STRATEGIES TO IMPROVE PLASMA CIRCULATION OF NANOPARTICLES

Author : Ameya R. Kirtane

Page Nos : 7 - 18

Cite Article :

Kirtane AR. Strategies to Improve Plasma Circulation of Nanoparticles. NUJPS. 2014;1(1):1–18.

Abbreviations :

Mononuclear phagocytic system, MPS; reticuloendothelial system, RES; poly-(ethylene glycol), PEG; red blood cells, RBCs; monosialylganglioside, GM1; poly- (vinyl pyrolidone), PVP; poly-(acrylamide), PAA; poly-(acryloylmorpholine), PAcM; poly- (2-methyl-2-oxazoline), PMOZ; poly-(2-ethyl-2-oxazoline), PEOZ; poly-(lactide-coglycolide), PLGA; accelerated blood clearance, ABC

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Introduction

Nano-sized carriers are rapidly emerging
as a useful platform for drug delivery [1,
2]. While nanoparticles offer several
advantages over other drug delivery
systems, these advantages can be realized
only if the carriers are maintained in
circulation for prolonged time intervals.
For example, nanoparticles are extensively
used to achieve targeted drug delivery [3,
4]. However, when the target has a limited
blood supply or is at a very low
abundance, nanoparticles need to circulate
through the target several times in order to
accumulate at high enough concentrations
[5, 6]. Nanoparticles are also used as
controlled-drug delivery vehicles to sustain
high drug levels in the plasma. This too,
can be achieved only if the nanoparticles
remain in circulation for prolonged periods
of time [7]. Hence, there is a considerable
interest in improving the plasma residence
time of nanoparticles. Once administered,
nanoparticles are rapidly eliminated from
blood circulation via a two-step process
[8]. In the first step serum proteins, of the
complement system, are adsorbed on the
surface of nanoparticles. This process is
known as opsonization and is vital for the
elimination of nanoparticles [8-10].
Following opsonization, nanoparticles are
engulfed by circulating macrophages or
macrophages residing in the liver and
spleen (referred to as mononuclear
phagocytic system (MPS) or the
reticuloendothelial system (RES)) [8, 9,
11]. Hence, in order to prolong the
circulation time of nanoparticles, it is

essential to subvert either of the two
processes.
There have been several attempts to
increase the blood circulation of
nanoparticles [12, 13]. Initial attempts
were directed towards altering the surface
of particles [14]. This led to the use of nonionic
hydrophilic polymers [15]. This has
been the most widely used strategy till
date. Some researchers have also shown
that changing the physicochemical
properties of the core of the nanoparticles,
like size and shape, can also increase their
circulation half-life [16]. Recent advances
in synthetic techniques have inspired
researchers to develop nanoparticles that
mimic blood cells [17]. These approaches
have also helped prolong the plasma
residence of nanoparticles.
In this short review, I have outlined a
various strategies that have been used
successfully to produce long circulating
nanomedicine. I focus, primarily, on the
rationale underlying the use of each
technique, their advantages and potential
shortcomings.
Modifying nanoparticle morphology to
enhance circulation time
Particle size
Particle size is an important determinant of
the plasma circulation time of
nanoparticles [18]. Hence, altering the
particle size can be an effective technique
to prolong plasma circulation. However,
unlike other strategies discussed in this
review, the choice of particle size iso.

peculiar [19]. Nanoparticles are eliminated
from plasma circulation via macrophage
uptake, distribution to peripheral organs, or
urinary excretion. Alterations in particle
size can affect each of these processes
[19]. Hence, careful consideration of
particle size is required. The particle size at
which all the three processes are
minimized will result in the highest
circulation time for nanoparticles. For
spherical nanoparticles, macrophage
uptake increases with an increase in
hydrodynamic diameter. Fang et al.
compared protein adsorption on 80, 170
and 240 nm nanoparticles incubated in
mouse serum [20]. They found that 240 nm
nanoparticles showed a 6-fold higher
protein adsorption as compared to 80 nm
particles. Consequently, larger
nanoparticles had a higher macrophage
uptake in vitro. Finally, 80 nm
nanoparticles also showed a longer
elimination half-life as compared to 170
and 240 nm nanoparticles [20]. Papović et
al. compared the circulation half-life of 12,
60 and 125 nm nanoparticles [21]. They
also found that the smallest particles had
the longest elimination half-life. These
results indicate that smaller particles are
more efficient at evading the RES [21].
While smaller nanoparticles show lower
RES uptake, decreasing the particle size
below a certain threshold may result in
elimination of particles through other
mechanisms [19]. For example, Liu et al.
showed that decreasing particle size below
50 nm increases their accumulation in the
liver [22]. As hepatic capillaries are
fenestrated (pore size ~75 nm), they are
permissive to the entry of extremely small

nanoparticles. Nanoparticles below 5.5 nm
can be filtered through renal filtration [23].
Choi and colleagues showed that the
circulation half-life of 8 nm quantum dots
was longer than that of 4 nm quantum dots.
This increased plasma residence for larger
particles was because of the lower renal
clearance of the larger nanoparticles [23].
Figure 1 shows the half-life of
nanoparticles of various sizes [23, 24]. For
extremely small particles (<20 nm), the
half-life increases with an increase in
particle size. The half-life reaches a peak at
an intermediate particle size and then
declines with a further increase in particle
size. However, since RES uptake is often
the major mechanism of particle
elimination and most studies utilize
particles in the size range of ~100 nm,
smaller nanoparticles are generally
considered favourable [24].

test
Figure 1: Half-life of various sized
nanoparticles
The figure shows plasma half-life of nanoparticles of
different sizes. For extremely small nanoparticles,
renal clearance decreases with an increase in particle
size. This leads to enhanced circulation times. For
particles larger than 20 nm, RES uptake increases
with an increase in particle size. Thus half-life
decreases with an increase in particle size. Data was
reported in [23, 24]. Adapted with permission from
[23, 24].

