annurev-physchem-040513-103718

PC66CH23-Nie ARI 12 January 2015 10:54

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Physical Chemistry ofNanomedicine: Understandingthe Complex Behaviors ofNanoparticles in VivoLucas A. Lane,1 Ximei Qian,1 Andrew M. Smith,2

and Shuming Nie1,3

1Departments of Biomedical Engineering and Chemistry, Emory University and GeorgiaInstitute of Technology, Atlanta, Georgia 30322; email: [email protected] of Bioengineering, University of Illinois at Urbana-Champaign, Urbana,Illinois 618013College of Engineering and Applied Sciences, Nanjing University, Nanjing,Jiangsu Province 210093, China; email: [email protected]

Annu. Rev. Phys. Chem. 2015. 66:521–47

The Annual Review of Physical Chemistry is online atphyschem.annualreviews.org

This article’s doi:10.1146/annurev-physchem-040513-103718

Copyright c© 2015 by Annual Reviews.All rights reserved

Keywords

active targeting, passive targeting, antifouling, molecular imaging,fluorescent dyes, oncology, image-guided surgery

Abstract

Nanomedicine is an interdisciplinary field of research at the interface ofscience, engineering, and medicine, with broad clinical applications rangingfrom molecular imaging to medical diagnostics, targeted therapy, and image-guided surgery. Despite major advances during the past 20 years, there arestill major fundamental and technical barriers that need to be understoodand overcome. In particular, the complex behaviors of nanoparticles underphysiological conditions are poorly understood, and detailed kinetic andthermodynamic principles are still not available to guide the rational designand development of nanoparticle agents. Here we discuss the interactionsof nanoparticles with proteins, cells, tissues, and organs from a quantitativephysical chemistry point of view. We also discuss insights and strategies onhow to minimize nonspecific protein binding, how to design multistage andactivatable nanostructures for improved drug delivery, and how to use theenhanced permeability and retention effect to deliver imaging agents forimage-guided cancer surgery.

521

Review in Advance first posted online on January 19, 2015. (Changes may still occur before final publication online and in print.)

Changes may still occur before final publication online and in print

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1. INTRODUCTION

The design and development of nanometer-sized structures and systems for biomedical applica-tions are of broad current interest in science, engineering, and medicine (1–4). The basic rationaleis that nanometer-sized particles have functional and structural properties that are not availablefrom either discrete molecules or bulk materials (3). When conjugated with biomolecular affinityligands, such as antibodies, peptides, or small molecules, these nanoparticles can be used to detectmolecular biomarkers and tumor cells at high sensitivity and specificity (5–7). Nanoparticles alsohave large surface areas for the attachment of multiple diagnostic (e.g., optical, radioisotopic, ormagnetic) and therapeutic (e.g., anticancer) agents. Recent advances have led to the developmentof biodegradable nanostructures for drug delivery (8–12), iron oxide nanocrystals for magneticresonance imaging (13, 14), and luminescent quantum dots for multiplexed molecular diagnosisand in vivo imaging (15–21). At present, however, there are still major fundamental and tech-nical barriers that need to be understood and overcome. These problems include the complexinteractions between nanoparticles and biological systems in vivo, the rapid uptake and clearanceof nanoparticles by the reticuloendothelial system (RES) organs (e.g., the liver and spleen), ac-tive versus passive targeting, and the limited penetration of nanoparticles into solid tumors (seeFigure 1). In fact, the complex behaviors of nanoparticles under physiological conditions are stillpoorly understood, and detailed kinetic and thermodynamic principles are not available to guidethe rational design and development of imaging and therapeutic nanoparticle agents.

Blood Liver

Kidney Tumor

Figure 1Schematic diagram showing the complex behaviors of nanoparticles under in vivo conditions. Upon systemic injection, nanoparticlesencounter several physiological behaviors before they can reach the intended targets, including protein adsorption and opsonization inthe blood, uptake by the liver and other reticuloendothelial organs, renal excretion, extravasation across leaky vasculatures (often foundin solid tumors), and binding to receptors on diseased cells, leading to subsequent internalization.

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In this article, we discuss the physical chemistry principles for understanding nanoparticleinteractions with blood proteins, cells, tissues, and organs. These principles provide important in-sights into major in vivo processes, such as nanoparticle uptake, transport, organ distribution, anddegradation. In particular, we discuss new strategies for designing nanoparticles that are resistantto protein binding, the use of multistage and so-called smart nanoparticles to overcome fundamen-tal barriers, the different pharmacokinetic properties for imaging versus therapeutic nanoparticles,and the use of passive and active targeting agents for image-guided surgery of naturally occur-ring tumors in dogs as well as pilot clinical studies in humans. There are still major challengesin developing safe and effective nanoparticle agents for biomedical applications, and systematictoxicological studies of nanoparticle distribution, excretion, metabolism, and pharmacokineticsare urgently needed. At the same time, there are also compelling opportunities in developingnew and innovative technologies for the treatment of cancer and cardiovascular, neurological,and infectious diseases. As discussed in more detail in Section 2, a quantitative and mechanisticunderstanding of the complex in vivo behaviors of nanoparticles is essential to this research anddevelopment effort.

2. NANOPARTICLE-PROTEIN INTERACTIONS

Nanoparticle interactions with proteins play a critical role in their biomedical behavior becauseproteins compose 75% of the dry weight in the human body and >90% of the dry weight ofblood plasma. Nanoparticles are often delivered to a patient through intravenous administration,and upon exposure to the blood, they immediately encounter a complex and crowded mixture ofions, small molecules, proteins, and cells. The key initial interactions with blood components arethrough physical association with plasma proteins, often called opsonization or biofouling (22–26).How the nanoparticles interact in this mixture dictates whether they can provide a useful diagnosticor therapeutic effect in specific tissues and organs. A high-affinity association with proteins isundesirable, as it masks the targeting or molecular recognition properties of the nanoparticle.This process leads to a shell of adsorbed proteins on the particle surface called a corona (22). Theadsorbed proteins themselves have biomolecular functionalities that can alter the surface of thenanoparticle. For example, the adsorbed proteins often denature and change their physiochemicalproperties, thus altering the particle destination in the body (23). The most frequent proteinsinvolved are globular albumins, fibronectin, complement proteins, fibrinogen, immunoglobulins,and apolipoproteins (24–26). Because these proteins exist at high concentrations in the blood (27),a corona can develop rapidly owing to the high frequency of collisions between the proteins andparticles, even when the association or binding affinity is weak. Based on approximations fromkinetic theory, the frequency of collisions ( fcollision) between a nanoparticle and a protein in theblood can be described as

fcollision = RT1,500η

· (rNP + rP)2

rNP · rP· c P, (1)

where R is the gas constant; T is the temperature; η is the blood viscosity; rNP and rP are the radiiof the nanoparticle and protein, respectively; and cP is the concentration of protein (28, 29). In theblood, fcollision is on the order of 106 s−1, so upon administration, a nanoparticle in the circulationwould meet its first protein in tenths of a microsecond and would experience millions of collisionswith each passing second. Indeed, some nanoparticles have been observed to develop a proteincorona almost instantaneously when immersed in blood serum (30).

