Clearance of Pathological Antibodies Using Biomimetic Nanoparticles

Clearance of pathological antibodies using biomimetic nanoparticles

Jonathan A. Coppa,b, Ronnie H. Fanga,b, Brian T. Luka,b, Che-Ming J. Hua,b, Weiwei Gaoa,b, Kang Zhanga,c, and Liangfang Zhanga,b,1

aDepartment of Nanoengineering, bMoores Cancer Center, and cDepartment of Ophthalmology and Shiley Eye Center, University of California, San Diego, La Jolla, CA 92093

Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved August 8, 2014 (received for review July 1, 2014) Pathological antibodies have been demonstrated to play a key role in is treated much the same way, with discontinuance of the offending type II immune hypersensitivity reactions, resulting in the destruction drug and, much more often than in AIHA, performance of blood of healthy tissues and leading to considerable morbidity for the transfusions (15, 16). A subsequent limitation of repeated trans- patient. Unfortunately, current treatments present significant iatro- fusions of packed RBCs is that although they revive tissue perfu- genic risk while still falling short for many patients in achieving clinical sion, they carry the risks of hemolytic transfusion reactions, the remission. In the present work, we explored the capability of target formation of alloantibodies, and iron toxicity (17–19). cell membrane-coated nanoparticles to abrogate the effect of patho- It has previously been shown that mammalian cellular mem- logical antibodies in an effort to minimize disease burden, without the need for drug-based immune suppression. Inspired by antibody-driven brane, from both nucleated and nonnucleated cells, can be fused pathology, we used intact RBC membranes stabilized by biodegrad- onto polymeric nanoparticle substrates to form stable core/shell able polymeric nanoparticle cores to serve as an alternative target for structures (20, 21). These particles have been shown to retain and pathological antibodies in an antibody-induced anemia disease model. present natural cell membrane and surface antigens (22), which Through both in vitro and in vivo studies, we demonstrated efficacy of bare the target epitopes involved in antibody-mediated cellular RBC membrane-cloaked nanoparticles to bind and neutralize anti-RBC clearance found in AIHA and DIA. To demonstrate the in- polyclonal IgG effectively, and thus preserve circulating RBCs. terception of pathological antibodies, we used RBC membrane- cloaked nanoparticles, herein denoted RBC antibody nanosponge(s) nanomedicine | immune therapy | type II hypersensitivity reaction | (ANS), to serve as alternative targets for anti-RBC antibodies autoantibody and preserve circulating RBCs (Fig. 1). Unlike conventional immune therapy, these biomimetic nanoparticles have no drug pay- ype II immune hypersensitivities are driven by pathological load to suppress normal lymphocytes or immune effector cells. Tantibodies targeting self-antigens, either naturally occurring Additionally, unlike blood transfusions, which serve as a replace- or due to exposure to an exogenous substance present on the cel- ment therapy, the RBC-ANS serves to deplete circulating anti- lular exterior or ECM. This disease type makes up many of the most prevalent autoimmune diseases, including pernicious anemia, Grave body levels, without contributing further toxic metabolites due to disease, and myasthenia gravis, as well as autoimmune hemolytic the hemolysis of transfused cells. Moreover, it has been demon- anemia (AIHA) and immune thrombocytopenia (1–4). In addition, strated in animal models of autoimmune diseases that the primary these diseases may occur after the administration of a new drug or target antigens can vary and shift over the course of the diseases following certain infections. Currently, therapies for these immune- (23). Exploiting target cell membranes in their entirety over- mediated diseases remain relatively nonspecific via broad immune comes the