protein- and cell-repelling properties of inerthydrophilic polymers, to enable escape from the MPS. Poly(ethylene glycol) (PEG) has been particularly useful in this regard, and it alsoexerts positive effects in other blood compatibility parameters, being correlated with decreased hemolysis, thrombogenicity, complementactivation and protein adsorption, due to its uncharged and hydrophilic nature. However, PEGylated nanocarriers are commonly found in theliver and spleen, the major MPS organs. In fact, a hydrophilic and cell-repelling delivery system is not always beneficial, as it might decreasethe interaction with the target cell and hinder drug release. Here, a full scope of the immunological and biochemical barriers is presentedalong with some selected examples of alternatives to PEGylation. We present a novel conceptual approach that includes virulence factors forthe engineering of bioactive, immune system-evasive stealth nanocarriers.

From the Clinical Editor: The efficacy of nanocarrier-based drug-delivery systems is often dampened by the Mononuclear PhagocyteSystem (MPS). Current approaches to circumvent MPS rely on protein- and cell-repelling properties of inert hydrophilic polymers,including PEG. This paper discusses the full scope of the immunological and biochemical barriers along with selected examples of alternativesto PEGylation.© 2011 Elsevier Inc. All rights reserved.

Key words: Nanoparticles; Drug delivery; Nanocarriers; Stealth; Virulence factors

In drug- and gene-targeted delivery using intravenousnanocarriers, nanoparticles (NPs) may encounter several specificbarriers that might prevent them from reaching the intended target(Figure 1). The Mononuclear Phagocyte System (MPS) consistsof blood and tissue cells with the capacity to uptake foreignparticles,1 therefore limiting the amount of nanocarrier payloadthat reaches the target. The current stealth technologies used tomake NPs unrecognizable by MPS only delay the inevitableuptake by this system.2 Therefore the discovery of efficient waysto prolong NP circulation times by reducing MPS-mediatednanocarrier clearance is of major importance to nanomedicine.2

This work was supported in whole or part by projects “Inbiomed” (2009/063) funded by Xunta de Galicia, Spain, “Immunonet” (SOE1/P1/E014)funded by SUDOE-Feder, Spain; and FCT/MCTES (REQUIMTE and GrantPTDC/QUI/64484/2006) and Luso-American Foundation (FLAD), Portugal.

⁎Corresponding author: REQUIMTE, Departamento de Química,Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa,2829-516 Caparica, Portugal.

E-mail address: [email protected] (R. Franco).

Please cite this article as: M. Cavadas, Á. González-Fernández, R. Franco, PatNanomedicine: NBM 2011;7:730-743, doi:10.1016/j.nano.2011.04.006

1549-9634/$ – see front matter © 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.nano.2011.04.006

Adsorption of plasma proteins and complement-systemactivation by NPs result in enhanced opsonophagocytic uptake,but they also present other specific problems. Interactions of NPswith blood proteins, which have specific trafficking signals, candivert NPs from their intended targets. On the other hand, NP-blood protein interactions might also present an opportunity for amore effective payload delivery as, e.g., apolipoproteins,involved in the metabolism of cholesterol and other lipids, arecommonly adsorbed on NP surfaces, allowing NPs to potentiallyutilize surface receptors for apolipoproteins to enter the cell.3

Complement activation, which has been observed with somenanocarriers, has the potential to induce leukocyte chemotaxesand local inflammatory reactions. Hemolysis and thrombogeni-city are two additional NP-blood interaction-related problemsthat have been observed in some systems. Therefore, the idealintravenous nanocarrier should be nonhemolytic, nonthrombo-genic, noncomplement activating and invisible to the immunesystem (“stealth”). Nonspecific interactions with plasma pro-teins should also be avoided because these might routenanocarriers away from their targets. Testing for all these

hogen-mimetic stealth nanocarriers for drug delivery: a future possibility.

mailto:[email protected]

http://dx.doi.org/10.1016/j.nano.2011.04.006

http://dx.doi.org/10.1016/j.nano.2011.04.006

Figure 1. Constraints for the intravenous use of nanocarrier DDS.

731M. Cavadas et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 730–743

blood compatibility parameters has been recommended asstandard procedures in preclinical trials1,4,5 and the interna-tionally recognized standard ISO-10993 recommends several invitro tests to examine the hematocompatibility of medicaldevices in relation to hemolysis, thrombogenicity (includingeffects on platelets) and complement activation.6

Until now, NPs have been generally investigated in terms oftheir physicochemical properties, drug-loading ability, in vitrotoxicity and relatively simple in vivo tests. However, otherimportant issues, such as the specific interaction of these NPswith human organs, tissues, cells, biomolecules or their effect onhuman metabolism have not been properly addressed. Furtherstudies, analyzing specific responses induced by bioactivenanomaterials instead of nonspecific responses, are requiredfor the wider application of NPs in drug delivery.7

Polyethylene glycol (PEG) is a hydrophilic and unchargedlinear polyether diol [HO–(CH2–CH2–O)n–H where n is thedegree of polymerization].8-10 The coating of NP surfaces with alayer of hydrophilic polymers is a strategy to avoid nonspecificinteractions between NPs and blood proteins or cells, thereforedecreasing the clearance levels by MPS cells11,12 that drive NPaccumulation in nontarget tissues. Thus, PEG molecules on thesurface of NPs can reduce the adsorption of opsonins and otherserum proteins by a mechanism known as the “stericstabilization”11 or “steric repulsion”12 effect. It has beenhypothesized that the mobile and flexible PEG molecules onthe NP surface could form a dynamic molecular “cloud” over theparticle inducing a repulsive effect, making it energeticallyunfavorable for proteins to adsorb to the PEG molecules.12

The capacity of PEGylated materials to circulate for longerperiods “under the radar” of or undetected by the body's defensemechanisms have led to the introduction of the term “stealth”

NPs, likened to “stealth bombers.” Thus, PEG is commonly usedin a wide range of intravenous human pharmaceutical formula-tions, and it has been so far perceived to be immunologically safe(although it can be antigenic), being eliminated in an intact formby the kidneys (for PEGs N 20,000 Mw).10

Intravenous nanocarriers can face challenges similar to thoseof blood-dwelling pathogens, especially in the field of immuneevasion. Virulence factors are pathogen molecules that enable amicroorganism to establish itself on or in a host of a particularspecies, enhancing its potential to cause disease.13 The use ofvirulence factors in nanocarriers might overcome some limita-tions of current stealthing technologies. The observation thatcertain pathogens like schistosomes might live for decades inhost blood vessels while exposed to the host's immune systemwithout being detected,14 provides a very good example ofspecific immune evasion.

