Annurev Physchem 040513 103718

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12 January 2015
D V A
Physical Chemistry of
Nanomedicine: Understanding
the Complex Behaviors of
Nanoparticles in Vivo
Lucas A. Lane,1 Ximei Qian,1 Andrew M. Smith,2
and Shuming Nie1,3
1
Departments of Biomedical Engineering and Chemistry, Emory University and Georgia

Institute of Technology, Atlanta, Georgia 30322; email: snie@emory.edu
Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana,

Illinois 61801
College of Engineering and Applied Sciences, Nanjing University, Nanjing,

Jiangsu Province 210093, China; email: snie@nju.edu.cn
Annu. Rev. Phys. Chem. 2015. 66:52147
Keywords
The Annual Review of Physical Chemistry is online at

physchem.annualreviews.org
active targeting, passive targeting, antifouling, molecular imaging,

uorescent dyes, oncology, image-guided surgery
This articles doi:

10.1146/annurev-physchem-040513-103718
c 2015 by Annual Reviews.
Copyright
All rights reserved
Abstract
Nanomedicine is an interdisciplinary eld of research at the interface of
science, engineering, and medicine, with broad clinical applications ranging
from molecular imaging to medical diagnostics, targeted therapy, and imageguided surgery. Despite major advances during the past 20 years, there are
still major fundamental and technical barriers that need to be understood
and overcome. In particular, the complex behaviors of nanoparticles under
physiological conditions are poorly understood, and detailed kinetic and
thermodynamic principles are still not available to guide the rational design
and development of nanoparticle agents. Here we discuss the interactions
of nanoparticles with proteins, cells, tissues, and organs from a quantitative
physical chemistry point of view. We also discuss insights and strategies on
how to minimize nonspecic protein binding, how to design multistage and
activatable nanostructures for improved drug delivery, and how to use the
enhanced permeability and retention effect to deliver imaging agents for
image-guided cancer surgery.
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1. INTRODUCTION

The design and development of nanometer-sized structures and systems for biomedical applications are of broad current interest in science, engineering, and medicine (14). The basic rationale
is that nanometer-sized particles have functional and structural properties that are not available
from either discrete molecules or bulk materials (3). When conjugated with biomolecular afnity
ligands, such as antibodies, peptides, or small molecules, these nanoparticles can be used to detect
molecular biomarkers and tumor cells at high sensitivity and specicity (57). Nanoparticles also
have large surface areas for the attachment of multiple diagnostic (e.g., optical, radioisotopic, or
magnetic) and therapeutic (e.g., anticancer) agents. Recent advances have led to the development
of biodegradable nanostructures for drug delivery (812), iron oxide nanocrystals for magnetic
resonance imaging (13, 14), and luminescent quantum dots for multiplexed molecular diagnosis
and in vivo imaging (1521). At present, however, there are still major fundamental and technical barriers that need to be understood and overcome. These problems include the complex
interactions between nanoparticles and biological systems in vivo, the rapid uptake and clearance
of nanoparticles by the reticuloendothelial system (RES) organs (e.g., the liver and spleen), active versus passive targeting, and the limited penetration of nanoparticles into solid tumors (see
Figure 1). In fact, the complex behaviors of nanoparticles under physiological conditions are still
poorly understood, and detailed kinetic and thermodynamic principles are not available to guide
the rational design and development of imaging and therapeutic nanoparticle agents.
Blood
Liver
Kidney
Tumor
Figure 1
Schematic diagram showing the complex behaviors of nanoparticles under in vivo conditions. Upon systemic injection, nanoparticles
encounter several physiological behaviors before they can reach the intended targets, including protein adsorption and opsonization in
the blood, uptake by the liver and other reticuloendothelial organs, renal excretion, extravasation across leaky vasculatures (often found
in 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 nanoparticle
interactions with blood proteins, cells, tissues, and organs. These principles provide important insights into major in vivo processes, such as nanoparticle uptake, transport, organ distribution, and
degradation. In particular, we discuss new strategies for designing nanoparticles that are resistant
to protein binding, the use of multistage and so-called smart nanoparticles to overcome fundamental 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 occurring tumors in dogs as well as pilot clinical studies in humans. There are still major challenges
in developing safe and effective nanoparticle agents for biomedical applications, and systematic
toxicological studies of nanoparticle distribution, excretion, metabolism, and pharmacokinetics
are urgently needed. At the same time, there are also compelling opportunities in developing
new 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 mechanistic
understanding of the complex in vivo behaviors of nanoparticles is essential to this research and
development effort.
2. NANOPARTICLE-PROTEIN INTERACTIONS
Nanoparticle interactions with proteins play a critical role in their biomedical behavior because
proteins compose 75% of the dry weight in the human body and >90% of the dry weight of
blood 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 of
ions, small molecules, proteins, and cells. The key initial interactions with blood components are
through physical association with plasma proteins, often called opsonization or biofouling (2226).
How the nanoparticles interact in this mixture dictates whether they can provide a useful diagnostic
or therapeutic effect in specic tissues and organs. A high-afnity association with proteins is
undesirable, 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). The
adsorbed proteins themselves have biomolecular functionalities that can alter the surface of the
nanoparticle. For example, the adsorbed proteins often denature and change their physiochemical
properties, thus altering the particle destination in the body (23). The most frequent proteins
involved are globular albumins, bronectin, complement proteins, brinogen, immunoglobulins,
and apolipoproteins (2426). 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 and
particles, even when the association or binding afnity is weak. Based on approximations from
kinetic theory, the frequency of collisions ( fcollision ) between a nanoparticle and a protein in the
blood can be described as
(rNP + rP )2
RT
fcollision =
c P,
(1)
1,500
rNP rP
where R is the gas constant; T is the temperature; is the blood viscosity; rNP and rP are the radii
of the nanoparticle and protein, respectively; and cP is the concentration of protein (28, 29). In the
blood, fcollision is on the order of 106 s1 , so upon administration, a nanoparticle in the circulation
would meet its rst protein in tenths of a microsecond and would experience millions of collisions
with each passing second. Indeed, some nanoparticles have been observed to develop a protein
corona almost instantaneously when immersed in blood serum (30).
Whether these collisions yield a fouled surface depends on the balance of the adsorption and
desorption rates of the proteins on the particle surface and how strongly the protein is bound
to the surface. The rates at which adsorption and desorption processes occur are proportional
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to their corresponding rate constants (kads and kdes , respectively). The value of kads depends on
the frequency of collisions between the proteins and nanoparticle ( fcollision ; Equation 1) and the
activation energy of adsorption (Eads ):