Whether these collisions yield a fouled surface depends on the balance of the adsorption anddesorption rates of the proteins on the particle surface and how strongly the protein is boundto the surface. The rates at which adsorption and desorption processes occur are proportional

www.annualreviews.org • Physical Chemistry of Nanomedicine 523


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to their corresponding rate constants (kads and kdes, respectively). The value of kads depends onthe frequency of collisions between the proteins and nanoparticle ( fcollision; Equation 1) and theactivation energy of adsorption (Eads):

kads ∝ fcollision exp(

− Eads

RT

). (2)

The value of kdes depends on the binding strength of the protein to the surface (the depth of thepotential energy well, Edes):

kdes ∝ exp(

− Edes

RT

). (3)

The ratio of kdes to kads is the equilibrium dissociation constant (Kd), which describes the proteinbinding affinity for the nanoparticle surface (31):

Kd = kdes

kads= exp

(�Gads

RT

). (4)

At equilibrium, the net Gibbs free energy for a protein adsorption event (�Gads = Eads − Edes)governs the degree to which proteins will remain on the particle surface. Protein interactions thatare weak and/or infrequent have a large positive �Gads and a large dissociation constant, indicatingrapid desorption. Protein interactions that are strong and/or frequent have large, negative �Gads

values (small Kd); in this case, proteins have a low probability of desorption from the surface andyield a fouled particle surface. Thus, major efforts have been made to maximize Eads and minimizeEdes to reduce biofouling.

To understand how the adsorption/desorption equilibrium can be modulated, we find it usefulto examine energy diagrams depicting the distance-dependent interaction between a nanoparti-cle and the surrounding proteins (see Figure 2). These diagrams are derived from the DLVO(Derjaguin-Landau-Verwey-Overbeek) theory of charge-stabilized or polymer-stabilized colloids(32). Figure 2b shows a typical free energy diagram for an interacting nanoparticle and proteinseparated by a surface-to-surface distance d. Negative free energy indicates stable, attractive inter-actions, whereas positive free energy indicates a net repulsion. The shape of the free energy curvecan be quite complex because the free energy is a sum of a large number of attractive and repulsiveforces from distinct chemical functionalities on the surfaces of protein and particles. Proteins, inparticular, have very complex interaction potentials because of the chemical diversity of amino acidresidues, which can exhibit strong or weak electrostatic, van der Waals, or hydrophobic forces.At large separation distances (large d ), the interaction energy is nearly zero until the separationdistance is close enough to reach a regime called the secondary minimum. This minimum resultsfrom interactions between the solvation shells and the terminal chemical groups tethered to theprotein or particle surface, yielding a weak net attraction that can be dissociated if the particlesseparate, as the depth is similar to the thermal energy (kT ). This minimum leads to a dynamicequilibrium state of easily exchanged proteins around a nanoparticle, known as a soft corona(30, 33).

At closer separation distances, the net attraction diminishes and becomes dominated by repul-sive forces at the adsorption barrier either because of electrostatic repulsion between like-chargedparticles or because of the loss of the flexibility of molecular domains on the surface. This loss ofconformational (rotational) flexibility creates local order, decreases entropy, and yields positive(unfavorable) free energy for protein-particle interactions. The height of this energy barrier withrespect to the secondary minimum is equivalent to Eads in Equation 2. If this energy barrier canbe overcome, then the particle separation distance can be further reduced to reach the most stablestate, the primary minimum. Here the particle surfaces are in physical contact with an interaction

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Energy

Energy

Energy

ΔGads < 0

Fouled particle

ΔGads > 0

Nonfouled particle

d

d

d

d

Born repulsion Adsorption barrier

Primary minimum Secondary minimum

e– e–

+ +Unfavorablestates

Favorablestates

Eads

Eads

Edes

Edes

ΔGads < 0

ΔGads > 0

a c

b d

Figure 2(a) Schematic illustration of the interparticle distance, d, defined as the distance from the nanoparticle surface to the protein surface.(b) Plot of a typical potential energy curve between a nanoparticle and a protein molecule, with the prominent features highlighted anddiscussed in the main text. (c) Potential energy plot of a particle with a deep primary energy well for adsorption and a small barrier forprotein adsorption, leading to surface fouling that is thermodynamically stable. (d ) Potential energy plot of a nanoparticle with a smallprimary well and a high-energy barrier for protein adsorption, leading to surface fouling that is thermodynamically unstable.

strength equivalent to Edes in Equation 3. Proteins that reach the primary minimum make up ahard corona (34) and are slow to exchange owing to the adsorption barrier, requiring several hoursto equilibrate in serum (30). Any further reduction in the separation distance is restricted becauseof the physical dimensions of the two objects, a highly repulsive regime called Born repulsion,resulting from excessive overlap between electron clouds of the interacting protein-particle pair(35–38).

2.1. Antifouling Coatings

The key to minimizing fouling is to offset the attractive potential between a nanoparticle andthe proteins by using surface chemical modifications designed to increase the adsorption barrier(Eads) and decrease the depth of the primary minimum (Edes). Coatings that are resistant to proteinadsorption are often electrostatically nearly neutral and exhibit a high degree of surface flexibilityand entropy. However, it is important to note that all nanoparticle coatings have a substantialsecondary minimum, as repulsive forces generally operate on short length scales, which dropexponentially from the surface (32, 39, 40); thus, binding by proteins at a larger separation distanceis likely unavoidable, but its role or significance under in vivo conditions is not clear. The depth ofthe secondary minimum is proportional to the surface area and the polarizability of the interactingentities (41), so it may be possible to eliminate its formation simply by using smaller particlesizes, yielding a smaller number of geometrically possible interactions for a lower net energyof attraction. Indeed, recent work has shown that semiconductor nanocrystals with the smallest



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hydrodynamic dimensions (4–8 nm) can reduce the nonspecific binding of proteins to very lowlevels (42).

2.2. Neutral and Zwitterionic Coatings

Nanoparticles coated with molecules that have a net electrostatic charge at physiological pH (e.g.,carboxyls and amines) are known to foul with proteins much more rapidly than those coated withelectrostatically neutral groups (e.g., hydroxyls and ethers) (43). This is largely a result of the highstrength and long-distance nature of Coulombic forces. Most proteins in the blood, and in mosttissues and cells in general, have a small net negative charge at neutral pH, measured as a zetapotential near −5 to −10 mV or an isoelectric pH of −5 to −6. Therefore, it is not surprising thatnanoparticles with a net cationic charge rapidly foul in biological fluids and that nanoparticles witha near-neutral electrostatic charge or a small negative charge are the most resistant to fouling (44).However, it is surprising that highly anionic nanoparticles also rapidly foul. Both experimentaland molecular dynamic simulations have shown that this effect is the result of local microdomainsof cationic charges on protein surfaces arising from tertiary structures comprising high localconcentrations of lysine and arginine (45), which can outweigh the net anion-anion repulsionbetween the two particles in close proximity (46). Thus, any high-magnitude surface charge canyield a high magnitude of Edes with proteins. Interestingly, the net surface charge and the chemicalidentity of surface groups also dictate the specific classes of bound proteins (47).