The advantages of nanocarrier drug-deliverysystems (DDS)

One of the main reasons for the lack of efficacy of somecurrently available medicines is their poor delivery to theintended site of action. In addition, nearly half of all newchemically based drugs are insoluble or poorly soluble in water,a factor that also contributes to their lower efficacy. Both factorsmight lead to increased dosages, with the concomitant increaseof side effects.15 Bionanotechnology can help overcome theseobstacles. Improved solubility can be obtained with NPformulations that facilitate the absorption of insoluble com-pounds and also by using smaller particles featuring the activeingredient and improving the rate of dissolution.15 Another

Table 1

Parameters of NPs' blood compatibility

Parameter Generally observed behaviors of NPs

Hemolysis Positive surface charge and increased surfactantsurface character correlated with increasedhemolysis.23-27

PEG imparts neutral charge and hydrophiliccharacter to NPs; correlated with decreasedhemolysis.5,27

Thrombogenicity Negative surface charge correlates with increasedblood coagulation and platelet activation/aggregation.5,28

PEG imparts a neutral charge to NPs; correlatedwith decreased blood coagulation and plateletactivation/aggregation.5,29,30

Interaction withblood proteins

Neutral particles have slower protein surfacecoverage rates than charged particles.31

Hydrophobicity influences the kinetics andequilibrium of opsonization as well as theamount and identities of bound proteins; size,morphology, shape and surface curvatureinfluences the amount of bound proteins but notits identity.31

PEG imparts a neutral charge and hydrophiliccharacter to NPs; hydrophilicity and sterichindrance correlates with decreased proteinbinding.8,9,32-34

NP properties can affect protein conformation,which in turn can have an effect on the interactionof these proteins with cellular components.35,36

Complement activation Charged surfaces and larger NPs correlate withincreased complement activation.1,37,38

PEG imparts a neutral charge to NPs; correlates

732 M. Cavadas et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 730–743

advantage could be improved pharmacokinetics, with sustainedrelease profiles for longer times, increasing the patient'scompliance with drug regimes.16 For an improved biodistribu-tion, NPs can be combined with ligands (such as antibodies,peptides, carbohydrates) for targeted drug delivery, allowing abetter drug efficiency at lower dosages, translating into fewerside effects.1

As in the treatment of tuberculosis, in which very toxic drugsare used, targeted delivery and sustained release afforded byextended nanocarrier circulations times could improve patientcompliance with the drug regimes, as lower dosages could beused for an equivalent therapeutic efficiency.

Although drugs are usually of very small size and areusually inert to the immune system (with the exception ofallergic reactions), drug-NP complexes can be recognized bythe immune cells, due to their size and due to features that aresimilar to those of some pathogens. Thus, except when specificuptake of these complexes by immune cells is required (forinstance, in the development of vaccines), the ideal nanocarrier-drug complex should:

(i) ensure that the drug arrives and acts preferentially at theselected target, reducing systemic levels of the drug15

(ii) be stable in the complex biological environment withan extended blood circulation lifetime, maximizingdrug action1

(iii) be nontoxic to blood cellular components1

(iv) increase the solubility, and therefore the bioavailabilityof insoluble drugs15

(v) be invisible to the immune system.1,5,17
with decreased complement activation. Sterichindrance at NP surface avoids complementdeposition.1,8,34,38-40
Cellular uptake NP surface curvature, polymer size, polymersurface configuration (“loop” versus “brushlike”configuration), coating thickness and accessibilityof reactive groups influence complementactivation.1,38,40

Larger NPs are more rapidly internalized andhave a higher phagocytic index.1,38,41

Increased lipophilicity and surface charge, and alower aspect ratio correlate with increaseduptake.25,41-44

PEG imparting the NP with cell-repealingproperties has the ability to increase bloodcirculation time.8,16,28,45

Constraints for the intravenous use of nanocarrier DDS

The viability of a nanocarrier for intravenous applicationsdepends on how its characteristics affect several blood-compatibility parameters (Figure 1).5 The gap between mostendothelial cells is less than 2 nm and that restricts the NPs'ability to leave the circulation. However, some tissues havelarger gaps.18 The hydrodynamic diameters of long circulatingNPs should be engineered with the awareness that particles up to30 nm are likely to be excreted by the kidneys, NPs that aresmaller than 150 nm can potentially leave the circulation in theliver and NPs bigger than 200 nm are more likely to becometrapped in the spleen, due to its distinctive filtration system.More details on this issue can be found in other reports.19-22

Hemolysis

Intravenously injected NPs are likely to interact witherythrocytes prior to encounters with immune cells becausethey occupy a large blood-volume fraction.1 A NP that triggershemolysis may adsorb some of the released hemoglobin and/ormay adhere to cell debris, which in turn increases its eliminationby macrophages through phagocytosis (phosphatidylserine-mediated via scavenger receptor).1 Careful manipulation of theNP surface properties can circumvent this problem (Table 1).

