Red blood cell-mimicking synthetic

biomaterial particles
Nishit Doshia,1, Alisar S. Zahra,1,2, Srijanani Bhaskarb, Joerg Lahannb,c,3, and Samir Mitragotria,3
aDepartment of Chemical Engineering, University of California, Santa Barbara, CA 93106; and Departments of bMacromolecular Science and Engineering
and cChemical Engineering and Materials Science and Engineering, University of Michigan, Ann Arbor, MI 48109

Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved October 29, 2009 (received for review June 25, 2009)

biomimetic drug delivery erythrocyte imaging nanotechnology

iomaterials provide a technological platform for launching

biomedical applications in drug delivery, medical imaging,
and regenerative medicine (1, 2). Several biomaterials including
polymeric nanoparticles and liposomes have been developed for
applications in drug delivery, some of which are already available
in the market (35). These biomaterials enhance the therapeutic
benefit of drugs via sustained release, reduced side-effects, and
effective targeting (6). Various innovative strategies have been
designed and implemented to optimize materials used for drug
delivery (7, 8). These include synthesis of polymers to improve
biocompatibility (9), fabrication of particles with various morphologies to control pharmacokinetics (1012), modification of
particle surface with polyethylene glycol to improve circulation
(13), and functionalization of particles with peptides (14) and
aptamers (15) for targeted drug delivery.
While synthetic biomaterials used for drug delivery have been
significantly advanced in terms of functionality and diversity,
they fail to match the complexity and sophistication routinely
exhibited by innate biological entities. In this context, red blood
cells (RBCs), the most abundant cells in blood, represent a
remarkably engineered biological entity designed for complex
biological functionality including oxygen delivery (16). RBCs
possess unique physical and chemical properties in terms of size,
shape, flexibility, and chemical composition, all of which are
essential to their biological functions (17, 18). Inspired by the
unique ability of these cells to perform complex tasks and
motivated by the need to design particles that adopt the sophistication exhibited by biological entities, we sought to design
synthetic carriers that mimic the key structural attributes of
RBCs including size, shape, and mechanical properties, yet offer
engineering control required in synthetic carriers. Herein, we
report the synthesis, initial characterization, and illustration of

www.pnas.orgcgidoi10.1073pnas.0907127106

biomedical applications of RBC-like particles. These particles

provide a path to bridge the gap between synthetic materials and
biological entities.
Results and Discussion
The structure of RBCs is characterized by several unique properties
including biconcave discoidal shape and mechanical flexibility that
have so far been unmatched by synthetic particles, which are
typically spherical and stiff. Unique structural properties of RBCs
allow them to routinely pass through ultrathin capillaries smaller
than their own diameter and sinusoidal slits in the spleen. The
biconcave discoidal shape also provides a favorable surface areato-volume ratio and allows RBCs to undergo marked deformations
while maintaining a constant surface area (18). The unique morphological properties of RBCs are achieved by a well-orchestrated
series of biochemical events. RBCs originate as spherical reticulocytes, which make a transition into the biconcave shape during
maturation over a period of 23 days (19).
Recreation of the complex morphology of RBCs in a synthetic
system has proved challenging using currently established techniques (20). We adopted a biomimetic strategy to prepare
RBC-shaped particles. In nature, initial spherical reticulocytes,
which have an elastic modulus of 3 MPa undergo a 100- to
1,000-fold reduction in elastic modulus and simultaneous change
in shape to form discoidal RBCs (21). Mimicking the genesis of
mature RBCs, we start with spherical polymeric particles, for
example, polystyrene microspheres with high elastic modulus,
and use them as a template to induce the change in shape and
mechanical properties to form RBC-like particles (Fig. 1).
Changing the shape of a solid polystyrene microparticle into an
RBC-shaped object, however, is quite challenging. We hypothesized that hollow polystyrene particles, upon solvent or heatinduced fluidization, can collapse into an RBC shape. For this
purpose, hollow polystyrene spheres (1-m diameter, 400-nm
shell thickness) were used. Although polystyrene, in its own
right, should not be considered a biocompatible polymer, the
commercial availability of hollow polystyrene spheres makes
them excellent model particles, which can serve as a starting
point. Layer-by-layer (LbL) self-assembly technique was used to
electrostatically deposit cationic and anionic polymers on the
particle surface (22). Initially, BSA and poly(allylamine hydrochloride) (PAH) were chosen as the polyanion and polycation,
respectively. The stepwise adsorption of BSA and PAH onto
Author contributions: N.D., A.S.Z., and S.M. designed research; N.D., A.S.Z., and S.B.
performed research; S.B. and J.L. contributed new reagents/analytic tools; N.D., A.S.Z., S.B.,
J.L., and S.M. analyzed data; and N.D., A.S.Z., S.B., J.L., and S.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1N.D.

and A.S.Z. contributed equally to this work.