Eads
.
(2)
kads fcollision exp
RT
The value of kdes depends on the binding strength of the protein to the surface (the depth of the
potential energy well, Edes ):

Edes
.
(3)
kdes exp
RT

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

kdes
Gads
.
(4)
= exp
Kd =
kads
RT
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 that
are weak and/or infrequent have a large positive Gads and a large dissociation constant, indicating
rapid 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 and
yield a fouled particle surface. Thus, major efforts have been made to maximize Eads and minimize
Edes to reduce biofouling.
To understand how the adsorption/desorption equilibrium can be modulated, we nd it useful
to examine energy diagrams depicting the distance-dependent interaction between a nanoparticle 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 protein
separated by a surface-to-surface distance d. Negative free energy indicates stable, attractive interactions, whereas positive free energy indicates a net repulsion. The shape of the free energy curve
can be quite complex because the free energy is a sum of a large number of attractive and repulsive
forces from distinct chemical functionalities on the surfaces of protein and particles. Proteins, in
particular, have very complex interaction potentials because of the chemical diversity of amino acid
residues, 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 separation
distance is close enough to reach a regime called the secondary minimum. This minimum results
from interactions between the solvation shells and the terminal chemical groups tethered to the
protein or particle surface, yielding a weak net attraction that can be dissociated if the particles
separate, as the depth is similar to the thermal energy (kT ). This minimum leads to a dynamic
equilibrium 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 repulsive forces at the adsorption barrier either because of electrostatic repulsion between like-charged
particles or because of the loss of the exibility of molecular domains on the surface. This loss of
conformational (rotational) exibility creates local order, decreases entropy, and yields positive
(unfavorable) free energy for protein-particle interactions. The height of this energy barrier with
respect to the secondary minimum is equivalent to Eads in Equation 2. If this energy barrier can
be overcome, then the particle separation distance can be further reduced to reach the most stable
state, the primary minimum. Here the particle surfaces are in physical contact with an interaction
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Gads < 0
Fouled particle
c
Energy
Eads Edes
d
Gads < 0
Gads > 0
Nonfouled particle
e
+
Eads
Energy
Unfavorable
states
e
+
Energy

Born repulsion Adsorption barrier
Edes
Gads > 0
d
Favorable
states
Primary minimum Secondary minimum
Figure 2
(a) Schematic illustration of the interparticle distance, d, dened 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 and
discussed in the main text. (c) Potential energy plot of a particle with a deep primary energy well for adsorption and a small barrier for
protein adsorption, leading to surface fouling that is thermodynamically stable. (d ) Potential energy plot of a nanoparticle with a small
primary 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 a
hard corona (34) and are slow to exchange owing to the adsorption barrier, requiring several hours
to equilibrate in serum (30). Any further reduction in the separation distance is restricted because
of 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
(3538).
2.1. Antifouling Coatings

The key to minimizing fouling is to offset the attractive potential between a nanoparticle and
the proteins by using surface chemical modications designed to increase the adsorption barrier
(Eads ) and decrease the depth of the primary minimum (Edes ). Coatings that are resistant to protein
adsorption are often electrostatically nearly neutral and exhibit a high degree of surface exibility
and entropy. However, it is important to note that all nanoparticle coatings have a substantial
secondary minimum, as repulsive forces generally operate on short length scales, which drop
exponentially from the surface (32, 39, 40); thus, binding by proteins at a larger separation distance
is likely unavoidable, but its role or signicance under in vivo conditions is not clear. The depth of
the secondary minimum is proportional to the surface area and the polarizability of the interacting
entities (41), so it may be possible to eliminate its formation simply by using smaller particle
sizes, yielding a smaller number of geometrically possible interactions for a lower net energy
of attraction. Indeed, recent work has shown that semiconductor nanocrystals with the smallest
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hydrodynamic dimensions (48 nm) can reduce the nonspecic binding of proteins to very low
levels (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 with
electrostatically neutral groups (e.g., hydroxyls and ethers) (43). This is largely a result of the high
strength and long-distance nature of Coulombic forces. Most proteins in the blood, and in most
tissues and cells in general, have a small net negative charge at neutral pH, measured as a zeta
potential near 5 to 10 mV or an isoelectric pH of 5 to 6. Therefore, it is not surprising that
nanoparticles with a net cationic charge rapidly foul in biological uids and that nanoparticles with
a 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 experimental
and molecular dynamic simulations have shown that this effect is the result of local microdomains
of cationic charges on protein surfaces arising from tertiary structures comprising high local
concentrations of lysine and arginine (45), which can outweigh the net anion-anion repulsion
between the two particles in close proximity (46). Thus, any high-magnitude surface charge can
yield a high magnitude of Edes with proteins. Interestingly, the net surface charge and the chemical
identity of surface groups also dictate the specic classes of bound proteins (47).
Two types of functional groups have been reported to substantially reduce nonspecic binding
when compared with coatings with a net charge. Hydroxyls, compared with amines and carboxylic
acids, are uncharged at neutral pH and simultaneously can serve as a hydrogen bond donor and
acceptor, allowing a large degree of hydration (48). Similarly, it is possible to create surfaces that
are 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 amino
acid residues, as well as the phospholipid surface of cellular membranes (49). These coatings are
particularly promising owing to the formation of a hydrogen-bonding network with a locally dense
region of counterions (50, 51). Various nanoparticles have been generated with coatings containing
phosphatidylcholine groups (anionic phosphate and cationic ammonium), sulfobetaines (anionic
sulfate and cationic ammonium), and carboxybetaine (anionic carboxylate and cationic ammonium)
(52). These coatings have been shown to greatly reduce nonspecic protein adsorption on both
macroscopic and microscopic surfaces (5355).
2.3. Steric Repulsion