Interaction with blood proteins

In many cases, cell adhesion to biomaterial surfaces ismediated by a layer of adsorbed blood proteins, such as immuno-globulins (Igs), complement products, vitronectin, apolipoproteinE, fibrinogen and fibronectin.35 The underlying surface-chemicalproperties influence the type, quantity and activity of theadsorbed proteins,31,35 and consequently will induce diversecellular responses depending on surface chemistries.35,46,47

A very large surface-to-volume ratio is one of the mostimportant characteristics of nano-sized materials. As the NP'ssurface starts to curve, there is an observed decrease in the degreeof protein coverage,48 potentially decreasing the affinity of some


proteins to a point where adsorption no longer occurs3,49 andleading to new opportunities of differential protein adsorption.

The concept of a protein corona has been established todescribe the structure that forms when NPs come into contactwith biological fluids.3,50 The NP-protein interactions are pairspecific, which means that for each NP there will be a range ofequilibrium constants (one for each protein) that represent thedifferent (and competitive) binding mechanisms that arepresent.3 The proteins will be associated with a particle as asoft ‘corona,’ rather than a solid fixed layer. Proteins at highconcentrations and with high association rates are expected tooccupy the surface of the NP initially, but later they could bereplaced by proteins at lower concentrations with slowerexchange rates and/or higher affinities.31,48 Thus, the entireprocess of competitive adsorption of proteins onto a limitedsurface is based on abundance, affinities and incubation time,collectively known as the “Vroman Effect.”31-45

In general, the most important driving forces for proteinadsorption are the electrostatic and hydrophobic interactions,possibly combined with the entropic gain of conformationalchanges of proteins during adsorption to the NP surface.51 Anoverview of how different NP characteristics, includingPEGylation, affect interactions with blood proteins, is summa-rized in Table 1.

Thrombogenicity

During blood clotting, soluble fibrinogen is converted to anetwork of insoluble fibrin fibers, and platelets become activated,exhibiting surface receptors that bind fibrin. A plug is thenformed, preventing blood loss in injury sites. Platelets becomeactivated in response to thrombin (an activated coagulationfactor), collagen (exposed to blood only after an injury) or plateletactivation factor (PAF), a cytokine produced by neutrophils. Theactivation of the coagulation cascade is initiated when injuryexposes a tissue factor (TF)-bearing cell to the blood flow(extrinsic pathway) or through contact activation on negativelycharged surfaces (intrinsic pathway).52,53 As for hemolysis,surface properties of the NPs should be carefully manipulatedto avoid undesirable thrombogenic effects (see Table 1).

Researchers have suggested that some NPs can induceplatelet aggregation via nontraditional pathways (not requiringthromboxane A2 and adenosine diphosphate (ADP) release),possibly rendering common anticoagulant therapeutics lessefficient in reducing NP-mediated platelet aggregation.54

It is generally perceived that the time of residence of a NP inthe bloodstream mainly depends on its capacity to evade MPScells, but Movat et al clearly demonstrated that phagocytosis ofNPs by platelets themselves precedes platelet activation andaggregation, thus suggesting redistribution to a blood clot asanother potential mechanism affecting NP blood life.28,55

Complement system

Complement is a term used to describe a group of bloodproteins linked in a zymogen-activation cascade that removespathogens from the body.13,56 Its main functions are theformation of pores on the pathogen surfaces and mediation ofinflammation. The complement system can be activated through

three different pathways: classical, alternative and lectin,13,56

these pathways differing in the way they are triggered.In all these complement-activating pathways, the central

event is the proteolysis of the protein C3 and the subsequentattachment of the proteolysis product (the opsonin C3b) tomicrobial cell surfaces or to immune complexes at the site ofcomplement activation.

Besides performing the function of cellular lyses through theformation of the membrane attack complex, many of thecomplement products have several immunological roles, such asthe induction of platelet aggregation,13 B-cell activation,13 recruit-ment of inflammatory cells and induction of phagocytosis.13

NP-induced complement activation has been shown in somecases,39,57,58 and in addition to inflammation, the activation of thecomplement cascade may result in rapid clearance of the NPsfrom the systemic circulation via complement receptor (CR)-mediated phagocytosis by MPS cells.1 Careful manipulation oftheNP surface characteristics could also circumvent this problem,because the analysis of several delivery systems has providedevidence of general trends for NP complement-activatingbehavior according to surface properties, as described in Table 1.

NP uptake by mammalian cells

Nanoparticles can stimulate and/or suppress immune responsesand their compatibility with the immune system is largelydetermined by their surface chemistry. The immuno-stimulatoryproperties of NPs have roles in adjuvant properties, inflammatoryresponses, antigenicity and finally recognition and uptake bymammalian cells.We focus onNP uptake by theMPS as themajorhindrance to the widespread utilization of intravenous nanocarriersystems. A detailed description of other NP–immune cellinteractions can be found elsewhere.5,17 Although the mechanismof colloidal NP uptake by mammalian cells is still far from beingcompletely understood,5 some general trends regarding theinfluence of size, form and physicochemical surface characteristicshave been observed and are summarized in Table 1.