2Present

address: Schepens Eye Research Institute, Harvard Medical School, Boston, MA

02114.
3To

whom correspondence may be addressed. E-mail: samir@engineering.ucsb.edu or

lahann@umich.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/

0907127106/DCSupplemental.

PNAS December 22, 2009 vol. 106 no. 51 2149521499

ENGINEERING

Biomaterials form the basis of current and future biomedical

technologies. They are routinely used to design therapeutic carriers, such as nanoparticles, for applications in drug delivery. Current
strategies for synthesizing drug delivery carriers are based either
on discovery of materials or development of fabrication methods.
While synthetic carriers have brought upon numerous advances in
drug delivery, they fail to match the sophistication exhibited by
innate biological entities. In particular, red blood cells (RBCs), the
most ubiquitous cell type in the human blood, constitute highly
specialized entities with unique shape, size, mechanical flexibility,
and material composition, all of which are optimized for extraordinary biological performance. Inspired by this natural example,
we synthesized particles that mimic the key structural and functional features of RBCs. Similar to their natural counterparts,
RBC-mimicking particles described here possess the ability to carry
oxygen and flow through capillaries smaller than their own diameter. Further, they can also encapsulate drugs and imaging agents.
These particles provide a paradigm for the design of drug delivery
and imaging carriers, because they combine the functionality of
natural RBCs with the broad applicability and versatility of synthetic drug delivery particles.

Fig. 1. Synthesis technique of RBC-mimicking particles. (A) RBC-shaped particles prepared from hollow PS template. Complementary layers of proteins and
polyelectrolytes were deposited by LbL technique on the template surface followed by cross-linking of the layers to increase stability. PS core was dissolved to
yield RBC-shaped particles, which can be loaded with therapeutic and imaging agents. (B) Biocompatible RBC-mimicking particles prepared from PLGA template
particles. PLGA RBC-shaped templates were synthesized by incubating spheres synthesized from electrohydrodynamic jetting in 2-propanol. LbL coating on
template, protein cross-linking, and dissolution of template core yielded biocompatible sRBCs.

template particles is mediated by hydrophobic and electrostatic

interactions. After the adsorption of multiple layers, the shell
was cross-linked using glutaraldehyde to provide stability to the
particles. The template core was then exposed to tetrahydrofuran (THF) to induce collapse and formation of RBC-shaped
particles (Fig. 2A). The collapse is induced by two factors;
fluidization and partial solubilization of the polymer core and
the build-up of an osmotic gradient across the shell due to the
presence of solvent on the outside and water on the inside of the
shell.
We next prepared RBC-like particles of similar morphology,
but comprised of proteins innate to RBCs, such as hemoglobin
(Hb), which is the main constituent of RBCs and is approximately 92% by dry weight (23). Hb is a tetramer with each chain
noncovalently bound to each other. The protein further carries
one heme group, to which oxygen and other small molecules can
bind reversibly. In this case, poly(4-styrene sulfonate) (PSS) and
Hb were used as complementary polyelectrolytes for the LbL
assembly to yield RBC-shaped particles (Fig. 2B). Alternatively,
Hb was adsorbed on to the surface of the template particles,
cross-linked with glutaraldehyde followed by the dissolution of
the core. The morphology of the particles was found to be similar
to those of the LbL particles (Fig. 2C). The methods described
here yield soft and synthetic RBC-mimicking particles, which we