Two types of functional groups have been reported to substantially reduce nonspecific bindingwhen compared with coatings with a net charge. Hydroxyls, compared with amines and carboxylicacids, are uncharged at neutral pH and simultaneously can serve as a hydrogen bond donor andacceptor, allowing a large degree of hydration (48). Similarly, it is possible to create surfaces thatare net neutral by using a balanced ratio of anionic and cationic charges (zwitterionic coatings),mimicking the natural composition of protein surfaces with balanced acidic and basic aminoacid residues, as well as the phospholipid surface of cellular membranes (49). These coatings areparticularly promising owing to the formation of a hydrogen-bonding network with a locally denseregion of counterions (50, 51). Various nanoparticles have been generated with coatings containingphosphatidylcholine groups (anionic phosphate and cationic ammonium), sulfobetaines (anionicsulfate and cationic ammonium), and carboxybetaine (anionic carboxylate and cationic ammonium)(52). These coatings have been shown to greatly reduce nonspecific protein adsorption on bothmacroscopic and microscopic surfaces (53–55).

2.3. Steric Repulsion

Whereas neutral coatings are beneficial to reduce the depth of the primary minimum by modulat-ing the enthalpy of interaction, they provide little benefit with regard to a protective adsorptionbarrier. Thus, an alternative and complementary approach is to coat nanoparticles with flexible,hydrophilic polymers, which effectively shield the surface through steric repulsion. The com-bination of flexibility and a strong interaction with water molecules leads to a large number ofmolecular configurations and degrees of freedom (high entropy). Fouling through the binding of abulky protein would necessarily reduce the degrees of freedom of the coating and reduce entropy,which is unfavorable (39), increasing the adsorption barrier height. These polymers are usuallylinear or branched chains, tethered to the nanoparticle surface through one end to act as entropicsprings that provide an outward repulsive force upon compression by an approaching protein(39, 56). Such repulsions are short ranged and strongly depend on the molecular weight (57):Longer chains lose more degrees of freedom upon compression and thus have a greater restor-ing force, although only marginal improvements are achieved at molecular weights beyond 3,500

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Da (58). The model polymer for this mechanism, polyethylene glycol (PEG), has been the stapleof most nonfouling coatings since it was discovered almost 40 years ago and used to attenuatethe immunogenicity of foreign proteins in the blood (59). PEG is widely used to stabilize a largevariety of nanoparticles, such as liposomes (60, 61), polymeric particles (62), micelles (63), andinorganic nanocrystals (e.g., gold, iron oxide, and quantum dot particles) (64–66), that have rigidsurfaces and low intrinsic surface flexibility. In addition, various other hydrophilic polymers havebeen found to exhibit good nonfouling behavior (67), including polyoxazolines (68), HPMA (69),and polysaccharides, such as chitosan (70), dextran (71), and hyaluronic acid (72).

We note, however, that polymeric coatings typically add an additional 4–10 nm of radial size,preventing the production of sub-10-nm particles, which have been observed to have some of themost unique biophysical behaviors. In addition, some polymeric coatings induce recognition bythe immune system, causing efficient removal as the body recognizes these foreign materials. Thishas been observed with PEG-coated liposomes, leading to rapid blood clearance upon multipleintravenous injections (73) owing to the binding of PEG-specific IgM antibodies produced duringthe first administered dose (73). Additionally, in drug screenings of a PEGylated product, it wasfound that one-quarter of the patient population previously produced anti-PEG antibodies (74),likely developed from the widespread use of PEG in cosmetic and hygiene products. This moti-vates the development of replacements for PEG coatings that do not induce immunological reac-tions. For example, poly–amino acid coatings prepared by a mixture of hydroxylethyl-glutamateand hydroxylethyl-asparagine residues were observed to resist accelerated blood clearance afterrepeated injections (75).

3. NANOPARTICLE-CELL INTERACTIONS

In comparison with rapid protein adsorption on nanoparticles, cell-nanoparticle interactions areoften limited by the low concentration of cells and the lower diffusion kinetics of nanoparticles.Nonetheless, uptake by white blood cells in the circulation and by resident macrophages in theliver and spleen can efficiently remove nanoparticles that have been opsonized, displaying epi-topes that are recognized by the cells as markers for clearance. The free energy diagrams fornanoparticle-cell interactions can have features that are similar to those presented in Figure 2(76), as the cell surface is rich with proteins and is slightly negative in charge. Similarly to pro-teins, charged particles associate with cells more rapidly than neutral particles (48), with cationicparticles exhibiting the greatest stickiness to the anionic cell membranes (77). Nanoparticles withgreater hydrophobic character also demonstrate more rapid association with cells and uptake,likely because of the interaction with lipophilic domains of the plasma membrane (78). Addition-ally, nonspecific associations with cells are similarly minimized through the use of zwitterionicand neutral coatings and hydrophilic flexible polymers (79).

Cellular binding and uptake of nanoparticles are strongly dependent on whether the nanoparti-cles have already been fouled by proteins. In fact, this attribute has been quite difficult to study as itis often not clear if the binding event is mediated by the targeting protein attached to the nanopar-ticle or mediated by fouling proteins bound to the nanoparticle and/or targeting ligand. Even forstudies in which cell cultures are prepared with media free from exogenous proteins, cells contin-ually secrete their own proteins, which can adsorb to the nanoparticle surface locally at the plasmamembrane to facilitate adhesion and uptake. Although nanoparticles that are nearly neutral andresistant to protein fouling usually have enhanced cellular association once they become fouled, ithas been observed that charged and/or hydrophobic nanoparticles will exhibit lower cellular asso-ciation once they are fouled with a protein corona (80). This lowering of adhesion rates may arisefrom an electrostatic charge reduction and masking of hydrophobic domains by steric repulsion



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provided by the proteins. Once fouled, particles may associate with cells through interactions thatare more specific, involving epitope binding of the adsorbed protein to its corresponding receptoron the cell membrane. Recent work by Walkey et al. (47) has identified protein corona fingerprints,suggesting that hyaluronan receptors are the major mediators of nanoparticle-cell interactions.