The MPS is a cell family capable of foreign materialphagocytosis. The MPS bone marrow-committed precursorsdifferentiate into resident phagocytes, which are responsible forthe majority of NP clearance from circulation2 (e.g., Kupffercells in liver, Langerhans cells in skin, and macrophages inspleen and bone marrow).19,59

Endocytosis is a highly regulated process of macromoleculeand particle internalization with at least 11 endocytic routesdescribed.60 Mannose-, complement- and Fcγ-receptor mediatedphagocytosis have been involved in manosylated chitosanNPs,61 lipid nanocapsules38 and fullerene derivative62 uptake,respectively. Scavenger receptors were involved in the uptake ofpolystyrene NPs63 as well as dextran-coated superparamagneticiron oxide NPs (SPIONs)64 and polylysine-coated gold NPs.65-68

Drawbacks of the steric stabilization approach

PEG coatings only delay NP phagocytosis

Although the presence of PEG delays the rate of nanocarrierphagocytosis, their final destination is always MPS clearance,


mainly in the spleen or the liver.69 For instance, naked poly(ɛ-caprolactone) NPs have a very rapid and extensive sequestrationin the MPS tissues; typically 90% of NPs are captured byphagocytosis in the first 5 minutes after injection. Conversely,only 45% of PEGylated NPs were found in the MPS 5 minutesafter injection.8 Moreover, naked poly(ɛ-caprolactone) NPsaccumulated more extensively in the liver, whereas for PEGylatedNPs, this final accumulation shifted toward the spleen.8 Coatingof NPs with plasma proteins that mediate binding to cell receptorsin the MPS organs is thought to be responsible for thisphenomenon, although the molecular mechanisms of the uptakeof NPs by the MPS are not fully understood.2,5,58

Several research groups have reported that the repeated,intravenous injections of PEGylated liposomes can generateimmune responses and elicit specific antibodies directed againstPEG, resulting in the loss of their long-circulating characteristicsand leading to accumulation in liver.70-72 According tosuggestions by Kiwada and collaborators,72 anti-PEG IgMantibodies are generated after the administration of PEGylatedliposomes, which subsequently activate the complement system,leading to opsonization and enhanced uptake of the liposomes byKupffer cells.72

In 2006, Ganson et al73 reported the results of a Phase I trial ofPEG-uricase, a treatment developed for patients with chronic goutwho are intolerant to available therapy for controllinghyperuricemia,73 based on promising preclinical results obtainedwith mice.74 These kind of protein derivatives were designed totake advantage of the PEG's protein- and cell-repelling propertiesand can be regarded as first-generation “nanomedicines.”75

However, in human studies, the circulating time and efficacy ofPEG–uricase decreased in several subjects by the induction of IgMand IgG antibodies against PEG.70 Similarly, Armstrong et al havereported the rapid drug clearance due to anti-PEG antibodies onpatients with leukemia receiving asparaginase covalently attachedto PEG.76 It was also reported that stealth NPs (PEGylatedliposomes34 and dextran-coated SPIONs in vivo77) can beremoved from circulation by opsonin-independent pathways.

PEGylation triggering of hypersensitive reactions

The previous observations indicate that the general assump-tion that PEG is a nonimmunogenic agent78 is not always provento be true. Moreover, two independent research groups usingliposomes containing antisense oligodeoxynucleotides79,80 havefound rapid plasma elimination of liposomes administered insubsequent injections and acute hypersensitive reactions, whichwere not caused by excessive systemic cytokine release orsignificant elevations in plasma histamine.11,79,80

Hypersensitive reactions have been traditionally categorizedin four types: acute reactions mediated by IgE (Type I), by IgG(Type II), by immune complexes (Type III) or delayedresponses by T cells (Type IV).81 However, many acuteallergic reactions whose symptoms fit Type I, do not involvespecific IgE and are called “anaphylactoid” or “pseudoallergic”reactions, which may represent up to 77% of all immune-mediated immediate hypersensitive reactions.81,82 Much evi-dence suggests that these reactions have a common triggermechanism, namely the activation of the complement cascade,

leading to the designation of “complement activation-relatedpseudoallergy” (CARPA), a new subdivision within the TypeI hypersensitive reactions.81,83,84

Activation of complement produces pro-inflammatory prod-ucts, such as C3a and C5a, which bind to leukocytes, mast cellsand macrophages. These cells release inflammatory mediatorssuch as histamine and pro-inflammatory cytokines, which in turnalter vascular permeability, induce smooth-muscle contractionand cause inflammatory cell migration.85,86

Agents that are known to cause pseudoallergic reactionsinclude liposomal drugs (PEGylated liposomal doxorubicin(DOX), liposomal amphotericin B and liposomal daunorubicincitrate) and micellar solvents (e.g., polyethoxylated castor oil inpaclitaxel formulations).81,83,84 Several studies have providedevidence for the involvement of PEG in CARPA. Incubation ofhuman serum with PEGylated liposomal DOX increased theactivation of complement.81 Complement activation was alsodetected in humans using 99mTc-labeled PEGylatedliposomes81 and with PEGylated liposomal DOX in bothhumans87 and pigs.88

Moreover, Hamad et al have recently examined the role ofPEGylated carbon nanotubes (CNT) in the induction of adversecellular reactions. The authors demonstrated that two differentfunctionalized PEG-CNT were able to activate complement, inspite of their protective PEG coating.10,16

The hydrophilic polymer coating and hindrance of drug release,interaction with target cell and lysosomal escape

For extracellular delivery purposes, when the payload is asmall and membrane diffusible molecule, the delay in thedestabilization of the carrier due to its steric protection may limitthe availability of the drug in the target region.11 In intracellulardelivery, when the payload cannot cross the cellular membrane,as in the case of proteins or nucleic acids, the polymer coatingavoids cellular interaction. Positively charged surfaces interactwith negatively charged membranes and are internalized viaendocytosis but are prone to rapid clearance and aggregation dueto electrostatic interactions with blood components11 (see Table1). As an alternative, targeting ligands are being coupled to theterminal end of the PEG chains. However, coupling the ligand tothe distal end of the polymer chains can lead to an acceleratedremoval of the targeted particles from the circulation, especiallyat higher ligand densities.89 Moreover, the presence of targetingligands may evoke immune responses prohibiting repeateddosing of the formulation.90

Additionally, it is often required that drug contents are deli-vered to the cytoplasm after particle internalization viaendocytosis.90 The presence of degradative enzymes and low pHin the lysosome and the fact that some drugs might be hydrophilicor too large to cross the lysosomal membrane can be a problem,particularly when delivering proteins or nucleic acids to the cells.11

The steric stabilization approach: alternativesto PEGylation

From the above discussion it is clear that the characteristics ofan effective DDS are becoming more stringent. Some


alternatives to PEGylation have been designed (e.g., poly(oxazoline), polyvinyl alcohol, poly(glycerol), poly-N-vinylpyr-rolidone and poly(amino acids)11), that successfully mimic thestealth capability of PEG, but present no conceptual advance, astheir eluding detection relies on the steric repulsion effect.Recently, some steps have been taken toward use of “smarter”stealth technologies as described below.