refer to as sRBCs with the recognition that these particles mimic

key but not necessarily all features of natural RBCs.
Having demonstrated the feasibility of preparing sRBCs using
hollow polystyrene templates, we sought to address two challenges that are associated with the use of polystyrene as a
template. The size of RBC-like particles fabricated from PS
templates was limited by the commercial availability of 1-m
hollow particles as opposed to natural RBCs, which are 7 m
in diameter. Moreover, PS is not biocompatible, and hence any
residual polymer will have the potential to render the particles
nonbiocompatible. To address these challenges, polystyrene was
replaced by poly(lactic acid-co-glycolide) (PLGA). PLGA is
biocompatible and biodegradable, and the size of PLGA particles can be controlled during particle synthesis (9). We first
prepared RBC-shaped template PLGA particles (7 2 m). For
this purpose, spherical PLGA particles of appropriate sizes were
prepared using the electrohydrodynamic jetting process (24),
and these particles were incubated in 2-propanol to induce
formation of RBC-shaped PLGA template particles (Fig. 3A).
The precise reason why incubation of PLGA particles with
2-propanol induces formation of RBC-shaped particles is unclear, although it may possibly originate from partial fluidization
of PLGA due to 2-propanol and subsequent particle collapse.
Smaller template particles (3 1.5 m) were also prepared using
the same technique to illustrate the control over size using

Fig. 2. SEM micrographs of RBC-mimicking particles synthesized using hollow PS template particles. (A) BSA/PAH was deposited on template particles by LbL
technique, and the layers were cross-linked. Particles were exposed to THF to yield sRBCs. Inset shows close up. (B) Hb/PSS-based sRBCs prepared by LbL technique.
(C) sRBCs prepared by adsorption of Hb on template particles. (Scale bars, 1 m.) (Inset, 500 nm.)
21496 www.pnas.orgcgidoi10.1073pnas.0907127106

Doshi et al.

PLGA particles. These templates were used to yield soft, proteinbased biocompatible particles using the modified LbL technique
described above. Because PSS is not biocompatible, it was also
replaced with BSA in the shell. Nine alternate layers of either
Hb/BSA or PAH/BSA were assembled on the templates, the
layers were cross-linked, and the underlying PLGA core was
removed using 1:2 2-propanol:THF to form sRBCs (Fig. 3B,
PAH/BSA sRBCs; see SI Text and Fig. S1 for images of sRBCs
made from Hb/BSA). The choice of solvent was important, and
deviation from this solvent mixture led either to incomplete
dissolution (excess 2-propanol) or complete collapse (excess
THF). sRBCs synthesized by this method demonstrate close
resemblance to natural RBCs (Fig. 3 B, sRBCs, and C, mouse
RBCs).
sRBCs were found to be flexible owing to the dissolution of
the template PLGA core, which leaves behind a soft protein shell
(Fig. 4A). The elastic modulus of sRBCs was measured using
atomic force microscopy (AFM). AFM has been previously used
to measure elastic modulus of soft materials, such as LbL films,
hollow protein particles, and platelets, and a wide range of elastic
moduli have been reported for LbL structures in the range of 10
kPa to 100 MPa depending on several parameters including the
template/shell materials, shell density, shell cross-linking, and
pH, among many others (2527). The elastic modulus of sRBCs
was obtained from force-indentation curves obtained by inducing deformations comparable to the capsule wall thickness,
where the elastic response is expected. The typical loadingunloading cycle used for this study and the corresponding force
curves obtained for sRBCs can be found in the SI Text and Fig.
S2. The elastic modulus of sRBCs (92.8 42 kPa) was found to
be four orders of magnitude lower than that of PLGA template
particles (1.6 0.6 GPa) and of the same order of magnitude as
that of natural RBCs. The elastic modulus of mouse RBCs was

Fig. 4. Mechanical properties of sRBCs measured using AFM. (A) Comparison of

elastic modulus of sRBCs with mouse RBCs and PLGA particles (*, P 0.001, n
5). (B) sRBCs (7 2 m) flowing through glass capillary (5-m inner diameter). The
image also shows a particle outside the capillary. (Scale bar, 5 m.)

Doshi et al.