varying antigen specificities and presents a previously suppression (5). For instance, comprehensive immune suppression unidentified approach in intercepting the autoreactive antibody through systemic glucocorticoids (i.e., prednisone, methylpredniso- mechanism of type II immune hypersensitivity reactions. lone), cytotoxic drugs (i.e., cyclophosphamide, methotrexate, aza- thioprine), and monoclonal antibodies (i.e., rituximab, belimumab, Significance infliximab) dominate treatment regimens to prevent further tissue destruction (6–8). Although this approach to therapy is effective for The selective depletion of disease-causing antibodies using some patients in achieving remission, its efficacy remains variable nanoparticles offers a new model in the management of type II and there is a well-established risk of adverse side effects, high- immune hypersensitivity reactions. The demonstration of patho- lighting the need for better tailored therapies (9, 10). physiologically inspired nanoengineering serves as a valuable The development of nanoparticle therapeutics has sparked prototype for additional therapeutic improvements with the goal new hope for the treatment of various important human dis- of minimizing therapy-related adverse effects. Through the use eases. Herein, we demonstrate the application of a biomimetic of cell membrane-cloaked nanoparticles, nanoscale decoys with nanoparticle for the clearance of pathological antibodies using strong affinity to pathological antibodies can be administered to an established murine model of antibody-induced anemia (11). disrupt disease processes in a minimally toxic manner. These This disease may be idiopathic, as in AIHA, or drug induced, as biomimetic nanoparticles enable indiscriminate absorption of in drug-induced anemia (DIA). In both cases, however, auto- pathological antibodies regardless of their epitope specificities. antibodies attack surface antigens present on RBCs. Therapy for This particular approach offers much promise in treating anti- AIHA is relatively standardized, with patients starting on sys- body-mediated autoimmune diseases, in which target antigens MEDICAL SCIENCES temic steroids and escalating to cytotoxic drugs and B cell- on susceptible cells can vary from patient to patient. depleting monoclonal antibodies, and then possibly splenectomy based on patient response to therapy (12, 13). The shortcoming Author contributions: J.A.C., R.H.F., B.T.L., C.-M.J.H., and L.Z. designed research; J.A.C., R.H.F., of suppressing the immune system with drug-based therapies B.T.L., C.-M.J.H., and W.G. performed research; J.A.C., R.H.F., B.T.L., C.-M.J.H., W.G., K.Z., and is the considerable iatrogenic risk associated with nonspecific L.Z. analyzed data; and J.A.C., R.H.F., B.T.L., C.-M.J.H., W.G., K.Z., and L.Z. wrote the paper. therapy and heightened susceptibility to severe infections fol- The authors declare no conflict of interest. lowing spleen removal (9, 10, 14). DIA, which can be the result This article is a PNAS Direct Submission. ENGINEERING of drug-hapten antibodies or drug-independent autoantibodies, 1To whom correspondence should be addressed. Email: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1412420111 PNAS | September 16, 2014 | vol. 111 | no. 37 | 13481–13486 Downloaded by guest on October 1, 2021 ANS was added simultaneously with anti-RBCs to 5 vol% RBC solution (Fig. 3 C and D). After separating the RBCs from any unbound antibodies and RBC-ANS, we measured fluorescent signal associated with the RBCs using flow cytometric analysis. Both preincubation and coincubation studies showed dose- dependent antibody neutralization. High binding ability and stability of RBC-ANS to anti-RBCs was shown in the preincubation neutralization experiment, which demonstrated an ∼60% reduction in RBC-bound antibodies with 100 μg of RBC- ANS and an ∼95% reduction with 1 mg of RBC-ANS compared with the negative control. Competitive coincubation showed