3.1. Multivalent Binding and Cellular Uptake

A common route of delivering nanoparticles to live cells is receptor-mediated endocytosis, inwhich affinity ligands conjugated on the surface of the particles bind to surface membrane recep-tors. For efficient cellular delivery, the particles must have a high affinity to the cell receptors andthen develop enough ligand-receptor binding pairs to overcome the energetic barrier of wrappingthe cellular membrane around the particle for internalization. Both the binding affinity and subse-quent cellular internalization can be enhanced by multivalent binding, in which multiple bindingevents occur between ligand-receptor pairs (81) (see Figure 3a). In multivalent binding, thereis also the possibility for the positive cooperativity of binding among the multiple complexes, asbinding of one ligand on the nanoparticle will localize neighboring ligands closer to other recep-tors, facilitating further binding events. The multivalent binding affinity (often called avidity) isdependent on both the monovalent binding affinity and the number of ligand-receptor binding

ca

b

K dmono

K dmulti

2

1

3

CYTOSOL

EXTRACELLULAR SPACE

Figure 3(a) Multivalent interactions, by forming multiple ligand-receptor bond pairs as opposed to a single bond, significantly increase theaffinity of nanoparticles to the cell membrane. (b) Nanoparticles with multiple affinity ligands increase the flux of receptors toward theparticle, leading to more binding events to gain the energy required to wrap the membrane around the particle. (c) A free nanoparticlewith affinity ligands (�) can contact the cell surface at which it binds multiple cell surface receptors to create energetically favorableconditions for membrane wrapping and endocytosis (� and �).

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pairs (valency) (82):

K multid = (K mono

d )βN . (5)

Here K multid is the observed dissociation constant including multivalent effects, K mono

d is the disso-ciation constant of a single ligand-receptor pair, β is the degree of cooperativity (β < 1, negativecooperativity; β = 1, no cooperativity; β > 1, positive cooperativity), and N is the number of affin-ity ligands bound to receptors. The positive cooperativity of the binding of multiple ligands candramatically enhance the observed dissociation constant from that of a single ligand-receptor com-plex. For example, nanoparticle-cell association enhancements have been observed experimentallyby using multiple folate receptor binding proteins, reaching values of 2,500–170,000-fold (83).Additionally, multivalent antiviral and anti-inflammation agents are known to have potencies thatare orders of magnitude higher than their monovalent counterparts (82). Multivalent interactionsmay occur for the adsorbed proteins within the nanoparticle corona as well for multiple adsorb-ing species presenting binding epitopes. Additionally, even sole adhesion molecules, such as theopsonin fibronectin, can contain multiple binding sites for cellular receptors (57).

The binding affinity to targeted cells is generally seen to increase with an increasing numberof ligands per particle, but there is a limit to the number of affinity ligands that can be used. Forexample, negative cooperativity would decrease the affinity of particles to cells with additionalligands as a result of steric crowding, for which each additional surface ligand will lower theparticle affinity to the cell by limiting the conformational freedom the ligand needs to effectivelybind to its target (84, 85). Another caution in applying multivalency involves the need to ensurethat the stealth properties offered by the nonfouling molecules are not lost owing to the completesurface coverage of affinity ligands (86). Such loss of stealth behavior leads to increased nonspecificinteractions with the proteins and cells and an increase in nanoparticle accumulation in the liverand spleen (87). Thus, optimization is needed to find the maximum allowable number of ligandsfor the greatest target affinity while not increasing nonspecific interactions.

3.2. Membrane Wrapping and Endocytosis

Once the nanoparticle has become associated with the cell surface, the cell membrane can start towrap around the particle to form a vesicle for engulfment (see Figure 3b). For the wrapping processto occur, there needs to be sufficient ligand-receptor bond pairs to overcome the energetic barrierof membrane bending (88, 89). Theoretical analysis of the thermodynamics of this process providestwo quantitative expressions for the minimum density of ligand-receptor bond pairs (ρl-r,min)for particle uptake and the length of time for the membrane to wrap around the nanoparticle (τw)(88, 89). The important parameters are the size of the particle (Rp), the diffusivity of the receptorson the membrane (Dr), the elastic modulus of the membrane (Ebend), and the energy of the bindingbetween the nanoparticle and membrane (Ebind), which can be a combination of the bindingenergies of the ligand-receptor pairs along with electrostatic and van der Waals interactions. Theproportionalities based on these parameters are

τw ∝ R2p

Dr, (6)

ρbond,min ∝ Ebend

R2p Ebind

. (7)

From this analysis, it can be seen that smaller particles are wrapped in less time but require a higherligand density to overcome a greater membrane bending energy (owing to the higher curvatureof smaller particles). Conversely, larger particles will require more time for the membrane to



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cover the surface but will have less energetic costs from bending the membrane owing to lowercurvatures. However, increasing the ligand density for greater multivalent binding can enhancethe rates at which receptors cluster to the binding site (90), leading to decreased wrapping timesand higher rates of cellular internalization for both large and small particles. From numericalresults of the energy balance, an optimal particle radius is approximately 50 nm, which has theshortest wrapping time (88, 89, 91). Experimentally, this size has been observed to show optimaluptake (88, 89). However, most experimental studies have been performed in two-dimensional invitro environments, in which larger particles are observed to have higher uptake rates, possiblybecause of higher sedimentation velocities, which increase their local concentration to the cellsresiding at the bottom of the well. In the case in which cells are suspended above the well floor,smaller particles (which are more buoyant) are observed to have higher uptake rates (92).

3.3. Direct Internalization

Another route to internalization is by penetrating the membrane without vesicle formation,thereby allowing nanoparticles to be delivered directly to the cytosol. One possible method fordirect delivery to the cytosol is to destabilize the membrane structure. Particles with surfaces ofmultiple cationic head groups can attract so many anionic phospholipids from the cell membranethat the appearance of holes has been observed (93). Theoretical studies have shown that positivelycharged particles that are larger in diameter than the thickness of the cell membrane can attractphospholipids to form a bilayer coating (94). Cell-penetrating peptides offer another approach forthe direct internalization of nanoparticles, but the exact mechanisms are still a matter of debate(95, 96). Alternatively, particles having alternating hydrophobic and anionic ligands can penetratemembranes without bilayer disruption (97). Computer simulations of these particles describe theirmechanism of internalization as a lowering of the free energy of insertion of the nanoparticle intothe membrane, which allows quick insertion and withdrawal from cellular membranes (98). Uti-lizing such ligand structures may allow quick and efficient delivery of drug payloads directly tothe cytosol.

3.4. Intracellular Trapping and Escape

The internalized nanoparticles are often trapped in intracellular organelles, such as endosomes andlysosomes, which have acidic pHs and contain degradative enzymes (99) (see Figure 3c). This en-vironment is detrimental to therapeutic agents because the drugs are not available for binding theirtargets and are subject to enzymatic degradation. Under certain conditions, endocytotic vesiclesmay not develop the harsh conditions of lysosomes (99), and particles in vesicles may be recycledback to the cell exterior or exocytosed (100, 101). Therefore, much research effort is currently de-voted to determining what parameters affect the internalization pathway to avoid lysosomal degra-dation and/or escaping endosomal vesicles to the cytosol. Particles with varying degrees of sizes andcharges have been investigated to determine what conditions favor internalization methods that arenot directed toward lysosomal pathways (102, 103), but there is no consensus on the optimal con-ditions for such nondegradative endosomal pathways (99). One approach for lysosomal escape usesthe proton sponge effect, in which particles are designed to absorb protons upon the acidification ofthe vesicle, disrupting the membrane by increasing osmotic pressure (104); another approach useslight-sensitive molecules that can be activated to generate reactive oxygen species, which degradethe vesicle components (105). The membrane-penetration approaches using peptides also offerdirect methods for the cytosol entrance, although lack the selectivity between normal and diseasedcells provided by the use of affinity ligands. The combination of ligands that allow direct entry

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into cells and those that target upregulated receptors may provide an optimal solution for drugdelivery into cells, although how these mechanisms affect each other needs further investigation.