found to be 15.2 3.5 kPa, which is consistent with the values

reported in literature (21). Further studies are required to
facilitate a detailed comparison of various mechanical properties
of sRBCs and natural RBCs; however, the data in Fig. 4A clearly
indicate that sRBCs are far closer to natural RBCs than to
routine polymer particles with respect to mechanical properties.
The flexibility of sRBCs (7 2 m) was confirmed by flowing
them through narrow glass capillaries (5-m inner diameter) and
visualizing the stretching (Fig. 4B, two sRBCs, one inside the
capillary and one outside the capillary). Whereas the particle
outside the capillary is symmetric and circular, the particle inside
the capillary is stretched due to flow (Fig. 4B). The average
aspect ratio of stretching was found to be 170 20% (n 20).
See Fig. S3 for more images of particles flowing through the
capillary. Further, particles were able to regain their discoidal
shape upon exiting the capillary, confirming the reversible
nature of the shape deformation. Thus, similar to their natural
counterpart, sRBCs maintain the ability to flow through channels smaller than their resting diameter and stretch in response
to flow. Further detailed studies of the kinetics of shape transition while passing through the capillaries are necessary to gain
further insight into the mechanical flexibility of sRBCs.
sRBCs reported in this study have numerous biomedical
applications. Because the primary function of natural RBCs is to
deliver oxygen to the various tissues of the body, we assessed the
ability of sRBCs to bind oxygen (Fig. 5A). Cross-linking and
exposure to solvent during particle preparation leads to deactivation of Hb, thereby limiting its oxygen carrying capacity (Fig.
5A, sRBC without Hb). To enhance oxygen carrying capacity of
sRBCs, particles were further fortified with additional, uncrosslinked Hb (see Materials and Methods). This procedure resulted
in high oxygen binding levels (Fig. 5A, sRBC with Hb, t 0)
compared to the positive control, which was mouse blood.
Approximately 90% of this oxygen carrying capacity was retained even after 1 week (Fig. 5A, sRBC with Hb, t 1 week).
Included is a negative control, BSA-coated particles, which
showed no ability to bind oxygen [Fig. 5A, () control]. See Fig.
S4 for visual confirmation of oxygen carrying capacity.
sRBCs are also excellent candidates for delivery of drugs,
especially in the vascular compartment. These particles can be
loaded with drugs by incubation in solutions containing the drug.
A model molecule, Texas-Red-conjugated dextran (3 and 10 kDa
molecular weight) was loaded into the sRBCs by direct incubation. Both molecules penetrated in the interior of the sRBCs.
Dextran was subsequently released from these particles in a
controlled manner (see SI Text and Fig. S5). Once the release of
dextran was confirmed, controlled release of a therapeutic drug
heparin (1015 kDa) was tested. Heparin is widely used as an
anti-coagulant for the treatment of thrombosis (28). Parenteral
administration of heparin can result in severe side effects such
as heparin-induced thrombocytopenia, elevation of serum aminotransferase levels, hyperkalemia, alopecia, and osteoporosis
PNAS December 22, 2009 vol. 106 no. 51 21497

ENGINEERING

Fig. 3. SEM images of biocompatible sRBCs. (A) RBC-shaped PLGA templates fabricated by electrohydrodynamic jetting. (B) Biocompatible sRBCs prepared from
PLGA template particles by LbL deposition of PAH/BSA and subsequent dissolution of the polymer core. (C) Cross-linked mouse RBCs. sRBCs demonstrate striking
resemblance to the natural counterparts. Insets show close up images. (Scale bars, 5 m.) (Insets, 2 m.)

Fig. 5. Biomedical applications of sRBCs. (A) Oxygen carrying capacity of sRBCs demonstrated based on the chemiluminescence reaction of luminol.
Cross-linking and exposure to the organic solvent reduces the oxygen carrying capacity, but coating the sRBCs with uncross-linked Hb increased the
oxygen-binding capacity to levels comparable to mouse blood (S-RBC, t 0). Ninety percent of oxygen carrying capacity was retained after 1 week (S-RBC, t
1 wk). BSA-coated particles were included as negative control (*, P 0.01, n 3). (B) Controlled release of radiolabeled heparin from sRBCs over a period of
10 days (n 5). (C) TEM micrograph showing encapsulation of 30 nm iron oxide nanoparticles in RBC-shaped PLGA templates. The Inset shows PLGA particles
loaded with iron oxide nanoparticles before conversion into RBC-like templates. (Scale bars, 1 m.)