Fig. 1. Schematic representation of RBC-ANS neutralizing anti-RBC antibodies (anti-RBCs). (A) Anti-RBCs opsonize RBCs for extravascular hemolysis, via phagocytosis, as observed in AIHA and DIA. (B) RBC-ANS absorbs and neutralizes anti-RBCs, thereby protecting RBCs from phagocytosis.

Results We constructed RBC-ANS following a previously reported protocol (21), in which purified mouse RBC membrane was mechanically extruded with 100 nm of poly(lactic-co-glycolic acid) (PLGA) polymeric cores. The resulting nanoparticles revealed a core/shell structure under transmission electron microscopy (TEM) that corresponds to unilamellar membrane coatings over the nanoparticle cores (Fig. 2A). Physicochemical characterizations showed that upon RBC membrane coating, the nanoparticles had an ∼20-nm increase in diameter and a 10-mV increase in surface zeta-potential (Fig. 2B), which were consistent with the addition of RBC membrane to the particle surface (24). A mixture of RBC- ANS with rabbit anti-mouse RBC IgG antibodies (anti-RBCs) resulted in a diameter increase of ∼26 nm, which can be attributed to the association of the IgG with the RBC-ANS. Such association also resulted in surface charge shielding, as was evidenced by the 10-mV increase in the particle zeta-potential (Fig. 2B). To in- vestigate the binding capacity of RBC-ANS for anti-RBC better, 250 μg of RBC-ANS was incubated with fluorescently labeled anti- RBCs ranging from 1.75 to 500 μg. This titration assay demonstrated a plateau in particle-bound antibody fluorescent signal, or binding maximum, corresponding with an antibody mass of ∼27 μg, yielding a particle-to-antibody mass ratio of ∼9:1 (Fig. 2C). To evaluate the specificity of antibody–antigen binding, RBC-ANS was incubated with fluorescently labeled anti-RBCs or goat anti- mouse Fc IgG (anti-Fc, as a negative control) for 10 min at 37 °C. Fig. 2D shows that significantly higher binding signal was observed between RBC-ANS and anti-RBCs with very little nonspecific binding with anti-Fc. PEGylated (PLGA) nanoparticle(s) (PEG- NP) incubated with anti-RBCs served as a negative control and showed little retention of the antibody. Furthermore, binding affinity of anti-RBCs to RBC-ANS was nearly identical to that of an equivalent amount of RBC ghosts (Fig. 2E). In the presence of serum proteins, RBC-ANS still retained greater than 60% of its Fig. 2. In vitro characterization of RBC-ANS. (A) TEM image demonstrates anti-RBC binding capacity compared with when the incubation was the core/shell structure of RBC-ANS. (Scale bar: 150 nm.) (B) Size and surface performed in buffer alone (Fig. 2F). These results are indicative of zeta-potential of pure PLGA cores, RBC-ANS, and RBC-ANS bound with anti- relatively low nonspecific antibody–nanoparticle binding inter- RBCs. (C) RBC-ANS (250 μg) incubated with five serial dilutions of fluorescent actions and demonstrate the necessity for antigen–antibody con- anti-RBCs demonstrated particle saturation at ∼27 μg of antibody, corre- cordance to achieve neutralization. sponding to a particle/antibody mass ratio of ∼9:1. (D) Equivalent amounts To characterize the binding stability and competitive binding of RBC-ANS incubated with anti-RBCs or anti-Fc demonstrated significantly capacity further, we varied the amounts of RBC-ANS mixed with greater specific binding compared with nonspecific binding. The corresponding PEG-NP incubated with anti-RBCs served as a negative control. (E) a constant amount of fluorescent anti-RBCs in 5 vol% RBC solu- Comparison of anti-RBC binding kinetics with a fixed amount of RBC-ANS or tion. To assess in vitro binding stability, RBC-ANS was pre- RBC ghosts. (Inset) Relative binding capacity of RBC-ANS vs. RBC ghosts at incubated with anti-RBCs before mixing with 5 vol% RBC solution saturation. (F) Relative binding capacity of RBC-ANS in PBS vs. RBC-ANS in (Fig. 3 A and B), and to test competitive binding capacity, RBC- serum at saturation.

13482 | www.pnas.org/cgi/doi/10.1073/pnas.1412420111 Copp et al. Downloaded by guest on October 1, 2021 Fig. 3. In vitro dose-dependent neutralization and stability of RBC-ANS/anti-RBC binding. (A) Flow cytometry histograms of RBC-ANS (from left to right: 1,000 μg, 500 μg, 250 μg, 100 μg, 50 μg, and 0 μg) preincubated with 50 μg of FITC–anti-RBCs before mixing with 5 vol% RBC solution demonstrated dose- dependent neutralization of anti-RBCs. (B) Mean fluorescence intensity of samples in A.(C) Flow cytometry histograms of RBC-ANS (from left to right: 1,000 μg, 500 μg, 250 μg, 100 μg, 50 μg, and 0 μg) coincubated with 50 μgofFITC–anti-RBCs and 5 vol% RBC solution demonstrated dose-dependent, competitive neutralization of anti-RBCs. (D) Mean fluorescence intensity of samples in C.(E–I) Varying amounts of RBC-ANS (from E to I:0μg, 25 μg, 50 μg,100 μg, and 250 μg) were coincubated with 15.6 μg of anti-RBCs (primary antibody) and 5 vol% RBC solution, followed by adding an equivalent dose of anti-Fc (agglutinating secondary antibody). The samples were then imaged by light microscopy at 10× magnification, demonstrating dose-related inhibition of RBC agglutination by RBC-ANS. (Scale bar: 100 μm.)