Whereas neutral coatings are benecial to reduce the depth of the primary minimum by modulating the enthalpy of interaction, they provide little benet with regard to a protective adsorption
barrier. Thus, an alternative and complementary approach is to coat nanoparticles with exible,
hydrophilic polymers, which effectively shield the surface through steric repulsion. The combination of exibility and a strong interaction with water molecules leads to a large number of
molecular congurations and degrees of freedom (high entropy). Fouling through the binding of a
bulky 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 usually
linear or branched chains, tethered to the nanoparticle surface through one end to act as entropic
springs 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 restoring 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 staple
of most nonfouling coatings since it was discovered almost 40 years ago and used to attenuate
the immunogenicity of foreign proteins in the blood (59). PEG is widely used to stabilize a large
variety of nanoparticles, such as liposomes (60, 61), polymeric particles (62), micelles (63), and
inorganic nanocrystals (e.g., gold, iron oxide, and quantum dot particles) (6466), that have rigid
surfaces and low intrinsic surface exibility. In addition, various other hydrophilic polymers have
been 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 410 nm of radial size,
preventing the production of sub-10-nm particles, which have been observed to have some of the
most unique biophysical behaviors. In addition, some polymeric coatings induce recognition by
the immune system, causing efcient removal as the body recognizes these foreign materials. This
has been observed with PEG-coated liposomes, leading to rapid blood clearance upon multiple
intravenous injections (73) owing to the binding of PEG-specic IgM antibodies produced during
the rst administered dose (73). Additionally, in drug screenings of a PEGylated product, it was
found 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 motivates the development of replacements for PEG coatings that do not induce immunological reactions. For example, polyamino acid coatings prepared by a mixture of hydroxylethyl-glutamate
and hydroxylethyl-asparagine residues were observed to resist accelerated blood clearance after
repeated injections (75).
3. NANOPARTICLE-CELL INTERACTIONS
In comparison with rapid protein adsorption on nanoparticles, cell-nanoparticle interactions are
often 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 the
liver and spleen can efciently remove nanoparticles that have been opsonized, displaying epitopes that are recognized by the cells as markers for clearance. The free energy diagrams for
nanoparticle-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 proteins, charged particles associate with cells more rapidly than neutral particles (48), with cationic
particles exhibiting the greatest stickiness to the anionic cell membranes (77). Nanoparticles with
greater hydrophobic character also demonstrate more rapid association with cells and uptake,
likely because of the interaction with lipophilic domains of the plasma membrane (78). Additionally, nonspecic associations with cells are similarly minimized through the use of zwitterionic
and neutral coatings and hydrophilic exible polymers (79).
Cellular binding and uptake of nanoparticles are strongly dependent on whether the nanoparticles have already been fouled by proteins. In fact, this attribute has been quite difcult to study as it
is often not clear if the binding event is mediated by the targeting protein attached to the nanoparticle or mediated by fouling proteins bound to the nanoparticle and/or targeting ligand. Even for
studies in which cell cultures are prepared with media free from exogenous proteins, cells continually secrete their own proteins, which can adsorb to the nanoparticle surface locally at the plasma
membrane to facilitate adhesion and uptake. Although nanoparticles that are nearly neutral and
resistant to protein fouling usually have enhanced cellular association once they become fouled, it
has been observed that charged and/or hydrophobic nanoparticles will exhibit lower cellular association once they are fouled with a protein corona (80). This lowering of adhesion rates may arise
from 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 that
are more specic, involving epitope binding of the adsorbed protein to its corresponding receptor
on the cell membrane. Recent work by Walkey et al. (47) has identied protein corona ngerprints,
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, in
which afnity ligands conjugated on the surface of the particles bind to surface membrane receptors. For efcient cellular delivery, the particles must have a high afnity to the cell receptors and
then develop enough ligand-receptor binding pairs to overcome the energetic barrier of wrapping
the cellular membrane around the particle for internalization. Both the binding afnity and subsequent cellular internalization can be enhanced by multivalent binding, in which multiple binding
events occur between ligand-receptor pairs (81) (see Figure 3a). In multivalent binding, there
is also the possibility for the positive cooperativity of binding among the multiple complexes, as
binding of one ligand on the nanoparticle will localize neighboring ligands closer to other receptors, facilitating further binding events. The multivalent binding afnity (often called avidity) is
dependent on both the monovalent binding afnity and the number of ligand-receptor binding
c
EXTRACELLULAR SPACE
1
mono
Kd
multi
Kd
CYTOSOL
Figure 3
(a) Multivalent interactions, by forming multiple ligand-receptor bond pairs as opposed to a single bond, signicantly increase the
afnity of nanoparticles to the cell membrane. (b) Nanoparticles with multiple afnity ligands increase the ux of receptors toward the
particle, leading to more binding events to gain the energy required to wrap the membrane around the particle. (c) A free nanoparticle
with afnity ligands () can contact the cell surface at which it binds multiple cell surface receptors to create energetically favorable
conditions for membrane wrapping and endocytosis ( and ).
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pairs (valency) (82):