Shedding approaches

The shedding principle consists on a loss of the coating uponactivation after arrival at the target site. Ideally, the nanocarriershould remain stable during circulation and extracellularactivation; once taken up by the cells, it should readily releasethe drug according to the spatiotemporal needs.91 Ligands thatmediate internalization via specific receptors and positivelycharged surfaces can be protected by the hydrophilic polymerduring circulation and exposed when they enter the target tissueby shedding of the hydrophilic polymer.11 This shedding isachieved by a specific stimulus at the site of action to ensure thatthe ability to interact with cells is achieved only at the intendedlocation, e.g., poly(hydroxyalkyl L-asparagine)-coated lipo-somes can be cleaved in vitro by cathepsin B, a lysosomalenzyme that is also present extracellularly in tumors andinflammatory tissue.11

Lysosomal escape can be mediated using fusogenicliposomes that contain the lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.92 These liposomes are not stealthagents per se, and the presence of a PEG layer diminishesthe fusogenic potential and the cytoplasmatic deliveryefficiency.11 A possible alternative would be the sheddingof the PEG once in the lysosome to restore the fusogenicpotential. Although several research groups claim tailoredproperties of their shedding systems, most shedding studieswere performed in vitro with lack of demonstration under invivo conditions.11

Poloxamers

An interesting system for stealth polymers, which have beenused as the outer layer in core/shell NPs,7 are poloxamers, aspecific class of block copolymers, composed of hydrophilicpoly(ethylene oxide) and hydrophobic poly(propylene oxide)blocks arranged in a A-B-A triblock structure. In aqueoussolutions at concentrations above critical micelle concentration,these amphiphilic copolymers self-assemble into micelles andhave the capability of incorporating into biological membraneswith subsequent translocation into the cells.93

Some side effects have been reported, such as adverse non-IgE-mediated hypersensitivity reactions following intravenousinjection of poloxamer 188-based pharmaceuticals, presumablyvia complement activation, as an intrinsic property of thepolymer.81,85 Additionally, an enhanced uptake by MPS cellsupon surface coating with poloxamers has been observed forpolybutylcyanoacrylate NPs.42

Polysaccharides, polypeptides and poly (acids)

Polymeric materials for delivery purposes must be biocom-patible and preferably also biodegradable.7 To this aim, poly

(lactic acid), poly(glycolic acid), polycaprolactone, polysac-charides, poly(acrylic acid) family and proteins or polypeptidesare being applied.7,94,95 Some of them are natural physiolog-ical materials, biocompatible and biodegradable,69 highlystable, safe, nontoxic and hydrophilic,7 with abundant re-sources in nature and low processing cost (e.g., algal alginate,plant pectin, microbial dextran and animal chitosan).94 Amongthe most common polysaccharides used for NP coatings, somehave been shown to decrease the uptake of NPs by the MPS,namely, heparin-coated poly (methyl methacrylate),96 mono-crystalline iron oxides coated with a dextran layer,97 liposomescoated with sialic acid,98 pullulan, dextran and glycolipids.69

On the other hand, because macrophages have cellularreceptors that mediate the uptake of polysaccharides, thecoating of NPs has been shown to increase the endocyticuptake,64,65 a feature that is useful only when delivery toimmune cells is intended.61

Considering the clinical safety of polysaccharide-coatedNPs, precautions for the intravenous administration arerecommended for a commercial SPIO colloid used in MRimaging to avoid anaphylactic-like or hypersensitivity re-actions related to dextran.99 Accordingly, cyclodextrin-containing polycation-based NPs are complement activators.37

Depending on the polymer configuration, dextran andchitosan coatings, these elements could behave as comple-ment inhibitors or activators.40 In fact, it is well known thatthe alternative complement pathway could be triggered byanionic polymers (dextran sulfate) and by pure carbohydrates(agarose, inulin).13 Not surprisingly, heparin-coated NPsdramatically inhibit complement activation, because heparinis known to increase the activity of the inhibitory hostprotein H.96,100

Albumin

Albumin is biodegradable, biocompatible, nontoxic and non-immunogenic,95 allowing albumin-based nanocarriers to berecognized as self-entities by the immune surveillance. Physi-ologically it transports both endogenous and exogenoushydrophilic components through noncovalent reversible in-teractions, releasing its cargo at the cell surface.101-103

Additionally it has preferential uptake in tumors and inflamedtissue95 and facilitates extravascular transcytosis of bound andunbound components.104 Regarding drug manufacturing, albu-min is widely available and extremely robust, being soluble inwater (and 40% ethanol), acidic, stable in a wide pH range (pH4–9) and at high temperatures (60°C up to 10 hours) withoutdeleterious effects.95

NP albumin-bound paclitaxel, prepared by high-pressurehomogenization of paclitaxel in the presence of humanserum albumin (HSA),104,105 was the first FDA-approvedand commercially available102 albumin NP-based product foroncology therapy. In 2005, a randomized, phase III trial in460 women with metastatic breast cancer demonstrated animproved efficacy and safety profile with albumin NP-paclitaxel, in comparison with standard paclitaxelformulation.102,106 This technology avoids the hypersensitiv-ity reactions that require steroid and antihistamine


premedication, as well as the neutropenia and prolonged andsometimes severe peripheral neuropathy associated withpolyethoxylated castor oil, the delivery vehicle in earlierpaclitaxel formulations.102 Albumin NPs are a promisingDDS, because although solvent-based hydrophobic chemo-therapy has been a primary tool for oncologists, in additionto the toxicity of the active agent, the solubilizers in thedelivery vehicles also exhibited serious toxicity effects.107