Particle shape
There is very limited information available
about the performance of non-spherical
nanoparticles. This, in part, was due to lack
of techniques for the synthesis of nonspherical
nanoparticles. However, in recent
years, synthesis and characterization of
non-spherical nanoparticles has gained
tremendous interest and has become a
subject of extensive research [16, 25-27].
Geng and co-workers were the first to
show that non-spherical nanoparticles have
superior pharmacokinetics and anti-tumour
efficacy as compared to spherical
nanoparticles [28]. This thesis studied the
plasma circulation of filamentous micelles
composed of block co-polymers of poly-
(ethylene glycol) (PEG) and poly-
(ethylethylene) and that of poly-(ethylene
glycol) and poly-(caprolactone). In a
mouse model, filamentous micelles had a
significantly longer half-life (~144 hours)
than spherical polymerosomes (half-life
~24 hours). The effect of particle size of
filomicelles was also studied. Interestingly,
filomicelles having an initial length of 8
μm (equivalent to the diameter of red
blood cells (RBCs)) had the longest
circulation half-life. Filomicelles with a
shorter or longer lengths were eliminated
faster [28]. While the study by Geng et al.
provided the first insight into the biological
performance of non-spherical
nanoparticles, it analysed only one particle
shape. A mechanistic understanding of this
interaction was first provided by
Champion et

ellipsoids, elliptical discs and rectangular
discs. The authors found that the initiation
and completion of phagocytosis were
strongly affected by the morphology of the
nanoparticles. The orientation of
nanoparticles relative to the macrophages
dictated the initiation of the process. The
volume of nanoparticles (a function of
particle size) dictated the completion of
this process. For example, if the alignment
of elliptical discs was perpendicular to the
approaching surface of the macrophage,
the process of phagocytosis did not initiate
at all. However, if the elliptical disc was
aligned parallel to the macrophage surface,
phagocytosis initiated spontaneously. The
completion of this process, however,
depended on the volume of the particle
relative to the macrophage [29]. Hence,
particle shapes that minimize initiation of
phagocytosis will have the longest
circulation half-life.
Following these studies, there have been a
number of experiments which have shown
that certain non-spherical nanoparticles
have superior pharmacokinetics, efficacy
and targeting abilities as compared to
spherical nanoparticles [30-33]. However,
spherical nanoparticles are still preferred
and more popular owing to their ease of
synthesis.
Surface functionalization of
nanoparticles with hydrophilic polymers
Opsonization and subsequent macrophage
uptake of nanoparticles are known to be
triggered by the hydrophobicity and charge
of the surface of nanoparticles [9, 34].
Hence, initial attempts to decrease MPS
mediated nanoparticle uptake were

directed towards masking the surface
hydrophobicity and charge of
nanoparticles. This has been achieved by
using non-ionic hydrophilic polymers,
physically adsorbed or chemically grafted,
on the surface of nanoparticles.
The use of amphiphilic polymers was first
suggested by Illum et al. [14]. In this
study, polystyrene microparticles were
surface functionalized with poloxamer
338, a tri-block co-polymer consisting of a
central hydrophobic chain of poly-
(propylene oxide), flanked by two
hydrophilic chains of poly-(ethylene
oxide). The authors showed that poloxamer
decreased the rapid initial uptake of
microparticles into the liver. This resulted
in higher plasma concentrations at early
time points. Although the hepatic uptake of
particles was decreased considerably, there
was no significant increase in terminal
half-life of the surface coated particles
[14].
In another study, Allen et al. hypothesized
that increasing surface hydrophilicity and
mimicking the cell surface of blood cells
may enhance the circulation time of
nanoparticles [35]. Earlier studies
investigating the long circulation times of
RBCs had highlighted the role of surface
sialic acid moieties [36]. To replicate this,
the liposome surface was modified with
sialic acid by incorporating
monosialylganglioside (GM1) into the
liposome bilayer. The authors found that
incorporation of GM1 in the lipid bilayer
significantly decreased their hepatic
uptake. As the molar ratio of GM1 in the
lipid bilayer increased, RES uptake of

liposomes decreased. However, increasing
the amount of GM1 beyond a certain
concentration compromised the stability of
the lipid membrane [35]. This limitation
could be overcome by using lipids, like
sphingomyelin, which showed greater
intermolecular interactions [37]. Such
lipids increased the rigidity of the liposome
membrane and allowed higher
incorporation of GM1 [35]. Gabizon et al.
later compared the tumour accumulation of
non-functionalized liposomes and GM-1
functionalized liposomes in a mouse model
of lymphoma [38]. GM-1 functionalized
liposomes showed 25-fold increase in
tumour levels as compared to the nonfunctionalized
liposomes [38]. As GM-1
functionalized liposomes partially evaded
the RES system, these liposomes were
referred to as “stealth” liposomes.
Poly-(ethylene glycol)
Though GM-1 provided excellent stealth
properties to nanocarriers, it was required
in large quantities. Additionally,
reproducible synthesis of this polymer was
also challenging [39]. Hence, there was
tremendous interest in developing alternate
polymers to substitute GM1. Klibanov et
al. used poly-(ethylene glycol) (PEG) to
increase the circulation of liposomes [40].
This strategy had been successfully
implemented to prolong the circulation
half-life of proteins [41]. In agreement
with those reports, the authors found that
PEG functionalized liposomes (half-life: 5
hours) had a significantly higher plasma
residence time than non-functionalized
liposomes (half-life: 0.5 hours). GM-1
functionalized liposomes had an

intermediate half-life of 1.5 hours [40].
Papahadjopoulos et al. later confirmed
these results in a mouse model of colon
carcinoma [42]. PEG liposomes had an
extended plasma half-life (~5 fold higher
than the non-functionalized liposomes) and
showed lower accumulation in the major
RES organs, liver and spleen.
Consequently, there was higher tumour
accumulation of PEG-liposomes as
compared to the plain liposomes [42].