a reduction of RBC-bound antibody signal by ∼40% and ∼80% at between the control and treatment groups showed that anti- equivalent RBC-ANS doses, respectively. To correlate dose de- RBCs preincubated with RBC-ANS was less effective in inducing pendence to relevant diagnostic parameters clinically, we com- an anemic response (Fig. 4). Mice in the treatment group pos- pleted an Ig agglutination test, which is equivalent to the qualitative sessed a higher RBC count, hemoglobin content, and hemat- direct antiglobulin test that is a gold standard laboratory diagnostic ocrit throughout the duration of the study. All parameters were test often used in the diagnosis of AIHA (25, 26). By varying the consistent with control mice that had not been challenged with dose of RBC-ANS from 0 to 250 μg, we demonstrated a dose- anti-RBCs but had their blood drawn daily. This result suggests dependent neutralization of anti-RBCs (primary antibody) as evi- that the anti-RBCs were trapped by the RBC-ANS and were denced by the progressive decrease of RBC agglutination upon precluded from binding to circulating RBCs. The experiment addition of an agglutinating secondary antibody (Fig. 3 E–I). demonstrates the feasibility of using target cell-mimicking After confirming in vitro that RBC-ANS could selectively bind nanoparticles to neutralize pathological antibodies. anti-RBCs, we next assessed the ability of the particles to retain To validate the clinical relevance of the RBC-ANS further, we antibodies durably in vivo. A previously described anemia disease administered daily injections of low-dose anti-RBCs i.p. to main- model, induced through i.p. injection of anti-RBCs, was used in tain a sustained level of the antibodies for anemia progression. the study (11). Five hundred micrograms of anti-RBCs, a suffi- RBC-ANS was injected i.v. with the aim of neutralizing the cir- cient amount to induce acute anemia, was injected i.p. into mice in culating antibodies and retarding anemia development. PEG-NP the control group. Following the injection, the antibodies could of analogous size was also administered as a control. Mice were diffuse across the peritoneal membrane, bind to circulating RBCs, divided into RBC-ANS plus anti-RBC, PEG-NP plus anti-RBC, and induce their clearance. Mice in the treatment group received and PBS plus anti-RBC groups. All mice received 200 μg of anti- the same dose of anti-RBCs incubated beforehand for 5 min at RBCs daily through i.p. injection, followed by i.v. injection of 2 mg 37 °C with 5 mg of RBC-ANS. The relevant clinical parameters of either RBC-ANS, PEG-NP, or PBS daily for 4 d. Blood was used for monitoring anemia responses, including RBC count, he- obtained daily for the duration of the experiment to assess RBC moglobin level, and hematocrit, of each group were then assessed count, hemoglobin level, and hematocrit. Starting from day 2, sig- daily for 4 d. Comparison of the hematological parameters nificant benefit in anemia-related parameters was observed in the MEDICAL SCIENCES

Fig. 4. In vivo binding stability of RBC-ANS and anti-RBCs. Mice (n = 6) were i.p. injected with 500 μg of anti-RBCs preincubated with 5 mg of RBC-ANS (red), 500 μg of anti-RBCs alone (blue), or PBS (black). Blood was collected daily to monitor RBC count (A, million cells per microliter), hemoglobin level (B, grams ENGINEERING per deciliter), and hematocrit (C, %) of the mice.