There are several other reports on albumin-encapsulateddrugs for cancer treatment,95,102,103,107 including albumin-docetaxel NPs and albumin-rapamycin NPs,102 and the “low-cost” process for synthesizing DOX–BSA–dextran NPs.104

Immune evasion mechanisms

Here we aim to exploit some of the parallel strategiesdeveloped by different classes of microorganisms when facing aparticular host immunity challenge, toward the engineering ofpathogen-mimetic stealth nanocarriers.108

An illustrative example of a widely used strategy is themolecular masking mechanism in which pathogens display hostmolecules on their surface, assuming the appearance of the hostcells, and sterically hinder the immune recognition of theirsurface-exposed antigens.14,109,110 In some aspects, albuminNPs can be regarded as the translation of this mechanism intonovel DDS.

Host targets, virulence factors and microorganisms involvedin these mechanisms include: plasminogen,111 albumin,112

fibronectin103 and non specific Igs113-115 bound by M and M-like proteins at the surface of group A streptococcus (GAS);similar virulence factor-host molecule pairs are used by groupG116 and B streptococcus117 (GGS and GBS respectively);Igs,14,109 major histocompatibility complex molecules,109 blood-group antigens109 and albumin14 are known to be associated withthe schistosomes' surfaces, although the biochemical processremains to be elucidated in many instances.118,119

Phagocytosis inhibition

Pathogen recognition by phagocytes is mediated by aplethora of receptors whose signal transduction might triggerantimicrobial production, secretion of pro-inflammatory cyto-kines and also cytoskeleton rearrangements that lead tophagocytosis.13,61,113,114,120,121

Bacteria have developed strategies to interfere invirtually all steps of phagocytosis, namely macrophageadhesion, internalization, phagosomal sequestration andphagolysosome formation.120 We focus on adhesion andinternalization inhibiting mechanisms, and within these,those related to antibodies and complement will beaddressed in separate sections.

Translocated modulators

Some bacteria like Yersinia species and enteropathogenicEscherichia coli (EPEC)120-122 inject proteins into the hostphagocytic cells that disorganize their cytoskeleton, preventing

phagocytosis. Yersinia species prevent phagocytosis by injectingYop (for Yersinia outer proteins), to target actin within the hostcell.108,121,123 In Y. pseudotuberculosis, YopE is injected into thecytoplasm, promoting the disruption of actin filaments byinteraction with the Rho GTPases Rac, Rho and CDC42.121,123

YopH, YopO and YopT in Yersinia species108,121,123 and theEPEC secreted protein F (EspF)122 have also been involved inantiphagocytic activities.108,123

Secreted and surface-associated modulators

Group A streptococcus successfully evades neutrophilphagocytosis and killing to cause human infections, producingseveral immune modulators. Other strategies used by thesebacteria include streptococcal C5a peptidase (ScpA) specificcleaving of complement factor C5a, therefore inhibitingrecruitment of neutrophils to sites of infection.115 The coloca-lization of streptococcal inhibitor of complement (Sic), a proteinsecreted mostly by serotype M1 GAS, with erizin (a humanprotein that in neutrophils functionally links the cytoskeleton tothe plasma membrane), promotes a deficient cytoskeletonfunctioning with phagocytosis inhibition.124

GAS Mac is a secreted protein that interacts with CD16/CD11b at the neutrophil plasma membrane to inhibit FcγR-and CR-mediated phagocytosis and block subsequent ROSproduction and killing of GAS by human neutrophils. GASMac has homology to the α-subunit of human Mac-1(CD11b). Because leukocyte β2-integrin Mac-1 is involvedin the regulation of critical neutrophil functions, includingadhesion, migration, phagocytosis, cell signaling andNADPH-oxidase-mediated killing,13,115 GAS Mac can affectall these processes.

The M protein of GAS has extensive sequence variability,with more than 120 serotypes described.115 M protein facilitatesresistance to phagocytosis in part by binding to complement hostregulator proteins (C4b-binding protein, factor-H and factor H-like protein) thereby impeding the binding of C3b to the GASsurface. Binding of fibrinogen to the M protein also inhibitsdeposition of C3b.115 Additionally, C4BP- and additional IgAFc-binding regions on the M protein of serotype M22 werereported to cooperate in conferring phagocytosis resistance to S.pyogenes and could fully account for the antiphagocyticproperties of M22.125

Not only bacteria, but also viruses like herpes viruses and poxviruses can express surface proteins that mimic membranemolecules like CD200,126 a host regulator of immune tolerancethat delivers inhibitory signals to macrophages.127-129

Complement evasion mechanisms

Inactivation by proteases

ScpA and its GBS-analogous streptococcal C5a peptidase(ScpB) cleaves and inactivates C5a.115,130 The gram-negative bacterium P. aeruginosa secretes active proteasesin the form of alkaline protease and elastase that cleaveC3b and thus inhibit C3b deposition and complementactivation at the bacterial surface.13,131 The streptococcal


pirogenic exotoxin B (SpeB), produced by virtually allstrains of GAS,115 cleaves C3 and C3b, being an importantfactor in resisting clearance by phagocytosis and survival inhost blood.114

In the case of parasites, proteomic analyses in the outermembrane and gut of schistosomes have shown the presence ofantibodies and complement fragments,14 probably cleaved byschistosomal enzymes.14