Schematic diagrams of PEG
Figure 2: Schematic diagrams of PEG
configurations on the upper hemisphere
of a polymeric nanoparticle.
In (a), the low surface coverage of PEG chains leads
to the “mushroom” configuration where most of the
chains are located closer to the particles surface. In
(b), the high surface coverage and lack of mobility
of the PEG chains leads to the “brush” configuration
where most of the chains are extended away from
the surface. Reproduced with permission from [9]
PEG provides steric hindrance to the
adsorption of opsonins and hence
decreases the RES uptake of nanoparticles
[9, 43, 44]. Surface density of PEG is an
important variable that can affect the
efficiency of the polymer [45]. Best steric
hindrance is achieved at an intermediate

density [46]. At a low surface density, PEG
chains exist in a mushroom configuration,
while at a high surface density they exist in
a brush configuration (Figure 2). As
opsonins approach the nanoparticle
surface, they cause “brush”-shaped PEG
chains to compress. The compressed-high
energy conformation of PEG chains is not
favourable and as it returns to the preferred
elongated conformation, opsonins are
pushed away from the nanoparticle
surface. Hence, it is important that the
surface density of PEG is high enough that
they exist in a brush conformation.
However, if the surface density is too high,
PEG chains lose their flexibility and their
protective ability. At low surface coverage,
opsonins can get adsorbed in the space
between the PEG chains [9, 45, 47].
Many studies have dealt with the effect of
molecular weight on the efficacy of PEG
[45, 48-50]. Higher molecular weight PEG
provides better protection against
opsonization [44]. Longer PEG chains can
provide higher repulsive forces against the
adsorption of opsonins [44]. However,
functionalizing nanoparticles with
extremely large PEG moieties decreases
their interaction with the target cells as
well [43]. Additionally, larger hydrophilic
polymers are thermodynamically more
stable when they are free in solution [51].
Thus, preparing stable nanoparticle
formulations with larger PEGs is
challenging. Hence, the use of mid-sized
PEGs (molecular weight: 1000- 5000 Da)
is most common [52, 53]. The method of
PEGylation (surface adsorption vs.
chemical grafting) and the type of PEG
(branched vs. linear vs. star shaped) have

also been found to affect the efficiency of
PEG. [54-58]. However, these parameters
have not been discussed in this review.
PEG is the preferred polymer for making
long circulating nanocarriers [6]. There are
several reasons for the popularity of PEG.
First, PEG is non-biodegradable. Hence, it
does not produce any toxic metabolites and
is easily cleared from blood circulation via
renal filtration. Additionally, synthesis of
PEG polymers with a low polydispersity
index is relatively simple. This allows a
precise control of the characteristics of the
final product. Moreover, PEG chains are
flexible. Hence a very small mole% of
PEG is required to cover the entire
nanoparticle surface and provide stealth
properties to the formulation [6, 47].
Finally, the two terminal groups of PEG
can be tailored to specification. It is
common to conjugate one end group with a
hydrophobic polymer (to allow docking on
the lipophilic surface of the nanoparticle)
and the other end group to targeting
moieties [3, 52, 53].
Other polymers
Due to the success of PEG, there was
considerable interest in investigating other
hydrophilic polymers for stealth properties.
Vinyl polymers like poly-(vinyl
pyrolidone) (PVP), poly-(acrylamide)
(PAA) and poly-(acryloylmorpholine)
(PAcM) were one of the first alternate
polymers studied [51, 59]. PVP, PAA and
PAcM were conjugated to a hydrophobic
phospholipid or acyl chain to produce
amphiphillic derivatives. Molecular
weights of the acyl linker and PVP or PAA

were optimized to provide highest
encapsulation efficiency and steric
protection. However, it was found that
these polymers performed similar to PEG
and did not provide any additional benefit
[51, 59].
Amphiphillic derivatives of poly-(2-
methyl-2-oxazoline) (PMOZ) and poly-(2-
ethyl-2-oxazoline) (PEOZ) have also been
investigated [60]. It was found that both,
PEOZ and PMOZ, delayed RES clearance
of liposomes in a rat model. The half-life
of PEOZ and PMOZ functionalized
liposomes was similar to that of PEG
functionalized liposomes [60]. Maruyama
and colleagues showed that polyglycerols
could also be used to delay RES uptake of
liposomes [61]. Amphiphillic derivatives
of polyglycerol were synthesized by
conjugating polyglycerol with
dipalmitoylphosphotidic acid. The
presence of polyglycerol moieties on the
surface of liposomes decreased their
uptake in major RES organs like the liver,
kidney and spleen. The protective ability of
polyglycerol increased with the degree of
polymerization of the polymer. However,
this study did not compare the polyglycerol
derivatives to PEG [61].
Some studies have also shown that
polysaccharides can be used effectively to
provide stealth properties to nanoparticles
[62]. In particular, dextran coating has
been widely used to protect iron oxide
nanoparticles against RES uptake [63].
Polysaccharides provide a unique
advantage over other polymer coatings.
Polysaccharides can be used as ‘active
targeting’ ligands, as many cells express

receptors for these molecules [62]. While
PEG and other synthetic polymers are
widely accepted [64-66], there are certain
disadvantages associated with them. Some
reports have shown the production of anti-
PEG IgM antibodies after the first dose of
PEGylated nanoparticles [67, 68]. This
significantly enhances the blood clearance
of the second dose of nanoparticles. In
fact, the second dose of PEG nanoparticles
are eliminated almost as rapidly as nonfunctionalized
nanoparticles. This
phenomenon, termed as accelerated blood
clearance (ABC) [67], can have significant
ramifications on the use of synthetic
polymers. It should be noted, however, that
ABC is observed only for a limited time
after the first dose of PEGylated
nanoparticles. Ishida et al. found that if the
second dose of PEG nanoparticles is
administered 3-7 days after the first, there
is 3-5-fold increase in the liver
accumulation of second-dose-nanoparticles
as compared to the first dose. However, if
the second dose is administered two weeks
after the first dose, there is no difference in
the liver accumulation of the nanoparticles
[69]. Thus allowing sufficient time
between consecutive doses of PEGylated
nanoparticles can help overcome the
problem of ABC.
Biomimetic approaches to enhance
plasma circulation of nanoparticles
RES mediated uptake of nanoparticles can
be significantly reduced by using
hydrophilic polymers. But a major fraction
of the dose is still found in the liver and
spleen. Additionally, immune reaction
upon repeated dosing limits the utility of