K dmulti = (K dmono ) N .
(5)
Here K dmulti is the observed dissociation constant including multivalent effects, K dmono is the dissociation constant of a single ligand-receptor pair, is the degree of cooperativity ( < 1, negative
cooperativity; = 1, no cooperativity; > 1, positive cooperativity), and N is the number of afnity ligands bound to receptors. The positive cooperativity of the binding of multiple ligands can
dramatically enhance the observed dissociation constant from that of a single ligand-receptor complex. For example, nanoparticle-cell association enhancements have been observed experimentally
by using multiple folate receptor binding proteins, reaching values of 2,500170,000-fold (83).
Additionally, multivalent antiviral and anti-inammation agents are known to have potencies that
are orders of magnitude higher than their monovalent counterparts (82). Multivalent interactions
may occur for the adsorbed proteins within the nanoparticle corona as well for multiple adsorbing species presenting binding epitopes. Additionally, even sole adhesion molecules, such as the
opsonin bronectin, can contain multiple binding sites for cellular receptors (57).
The binding afnity to targeted cells is generally seen to increase with an increasing number
of ligands per particle, but there is a limit to the number of afnity ligands that can be used. For
example, negative cooperativity would decrease the afnity of particles to cells with additional
ligands as a result of steric crowding, for which each additional surface ligand will lower the
particle afnity to the cell by limiting the conformational freedom the ligand needs to effectively
bind to its target (84, 85). Another caution in applying multivalency involves the need to ensure
that the stealth properties offered by the nonfouling molecules are not lost owing to the complete
surface coverage of afnity ligands (86). Such loss of stealth behavior leads to increased nonspecic
interactions with the proteins and cells and an increase in nanoparticle accumulation in the liver
and spleen (87). Thus, optimization is needed to nd the maximum allowable number of ligands
for the greatest target afnity while not increasing nonspecic interactions.
3.2. Membrane Wrapping and Endocytosis

Once the nanoparticle has become associated with the cell surface, the cell membrane can start to
wrap around the particle to form a vesicle for engulfment (see Figure 3b). For the wrapping process
to occur, there needs to be sufcient ligand-receptor bond pairs to overcome the energetic barrier
of membrane bending (88, 89). Theoretical analysis of the thermodynamics of this process provides
two quantitative expressions for the minimum density of ligand-receptor bond pairs (l -r,min)
for particle uptake and the length of time for the membrane to wrap around the nanoparticle ( w )
(88, 89). The important parameters are the size of the particle (Rp ), the diffusivity of the receptors
on the membrane (Dr ), the elastic modulus of the membrane (Ebend ), and the energy of the binding
between the nanoparticle and membrane (Ebind ), which can be a combination of the binding
energies of the ligand-receptor pairs along with electrostatic and van der Waals interactions. The
proportionalities based on these parameters are
w
bond,min
Rp2
Dr
Ebend
.
Rp2 Ebind
(6)
(7)
From this analysis, it can be seen that smaller particles are wrapped in less time but require a higher
ligand density to overcome a greater membrane bending energy (owing to the higher curvature
of 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 lower
curvatures. However, increasing the ligand density for greater multivalent binding can enhance
the rates at which receptors cluster to the binding site (90), leading to decreased wrapping times
and higher rates of cellular internalization for both large and small particles. From numerical
results of the energy balance, an optimal particle radius is approximately 50 nm, which has the
shortest wrapping time (88, 89, 91). Experimentally, this size has been observed to show optimal
uptake (88, 89). However, most experimental studies have been performed in two-dimensional in
vitro environments, in which larger particles are observed to have higher uptake rates, possibly
because of higher sedimentation velocities, which increase their local concentration to the cells
residing at the bottom of the well. In the case in which cells are suspended above the well oor,
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 for
direct delivery to the cytosol is to destabilize the membrane structure. Particles with surfaces of
multiple cationic head groups can attract so many anionic phospholipids from the cell membrane
that the appearance of holes has been observed (93). Theoretical studies have shown that positively
charged particles that are larger in diameter than the thickness of the cell membrane can attract
phospholipids to form a bilayer coating (94). Cell-penetrating peptides offer another approach for
the 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 penetrate
membranes without bilayer disruption (97). Computer simulations of these particles describe their
mechanism of internalization as a lowering of the free energy of insertion of the nanoparticle into
the membrane, which allows quick insertion and withdrawal from cellular membranes (98). Utilizing such ligand structures may allow quick and efcient delivery of drug payloads directly to
the cytosol.
3.4. Intracellular Trapping and Escape

