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)
Biomaterials form the basis of current and future biomedical
technologies. They are routinely used to design therapeutic carri-
ers, 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 extraor-
dinary biological performance. Inspired by this natural example,
we synthesized particles that mimic the key structural and func-
tional 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 diam-
eter. 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 syn-
thetic drug delivery particles.
biomimetic � drug delivery � erythrocyte � imaging � nanotechnology
B iomaterials provide a technological platform for launchingbiomedical 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 (3–5). 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 mor-
phologies to control pharmacokinetics (10–12), 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, f lexibility, 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 sophis-
tication 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
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 ofRBCs 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 area-
to-volume ratio and allows RBCs to undergo marked deformations
while maintaining a constant surface area (18). The unique mor-
phological properties of RBCs are achieved by a well-orchestrated
series of biochemical events. RBCs originate as spherical reticulo-
cytes, which make a transition into the biconcave shape during
maturation over a period of 2–3 days (19).
Recreation of the complex morphology of RBCs in a synthetic
system has proved challenging using currently established tech-
niques (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 hypoth-
esized that hollow polystyrene particles, upon solvent or heat-
induced 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 hydro-
chloride) (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.
www.pnas.org�cgi�doi�10.1073�pnas.0907127106 PNAS � December 22, 2009 � vol. 106 � no. 51 � 21495–21499
EN
G
IN
EE
RI
N
G
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 tetrahydrofu-
ran (THF) to induce collapse and formation of RBC-shaped
particles (Fig. 2A). The collapse is induced by two factors;
f luidization 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 approxi-
mately 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 chal-
lenges 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 parti-
cles 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 un-
clear, although it may possibly originate from partial f luidization
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. 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 loadedwith therapeutic and imaging agents. (B) Biocompatible RBC-mimicking particles prepared fromPLGA 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.
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 layerswere cross-linked. Particleswere exposed to THF to yield sRBCs. Inset shows close up. (B) Hb/PSS-based sRBCs preparedby LbL technique.
(C) sRBCs prepared by adsorption of Hb on template particles. (Scale bars, 1 �m.) (Inset, 500 nm.)
21496 � www.pnas.org�cgi�doi�10.1073�pnas.0907127106 Doshi et al.
PLGAparticles. These templates were used to yield soft, protein-
based 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�100MPa depending on several parameters including the
template/shell materials, shell density, shell cross-linking, and
pH, among many others (25–27). The elastic modulus of sRBCs
was obtained from force-indentation curves obtained by induc-
ing deformations comparable to the capsule wall thickness,
where the elastic response is expected. The typical loading-
unloading 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
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 chan-
nels smaller than their resting diameter and stretch in response
to flow. Further detailed studies of the kinetics of shape tran-
sition while passing through the capillaries are necessary to gain
further insight into the mechanical f lexibility 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 deacti-
vation 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, uncross-
linked 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 re-
tained 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.
Amodel molecule, Texas-Red-conjugated dextran (3 and 10 kDa
molecular weight) was loaded into the sRBCs by direct incuba-
tion. 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 (10–15 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 ami-
notransferase levels, hyperkalemia, alopecia, and osteoporosis
Fig. 3. SEM images of biocompatible sRBCs. (A) RBC-shapedPLGA templates fabricatedby 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-linkedmouse RBCs. sRBCs demonstrate striking
resemblance to the natural counterparts. Insets show close up images. (Scale bars, 5 �m.) (Insets, 2 �m.)
Fig. 4. Mechanical propertiesof sRBCsmeasuredusingAFM. (A) Comparisonof
elastic modulus of sRBCs with mouse RBCs and PLGA particles (*, P� 0.001, n�
5). (B) sRBCs (7�2�m)flowingthroughglass capillary (5-�minnerdiameter).The
image also shows a particle outside the capillary. (Scale bar, 5 �m.)
Doshi et al. PNAS � December 22, 2009 � vol. 106 � no. 51 � 21497
EN
G
IN
EE
RI
N
G
(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 require-
ment for this use is homogenous dispersion of the iron oxide
nanocrystals. As shown in Fig. 5C, transmission electron micros-
copy (TEM) images show well-distributed iron oxide particles in
the PLGA matrix. The Inset shows TEM image of a spherical
PLGA particle before shapemodification.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 treat-
ment 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 func-
tional properties of RBCs, have the potential to address some of
the key challenges faced by current drug delivery carriers. The
results presen
本文档为【Event-Related Potentials as Indices of Time Processing】,请使用软件OFFICE或WPS软件打开。作品中的文字与图均可以修改和编辑,
图片更改请在作品中右键图片并更换,文字修改请直接点击文字进行修改,也可以新增和删除文档中的内容。
该文档来自用户分享,如有侵权行为请发邮件ishare@vip.sina.com联系网站客服,我们会及时删除。
[版权声明] 本站所有资料为用户分享产生,若发现您的权利被侵害,请联系客服邮件isharekefu@iask.cn,我们尽快处理。
本作品所展示的图片、画像、字体、音乐的版权可能需版权方额外授权,请谨慎使用。
网站提供的党政主题相关内容(国旗、国徽、党徽..)目的在于配合国家政策宣传,仅限个人学习分享使用,禁止用于任何广告和商用目的。