this approach. Hence, alternate approaches
for engineering stealth nanoparticles are of
tremendous interest. Most nanoparticulate
systems show a circulation half-life of
~10-12 hours. In contrast, blood cells (like
erythrocytes) remain in circulation for
weeks. Thus, several studies are aimed at
generating nano-drug delivery systems that
mimic blood cells.
Modifying the surface of nanoparticles to
mimic blood cells
The mechanism of RES evasion by RBCs
is a subject of extensive research. Though
RBCs undergo opsonization they are not
engulfed by macrophages. RBCs employ
various self-markers to evade RES uptake.
These include complement receptor 1,
decay accelerating factor, C8 binding
protein, CD59 and CD47 [70]. CD47 is
expressed on the plasma membrane of
erythrocytes [71] and often upregulated on
circulating tumour cells and stem cells [72,
73]. CD47 interacts with signal receptor
protein α (SIRPα), an inhibitory receptor
found on the surface of macrophages.
Activation of SIRPα leads to an
intracellular cascade resulting in the
inactivation of myosin. This retards
macrophage mediated phagocytosis [73].
Hence, activating SIRPα on the
macrophage surface can reduce the
phagocytosis of nanoparticles.
Tsai et al. were the first to explore this
approach [74]. In this report, streptavidin
coated polystyrene microparticles were
conjugated to biotinylated CD47. In vitro
studies showed that, in spite of similar
levels of opsonization, CD47 coated

microparticles had a significantly lower
macrophage uptake than nonfunctionalized
microparticles. Additionally,
macrophage uptake decreased with an
increase in the surface density of CD47,
reaching a plateau at ~200 molecules/μm2
[74]. Rodriguez et al. tested whether
presence of CD47 on the surface of 160-
nm polystyrene nanoparticles increases
their circulation time in vivo [75]. The
authors found that CD47 functionalized
nanoparticles had a longer plasma
circulation as compared to the nonfunctionalized
nanoparticles. It is
interesting to note that CD47, unlike PEG,
has no effect on the opsonization process.
CD47 is involved in decreasing
phagocytosis via macrophages. Thus, in
principle, presence of PEG and CD47 can
offer a dual advantage. Consistent with this
hypothesis, the authors found that
PEGylated polystyrene nanoparticles
functionalized with CD47 had a longer
plasma residence time and showed lower
accumulation in the spleen as compared to
PEGylated polystyrene nanoparticles [75].
The presence of CD47 on the surface of
nanoparticles improves their plasma
circulation. However, the circulation time
of these nanoparticles is still not
comparable to blood cells. It is possible
that the activity of CD47, alone, cannot
provide nanoparticles with stealth
properties like blood cells. Surface density
of CD47 and the nature of interaction of
CD47 with SIRPα may also be different
for these artificial vessels. In other words,
it is difficult to recreate the complex
biochemistry of blood cells on the surface
of nanoparticles using a single ligand. To

overcome this issue, some recent studies
have proposed coating nanoparticles with
the entire plasma membrane of blood cells.

Schematic and actual
Figure 3: Schematic and actual
structures.

a, Schematic structure of toxin nanosponges and
their mechanism of neutralizing toxins. The
nanosponges consist of substrate-supported RBC
bilayer membranes into which toxins can
incorporate. After being absorbed and arrested by
the nanosponges, the toxins are diverted away from
their cellular targets, thereby avoiding target cells
and preventing toxin-mediated haemolysis. b, TEM
visualization of nanosponges mixed with a-toxin
(scale bar, 80 nm) and the zoomed-in view of a
single toxin-absorbed nanosponge (scale bar, 20
nm). The sample was negatively stained with uranyl
acetate before TEM imaging.
Reproduced with permission from [77]
Hu et al. coated poly-(lactide-co-glycolide)
(PLGA) nanoparticles with the plasma
membrane of RBCs [76]. The authors
confirmed that major proteins in the RBC
membrane were retained even after the
coating process. In mouse studies,
erythrocyte membrane coated
nanoparticles showed an elimination halflife
of ~40 hours while PEG nanoparticles
showed a half-life of ~15 hours. RBCcoated
nanoparticles were later used as
decoys to adsorb and eliminate blood

borne-toxins that can cause haemolysis
(Figure 3) [77]. In this study, the surface of
nanoparticles attracted the blood-borne
toxin, while the core of the nanoparticle
acted as a ‘sponge’ to absorb the toxic
chemical. This membrane-coating platform
has been applied successfully to other
nanoparticle systems as well [70, 78].
Parodi et al. coated nanoporous silicon
microparticles with leukocyte cell
membrane [79]. The camouflaged
microparticles, termed as leuko-like
vectors, showed significantly reduced
opsonization and macrophage uptake in
vitro as compared to plain microparticles.
Membrane coating also reduced the in vivo
hepatic uptake of the microparticles.
However, this advantage was transient.
While the initial hepatic uptake of coated
microparticles was lower, both coated and
uncoated microparticles showed similar
levels of hepatic uptake 40 minutes post
injection. This indicates that the particles
lose their coating rapidly and accumulate
in the liver. In spite of this, coated
microparticles showed a higher tumour
accumulation as compared to the uncoated
particles [79].
Blood cell mimicking nanoparticles show a
longer half-life than PEGylated
nanoparticles. However, most PEGylated
nanoparticles are produced using singlestep
processes [53, 66]. Thus PEGylation
is a simpler and more reproducible
technique than the techniques mentioned
above. Additionally, availability of
erythrocytes and leucocytes may also
significantly limit the translation of these
approaches.

Altering the core of nanoparticles to mimic
the flexibility of blood cells
Prolonged circulation of RBCs is also
attributed to the structure and flexibility of
these cells. Erythrocytes are too large to
pass through constricted capillaries of the
lungs. However, RBCs can pass through
capillaries that have a diameter as small as
5μm [80]. Similar to RBCs, circulating
tumour cells are also known to be more
flexible as compared to normal cells. This
flexibility allows circulating tumour cells
to extravasate into, intravasate out of
circulation and avoid getting trapped in
constricted capillaries [81]. Thus,
engineering the flexibility of long
circulating cells into nanoparticles may
allow longer plasma circulation.
Doshi and colleagues showed that the
shape and flexibility of RBCs can be built
into PLGA microparticles [82]. Spherical
PLGA particles were converted to discoid
biconcave structures using partial
fluidization. Using layer-by-layer coating,
bovine serum albumin and haemoglobin
were added to the surface of the discoid
microparticles. The particles were again
exposed to a solvent to fluidize the core
even further. The resultant particles were
extremely soft and had a remarkably
reduced elastic modulus. The
pharmacokinetics of these particles was
not studied in this report [82]. Merkel et al.
showed that the plasma residence of 2-
hydroxyethyl acrylate microparticles can
be changed by altering their bulk modulus
[83]. Microparticles were synthesized by
cross linking 2-hydroxyethyl acrylate with
poly-(ethylene glycol) diacrylate. Bulk