The internalized nanoparticles are often trapped in intracellular organelles, such as endosomes and
lysosomes, which have acidic pHs and contain degradative enzymes (99) (see Figure 3c). This environment is detrimental to therapeutic agents because the drugs are not available for binding their
targets and are subject to enzymatic degradation. Under certain conditions, endocytotic vesicles
may not develop the harsh conditions of lysosomes (99), and particles in vesicles may be recycled
back to the cell exterior or exocytosed (100, 101). Therefore, much research effort is currently devoted to determining what parameters affect the internalization pathway to avoid lysosomal degradation and/or escaping endosomal vesicles to the cytosol. Particles with varying degrees of sizes and
charges have been investigated to determine what conditions favor internalization methods that are
not directed toward lysosomal pathways (102, 103), but there is no consensus on the optimal conditions for such nondegradative endosomal pathways (99). One approach for lysosomal escape uses
the proton sponge effect, in which particles are designed to absorb protons upon the acidication of
the vesicle, disrupting the membrane by increasing osmotic pressure (104); another approach uses
light-sensitive molecules that can be activated to generate reactive oxygen species, which degrade
the vesicle components (105). The membrane-penetration approaches using peptides also offer
direct methods for the cytosol entrance, although lack the selectivity between normal and diseased
cells provided by the use of afnity 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 drug
delivery 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 particles, such as discs, rods, and hemispheres, have much slower rates of cellular uptake (thus longer
blood circulation times) than do isotropic spherical particles. Similar to the radius thresholds observed for spherical nanoparticles, rods can have radii below the minimum threshold and can have
large 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 some
studies, anisotropic particles have been observed to be taken up more rapidly than their spherical
counterparts (108, 109). This discrepancy is likely caused by variations in the ligand density and
experimental conditions. For example, afnity ligands may pack more efciently on elongated or
at surface particles, such as rods or cubes, which can increase driving forces for cellular internalization. Additionally, anisotropic nanoparticles are often synthesized by using hydrophobic ligands
that bind to the nanoparticles more strongly and are not completely removed during subsequent
steps, leading to erroneous interpretations of shape effects on cellular internalization.
Furthermore, exible and hydrophilic polymers, such as PEG, can interfere with receptor
binding and cellular internalization (see Figure 4a). In fact, a generally poor design for targeted
nanoparticles would involve afnity ligands directly attached to the particle surface and surrounded
by a PEG layer (Figure 4b). For PEG to impart a stealth behavior, it needs to have a molecular
weight of at least 2,000 Da at a grafting density high enough to extend the polymers into a brush
conguration (58, 110). Conversely, to expose surface-anchored targeting ligands for cell targeting, the PEG2000 molecules need to be sparse enough to adopt mushroom-like conformations,
which result in less nonspecic binding protection (111, 112). Therefore, a better design is to
tether the afnity ligand on the outer ends of the PEG chains. This situation benets from dense
PEG grafting densities as the brush conformation pushes the ligand to the outer surface, whereas
mushroom-like conformations will bury the ligand (see Figure 4c). It is also important that the
PEG length of the chain tethering the afnity ligand is similar to that of its unconjugated neighbors. If the ligand tethered chain is much longer than its neighbors, the extra length can fold into
the 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 ability
of 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 cancerous
tissue after intravenous injection. The tumor microenvironment differs from normal tissue in
various ways, some of which can be exploited for enhanced drug delivery through the use of
nanoparticle delivery agents (see Figure 5). There are several steps involved beyond just reaching
the tumor tissue, which include moving past the RES organs, crossing the vessel wall, traversing
through the tumoral interstitial space, binding to receptors on tumor cells, and internalization
(115). The ability of the nanoparticle to navigate these physiological barriers depends primarily
on the convective and diffusive transport properties, along with the chemical afnities between
the nanoparticle and the environment.
Convection and diffusion are the main transport mechanisms by which a nanoparticle crosses
the blood vessel walls (called extravasation). A mathematical model of the extravasation rate is
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Figure 4
Conformational effects of antifouling polymers on the binding ability of targeting molecules. (a) Targeting molecules anchored to the
nanoparticle surface are able to bind to their target if the surrounding polymers are sparsely grafted and adopt short mushroom-like
conformations. (b) With the use of higher grafting densities to push the polymers into a brush-like conformation, the particle is able to
resist biofouling but will block a surface-anchored molecule from binding to its target. (c) The ideal situation involves tethering the
targeting ligand to the end of the polymer chain and surrounding it by densely grafted polymers of the same length adopting a
brush-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 back
and bury the ligand, which hinders its ability to bind.
offered by the Staverman-Kedem-Katchalsky equation (116):
Js = P[CV CI ] + LP [(PV PI ) (V I )][1 F ]Clm .
(8)
Here Js is the net ux 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; is the osmotic reection coefcient; V and I are the osmotic pressures of the
vessel and interstitial areas, respectively; F is the solvent drag reection coefcient; and Clm is
the log mean of the vessel and interstitial particle concentrations. The equation is grouped by two
additive terms: The rst, presented in Equation 8, is particle ux from permeation/diffusion, and
the second is from convection.
In normal vessels, there is a balance between the hydrostatic pressure of the blood wanting to
push uid out and the opposing osmotic pressure from the higher plasma protein concentration
inside the vessel compared to the interstitial space [(PV PI ) (V I )]. These two effects cancel out the convective transport of nanoparticles for normal vessels, so nanoparticle extravasation
depends strongly on vessel permeability. Generally, particles that are larger than a few nanometers
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Figure 5
Schematic illustration of normal and leaky vasculatures, passive and active targeting, and transcytosis and exocytosis. (a) With small
pores within the vasculature, such as that of the gap junction of endothelial cells in normal tissue, extravasation of the particles is
inhibited, and they stay in the circulation. (b) In tumoral regions where there is leaky vasculature with large pores, particle extravasation
is facilitated, after which the particles can migrate through the interstitium. (c) Particles that are passively targeted, otherwise having no
afnity ligands for cell receptors, may perfuse the tissue, exhibiting cell-free channels. (d ) Actively targeted nanoparticles are likely to
be bound with the rst cells they encounter, signicantly slowing the transport within cell-free channels. (e) Passively targeted particles
have little mobility to pass cell-dense layers without a leaky or cell-free channel to diffuse in. ( f ) Actively targeted particles may be able
to 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 size
of endothelial gap junctions (117). Regions of high permeability in normal vessels exist at the
discontinuous capillaries found within the liver and spleen, which have wide openings (called fenestrations) between the endothelial cells lining the vessel wall (115). Thus, intravenously injected
particles display a biased accumulation toward tissues exhibiting vascular fenestrations that are
larger than the particle size. Particles in these regions of high permeability can potentially leave
these areas by returning to the vessel or by lymphatic drainage if not bound or taken up by the
cells. If particles are lacking a stealth coating or are otherwise opsonized, upon entering the highly
permeable regions of the liver and spleen, where there is a high local concentration of macrophage
cells, there will be a greater retention of particles within these organs. However, with good stealth
coatings, the particles are more likely to maintain longer circulation times and accumulate in the
liver and spleen at slower rates.
4.1. Passive Targeting