Copp et al. PNAS | September 16, 2014 | vol. 111 | no. 37 | 13483 Downloaded by guest on October 1, 2021 RBC-ANS–treatedgroupcomparedwithPEG-NPcontrolmice antibodies or cytotoxic drugs, with side effects of severe infection, and vehicle-only mice (Fig. 5). The inability of PEG-NP to prevent antibody transfusion reactions, and even the development of anemia further supports the antigen-specific clearance of anti-RBCs malignancies (10, 37). Given this landscape, it is meaningful to mediated by RBC-ANS as opposed to the preservation of RBCs via continue development of innovative therapeutic strategies to saturation of the mononuclear phagocyte system (27, 28). To help manage disease burden while minimizing iatrogenic risk. Nano- assess the safety of the RBC-ANS approach, we also examined the particles have already shown promise in reducing the risk of sys- autologous anti-RBC serum titers in mice 6 wk following RBC-ANS temic toxicity of chemotherapy while increasing efficacy both in treatment. ELISA assessment of autoantibodies against mouse emerging literature and clinically (38–40). We demonstrated that RBCs showed no observable elevation of autologous anti-RBC nanoparticles can be engineered to intercept binding between responses in mice receiving RBC-ANS treatment compared with pathological antibodies and their target cells to have a favorable the controls. The result confirms that the RBC-ANS/anti-RBC impact on disease status. This particular approach offers a unique complex does not potentiate a humoral immune response against therapeutic intervention for type II immune hypersensitivity particle-associated membrane antigens (Fig. 6). reactions by targeting a final pathological mechanism and presents an attractive alternative to broad-spectrum immune suppression. Discussion Through the stabilization of biological membrane on a poly- Autoimmune diseases, which include type II, type III, and type meric nanoparticle substrate, we unveiled the ability of cell IV immune hypersensitivity reactions, are known to attack al- membrane-coated nanoparticles to serve as an antibody decoy to most every body tissue, make up over 50 diseases, and contribute improve disease parameters. Our results indicate the ability of to over $65 billion in health care costs annually (29). AIHA was RBC-ANS to bind to anti-RBCs effectively and preclude its in- attributed to an autoantibody in 1904 by Donath and Land- teraction with RBCs. The therapeutic potential of the proposed steiner, and the mechanism of extravascular hemolysis was de- approach was validated in vivo with separate administration of scribed by Metchinkoff in 1905, making it the first disease known anti-RBCs and RBC-ANS via i.p. and i.v. routes, respectively. to be caused by this mechanism (30). Although the etiology is Although the RBC-ANS reduced the antibody-mediated anemic often idiopathic, it can be induced by drugs (cephalosporins, response, equivalent doses of PEG-NP of analogous physico- chemotherapies, and quinines), as well as by malignancies and chemical properties failed to moderate the effect of the anti- viral infections (25, 26, 30). Despite the differences in etiology, RBCs. The outcome of the in vivo study further indicates that the the final common disease pathway is the generation of antibodies improved hematological status upon RBC-ANS treatment was against RBC membrane components, typically rhesus group and mediated by specific antibody–antigen interaction, rather than glycophorins, by a B-lymphocyte population that has lost self- particle-mediated saturation of phagocytic cells (27, 28). We also tolerance to RBC surface antigen(s) (31). Most commonly, the established a lack of humoral response against the RBC mem- pathological mechanism is IgG-mediated attack that leads to the brane antigens following administration of RBC-ANS and anti- opsonization of RBCs for extravascular destruction by phagocytes. RBCs, which validates the safety of the approach, because the Alternatively, AIHA can also be induced by IgM-mediated RBC-ANS, in the presence of anti-RBCs, did not potentiate an attack on RBCs, which causes RBC intravascular hemolysis via RBC autoantibody immune response. It has been previously activation of the complement system (26, 30, 32, 33). Even reported that RBC membrane-coated nanoparticles are primarily though autoantibodies have long been recognized to play a sig- metabolized in the liver (21, 41), where particulate metabolism nificant role in the disease, to our knowledge, therapies specifi- generally promotes a tolerogenic immune response (42, 43). In cally directed at these pathological antibodies were not pre- addition, several reports have shown that antigen-laden polymeric viously explored. Existing AIHA therapy continues to target nanoparticles, in the absence of immune adjuvants, are immune- upstream disease mechanisms through reliance on broad im- tolerizing (44–46). Although rigorous immunological studies in mune suppression, blood transfusions, or splenectomy for re- more faithful AIHA animal models are warranted, the present fractory cases (12, 15). This treatment paradigm holds true for study exhibits the feasibility of applying cell membrane-coated other type II immune hypersensitivities, which are also man- nanoparticles for clearing pathological antibodies. Adding prom- aged with broad immune suppression, such as using systemic ise to the approach is the demonstration of both nucleated and glucocorticoids or cytotoxic drugs (3, 34). nonnucleated mammalian cell membranes that have been suc- Although efficacious for many patients, systemic steroids carry cessfully stabilized by nanoparticle cores (20, 21). This capacity to some of the highest risks of iatrogenic illness. Adverse effects of functionalize particles, with a variety of multiantigen membranes, therapy include steroid myopathy, nosocomial infection, aseptic offers a platform for the development of a robust line of therapies bone necrosis, accelerated osteoporosis, weight gain, metabolic against additional type II immune hypersensitivities. derangements, and Cushingoid appearance (35, 36). In addition Currently, the paradigm in targeted nanomedicine revolves to these side effects, if steroid therapy fails, a patient may need to around high-throughput screening for ligand-receptor recognition undergo surgery or systemic B-cell depletion with monoclonal and the subsequent nanoparticle functionalization with specific