modulus of the microparticles was
controlled by modifying the amount of
cross-linker in the formulation. The
authors found that by changing the
modulus from 63 kPa to 8 kPa, the
elimination half-life of the microparticles
increased from 3 hours to 93 hours. This
dramatic increase in half-life was
attributed to the deformability of the
microparticles and their ability to escape
constricted microvasculature. It is
interesting to note that though this study
utilized microparticles, the half-life
achieved was greater than some
nanoparticulate systems [83]. Haghgooie et
al. also showed that similar flexible
microparticles can be produced in a variety
of shapes and carrying different cargoes
[84].
Conclusion
The plasma circulation time of
nanoparticle is a key feature that dictates
the final performance of these carriers.
Rapid macrophage uptake of nanoparticles
severely reduces their circulation time.
There have been several attempts to reduce
RES uptake of nanoparticles. These
strategies include modifying particle size,
shape, surface characteristics etc. More
recently, several studies have attempted to
produce nanoparticles that better mimic
naturally occurring colloids like RBCs. In
spite of considerable improvement in the
plasma circulation of nanoparticles, most
systems still show a high accumulation in
the RES organs like the liver and spleen.
Hence, alternate strategies are still of
considerable interest in the field of medical
nanotechnology.

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Introduction

Nano-sized carriers are rapidly emerging
as a useful platform for drug delivery [1,
2]. While nanoparticles offer several
advantages over other drug delivery
systems, these advantages can be realized
only if the carriers are maintained in
circulation for prolonged time intervals.
For example, nanoparticles are extensively
used to achieve targeted drug delivery [3,
4]. However, when the target has a limited
blood supply or is at a very low
abundance, nanoparticles need to circulate
through the target several times in order to
accumulate at high enough concentrations
[5, 6]. Nanoparticles are also used as
controlled-drug delivery vehicles to sustain
high drug levels in the plasma. This too,
can be achieved only if the nanoparticles
remain in circulation for prolonged periods
of time [7]. Hence, there is a considerable
interest in improving the plasma residence
time of nanoparticles. Once administered,
nanoparticles are rapidly eliminated from
blood circulation via a two-step process
[8]. In the first step serum proteins, of the
complement system, are adsorbed on the
surface of nanoparticles. This process is
known as opsonization and is vital for the
elimination of nanoparticles [8-10].
Following opsonization, nanoparticles are
engulfed by circulating macrophages or
macrophages residing in the liver and
spleen (referred to as mononuclear
phagocytic system (MPS) or the
reticuloendothelial system (RES)) [8, 9,
11]. Hence, in order to prolong the
circulation time of nanoparticles, it is

essential to subvert either of the two
processes.
There have been several attempts to
increase the blood circulation of
nanoparticles [12, 13]. Initial attempts
were directed towards altering the surface
of particles [14]. This led to the use of nonionic
hydrophilic polymers [15]. This has
been the most widely used strategy till
date. Some researchers have also shown
that changing the physicochemical
properties of the core of the nanoparticles,
like size and shape, can also increase their
circulation half-life [16]. Recent advances
in synthetic techniques have inspired
researchers to develop nanoparticles that
mimic blood cells [17]. These approaches
have also helped prolong the plasma
residence of nanoparticles.
In this short review, I have outlined a
various strategies that have been used
successfully to produce long circulating
nanomedicine. I focus, primarily, on the
rationale underlying the use of each
technique, their advantages and potential
shortcomings.
Modifying nanoparticle morphology to
enhance circulation time
Particle size
Particle size is an important determinant of
the plasma circulation time of
nanoparticles [18]. Hence, altering the
particle size can be an effective technique
to prolong plasma circulation. However,
unlike other strategies discussed in this
review, the choice of particle size is

Particle shape
There is very limited information available
about the performance of non-spherical
nanoparticles. This, in part, was due to lack
of techniques for the synthesis of nonspherical
nanoparticles. However, in recent
years, synthesis and characterization of
non-spherical nanoparticles has gained
tremendous interest and has become a
subject of extensive research [16, 25-27].
Geng and co-workers were the first to
show that non-spherical nanoparticles have
superior pharmacokinetics and anti-tumour
efficacy as compared to spherical
nanoparticles [28]. This thesis studied the
plasma circulation of filamentous micelles
composed of block co-polymers of poly-
(ethylene glycol) (PEG) and poly-
(ethylethylene) and that of poly-(ethylene
glycol) and poly-(caprolactone). In a
mouse model, filamentous micelles had a
significantly longer half-life (~144 hours)
than spherical polymerosomes (half-life
~24 hours). The effect of particle size of
filomicelles was also studied. Interestingly,
filomicelles having an initial length of 8
μm (equivalent to the diameter of red
blood cells (RBCs)) had the longest
circulation half-life. Filomicelles with a
shorter or longer lengths were eliminated
faster [28]. While the study by Geng et al.
provided the first insight into the biological
performance of non-spherical
nanoparticles, it analysed only one particle
shape. A mechanistic understanding of this
interaction was first provided by
Champion et

ellipsoids, elliptical discs and rectangular
discs. The authors found that the initiation
and completion of phagocytosis were
strongly affected by the morphology of the
nanoparticles. The orientation of
nanoparticles relative to the macrophages
dictated the initiation of the process. The
volume of nanoparticles (a function of
particle size) dictated the completion of
this process. For example, if the alignment
of elliptical discs was perpendicular to the
approaching surface of the macrophage,
the process of phagocytosis did not initiate
at all. However, if the elliptical disc was
aligned parallel to the macrophage surface,
phagocytosis initiated spontaneously. The
completion of this process, however,
depended on the volume of the particle
relative to the macrophage [29]. Hence,
particle shapes that minimize initiation of
phagocytosis will have the longest
circulation half-life.
Following these studies, there have been a
number of experiments which have shown
that certain non-spherical nanoparticles
have superior pharmacokinetics, efficacy
and targeting abilities as compared to
spherical nanoparticles [30-33]. However,
spherical nanoparticles are still preferred
and more popular owing to their ease of
synthesis.
Surface functionalization of
nanoparticles with hydrophilic polymers
Opsonization and subsequent macrophage
uptake of nanoparticles are known to be
triggered by the hydrophobicity and charge
of the surface of nanoparticles [9, 34].
Hence, initial attempts to decrease MPS
mediated nanoparticle uptake were