Complement disguise

Complement regulator acquiring surface proteins (CRASP)attach soluble host-complement regulators to the surface of thepathogen, including Complement Factor H (CFH), factor H-like1 (CFHL-1), factor H-related 1 (CFHR-1) and C4b-bindingprotein (C4BP). The host-complement regulators CFH, CFHL-1and C4BP, bind to the surface of the pathogen, maintain cofactoractivity for inactivating C3b and C4b and thus restrictcomplement activation.131

The M protein of GAS binds CFH, CFHL-1 and C4BPavoiding the binding of C3b to their surface.115 There is alsoevidence that schistosomes, the causative agents of humanschistosomiasis, can somehow incorporate into their outermembrane the membrane-bound glycoprotein decay-accelerat-ing factor (DAF),132 a vertebrate complement regulator thatprevents the assembly or accelerates the decay of C3convertase in host cells.56 The same strategy has beenadopted by an extremely large number of pathogens,including bacteria, fungi, viruses, multicelullar eukaryoticorganisms and parasites.131

Other inactivating proteins

The staphylococcal complement inhibitor (SCIN) from S.aureus blocks all pathways of complement activation bybinding specifically to activator-bound convertases, but not tofree components. Following binding, surface-bound C3 con-vertases are stabilized and enzymatic activity is inhibited.130

This pathogen also expresses an extracellular fibrinogen-binding protein that binds simultaneously fibrinogen andC3b, altering its conformation, and inhibiting its depositiononto sensitized surfaces.133 Plasmin receptor, a Group Bstreptococcus surface protein, inhibits the biological effects ofC5a on human neutrophils by capturing and binding C5a withhigh affinity.131

Several viruses, including herpes simplex (HSV), cow-pox, vaccinia and variola virus, present gene productsresponsible for complement control activities that include C3and C3 convertase inactivation and inhibition of C9polymerization as well as IgG Fc receptors that avoid theclassical pathway of complement activation.134 This is thecase for the soluble gE-gI complexes, HSV-1 encodedglycoproteins (see below) that bind IgG and avoid theclassical pathway of complement activation.134

Schistosomes also have several proteins that serve asinactivating complement receptors. For example, paramyosinwas shown to bind in vitro to C1q and inhibit C1 and theclassical pathway of complement activation.135 The comple-ment C2 receptor-inhibiting trispanning is a receptor for C2

that interferes with the formation of C3 convertase in theclassical and lectin pathways.56 A 130 kDa parasite proteinwas reported to bind C3, inactivate it and ultimately limitthe damage mediated by all three complement pathways,because C3 is the common pivotal molecule.56

The anticomplement protein of Ixodes scapularis (ISAC) andI. ricinus (IRAC),130 and the complement inhibitor protein ofOrnithodoros moubata (OmCI),136 all of them human tickspecies, ectoparasites that feed with their mouthparts embeddedfor several days into the host skin, bind C5a with high affinity,leading to their clinical testing as biopharmaceuticals forcomplement-mediated diseases.

Subversion of antibody responses

Avoidance of Fc-dependent immune functions

A broad range of pathogens, including HSV-1,137 GAS,GBS138 and also the blood parasites schistosomes109,110

skillfully use FcR to avoid the consequences of immune-complex formation. Alternatively, such receptors might alsoscavenge exposed Fc domains after Fab binding to thepathogen's antigen. This antibody bipolar bridging wouldprevent subsequent Fc-dependent immune activation.134

The herpes simplex virus HSV-1 gE and gI glycopro-teins form a hetero-oligomer complex that binds bothmonomeric and aggregated IgG with high affinity onHSV-1 virions and on infected cells.137,139 In vitro, gE-gIprotects HSV-1-infected cells from antibody-dependentcellular cytotoxicity, granulocyte recognition and classicalpathway complement activation.134

The binding specificity for the CH2-CH3 domain is alsoexhibited by IgA-binding proteins from group A and Bstreptococcus. Presumably, this disrupts Fc-dependent functions,such as the ability to activate complement.138

In schistosome-derived infections, the host mounts antibodyresponses with specificity for a broad range of parasite antigensbut is incapable of clearing the established parasites.14

Paramyosin (Pmy) was identified as an IgG FcR,109,110 andother uncharacterized schistosome membrane proteins were alsoable to bind antibodies.110

Another efficient mechanism for avoiding Fc-dependentimmune functions is to cleave antigen-bound (Fab region-bound) IgG.111,113,114 In GAS, proteases SpeB and immuno-globulin G-degrading enzyme of S. pyogenes (IdeS) contributeto their escape from IgG-mediated phagocytosis.113

Deglycosidation and suppression of antibody production

Some pathogens decrease the immune control at thelevel of antibody mediated responses, using mechanismssuch as the removal of oligosaccharides from the antibodystructure. As an example, the endo-beta-N-acetylglucosami-nidase protein induces the deglycosidation of human IgGand is quite effective in blocking the uptake and killing ofGAS in blood.115 Another example is the suppression ofantibody production by B cells, a mechanism for which the

Figure 2. Proposal for a stealth nanocarrier presenting pathogen virulencefactors. Color codes describe the components of the nanocarrier. A“classical” nanocarrier is regarded as a three-component system, includingan unspecific stealthing moiety.


measles virus nucleocapsid protein is known to be quiteeffective in vitro.140

Conclusions and future perspectives

The challenge of creating effective ways to prolongnanocarrier circulation is of major and increasing importancein nanomedicine.2 Although PEGylated therapeutics have so farbeen the most widespread and reliable source of “stealth”medicines, alternative strategies should be developed asPEGylated therapeutics have displayed unexpected pharmaco-kinetic behavior upon repeated injection. Such behavior might bedue to the generation of IgM and IgG antibodies against PEG,which can compromise therapeutic efficacy or even generateundesirable side effects.70

A new paradigm of stealth nanocarriers is therefore proposed(Figure 2), in which virulence factors are bound at thenanocarrier surface to interact directly with phagocytic andcomplement elements. Our proposed improved nanocarrier isbioactive (a response modulator) in contrast with current inerthydrophilic polymeric coatings that indiscriminately avoidinteractions with proteins and cells. Nevertheless, the presenceof specific and unspecific stealthing strategies in a singlenanocarrier (Figure 2) could be complementary, with eachapproach imparting their positive features when simultaneouslypresent in the nanocarrier.