(29). The sRBCs showed high amounts of heparin loading (70 g

heparin per mg particles) and continuous release over a period
of several days in vitro (Fig. 5B).
sRBCs also have potential applications in medical imaging.
For example, iron oxide nanocrystals with an average diameter
of 30 nm were encapsulated inside the PLGA particles prepared
via electrohydrodynamic jetting. Incorporation of iron oxide
nanoparticles makes particles suitable as contrast agents for
magnetic resonance imaging (MRI) (30). An important requirement for this use is homogenous dispersion of the iron oxide
nanocrystals. As shown in Fig. 5C, transmission electron microscopy (TEM) images show well-distributed iron oxide particles in
the PLGA matrix. The Inset shows TEM image of a spherical
PLGA particle before shape modification. Magnetic particles are
currently being developed for a wide spectrum of applications
such as MRI contrast agents for diseases, such as atherosclerotic
plaque, targeted therapeutic delivery, and hyperthermia treatment for cancerous tumors (31). The interior of the particles
described here can be further engineered by the formation of
separate compartments using electrohydrodynamic co-jetting
process (24). At the same time, the surface can be engineered by
adsorption of additional proteins such as CD47, a ubiquitous
self-marker expressed on the surface of RBCs or modification of
the particle surface with hydrophilic polymers, such as PEG,
depending on the application.
In addition to preparing particles that mimic the shape and
properties of healthy RBCs, the technique reported here can also
be used to design particles that mimic the shape and properties
of diseased cells. For example, hereditary elliptocytosis is a
disease that leads to the formation of elliptical RBCs (32), a
shape that can be mimicked in our method (see SI Text and Fig.
S6). Other examples of diseased conditions where the shape of
RBCs is altered include spherocytosis and sickle-cell anemia.
Such disease cell mimicking particles can serve as synthetic
models to help elucidate the effect of transformation in physical
properties of RBCs in these disease conditions.
Drug delivery carriers, which mimic the structural and functional properties of RBCs, have the potential to address some of
the key challenges faced by current drug delivery carriers. The
results presented here demonstrate synthetic mimicry of many
key attributes of RBCs including the size, shape, elastic modulus,
ability to deform under flow, and oxygen-carrying capacity. In
addition, we report incorporation of additional functionalities
such as therapeutic and diagnostic agents in these carriers, which
enable further capabilities. Upon further confirmation of RBCmimicry through in vivo experiments focused on circulation and
biocompatibility, the particles reported here may open oppor21498 www.pnas.orgcgidoi10.1073pnas.0907127106

tunities in drug delivery, medical imaging, and the establishment

of improved disease models.
Materials and Methods
Materials. PSS (Mw 70 kDa), PAH (Mw 50 kDa), BSA, human Hb, PLGA with
a lactide:glycolide ratio of 85:15 (Mw 40 75 kDa), chloroform, N,Ndimethylformamide, heparin, luminol, sodium perborate, sodium carbonate,
2-propanol, toluene, PBS tablets, sodium citrate, and poly(vinyl alcohol) (PVA
fully hydrolyzed) were obtained from SigmaAldrich. Polybead hollow microspheres (5.21% solids, 1 m in diameter) were purchased from Polysciences.
Texas-Red-conjugated dextran (Mw 3 kDa, 10 kDa) and anti-fade agent were
purchased from Invitrogen. THF, mineral oil, and glycerol were purchased
from EMD Biosciences. Solvable was purchased from Perkin-Elmer. Dialysis
cassettes (MWCO 2500) were purchased from Thermo Scientific. Syringes (1
mL) were purchased from BD and 23 guage, 1.5-inch-long single capillary
stainless steel tip was from EFD. Iron oxide nanoparticles of 30-nm diameter
suspended in chloroform with oleic acid stabilization were purchased from
Ocean Nanotech. Filters (5 8 m) were purchased from Millipore.
Preparation of RBC-Mimicking Particles. LbL assembly was used to electrostatically adsorb proteins or polyelectrolytes (PEs) on the surface of hollow polystyrene particles. Proteins or PEs were incubated with template particles at a
concentration of 2 mg/mL in 0.5 M NaCl solution for 20 min on a shaker plate
at 350 rpm, followed by three washings (centrifugation and resuspension) in
0.5 M NaCl. For example, step-wise shell formation composed of BSA and
polycation PAH was performed until four bilayers were deposited onto the
polybead hollow microparticles (108 particles/mL) (BSA/PAH)4. Alternate layers of Hb and PSS were also used to construct the shell of RBC-mimicking
particles. Next, the layers were cross-linked using the following procedure.
Five hundred microliters 2.5% glutaraldehyde solution in 0.2 M sodium cacodylate buffer were added to protein-coated microparticles and left to incubate on a shaker plate for 1 h. Next, the particles were sonicated, and a stop
solution of 30 mM sodium borohydride was added to the particle solution for
30 min followed by three wash steps with 0.01 M PBS. The particle solution was
placed in a dialysis cassette in 0.01 M PBS. After the first hour in dialysis, 400
mL fresh 0.01 M PBS were added to the reservoir. After 24 h in the dialysis
cassette, the particle solution was removed and centrifuged. To dissolve the
polymeric core, cross-linked particles were incubated with THF, vortexed, and
then sonicated. Template polymeric particles were dissolved in THF for approximately12 h. Polybead oligomers were removed by washing with 1 mL
THF two times (vortexed and centrifuged). Particles were then washed four
times with 1 mL 0.5 M NaCl followed by overnight dialysis in 0.5 M NaCl.
Particles were finally resuspended in either 0.5 M NaCl or PBS or deionized
water to ensure complete removal of the solvent from the particles.
sRBCs were also prepared by deposition of only Hb layers by incubating the
particles with 2 mg/mL Hb for 4 h on a shaker plate followed by cross-linking using
5% glutaraldehyde as mentioned above. The polymeric template was then
dissolved using THF. For chemiluminescence experiments, sRBCs were fortified
with Hb by incubation with Hb solution (2 mg/mL) for 1 h. For sustained oxygen
carrying capacity experiments, sRBCs fortified with Hb were washed three times
with PBS and incubated in PBS for 7 days. The particles were washed again with
PBS before the oxygen carrying capacity was determined.