3.5. Effects of Particle Shape, Size, and Conformation

Dynamic modeling studies by Ferrari and coworkers (106, 107) have revealed that anisotropic par-ticles, such as discs, rods, and hemispheres, have much slower rates of cellular uptake (thus longerblood circulation times) than do isotropic spherical particles. Similar to the radius thresholds ob-served for spherical nanoparticles, rods can have radii below the minimum threshold and can havelarge lengths, with excessive wrapping times that are thermodynamically unfavored for engulfment.The combination of these effects can make a stronger barrier to cellular uptake. However, in somestudies, anisotropic particles have been observed to be taken up more rapidly than their sphericalcounterparts (108, 109). This discrepancy is likely caused by variations in the ligand density andexperimental conditions. For example, affinity ligands may pack more efficiently on elongated orflat surface particles, such as rods or cubes, which can increase driving forces for cellular internal-ization. Additionally, anisotropic nanoparticles are often synthesized by using hydrophobic ligandsthat bind to the nanoparticles more strongly and are not completely removed during subsequentsteps, leading to erroneous interpretations of shape effects on cellular internalization.

Furthermore, flexible and hydrophilic polymers, such as PEG, can interfere with receptorbinding and cellular internalization (see Figure 4a). In fact, a generally poor design for targetednanoparticles would involve affinity ligands directly attached to the particle surface and surroundedby a PEG layer (Figure 4b). For PEG to impart a stealth behavior, it needs to have a molecularweight of at least 2,000 Da at a grafting density high enough to extend the polymers into a brushconfiguration (58, 110). Conversely, to expose surface-anchored targeting ligands for cell target-ing, the PEG2000 molecules need to be sparse enough to adopt mushroom-like conformations,which result in less nonspecific binding protection (111, 112). Therefore, a better design is totether the affinity ligand on the outer ends of the PEG chains. This situation benefits from densePEG grafting densities as the brush conformation pushes the ligand to the outer surface, whereasmushroom-like conformations will bury the ligand (see Figure 4c). It is also important that thePEG length of the chain tethering the affinity ligand is similar to that of its unconjugated neigh-bors. If the ligand tethered chain is much longer than its neighbors, the extra length can fold intothe mushroom-like conformations, which bury the ligand (113) (see Figure 4d ). For example,when folate ligands are attached to PEG3400 and surrounded by PEG2000, the targeting abilityof the nanoparticles to cancer cells is lost (114).

4. IN VIVO NANOPARTICLE TRANSPORT AND TARGETING

The in vivo transport of nanoparticles has been explored in the context of targeting canceroustissue after intravenous injection. The tumor microenvironment differs from normal tissue invarious ways, some of which can be exploited for enhanced drug delivery through the use ofnanoparticle delivery agents (see Figure 5). There are several steps involved beyond just reachingthe tumor tissue, which include moving past the RES organs, crossing the vessel wall, traversingthrough the tumoral interstitial space, binding to receptors on tumor cells, and internalization(115). The ability of the nanoparticle to navigate these physiological barriers depends primarilyon the convective and diffusive transport properties, along with the chemical affinities betweenthe nanoparticle and the environment.

Convection and diffusion are the main transport mechanisms by which a nanoparticle crossesthe blood vessel walls (called extravasation). A mathematical model of the extravasation rate is



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cc

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Figure 4Conformational effects of antifouling polymers on the binding ability of targeting molecules. (a) Targeting molecules anchored to thenanoparticle surface are able to bind to their target if the surrounding polymers are sparsely grafted and adopt short mushroom-likeconformations. (b) With the use of higher grafting densities to push the polymers into a brush-like conformation, the particle is able toresist biofouling but will block a surface-anchored molecule from binding to its target. (c) The ideal situation involves tethering thetargeting ligand to the end of the polymer chain and surrounding it by densely grafted polymers of the same length adopting abrush-like conformation. This design resists biofouling while orienting the ligand on the outer surface, where it can bind to its target.(d ) However, if the ligand is tethered to a polymer that is much longer than its neighboring polymers, the extra length can fold backand bury the ligand, which hinders its ability to bind.

offered by the Staverman-Kedem-Katchalsky equation (116):

Js = P [CV − CI] + LP[(PV − PI) − σ (πV − πI)][1 − σF]�Clm. (8)

Here Js is the net flux of particles crossing the vascular wall; P is the permeability of the vessel;CV and CI are the vessel and interstitial space concentrations of the particles, respectively; LP

is the hydraulic conductivity of the vessel; PV and PI are the vascular and interstitial pressures,respectively; σ is the osmotic reflection coefficient; πV and π I are the osmotic pressures of thevessel and interstitial areas, respectively; σ F is the solvent drag reflection coefficient; and �Clm isthe log mean of the vessel and interstitial particle concentrations. The equation is grouped by twoadditive terms: The first, presented in Equation 8, is particle flux from permeation/diffusion, andthe second is from convection.

In normal vessels, there is a balance between the hydrostatic pressure of the blood wanting topush fluid out and the opposing osmotic pressure from the higher plasma protein concentrationinside the vessel compared to the interstitial space [(PV − PI) ∼ σ (πV −πI)]. These two effects can-cel out the convective transport of nanoparticles for normal vessels, so nanoparticle extravasationdepends strongly on vessel permeability. Generally, particles that are larger than a few nanometers

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ba

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Figure 5Schematic illustration of normal and leaky vasculatures, passive and active targeting, and transcytosis and exocytosis. (a) With smallpores within the vasculature, such as that of the gap junction of endothelial cells in normal tissue, extravasation of the particles isinhibited, and they stay in the circulation. (b) In tumoral regions where there is leaky vasculature with large pores, particle extravasationis facilitated, after which the particles can migrate through the interstitium. (c) Particles that are passively targeted, otherwise having noaffinity ligands for cell receptors, may perfuse the tissue, exhibiting cell-free channels. (d ) Actively targeted nanoparticles are likely tobe bound with the first cells they encounter, significantly slowing the transport within cell-free channels. (e) Passively targeted particleshave little mobility to pass cell-dense layers without a leaky or cell-free channel to diffuse in. ( f ) Actively targeted particles may be ableto travel beyond dense layers of cells by being taken up by the cells and transcytosed or exocytosed to the other side.

will have little to no permeability through most normal vasculatures as they exceed the pore sizeof endothelial gap junctions (117). Regions of high permeability in normal vessels exist at thediscontinuous capillaries found within the liver and spleen, which have wide openings (called fen-estrations) between the endothelial cells lining the vessel wall (115). Thus, intravenously injectedparticles display a biased accumulation toward tissues exhibiting vascular fenestrations that arelarger than the particle size. Particles in these regions of high permeability can potentially leavethese areas by returning to the vessel or by lymphatic drainage if not bound or taken up by thecells. If particles are lacking a stealth coating or are otherwise opsonized, upon entering the highlypermeable regions of the liver and spleen, where there is a high local concentration of macrophagecells, there will be a greater retention of particles within these organs. However, with good stealthcoatings, the particles are more likely to maintain longer circulation times and accumulate in theliver and spleen at slower rates.