The main clinical obstacles that need to be addressedwhen using these pathogen virulence factors in humanapplications are: (i) complement activation; (ii) allergicreactions; (iii) the effects of any antibodies on the therapeuticprocess that could lead to treatment resistance (namely,neutralization or changes in pharmakocynetics); and (iv),potential autoimmune responses induced by cross-reactive

antibodies or immune cells against modified endogenoushuman proteins.

For all biopharmaceuticals, the precise extent ofimmunogenicity of any protein product will depend onproduct purity, formulation, injection route, dosage, fre-quency of administration (i.e., acute vs. chronic), andimmune status of the patient population.141,142 Thequestion of whether immunogenicity in humans will restricttheir usage to only acute clinical indications will beproperly addressed by: (i) carefully designed human phaseI and II trials (there is a poor prognostic capacity ofanimal-based immunogenicity studies) and (ii) detailedanalysis of the efficiency parameters following multipletreatment protocols.142

Although there is yet no experimental support for theproposed immune-evasive nanocarriers, an ever-growing listof nonhuman-derived products is being used or tested forclinical applications, including the above-mentioned OmCIprotein for the treatment of complement-related diseases,136

secreted immunomodulatory viral proteins for the treatmentof inflammation-based disorders,142 CpG oligonucleotides asadjuvants to improve vaccination,143 viral NPs,144 cell-penetrating peptides (CPP), e.g., Tat and penetratin fromviral and insect sources, respectively, for intracellular deliveryof biologically active components.145 Recently, CPP havebeen reported to increase the uptake efficiency of quantumdot probes into living cells,146 and Tat conjugated toritonavir-loaded NPs have been tested for their efficiency inreducing HIV-1 replication in macrophages.147 Snake venompeptides provided the basis for the development ofangiotensin-converting enzyme inhibitors and gpIIbIIIa an-tagonists for the treatment of cardiovascular diseases.142 Inaddition, bacterial streptokinase/staphylokinase, bovine aden-osine deaminase, or salmon calcitonin, have been usedtransiently for acute therapeutic indications.142

In fact, all therapeutic proteins currently licensed foruse in humans, whether from human or nonhumansources, exhibit some degree of immunogenicity inpatients.148 Even recombinant insulin derived solely fromhumans can be limited for long-term chronic administrationprotocols.142 As small peptides are less prone to developimmune responses (e.g., antibodies), identifying the viru-lence-factor fragments involved in the immune evasivemechanism but not in its toxicity could reduce thepotential nanocarrier's immunogenicity.

Nevertheless, possible negative outcomes might be aminor concern in cases where other therapeutics haveconsistently failed, as in terminal cancer patients. Therefore,we project that increasing attention will be given to immune-evasive virulence factors, as templates for bioactive nano-carriers that specifically inhibit the uptake by MPS andcomplement activation. The applications foreseen for ananocarrier upgrade have to be considered on a case-by-case approach, depending on the outcomes expected. Forexample, in those drug formulations that trigger pseudoal-lergy, e.g., PEGylated liposomal DOX, attaching an activepeptide fragment from a complement-inhibitor protein suchas SCIN to the liposome's surface could decrease the

image of Figure 2

Figure 3. Possible experimental approach for the development of a stealth nanocarrier, presenting pathogen virulence factors. (1) Identification of theimmunological barrier. Complement activation by PEGylated liposomal DOX (represented schematically), is the exemplary immunological barrier to beovercome by virulence factors.87 (2) Identification of possible virulence factors. SCIN was selected due to its ability to inactivate C3 convertases, reducing thelevels of C3b (opsonin) and C5a (pro-inflamatory).130 The structure of SCIN is shown in ribbon representation, helices are colored blue (h1), magenta (h2), andorange (h3).149 (3) Active peptide identification, reducing the likeness of immunological reactions. In SCIN (ribbon representation, gray) the 18-residue segmentLeu31-Gly48 (ball and stick representation, green) is crucial for the complement inhibition activity.149 Images of the SCIN peptide were generated using UCSFChimera.150 (4) Active peptide-nanocarrier conjugation. (5) Upon conjugation the peptide should retain its inhibition activity in the relevant in vitro studies, e.g.,complement activation tests for the liposomal DOX as schematically represented. (6) Testing the in vivo effects. As represented schematically, an inhibition ofcomplement activation in the appropriate animal model should correlate with absence of pseudoallergy symptoms and increased blood half-life due to decreasedC3b-mediated MPS uptake. Such effect should shift the biodistribution from the MPS tissues towards the tumor, culminating in enhanced decrease of tumor size.


inflammatory complications and the clearance of theliposome-encapsulated drug, therefore increasing the treat-ment efficiency (Figure 3). Increasing the nanocarriercirculation times by avoiding the MPS uptake would beparticularly suitable for chronic treatment where sequentialdoses are required, as in cancer or autoimmune diseases, as

increased drug-circulation times would increase the overalltreatment compliance. Figure 3 illustrates a possibleexperimental approach for the development of an upgradednanocarrier, containing virulence factors inhibiting compo-nents. Complement activation by PEGylated liposomal DOX,and the use of the complement inhibitor SCIN, were selected

image of Figure 3


as case studies for our proposal on how a nanocarrierupgrade using virulence factors could deal with theimmunological barriers of the intravenous environment.

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Pathogen-mimetic stealth nanocarriers for drug delivery: a future possibility

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