Doshi et al.

Synthesis of PLGA Particles. The experimental setup used for electrohydrodynamic jetting is described elsewhere (33). Briefly, a 4.5% (wt/wt) solution of
PLGA in 97:3 (by volume) CHCl3: DMF was drawn in a syringe and pumped at
0.7 mL/h via a syringe pump (KDS100; KD Scientific). A single capillary was
connected to the tip of the syringe and further attached to the cathode of a
high-voltage supply (Gamma High Voltage Source). The voltage was controlled in the range of 5.7 6 kV. A square piece of aluminum foil was used as
the anode, which also acted as a collecting substrate. The distance between
the electrodes was maintained in the range of 2530 cm.
PLGA RBC-Shaped Template Particles. The particles obtained by electrohydrodynamic jetting were harvested from the substrate and incubated for 12 h in
2-propanol at room temperature (1 mL 2-propanol/2 mg particles). The particles were then centrifuged and resuspended in DI water containing 0.01%
Tween-20. Alternatively, collapsed template particles were prepared by the
use of higher flow rates during electrohydrodynamic jetting.

tion constituted 12% by weight of total PLGA. Flow rates from 0.08 0.1
mL/h and voltages in the range of 3.9 4.5 kV were used.
Mouse RBCs. Mouse blood was obtained by cardiac puncture, collected in
heparinized tubes and diluted in 4% sodium citrate buffer (pH 7.4). The RBCs
were isolated by centrifugation at 100 g for 3 min. These were then used for
the chemiluminescence experiments in appropriate concentrations. For scanning electron microscopy (SEM), the cells were cross-linked using 2% glutaraldehyde for 2 h and washed with sodium citrate buffer.
Methods of Characterizing sRBCs. These methods (confocal microscopy, electron microscopy, AFM, capillary flow experiments, chemiluminescence, and
controlled release) are described in the SI Text.
Note Added in Proof. The authors point to a recent report by Haghgooie et al.
(34) on synthesis of RBC-inspired, deformable soft hydrogel particles using
stop flow lithography, which was published after the submission of this
manuscript.

Iron Oxide Nanoparticle Encapsulation in PLGA RBC-Shaped Particles. For

encapsulation of iron oxide nanoparticles into the PLGA particles, electrohydrodynamic jetting was carried out using a 3.8 wt% PLGA in 95:5 CHCl3:DMF
(by vol), and 30 nm iron oxide nanoparticles with oleic acid surface stabiliza-

ACKNOWLEDGMENTS. We thank Dr. Alejandro Bonilla for assistance with

atomic force microscopy and Mansi Seth and Prof. Gary Leal for providing
capillaries and assistance with capillary flow experiments. This work was
supported by the National Heart Lung and Blood Institutes Program of
Excellence in Nanotechnology Grant 1UO1 HL080718, The National Center for
Research Resources shared instrumentation Grant 1S10RR017753 01 was
used for confocal laser-scanning microscopy, and the American Cancer Society
Grant 08 1556 (to J.L.).

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Doshi et al.

PNAS December 22, 2009 vol. 106 no. 51 21499

ENGINEERING

Similar procedure was adopted for the fabrication of particles from PLGA
template particles. Nine alternate layers of Hb/BSA or PAH/BSA were deposited, and the layers were cross-linked using glutaraldehyde. A mixture of THF
and 2-propanol of varying concentrations (10:1, 5:1, 2:1, and 1:1) was used to
dissolve the template PLGA particles.