4.1. Passive Targeting

Solid tumors are known to have highly permeable vasculatures, allowing nanoparticles to crossthe vessel walls into the interstitial space (see Figure 5b). This highly permeable vascular networkis created when tumors reach length scales at which the diffusive transport of oxygen is notsufficient to meet the metabolic needs of tumor cells. Therefore, the cells begin releasing factors



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to create neovascular networks to bring in the blood supply. These freshly created networkshave numerous fenestrations, which range from hundreds of nanometers to tens of micrometers,displaying permeability constants that are an order of magnitude greater than that of normal tissue(115). In addition to enhanced permeability from leaky vasculatures, large tumors generally lack afunctional lymphatic drainage system, thereby reducing particle clearance (118). The combinationof these conditions leads to an increased accumulation of circulating nanoparticles in the tumorinterstitium, which is called the enhanced permeation and retention (EPR) effect (119, 120).Because small drug molecules have high permeability and clearance within various tissues ofthe body, typically only 1 out of 1,000–100,000 injected drug molecules reaches its intendeddestination (121, 122). By having a size that is optimal for delivering drugs more preferentiallyto tumors through the EPR effect, local dose concentrations delivered by nanoparticles can be10–100 times that of the free drug (123). The use of the EPR effect to deliver nanoparticles invivo is known as passive targeting (see Figure 5c).

However, the leaky vasculature and inefficient lymphatic drainage of tumor tissues can leadto inefficient nanoparticle transport in solid tumors. Because there is no effective outlet for ex-travasated fluid in tumors, the interstitial fluid pressures are comparable to the vessel pressure(PV ∼ PI) (118). The interstitial osmotic pressure is increased as well because there is no effectivebarrier to keep the plasma proteins from building up in the tumor interstitial space (πV ∼ πI)(124). Thus, as with normal tissues, extravasation relies primarily on permeability. Furthermore,because of inefficient lymphatic drainage within the tumor interstitium, diffusion is the primarymode of transport once the particle has crossed the vascular wall. Favorable conditions for particlesto perfuse the tumoral area will include high vascularization throughout the tumor volume andlow-density extracellular matrices allowing higher particle diffusivity.

Methods to increase nanoparticle delivery to tumors include shortening the diffusion lengthand increasing the vessel area per unit volume of tissue (125), momentarily increasing the bloodpressure to promote enhanced extravasation of particles (126), and utilizing enzymes to degrade theextracellular matrix (127). Jain (128) demonstrated that vascular renormalization can increase theefficacy of cancer therapeutic drugs. The addition of vascular channels throughout the tumor willdecrease the particle diffusive distances to cells from a vessel and cause greater convection towardthe tumor core, which will decrease the interstitial pressure, leading to higher extravasation rates(129). The tumor perfusion of nanoparticles can also be enhanced by raising the vessel pressure viathe administration of the strong vasoconstrictor angiotensin II (126). As tumor blood vessels lacksmooth muscle cells, angiotensin II will constrict only normal blood vessels. As the blood flow ishindered in the normal vessels, greater blood flow will be directed to the tumor vessels, which main-tain the same dilation. However, we note that this effect is temporary, and the vessel and interstitialpressures will reverse flow to equilibrate, which may cause particles to re-enter the bloodstreamduring the process. The extracellular matrix of the tumor interstitium is a dense network of col-lagen fibers, which severely limits the diffusive mobility of particles. For the enhancement of thediffusive mobility within tumor tissues, the use of bacterial collagenase enzymes has been foundto increase the interstitial distribution of 75-nm viral particles by a factor of three (130).

4.2. Active Targeting

In comparison with the nonspecific EPR effect, active targeting uses affinity ligands, such asantibodies and peptides, to specifically bind to surface receptors expressed on target cell membranes(131) (see Figure 5d ). Recent work has shown that the use of tumor-targeting ligands is effectivein delivering imaging and therapeutic agents to solid tumors (132). For example, tumor-targetingstudies using fluorescently and radioactively labeled antibodies have demonstrated higher tumor

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uptake (measured on the basis of targeting agent per gram of tumor mass) for macromolecules andnanoparticles than their nontargeted controls (132). However, opposing evidence has also beenreported, indicating that the use of tumor-targeting ligands does not increase the total nanoparticleaccumulation in solid tumors, although it does increase receptor-mediated internalization andcould thus improve the therapeutic efficacy for cancer drugs that act on intracellular protein targets(133). Complicating this matter further, recent work has shown that the use of targeting ligandsmight even be detrimental because the exposed ligands can accelerate nanoparticle opsonization(adsorption of blood proteins) and blood clearance, leading to an overall reduction in tumornanoparticle uptake (134).

It is generally accepted that active targeting can aid in greater endocytosis of the nanoparticles astargeted ligands decrease the free energy of internalization. For instance, it has been observed thattransferrin-targeted nanoparticles had improved internalization within cancer cells (135). Thereare cases in which molecular therapeutics have poor uptake, in which active targeting is necessary,as in the delivery of nucleic acids such as small interfering RNA, to achieve efficacy (133, 136).Targeting may also be necessary for nanoparticles to locate to small tumors or micrometastases,where EPR delivery may be minimal (137). Conversely, with active targeting, it is possible thatparticles with high adhesion strengths to their target will be prevented from complete tumor per-fusion beyond vascular channels, as they will be stuck on the periphery of their first contact (138).However, for dense tumor spheroids having minimal pores, active targeting has been observedto have deeper tissue penetration than passive particles as the nanoparticle can travel through thetissue by endocytotic/exocytotic transport (139) (see Figure 5e,f ). Another possible outcome isthat, if these agents are highly localized and are then able to be either internalized or released tosurrounding cells in a timely fashion, there may be an additional bystander effect of cytotoxicity tocancer cells without high levels of target receptor expression (137). An additional concern is thattargeting molecules present on the nanoparticle surface may be more prone to nonspecific proteinadsorption and immune response, which may block the targeting ability and increase immunecell uptake, leading to decreased delivery of particles to the tumor than with passive targeting(86). In particular, monoclonal antibodies, which have widespread use in the construction of ac-tive targeted nanoparticles owing to high affinities to their cellular targets, have such drawbacksdue to their relatively large sizes and immunogenicity (140, 141). Thus, there is great interest indeveloping fully human antibodies and using fragments to retain the affinity and specificity of theparent molecule with less immunogenicity and smaller size (142, 143).