Solid tumors are known to have highly permeable vasculatures, allowing nanoparticles to cross
the vessel walls into the interstitial space (see Figure 5b). This highly permeable vascular network
is created when tumors reach length scales at which the diffusive transport of oxygen is not
sufcient 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 networks
have 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 a
functional lymphatic drainage system, thereby reducing particle clearance (118). The combination
of these conditions leads to an increased accumulation of circulating nanoparticles in the tumor
interstitium, which is called the enhanced permeation and retention (EPR) effect (119, 120).
Because small drug molecules have high permeability and clearance within various tissues of
the body, typically only 1 out of 1,000100,000 injected drug molecules reaches its intended
destination (121, 122). By having a size that is optimal for delivering drugs more preferentially
to tumors through the EPR effect, local dose concentrations delivered by nanoparticles can be
10100 times that of the free drug (123). The use of the EPR effect to deliver nanoparticles in
vivo is known as passive targeting (see Figure 5c).
However, the leaky vasculature and inefcient lymphatic drainage of tumor tissues can lead
to inefcient nanoparticle transport in solid tumors. Because there is no effective outlet for extravasated uid in tumors, the interstitial uid pressures are comparable to the vessel pressure
(PV PI ) (118). The interstitial osmotic pressure is increased as well because there is no effective
barrier to keep the plasma proteins from building up in the tumor interstitial space (V I )
(124). Thus, as with normal tissues, extravasation relies primarily on permeability. Furthermore,
because of inefcient lymphatic drainage within the tumor interstitium, diffusion is the primary
mode of transport once the particle has crossed the vascular wall. Favorable conditions for particles
to perfuse the tumoral area will include high vascularization throughout the tumor volume and
low-density extracellular matrices allowing higher particle diffusivity.
Methods to increase nanoparticle delivery to tumors include shortening the diffusion length
and increasing the vessel area per unit volume of tissue (125), momentarily increasing the blood
pressure to promote enhanced extravasation of particles (126), and utilizing enzymes to degrade the
extracellular matrix (127). Jain (128) demonstrated that vascular renormalization can increase the
efcacy of cancer therapeutic drugs. The addition of vascular channels throughout the tumor will
decrease the particle diffusive distances to cells from a vessel and cause greater convection toward
the 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 via
the administration of the strong vasoconstrictor angiotensin II (126). As tumor blood vessels lack
smooth muscle cells, angiotensin II will constrict only normal blood vessels. As the blood ow is
hindered in the normal vessels, greater blood ow will be directed to the tumor vessels, which maintain the same dilation. However, we note that this effect is temporary, and the vessel and interstitial
pressures will reverse ow to equilibrate, which may cause particles to re-enter the bloodstream
during the process. The extracellular matrix of the tumor interstitium is a dense network of collagen bers, which severely limits the diffusive mobility of particles. For the enhancement of the
diffusive mobility within tumor tissues, the use of bacterial collagenase enzymes has been found
to increase the interstitial distribution of 75-nm viral particles by a factor of three (130).

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4.2. Active Targeting

In comparison with the nonspecic EPR effect, active targeting uses afnity ligands, such as
antibodies and peptides, to specically 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 effective
in delivering imaging and therapeutic agents to solid tumors (132). For example, tumor-targeting
studies using uorescently 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 and
nanoparticles than their nontargeted controls (132). However, opposing evidence has also been
reported, indicating that the use of tumor-targeting ligands does not increase the total nanoparticle
accumulation in solid tumors, although it does increase receptor-mediated internalization and
could thus improve the therapeutic efcacy for cancer drugs that act on intracellular protein targets
(133). Complicating this matter further, recent work has shown that the use of targeting ligands
might even be detrimental because the exposed ligands can accelerate nanoparticle opsonization
(adsorption of blood proteins) and blood clearance, leading to an overall reduction in tumor
nanoparticle uptake (134).
It is generally accepted that active targeting can aid in greater endocytosis of the nanoparticles as
targeted ligands decrease the free energy of internalization. For instance, it has been observed that
transferrin-targeted nanoparticles had improved internalization within cancer cells (135). There
are 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 efcacy (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 that
particles with high adhesion strengths to their target will be prevented from complete tumor perfusion beyond vascular channels, as they will be stuck on the periphery of their rst contact (138).
However, for dense tumor spheroids having minimal pores, active targeting has been observed
to have deeper tissue penetration than passive particles as the nanoparticle can travel through the
tissue by endocytotic/exocytotic transport (139) (see Figure 5e,f ). Another possible outcome is
that, if these agents are highly localized and are then able to be either internalized or released to
surrounding cells in a timely fashion, there may be an additional bystander effect of cytotoxicity to
cancer cells without high levels of target receptor expression (137). An additional concern is that
targeting molecules present on the nanoparticle surface may be more prone to nonspecic protein
adsorption and immune response, which may block the targeting ability and increase immune
cell 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 active targeted nanoparticles owing to high afnities to their cellular targets, have such drawbacks
due to their relatively large sizes and immunogenicity (140, 141). Thus, there is great interest in
developing fully human antibodies and using fragments to retain the afnity and specicity of the
parent molecule with less immunogenicity and smaller size (142, 143).
5. NANOPARTICLE DESIGNS TO OVERCOME

PHYSIOLOGICAL BARRIERS
As discussed above, there are technical and fundamental barriers to in vivo nanoparticle delivery
and targeting, including biofouling, RES uptake, poor tissue penetration, and limited endosomal
release. These problems could be overcome or mitigated by the design of smart or intelligent nanostructures, 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 acidic
environments within tumoral areas (144). The concept here is to envelop a targeted nanoparticle
with an antifouling coating, which will prevent biological entities from accessing the targeting
ligands until the nanoparticle is delivered to the tumoral area, which then is cleaved away, exposing 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 liposomes, having targeting ligands that showed enhanced accumulation and penetration within tumor
tissues. Once the nanoparticles have accumulated within the tumoral area, another stimulus may
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Drug loading
Tumor environment
pH drop
PNIPAAM
coating

