Event-Related Potentials as Indices of Time Processing

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