Fig. 5. In vivo neutralization of anti-RBCs by RBC-ANS. Mice (n = 10) were i.p. injected with 200 μg of anti-RBCs on days 0, 1, 2, and 3. After each dose of the antibody, the mice received 2 mg of RBC-ANS (red), PEG-NP (black), or PBS (blue) via tail vein i.v. injection. Blood was collected daily to monitor RBC count (A, million cells per microliter), hemoglobin level (B, grams per deciliter), and hematocrit (C, %) of the mice.

13484 | www.pnas.org/cgi/doi/10.1073/pnas.1412420111 Copp et al. Downloaded by guest on October 1, 2021 (anti-RBCs; Rockland Antibodies and Assays) at 10 mg/mL was mixed with 3.0 μL of 10 mg/mL FITC (Thermo Scientific) in DMSO. The mixture was incubated at room temperature in the dark for 1 h and then run through a Sephadex G-25 column (Sigma–Aldrich) with deionized water to purify conjugated FITC–anti-RBCs for subsequent experiments. For the antibody retention study, 250 μg of DiD-loaded RBC-ANS was combined with six serial dilutions (500 μg, 250 μg, 125 μg, 31.25 μg, 7.81 μg, and 1.95 μg) of FITC- labeled antibody in triplicate in a Costar 96-well plate (Corning Unlimited). Before incubation, the samples’ fluorescence intensities were measured using a Tecan Infinite M200 reader (TeCan) to determine 100% signal of FITC (515 nm) and DiD (670 nm). Solutions were then incubated for 30 min at 37 °C, followed by spinning down in a Legend 21R Microcentrifuge (Thermo Scientific) at 21,200 × g for 5 min to collect pelleted RBC-ANS/anti-RBC complex. Samples were then washed three times in 1 mL of water, and their fluorescence intensity was remeasured to determine the signal intensity of FITC in relation to DiD. All DiD signals were greater than 90% of the original signal, ensuring minimal loss during washing steps. These steps were repeated at optimum concentrations of 250 μg of DiD-loaded RBC-ANS or 250 μg of DiD-loaded PEG-NP, combined with 7.8 μg of FITC–anti-RBC and Fig. 6. RBC-ANS does not elicit autoimmune antibodies against RBCs. Six 7.8 μg of FITC-conjugated anti-Fc (Rockland Antibodies and Assays) to de- weeks following administration, ELISA analysis of serum from mice receiving termine the specificity of RBC-ANS against anti-RBCs compared with control RBC-ANS plus anti-RBCs, anti-RBCs alone, or PBS (Blank) showed no observ- samples. To compare binding kinetics, serially diluted concentrations of able elevation of anti-RBC titer compared with the positive control. FITC–anti-RBCs (1, 0.5, 0.25, 0.125, 0.063, and 0.031 mg/mL) were incubated with a constant substrate concentration (0.25 mg/mL RBC-ANS or an equivalent amount of RBC ghosts). Final values were normalized to the maximum targeting molecules (47, 48). With regard to type II immune hy- binding observed at saturation. Binding capacity was expressed as a ratio of the fluorescent signals at saturation. To test binding capacity in serum, RBC- persensitivity reactions, such a functionalization process could prove – limited owing to the varying antigen specificities among pathological ANS was incubated with a saturated amount of FITC anti-RBCs in PBS or in the presence of 50 vol% FBS (Thermo Scientific). Values were expressed as antibodies from patient to patient, such as in the case of AIHA (11, a ratio of the fluorescent signals. 23, 45). Through the appropriate application of biological membranes, which possess the diversity of surface antigens susceptible to Competitive Binding Studies. RBC-ANS was prepared at 1 mg/mL in 1× Dulbecco’s pathological antibodies, biomimetic nanoparticles can be prepared PBS (Gibco) and serially diluted to make five solutions (1 mg/mL, 500 μg/mL, in a facile manner for selective immunomodulation. Furthermore, 250 μg/mL, 100 μg/mL, and 50 μg/mL) with 1× PBS as a control. For the drug-loaded cores or those made from different materials, such as preincubation study, these solutions were combined with 50 μg of anti-RBCs metallic or inorganic nanoparticles, can be used to create multi- and incubated