Laser
irradiation
Magnetic field
d
T
Alternating
magnetic field
Figure 6
Schematic illustration of smart nanoparticle systems to overcome delivery barriers. (a) A pH sheddable antifouling coating covers
afnity ligands in a nanoparticle until the particle reaches the tumor microenvironment, where it experiences a drop in pH and
subsequently sheds the antifouling coating, exposing the ligands for binding to their targets. (b) Gold cubes that are hollow and porous
when coated with temperature-sensitive polymers, such as NIPAAM, can be photothermally activated with laser light, which collapses
the polymer coating and allows the release of the enclosed drug. (c) Magnetic nanoparticles have the ability to be guided with magnetic
elds to the tumor site. (d ) An alternating magnetic eld can then be applied to thermally agitate the particles, thus raising the local
temperature to destroy the surrounding cells.
be employed for a rapid release of the drug payload. Polymers such as PNIPAAM, which phase
transitions from solvated extended coils to shrunken states upon heating, can act as temperatureactivated doors to the drugs encased within (146). The induction of temperature increases for
drug release can come from plasmonic nanoparticles, which efciently convert photon energy
to 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 when
irradiated with laser light. The particles were able to be loaded with drug molecules and then
coated with NIPAAM polymers, which allowed drug release from the pores on command upon
light activation (148). Alternatively, localization and therapy may be performed through a single
type of stimulus. For example, magnetic nanoparticles under the inuence of external magnetic
elds can be guided to the tumor site (149, 150). Once at the tumor site, an alternating magnetic
eld can be applied to agitate the nanoparticles to increase the local temperature, killing nearby
cancer cells through a process called magnetic ablation (151) (see Figure 6c,d ).
There have also been various constructions of nanoparticles with multiple stages of stimuli responses (152). Although multistimuli-responsive nanoparticles have recently sparked great
enthusiasm in research, we note that more functionality leads to more complexity in particle development. 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,
purication procedure, and nal yield. Additionally, the accumulation of the levels of functionality
may lead to interactions, which can lead to less desirable outcomes than if multiple monofunctional
particles were employed (153). Thus, a careful and thorough characterization of such particles is
warranted to inspect the complex surface properties from these interactions and their relationship
to the stability and immunogenicity of the particles within in vivo environments. An approach to
reduce complexity would be the use of materials that have inherent multifunctionality, such as
magnetic nanoparticles or drug molecules, which have an inherent physical property that can be
imaged.

5.1. pH-Triggered On/Off Probes

For cellular and in vivo cancer imaging, Gao and coworkers (154, 155) developed a class of activatable or smart nanoparticles based on the use of copolymer materials with ionizable tertiary amine
groups and covalently conjugated uorescence dyes. A novel nding is that the self-assembled
structures undergo a dramatic and sharp transition within the very narrow range of pH (often
less than 0.2 pH units). This pH-induced transition leads to rapid and complete dissociation of
the nanomicelles, and as a result, the covalently linked dyes change from a self-quenched off state
to a highly emissive, bright on state. This supersensitive and nonlinear response to external pH
provides a new strategy in targeting acidic organelles in cancer cells, as well as the acidic microenvironment in solid tumors. This feature is important in addressing the tumor heterogeneity
problem, a major challenge for various imaging and therapeutic approaches based on molecular or
receptor targeting. By targeting the more common hallmarks of tumors (i.e., the acidic habitat or
microenvironment and the growth of new blood vessels or angiogenesis), this work has opened up
exciting opportunities in detecting and potentially treating a broad range of human solid tumors.
Another feature is the signicant improvement in detection sensitivity because each nanoparticle
probe contains multiple copies of the dye, which are turned on (restored to uorescence) in an
all-or-none fashion, leading to amplied uorescence signals that are many times brighter than
single dye molecules. Of course, a major limitation of optical imaging is that the tissue penetration
of 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 endoscopic and image-guided surgery applications, in which the light is brought to the tissue and tumor
surfaces via an endoscope or a surgical incision. Overall, this class of pH-activated and supersensitive polymeric micelles has demonstrated a new concept in the design of novel nanoparticle probes
and is expected to have broad applications in cancer biology, endoscopic cancer screening, and
image-guided interventions.
5.2. Mothership Nanocarriers

Multistage or mothership nanocarriers are constructions in which a larger nanoparticle either
encases or is constructed from nanoparticles that are near an order of magnitude smaller, which
are released upon the dissolution or enzymatic degradation of the container or linkers (156159).
Such constructions address the fact that larger particles typically have longer circulation lifetimes
and are more effective in taking advantage of the EPR effect, whereas smaller nanoparticles, less
than 10 nm, are rapidly eliminated from the blood by renal excretion and are more effective in
perfusing tumoral tissue (160). One method to construct multistage particles is to use DNA to
link several 6-nm particles into a larger construct (159). Here, particles can efciently accumulate
to 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 agents
as they are able to effectively locate to the site to light up the tumoral area and then degrade for
rapid elimination from the body, thereby reducing potential toxicity concerns (161). To address
the different stages of transport in the tumor microenvironment, Wong et al. (158) presented a
multistage method in which nanoparticles could penetrate deep into tumor tissue. They encased
10-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, where
the gelatin is then degraded by enzymes, which release the quantum dots that exhibit better
penetration into the tumor parenchyma.
6. FIRST-IN-HUMAN CLINICAL STUDIES

Recent rst-in-human pilot studies have successfully used both passive and active targeting agents
for image-guided surgery of naturally occurring tumors in humans (162164). The specic tumor
types 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 moderately
leaky vasculatures, so the EPR effect provides a general means for the passive delivery of imaging agents (56-nm albumin-bound indocyanine green) to a broad range of human tumors, and
(b) uorescent dyes, such as uorescein, can be conjugated to targeting ligands, such as folate acid,
for specic 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 enrolled ve 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 the
clinical stage and operative approach prior to surgery. All enrolled patients were thought to have
limited disease, amenable to surgery, and no metastases (i.e., potentially curable). The median
tumor size was 2.3 cm (range of 1.89.1 cm) on preoperative imaging. Patients were injected
with indocyanine green prior to surgery. At the time of surgery, the body cavity was opened and
inspected. The results demonstrate that NIR imaging can identify tumors from normal tissues,
provides excellent tissue contrast, and facilitates the resection of tumors. However, in situations
in which there is signicant peritumoral inammation, NIR imaging with indocyanine green is
not helpful. This suggests that nontargeted NIR dyes that accumulate in hyperpermeable tissues
will have signicant limitations in the future, and receptor-specic NIR dyes may be necessary to
overcome this problem.
6.2. Clinical Studies of Active Fluorescent Agents