directed towards masking the surface
hydrophobicity and charge of
nanoparticles. This has been achieved by
using non-ionic hydrophilic polymers,
physically adsorbed or chemically grafted,
on the surface of nanoparticles.
The use of amphiphilic polymers was first
suggested by Illum et al. [14]. In this
study, polystyrene microparticles were
surface functionalized with poloxamer
338, a tri-block co-polymer consisting of a
central hydrophobic chain of poly-
(propylene oxide), flanked by two
hydrophilic chains of poly-(ethylene
oxide). The authors showed that poloxamer
decreased the rapid initial uptake of
microparticles into the liver. This resulted
in higher plasma concentrations at early
time points. Although the hepatic uptake of
particles was decreased considerably, there
was no significant increase in terminal
half-life of the surface coated particles
[14].
In another study, Allen et al. hypothesized
that increasing surface hydrophilicity and
mimicking the cell surface of blood cells
may enhance the circulation time of
nanoparticles [35]. Earlier studies
investigating the long circulation times of
RBCs had highlighted the role of surface
sialic acid moieties [36]. To replicate this,
the liposome surface was modified with
sialic acid by incorporating
monosialylganglioside (GM1) into the
liposome bilayer. The authors found that
incorporation of GM1 in the lipid bilayer
significantly decreased their hepatic
uptake. As the molar ratio of GM1 in the
lipid bilayer increased, RES uptake of

liposomes decreased. However, increasing
the amount of GM1 beyond a certain
concentration compromised the stability of
the lipid membrane [35]. This limitation
could be overcome by using lipids, like
sphingomyelin, which showed greater
intermolecular interactions [37]. Such
lipids increased the rigidity of the liposome
membrane and allowed higher
incorporation of GM1 [35]. Gabizon et al.
later compared the tumour accumulation of
non-functionalized liposomes and GM-1
functionalized liposomes in a mouse model
of lymphoma [38]. GM-1 functionalized
liposomes showed 25-fold increase in
tumour levels as compared to the nonfunctionalized
liposomes [38]. As GM-1
functionalized liposomes partially evaded
the RES system, these liposomes were
referred to as “stealth” liposomes.
Poly-(ethylene glycol)
Though GM-1 provided excellent stealth
properties to nanocarriers, it was required
in large quantities. Additionally,
reproducible synthesis of this polymer was
also challenging [39]. Hence, there was
tremendous interest in developing alternate
polymers to substitute GM1. Klibanov et
al. used poly-(ethylene glycol) (PEG) to
increase the circulation of liposomes [40].
This strategy had been successfully
implemented to prolong the circulation
half-life of proteins [41]. In agreement
with those reports, the authors found that
PEG functionalized liposomes (half-life: 5
hours) had a significantly higher plasma
residence time than non-functionalized
liposomes (half-life: 0.5 hours). GM-1
functionalized liposomes had an

intermediate half-life of 1.5 hours [40].
Papahadjopoulos et al. later confirmed
these results in a mouse model of colon
carcinoma [42]. PEG liposomes had an
extended plasma half-life (~5 fold higher
than the non-functionalized liposomes) and
showed lower accumulation in the major
RES organs, liver and spleen.
Consequently, there was higher tumour
accumulation of PEG-liposomes as
compared to the plain liposomes [42].

Schematic diagrams of PEG
Figure 2: Schematic diagrams of PEG
configurations on the upper hemisphere
of a polymeric nanoparticle.

In (a), the low surface coverage of PEG chains leads
to the “mushroom” configuration where most of the
chains are located closer to the particles surface. In
(b), the high surface coverage and lack of mobility
of the PEG chains leads to the “brush” configuration
where most of the chains are extended away from
the surface. Reproduced with permission from [9]
PEG provides steric hindrance to the
adsorption of opsonins and hence
decreases the RES uptake of nanoparticles
[9, 43, 44]. Surface density of PEG is an
important variable that can affect the
efficiency of the polymer [45]. Best steric
hindrance is achieved at an intermediate

density [46]. At a low surface density, PEG
chains exist in a mushroom configuration,
while at a high surface density they exist in
a brush configuration (Figure 2). As
opsonins approach the nanoparticle
surface, they cause “brush”-shaped PEG
chains to compress. The compressed-high
energy conformation of PEG chains is not
favourable and as it returns to the preferred
elongated conformation, opsonins are
pushed away from the nanoparticle
surface. Hence, it is important that the
surface density of PEG is high enough that
they exist in a brush conformation.
However, if the surface density is too high,
PEG chains lose their flexibility and their
protective ability. At low surface coverage,
opsonins can get adsorbed in the space
between the PEG chains [9, 45, 47].
Many studies have dealt with the effect of
molecular weight on the efficacy of PEG
[45, 48-50]. Higher molecular weight PEG
provides better protection against
opsonization [44]. Longer PEG chains can
provide higher repulsive forces against the
adsorption of opsonins [44]. However,
functionalizing nanoparticles with
extremely large PEG moieties decreases
their interaction with the target cells as
well [43]. Additionally, larger hydrophilic
polymers are thermodynamically more
stable when they are free in solution [51].
Thus, preparing stable nanoparticle
formulations with larger PEGs is
challenging. Hence, the use of mid-sized
PEGs (molecular weight: 1000- 5000 Da)
is most common [52, 53]. The method of
PEGylation (surface adsorption vs.
chemical grafting) and the type of PEG
(branched vs. linear vs. star shaped) have