for 2 min at 37 °C before the addition of 1 mL of washed 5 vol% functional formulations. We believe the demonstration of patho- mouse RBC solution. For the competitive coincubation study, RBC-ANS and physiologically inspired nanoengineering serves as a valuable anti-RBCs were added simultaneously to 1 mL of 5 vol% RBC solution. Each experiment was done in triplicate. Samples were allowed to incubate for prototype for additional therapeutic advances, offering the oppor- 10 min at 37 °C and then washed three times in 1× PBS to remove supernatant tunity for selective disease intervention while minimizing iatrogenic thoroughly and collect RBC pellets. Flow cytometry was used to measure the risks associated with many traditional drug-based therapies. FITC signal of the collected RBC population using a Becton Dickinson FACSCanto II. Flow cytometry data were analyzed using FlowJo software from Treestar. Materials and Methods Preparation of RBC-ANS. RBC-ANS was prepared following previously described RBC Agglutination Titration. The experiment was carried out per the manu- methods (21). Briefly, ∼100-nm PLGA polymeric cores were prepared using facturer’s instructions (Rockport Antibodies and Assays). Briefly, 100 μLof 0.67 dL/g of carboxy-terminated 50:50 poly(DL-lactide-co-glycolide) (LACTEL anti-RBCs (primary antibody) at 156 μg/mL was added to 100 μL of 5 vol% Absorbable Polymers) in a nanoprecipitation process. The PLGA polymer was washed RBCs in 1× PBS, along with 62.5 μL of RBC-ANS (250 μg, 100 μg, first dissolved in acetone at a concentration of 10 mg/mL. One milliliter of the 50 μg, 25 μg, or 0 μg) and incubated for 45 min at 37 °C. The RBC solution solution was then added rapidly to 3 mL of water. For fluorescently labeled was then washed three times by centrifuging the sample at 3,500 × g for formulations, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine per- 1 min and exchanging the supernatant with 1× PBS each time. One hundred chlorate (DiD; excitation/emission = 644/665 nm; Life Technologies) was loaded microliters of anti-Fc (agglutinating secondary antibody) at 156 μg/mL was into the polymeric cores at 0.1 wt%. The mixture was then stirred in open air added to each sample, which was then placed in an analog vortex mixer for 1 h and placed in a vacuum for another 3 h. The resulting nanoparticle (Fisher Scientific) at 625 rpm for 5 min and then spun down at 3,500 × g for solution was filtered using Amicon Ultra-4 Centrifugal Filters with a molecular 20 s. The sample was then resuspended using a pipette to disrupt the pellet. mass cutoff of 10 kDa (Millipore). RBC membrane coating was then completed For the negative control, 100 μL of 6% (wt/vol) BSA was used in lieu of by fusing RBC membrane vesicles with PLGA particles via sonication using an secondary antibody. All samples were then viewed via a light microscope at FS30D bath sonicator at a frequency of 42 kHz and a power of 100 W for 10× magnification and imaged via mounted camera. 2 min. The size and the zeta-potential of the resulting RBC-ANS were obtained from three dynamic light scattering measurements using a Malvern ZEN 3600 In Vivo Stability of RBC-ANS and Anti-RBC Binding. Following induction of Zetasizer, which showed an average hydrodynamic diameter of ∼100 nm and anemia via i.p. injection of anti-RBCs, we randomly assorted 12 CD-1 mice ∼115 nm before and after the membrane coating process, respectively. The (Charles River Laboratories) into two groups of six. The treatment group structure of RBC-ANS was examined with TEM. A drop of the RBC-ANS solution received 500 μg of anti-RBCs incubated with 5 mg of RBC-ANS for 5 min at at 100 μg/mL was deposited onto a glow-discharged, carbon-coated grid for 37 °C before injection, and anti-RBC only mice received anti-RBCs incubated 10 s and then rinsed with 10 drops of distilled water. A drop of 1 wt% uranyl in 1× PBS for 5 min. A control group of mice received injections of PBS only.