First-in-human results have also been reported from intraoperative tumor-specic uorescence
imaging by using uorescein-conjugated agents to actively target the folate receptor in both
ovarian cancer and lung cancer. In patients with lung cancer, the results showed that targeted
molecular imaging could identify 46 (92%) of the 50 lung adenocarcinomas and had no false
positive uptake in the chest. In vivo, prior to exposing the tumor, molecular imaging could only
locate 7 of the lesions. After dissecting the lung parenchyma, 39 more nodules could be detected.
Four nodules were not uorescent, and immunohistochemistry showed that these nodules did not
express FR. In the 46 positive nodules, tumor uorescence was independent of size, metabolic
activity, histology, and tumor differentiation. Tumors closer to the pleural surface were more
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CT
PET
Brightfield
NIR
Lung
cancer

Thymic
neoplasm
Carcinoid
Figure 7
Human clinical data comparing uorescent 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 underwent
resection of their tumors. Ex vivo, near-infrared (NIR) uorescence imaging demonstrated that the tumors were highly uorescent and
the surrounding organ had minimal background noise. The optical images were easy to interpret by the surgeon and facilitated the
identication of tumors. Spectroscopy demonstrated a signal-to-background ratio of 8:1 for the thymoma and 7.9:1 for the lung
carcinoid. Figure adapted from Holt et al. (162) with permission.
uorescent than tumors deep in the parenchyma. Additionally, in two cases, this strategy was able
to discover tumor nodules that were not located preoperatively or intraoperatively by standard
techniques. Taken together, these pilot clinical trials have demonstrated for the rst time that
targeted intraoperative imaging may lead to more complete surgical resections and potentially
better staging.
7. CONCLUDING REMARKS
In conclusion, we note that optimal nanoparticles designed for imaging and those for therapy usually have very different behaviors in vivo. Imaging agents should rapidly reach the target site and
then be eliminated from the body via renal clearance, degradation, or other fast pharmacokinetic
mechanisms (i.e., fast in and fast out). Also, for the detection and delineation of tumor margins
and residual tumor cells, imaging agents do not need to penetrate deeply into the tumor interior,
and accumulation at the tumor boundaries is sufcient. In contrast, drug therapy benets from
nanoparticles that have longer circulation times, are retained in tumors over an extended period of
time, and are able to penetrate the tumors interior (i.e., slow in and slow out). These opposing optimal conditions have raised concerns about the design of theranostic particles, which are particles
that contain both therapeutic and imaging agents (153). The rapid elimination of contrast agents
not 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 benets from
long circulation times for greater accumulation within the target. However, the combination of
both imaging and therapy within nanoparticles may be useful in tracking biodistributions. Looking into the future, there are several scientic issues and research directions that are particularly
promising but require concerted effort for success. (a) Researchers need to design and develop
stimuli-responsive and biodegradable nanoparticles to overcome nonspecic protein adsorption,
adverse organ uptake, and RES scavenging. (b) Combinatorial contrast agents (cocktail tracers)
should be developed for multicolor and molecularly specic detection of tumors, nerves, and blood
vessels for image-guided diagnostic or surgical procedures under minimally invasive conditions
(e.g., endoscopy and robotic surgery). (c) Strategies should be established to deliver therapeutic
nanoparticles into solid tumors beyond the rst few layers of vascular endothelial cells. (d ) We need
effective mechanisms to trigger the endosomal and lysosomal release of drug payloads inside targeted cells or organs. (e) Nanotoxicological studies including nanoparticle distribution, excretion,
metabolism, and pharmacokinetics and pharmacodynamics in large animal models such as cats
and dogs, which are most relevant to human physiology, should be done as well. ( f ) Nanoparticles
should be standardized and manufactured in compliance with FDA requirements.

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DISCLOSURE STATEMENT
One of the authors (S.N.) is a scientic consultant of Spectropath Inc., a company to further
develop and commercialize spectroscopic devices and agents for image-guided cancer diagnostics
and 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 Program
for a Pathway to Independence Award (K99CA154006 and R00CA153914).
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Review in Advance first posted online

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Departments of Biomedical Engineering and Chemistry, Emory University and Georgia

Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana,

College of Engineering and Applied Sciences, Nanjing University, Nanjing,

Annu. Rev. Phys. Chem. 2015. 66:52147

The Annual Review of Physical Chemistry is online at

active targeting, passive targeting, antifouling, molecular imaging,

This articles doi:

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

Born repulsion Adsorption barrier

2.1. Antifouling Coatings

2.2. Neutral and Zwitterionic Coatings

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

2.3. Steric Repulsion

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

3.1. Multivalent Binding and Cellular Uptake

pairs (valency) (82):

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

3.2. Membrane Wrapping and Endocytosis

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

3.3. Direct Internalization

3.4. Intracellular Trapping and Escape

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

3.5. Effects of Particle Shape, Size, and Conformation

4. IN VIVO NANOPARTICLE TRANSPORT AND TARGETING

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

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

Js = P[CV CI ] + LP [(PV PI ) (V I )][1 F ]Clm .

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

4.1. Passive Targeting

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

4.2. Active Targeting

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

5. NANOPARTICLE DESIGNS TO OVERCOME

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

5.1. pH-Triggered On/Off Probes

5.2. Mothership Nanocarriers

6. FIRST-IN-HUMAN CLINICAL STUDIES

6.1. Clinical Studies of Passive Fluorescent Agents

6.2. Clinical Studies of Active Fluorescent Agents

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

protein monolayer formation on colloidal nanoparticles. Nat. Nanotechnol. 4:57780

adsorption on polymeric nanoparticles: analysis by two-dimensional electrophoresis. Eur. J. Pharm.

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Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

liposomes in vitro based on sterol-PEG1300 anchors. Pharm. Res. 26:52938

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

Annu. Rev. Phys. Chem. 2015.66. Downloaded from www.annualreviews.org

www.annualreviews.org Physical Chemistry of Nanomedicine

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