also been found to affect the efficiency of
PEG. [54-58]. However, these parameters
have not been discussed in this review.
PEG is the preferred polymer for making
long circulating nanocarriers [6]. There are
several reasons for the popularity of PEG.
First, PEG is non-biodegradable. Hence, it
does not produce any toxic metabolites and
is easily cleared from blood circulation via
renal filtration. Additionally, synthesis of
PEG polymers with a low polydispersity
index is relatively simple. This allows a
precise control of the characteristics of the
final product. Moreover, PEG chains are
flexible. Hence a very small mole% of
PEG is required to cover the entire
nanoparticle surface and provide stealth
properties to the formulation [6, 47].
Finally, the two terminal groups of PEG
can be tailored to specification. It is
common to conjugate one end group with a
hydrophobic polymer (to allow docking on
the lipophilic surface of the nanoparticle)
and the other end group to targeting
moieties [3, 52, 53].
Other polymers
Due to the success of PEG, there was
considerable interest in investigating other
hydrophilic polymers for stealth properties.
Vinyl polymers like poly-(vinyl
pyrolidone) (PVP), poly-(acrylamide)
(PAA) and poly-(acryloylmorpholine)
(PAcM) were one of the first alternate
polymers studied [51, 59]. PVP, PAA and
PAcM were conjugated to a hydrophobic
phospholipid or acyl chain to produce
amphiphillic derivatives. Molecular
weights of the acyl linker and PVP or PAA

were optimized to provide highest
encapsulation efficiency and steric
protection. However, it was found that
these polymers performed similar to PEG
and did not provide any additional benefit
[51, 59].
Amphiphillic derivatives of poly-(2-
methyl-2-oxazoline) (PMOZ) and poly-(2-
ethyl-2-oxazoline) (PEOZ) have also been
investigated [60]. It was found that both,
PEOZ and PMOZ, delayed RES clearance
of liposomes in a rat model. The half-life
of PEOZ and PMOZ functionalized
liposomes was similar to that of PEG
functionalized liposomes [60]. Maruyama
and colleagues showed that polyglycerols
could also be used to delay RES uptake of
liposomes [61]. Amphiphillic derivatives
of polyglycerol were synthesized by
conjugating polyglycerol with
dipalmitoylphosphotidic acid. The
presence of polyglycerol moieties on the
surface of liposomes decreased their
uptake in major RES organs like the liver,
kidney and spleen. The protective ability of
polyglycerol increased with the degree of
polymerization of the polymer. However,
this study did not compare the polyglycerol
derivatives to PEG [61].
Some studies have also shown that
polysaccharides can be used effectively to
provide stealth properties to nanoparticles
[62]. In particular, dextran coating has
been widely used to protect iron oxide
nanoparticles against RES uptake [63].
Polysaccharides provide a unique
advantage over other polymer coatings.
Polysaccharides can be used as ‘active
targeting’ ligands, as many cells express

receptors for these molecules [62]. While
PEG and other synthetic polymers are
widely accepted [64-66], there are certain
disadvantages associated with them. Some
reports have shown the production of anti-
PEG IgM antibodies after the first dose of
PEGylated nanoparticles [67, 68]. This
significantly enhances the blood clearance
of the second dose of nanoparticles. In
fact, the second dose of PEG nanoparticles
are eliminated almost as rapidly as nonfunctionalized
nanoparticles. This
phenomenon, termed as accelerated blood
clearance (ABC) [67], can have significant
ramifications on the use of synthetic
polymers. It should be noted, however, that
ABC is observed only for a limited time
after the first dose of PEGylated
nanoparticles. Ishida et al. found that if the
second dose of PEG nanoparticles is
administered 3-7 days after the first, there
is 3-5-fold increase in the liver
accumulation of second-dose-nanoparticles
as compared to the first dose. However, if
the second dose is administered two weeks
after the first dose, there is no difference in
the liver accumulation of the nanoparticles
[69]. Thus allowing sufficient time
between consecutive doses of PEGylated
nanoparticles can help overcome the
problem of ABC.
Biomimetic approaches to enhance
plasma circulation of nanoparticles
RES mediated uptake of nanoparticles can
be significantly reduced by using
hydrophilic polymers. But a major fraction
of the dose is still found in the liver and
spleen. Additionally, immune reaction
upon repeated dosing limits the utility of

this approach. Hence, alternate approaches
for engineering stealth nanoparticles are of
tremendous interest. Most nanoparticulate
systems show a circulation half-life of
~10-12 hours. In contrast, blood cells (like
erythrocytes) remain in circulation for
weeks. Thus, several studies are aimed at
generating nano-drug delivery systems that
mimic blood cells.
Modifying the surface of nanoparticles to
mimic blood cells
The mechanism of RES evasion by RBCs
is a subject of extensive research. Though
RBCs undergo opsonization they are not
engulfed by macrophages. RBCs employ
various self-markers to evade RES uptake.
These include complement receptor 1,
decay accelerating factor, C8 binding
protein, CD59 and CD47 [70]. CD47 is
expressed on the plasma membrane of
erythrocytes [71] and often upregulated on
circulating tumour cells and stem cells [72,
73]. CD47 interacts with signal receptor
protein α (SIRPα), an inhibitory receptor
found on the surface of macrophages.
Activation of SIRPα leads to an
intracellular cascade resulting in the
inactivation of myosin. This retards
macrophage mediated phagocytosis [73].
Hence, activating SIRPα on the
macrophage surface can reduce the
phagocytosis of nanoparticles.
Tsai et al. were the first to explore this
approach [74]. In this report, streptavidin
coated polystyrene microparticles were
conjugated to biotinylated CD47. In vitro
studies showed that, in spite of similar
levels of opsonization, CD47 coated