5. NANOPARTICLE DESIGNS TO OVERCOMEPHYSIOLOGICAL BARRIERS

As discussed above, there are technical and fundamental barriers to in vivo nanoparticle deliveryand targeting, including biofouling, RES uptake, poor tissue penetration, and limited endosomalrelease. These problems could be overcome or mitigated by the design of smart or intelligent nano-structures, such as stimuli-responsive nanoparticles and multistage/mothership delivery vehicles.One strategy is the use of pH sheddable coatings, which can respond to the slightly more acidicenvironments within tumoral areas (144). The concept here is to envelop a targeted nanoparticlewith an antifouling coating, which will prevent biological entities from accessing the targetingligands until the nanoparticle is delivered to the tumoral area, which then is cleaved away, expos-ing the ligands and allowing for binding to the intended cellular targets (see Figure 6a). Kale &Torchilin (145) have developed such a system with pH detachable PEG outer layers upon lipo-somes, having targeting ligands that showed enhanced accumulation and penetration within tumortissues. Once the nanoparticles have accumulated within the tumoral area, another stimulus may



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Alternatingmagnetic field

ΔT

d

Drug loading

Laserirradiation

PNIPAAMcoating

Magnetic fieldc

Tumor environmentpH drop

a b

Figure 6Schematic illustration of smart nanoparticle systems to overcome delivery barriers. (a) A pH sheddable antifouling coating coversaffinity ligands in a nanoparticle until the particle reaches the tumor microenvironment, where it experiences a drop in pH andsubsequently sheds the antifouling coating, exposing the ligands for binding to their targets. (b) Gold cubes that are hollow and porouswhen coated with temperature-sensitive polymers, such as NIPAAM, can be photothermally activated with laser light, which collapsesthe polymer coating and allows the release of the enclosed drug. (c) Magnetic nanoparticles have the ability to be guided with magneticfields to the tumor site. (d ) An alternating magnetic field can then be applied to thermally agitate the particles, thus raising the localtemperature to destroy the surrounding cells.

be employed for a rapid release of the drug payload. Polymers such as PNIPAAM, which phasetransitions from solvated extended coils to shrunken states upon heating, can act as temperature-activated doors to the drugs encased within (146). The induction of temperature increases fordrug release can come from plasmonic nanoparticles, which efficiently convert photon energyto heat when irradiated with near-infrared (NIR) light (147, 148) (see Figure 6b). For example,Yavuz et al. (148) developed porous, hollow cubes of gold that can raise local temperatures whenirradiated with laser light. The particles were able to be loaded with drug molecules and thencoated with NIPAAM polymers, which allowed drug release from the pores on command uponlight activation (148). Alternatively, localization and therapy may be performed through a singletype of stimulus. For example, magnetic nanoparticles under the influence of external magneticfields can be guided to the tumor site (149, 150). Once at the tumor site, an alternating magneticfield can be applied to agitate the nanoparticles to increase the local temperature, killing nearbycancer cells through a process called magnetic ablation (151) (see Figure 6c,d ).

There have also been various constructions of nanoparticles with multiple stages of stim-uli responses (152). Although multistimuli-responsive nanoparticles have recently sparked greatenthusiasm in research, we note that more functionality leads to more complexity in particle de-velopment. Each level of additional functionality will lead to another step in the synthesis of the

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particle, which may lead to higher costs, depending on the involvement of the extra synthesis step,purification procedure, and final yield. Additionally, the accumulation of the levels of functionalitymay lead to interactions, which can lead to less desirable outcomes than if multiple monofunctionalparticles were employed (153). Thus, a careful and thorough characterization of such particles iswarranted to inspect the complex surface properties from these interactions and their relationshipto the stability and immunogenicity of the particles within in vivo environments. An approach toreduce complexity would be the use of materials that have inherent multifunctionality, such asmagnetic nanoparticles or drug molecules, which have an inherent physical property that can beimaged.

5.1. pH-Triggered On/Off Probes

For cellular and in vivo cancer imaging, Gao and coworkers (154, 155) developed a class of activat-able or smart nanoparticles based on the use of copolymer materials with ionizable tertiary aminegroups and covalently conjugated fluorescence dyes. A novel finding is that the self-assembledstructures undergo a dramatic and sharp transition within the very narrow range of pH (oftenless than 0.2 pH units). This pH-induced transition leads to rapid and complete dissociation ofthe nanomicelles, and as a result, the covalently linked dyes change from a self-quenched off stateto a highly emissive, bright on state. This supersensitive and nonlinear response to external pHprovides a new strategy in targeting acidic organelles in cancer cells, as well as the acidic mi-croenvironment in solid tumors. This feature is important in addressing the tumor heterogeneityproblem, a major challenge for various imaging and therapeutic approaches based on molecular orreceptor targeting. By targeting the more common hallmarks of tumors (i.e., the acidic habitat ormicroenvironment and the growth of new blood vessels or angiogenesis), this work has opened upexciting opportunities in detecting and potentially treating a broad range of human solid tumors.Another feature is the significant improvement in detection sensitivity because each nanoparticleprobe contains multiple copies of the dye, which are turned on (restored to fluorescence) in anall-or-none fashion, leading to amplified fluorescence signals that are many times brighter thansingle dye molecules. Of course, a major limitation of optical imaging is that the tissue penetrationof the light beam is limited to a few millimeters mainly because of light scattering and absorption.However, this problem can be mitigated by adapting optical contrast agents and devices for endo-scopic and image-guided surgery applications, in which the light is brought to the tissue and tumorsurfaces via an endoscope or a surgical incision. Overall, this class of pH-activated and supersensi-tive polymeric micelles has demonstrated a new concept in the design of novel nanoparticle probesand is expected to have broad applications in cancer biology, endoscopic cancer screening, andimage-guided interventions.

5.2. Mothership Nanocarriers

Multistage or mothership nanocarriers are constructions in which a larger nanoparticle eitherencases or is constructed from nanoparticles that are near an order of magnitude smaller, whichare released upon the dissolution or enzymatic degradation of the container or linkers (156–159).Such constructions address the fact that larger particles typically have longer circulation lifetimesand are more effective in taking advantage of the EPR effect, whereas smaller nanoparticles, lessthan 10 nm, are rapidly eliminated from the blood by renal excretion and are more effective inperfusing tumoral tissue (160). One method to construct multistage particles is to use DNA tolink several 6-nm particles into a larger construct (159). Here, particles can efficiently accumulateto the tumor site, where the DNA linkers are subsequently degraded, and the smaller particles



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are then excreted from the specimen. Particles that follow this design are ideal for imaging agentsas they are able to effectively locate to the site to light up the tumoral area and then degrade forrapid elimination from the body, thereby reducing potential toxicity concerns (161). To addressthe different stages of transport in the tumor microenvironment, Wong et al. (158) presented amultistage method in which nanoparticles could penetrate deep into tumor tissue. They encased10-nm quantum dots by a 100-nm gelatin matrix with an added layer of PEG to impart stability.The larger nanoparticle stage is effectively taken up by the EPR effect to the tumor region, wherethe gelatin is then degraded by enzymes, which release the quantum dots that exhibit betterpenetration into the tumor parenchyma.