microparticles had a significantly lower
macrophage uptake than nonfunctionalized
microparticles. Additionally,
macrophage uptake decreased with an
increase in the surface density of CD47,
reaching a plateau at ~200 molecules/μm2
[74]. Rodriguez et al. tested whether
presence of CD47 on the surface of 160-
nm polystyrene nanoparticles increases
their circulation time in vivo [75]. The
authors found that CD47 functionalized
nanoparticles had a longer plasma
circulation as compared to the nonfunctionalized
nanoparticles. It is
interesting to note that CD47, unlike PEG,
has no effect on the opsonization process.
CD47 is involved in decreasing
phagocytosis via macrophages. Thus, in
principle, presence of PEG and CD47 can
offer a dual advantage. Consistent with this
hypothesis, the authors found that
PEGylated polystyrene nanoparticles
functionalized with CD47 had a longer
plasma residence time and showed lower
accumulation in the spleen as compared to
PEGylated polystyrene nanoparticles [75].
The presence of CD47 on the surface of
nanoparticles improves their plasma
circulation. However, the circulation time
of these nanoparticles is still not
comparable to blood cells. It is possible
that the activity of CD47, alone, cannot
provide nanoparticles with stealth
properties like blood cells. Surface density
of CD47 and the nature of interaction of
CD47 with SIRPα may also be different
for these artificial vessels. In other words,
it is difficult to recreate the complex
biochemistry of blood cells on the surface
of nanoparticles using a single ligand. To

overcome this issue, some recent studies
have proposed coating nanoparticles with
the entire plasma membrane of blood cells.

Schematic and actual
Figure 3: Schematic and actual
structures.
a, Schematic structure of toxin nanosponges and
their mechanism of neutralizing toxins. The
nanosponges consist of substrate-supported RBC
bilayer membranes into which toxins can
incorporate. After being absorbed and arrested by
the nanosponges, the toxins are diverted away from
their cellular targets, thereby avoiding target cells
and preventing toxin-mediated haemolysis. b, TEM
visualization of nanosponges mixed with a-toxin
(scale bar, 80 nm) and the zoomed-in view of a
single toxin-absorbed nanosponge (scale bar, 20
nm). The sample was negatively stained with uranyl
acetate before TEM imaging.
Reproduced with permission from [77]
Hu et al. coated poly-(lactide-co-glycolide)
(PLGA) nanoparticles with the plasma
membrane of RBCs [76]. The authors
confirmed that major proteins in the RBC
membrane were retained even after the
coating process. In mouse studies,
erythrocyte membrane coated
nanoparticles showed an elimination halflife
of ~40 hours while PEG nanoparticles
showed a half-life of ~15 hours. RBCcoated
nanoparticles were later used as
decoys to adsorb and eliminate blood

borne-toxins that can cause haemolysis
(Figure 3) [77]. In this study, the surface of
nanoparticles attracted the blood-borne
toxin, while the core of the nanoparticle
acted as a ‘sponge’ to absorb the toxic
chemical. This membrane-coating platform
has been applied successfully to other
nanoparticle systems as well [70, 78].
Parodi et al. coated nanoporous silicon
microparticles with leukocyte cell
membrane [79]. The camouflaged
microparticles, termed as leuko-like
vectors, showed significantly reduced
opsonization and macrophage uptake in
vitro as compared to plain microparticles.
Membrane coating also reduced the in vivo
hepatic uptake of the microparticles.
However, this advantage was transient.
While the initial hepatic uptake of coated
microparticles was lower, both coated and
uncoated microparticles showed similar
levels of hepatic uptake 40 minutes post
injection. This indicates that the particles
lose their coating rapidly and accumulate
in the liver. In spite of this, coated
microparticles showed a higher tumour
accumulation as compared to the uncoated
particles [79].
Blood cell mimicking nanoparticles show a
longer half-life than PEGylated
nanoparticles. However, most PEGylated
nanoparticles are produced using singlestep
processes [53, 66]. Thus PEGylation
is a simpler and more reproducible
technique than the techniques mentioned
above. Additionally, availability of
erythrocytes and leucocytes may also
significantly limit the translation of these
approaches.

Altering the core of nanoparticles to mimic
the flexibility of blood cells
Prolonged circulation of RBCs is also
attributed to the structure and flexibility of
these cells. Erythrocytes are too large to
pass through constricted capillaries of the
lungs. However, RBCs can pass through
capillaries that have a diameter as small as
5μm [80]. Similar to RBCs, circulating
tumour cells are also known to be more
flexible as compared to normal cells. This
flexibility allows circulating tumour cells
to extravasate into, intravasate out of
circulation and avoid getting trapped in
constricted capillaries [81]. Thus,
engineering the flexibility of long
circulating cells into nanoparticles may
allow longer plasma circulation.
Doshi and colleagues showed that the
shape and flexibility of RBCs can be built
into PLGA microparticles [82]. Spherical
PLGA particles were converted to discoid
biconcave structures using partial
fluidization. Using layer-by-layer coating,
bovine serum albumin and haemoglobin
were added to the surface of the discoid
microparticles. The particles were again
exposed to a solvent to fluidize the core
even further. The resultant particles were
extremely soft and had a remarkably
reduced elastic modulus. The
pharmacokinetics of these particles was
not studied in this report [82]. Merkel et al.
showed that the plasma residence of 2-
hydroxyethyl acrylate microparticles can
be changed by altering their bulk modulus
[83]. Microparticles were synthesized by
cross linking 2-hydroxyethyl acrylate with
poly-(ethylene glycol) diacrylate. Bulk

modulus of the microparticles was
controlled by modifying the amount of
cross-linker in the formulation. The
authors found that by changing the
modulus from 63 kPa to 8 kPa, the
elimination half-life of the microparticles
increased from 3 hours to 93 hours. This
dramatic increase in half-life was
attributed to the deformability of the
microparticles and their ability to escape
constricted microvasculature. It is
interesting to note that though this study
utilized microparticles, the half-life
achieved was greater than some
nanoparticulate systems [83]. Haghgooie et
al. also showed that similar flexible
microparticles can be produced in a variety
of shapes and carrying different cargoes
[84].
Conclusion
The plasma circulation time of
nanoparticle is a key feature that dictates
the final performance of these carriers.
Rapid macrophage uptake of nanoparticles
severely reduces their circulation time.
There have been several attempts to reduce
RES uptake of nanoparticles. These
strategies include modifying particle size,
shape, surface characteristics etc. More
recently, several studies have attempted to
produce nanoparticles that better mimic
naturally occurring colloids like RBCs. In
spite of considerable improvement in the
plasma circulation of nanoparticles, most
systems still show a high accumulation in
the RES organs like the liver and spleen.
Hence, alternate strategies are still of
considerable interest in the field of medical
nanotechnology.

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