6. FIRST-IN-HUMAN CLINICAL STUDIES

Recent first-in-human pilot studies have successfully used both passive and active targeting agentsfor image-guided surgery of naturally occurring tumors in humans (162–164). The specific tumortypes studied include breast, lung, ovarian, and pancreatic cancers. Although still preliminary,the results have helped to clarify two important issues: (a) Most human tumors have moderatelyleaky vasculatures, so the EPR effect provides a general means for the passive delivery of imag-ing agents (5–6-nm albumin-bound indocyanine green) to a broad range of human tumors, and(b) fluorescent dyes, such as fluorescein, can be conjugated to targeting ligands, such as folate acid,for specific targeting and high-contrast imaging of human tumors.

6.1. Clinical Studies of Passive Fluorescent Agents

In preliminary clinical trials, Singhal and coworkers (162) of the University of Pennsylvania en-rolled five patients undergoing surgery for resection of three lung nodules, one chest wall mass,and one anterior mediastinal mass (see Figure 7). Two surgeons reached a consensus about theclinical stage and operative approach prior to surgery. All enrolled patients were thought to havelimited disease, amenable to surgery, and no metastases (i.e., potentially curable). The mediantumor size was 2.3 cm (range of 1.8–9.1 cm) on preoperative imaging. Patients were injectedwith indocyanine green prior to surgery. At the time of surgery, the body cavity was opened andinspected. The results demonstrate that NIR imaging can identify tumors from normal tissues,provides excellent tissue contrast, and facilitates the resection of tumors. However, in situationsin which there is significant peritumoral inflammation, NIR imaging with indocyanine green isnot helpful. This suggests that nontargeted NIR dyes that accumulate in hyperpermeable tissueswill have significant limitations in the future, and receptor-specific NIR dyes may be necessary toovercome this problem.

6.2. Clinical Studies of Active Fluorescent Agents

First-in-human results have also been reported from intraoperative tumor-specific fluorescenceimaging by using fluorescein-conjugated agents to actively target the folate receptor in bothovarian cancer and lung cancer. In patients with lung cancer, the results showed that targetedmolecular imaging could identify 46 (92%) of the 50 lung adenocarcinomas and had no falsepositive uptake in the chest. In vivo, prior to exposing the tumor, molecular imaging could onlylocate 7 of the lesions. After dissecting the lung parenchyma, 39 more nodules could be detected.Four nodules were not fluorescent, and immunohistochemistry showed that these nodules did notexpress FRα. In the 46 positive nodules, tumor fluorescence was independent of size, metabolicactivity, histology, and tumor differentiation. Tumors closer to the pleural surface were more

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CT PET Brightfield NIR

Lungcancer

Thymicneoplasm

Carcinoid

Figure 7Human clinical data comparing fluorescent imaging with computed tomography (CT) scans and positron emission tomography (PET)scans of thymic neoplasms and lung carcinomas (not to scale). Patients were injected with indocyanine green and then underwentresection of their tumors. Ex vivo, near-infrared (NIR) fluorescence imaging demonstrated that the tumors were highly fluorescent andthe surrounding organ had minimal background noise. The optical images were easy to interpret by the surgeon and facilitated theidentification of tumors. Spectroscopy demonstrated a signal-to-background ratio of 8:1 for the thymoma and 7.9:1 for the lungcarcinoid. Figure adapted from Holt et al. (162) with permission.

fluorescent than tumors deep in the parenchyma. Additionally, in two cases, this strategy was ableto discover tumor nodules that were not located preoperatively or intraoperatively by standardtechniques. Taken together, these pilot clinical trials have demonstrated for the first time thattargeted intraoperative imaging may lead to more complete surgical resections and potentiallybetter staging.

7. CONCLUDING REMARKS

In conclusion, we note that optimal nanoparticles designed for imaging and those for therapy usu-ally have very different behaviors in vivo. Imaging agents should rapidly reach the target site andthen be eliminated from the body via renal clearance, degradation, or other fast pharmacokineticmechanisms (i.e., fast in and fast out). Also, for the detection and delineation of tumor marginsand residual tumor cells, imaging agents do not need to penetrate deeply into the tumor interior,and accumulation at the tumor boundaries is sufficient. In contrast, drug therapy benefits fromnanoparticles that have longer circulation times, are retained in tumors over an extended period oftime, and are able to penetrate the tumor’s interior (i.e., slow in and slow out). These opposing op-timal conditions have raised concerns about the design of theranostic particles, which are particlesthat contain both therapeutic and imaging agents (153). The rapid elimination of contrast agentsnot only reduces potential toxicity, but also provides a higher signal-to-noise ratio in the target site



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from the reduction of the background signal. Conversely, drug delivery to tumors benefits fromlong circulation times for greater accumulation within the target. However, the combination ofboth imaging and therapy within nanoparticles may be useful in tracking biodistributions. Look-ing into the future, there are several scientific issues and research directions that are particularlypromising but require concerted effort for success. (a) Researchers need to design and developstimuli-responsive and biodegradable nanoparticles to overcome nonspecific protein adsorption,adverse organ uptake, and RES scavenging. (b) Combinatorial contrast agents (cocktail tracers)should be developed for multicolor and molecularly specific detection of tumors, nerves, and bloodvessels for image-guided diagnostic or surgical procedures under minimally invasive conditions(e.g., endoscopy and robotic surgery). (c) Strategies should be established to deliver therapeuticnanoparticles into solid tumors beyond the first few layers of vascular endothelial cells. (d ) We needeffective mechanisms to trigger the endosomal and lysosomal release of drug payloads inside tar-geted cells or organs. (e) Nanotoxicological studies including nanoparticle distribution, excretion,metabolism, and pharmacokinetics and pharmacodynamics in large animal models such as catsand dogs, which are most relevant to human physiology, should be done as well. ( f ) Nanoparticlesshould be standardized and manufactured in compliance with FDA requirements.

DISCLOSURE STATEMENT

One of the authors (S.N.) is a scientific consultant of Spectropath Inc., a company to furtherdevelop and commercialize spectroscopic devices and agents for image-guided cancer diagnosticsand surgery.

ACKNOWLEDGMENTS

We thank the National Institutes of Health for grant support (R01CA163256, RC2CA148265,and HHSN268201000043C to S.N.). A.M.S. also acknowledges the NCI Nano-Alliance Programfor a Pathway to Independence Award (K99CA154006 and R00CA153914).

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