acetate stain was added to the grid. The sample was then imaged using an FEI A few drops of blood were collected from each mouse before injections on MEDICAL SCIENCES 200 kV Sphera microscope. PEG-NP was prepared using poly(ethylene glycol) day 0 to establish starting blood counts, and this procedure was repeated on methyl ether-block-poly(lactide-co-glycolide) (PEG-PLGA; Sigma–Aldrich). The each day of the experiment. Samples were stored in potassium-EDTA PEG-PLGA polymer was dissolved in acetone at 10 mg/mL, and 1 mL of solution Microvette tubes (Sarstedt) and vigorously mixed to prevent clotting. Sam- was added to 3 mL of water. For fluorescently labeled formulations, DiD was ples were then run on the same day using a Drew Scientific Hemavet 950 loaded into the polymeric cores at 0.1 wt%. The mixture was then stirred in (Erba Diagnostics), and RBC count, hemoglobin level, and hematocrit were open air for 1 h and subsequently placed in a vacuum for another 3 h. recorded daily.

RBC-ANS Binding Capacity and Specificity Studies. Antibodies were first la- In Vivo Neutralization of Circulating Anti-RBCs by RBC-ANS. Using the esta- ENGINEERING beled with FITC. Specifically, 100 μL of polyclonal rabbit anti-mouse RBC IgG blished i.p. model for antibody delivery, 30 mice were randomized to three

Copp et al. PNAS | September 16, 2014 | vol. 111 | no. 37 | 13485 Downloaded by guest on October 1, 2021 groups of 10 mice. Each group of mice received a 100-μL i.p. injection of added in a sequence of six 1:5 dilutions. HRP-conjugated goat anti-mouse 2 mg/mL anti-RBCs on days 0, 1, 2, and 3. The treatment group also received antibody IgG (Biolegend) was used to probe for bound antibodies. The plate a tail vein i.v. injection of 200 μL of RBC-ANS (10 mg/mL) in 1× PBS within was developed using 3,3′,5,5′-tetramethylbenzidine substrate, and 1 M HCl 30 min of i.p. antibody delivery. The PEG-NP group received an equivalent was used to stop the reaction. Absorbance was measured at 450 nm. i.v. dose of PEG-NP, and the anti-RBC only group received 200 μL PBS via i.v. injection. The RBC count, hemoglobin level, and hematocrit of each sample ACKNOWLEDGMENTS. This work is supported by the National Institute of were recorded on days 0, 1, 2, 3, and 4. Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (NIH) under Award R01DK095168. J.A.C. is supported by a Howard Anti-RBC Autoimmune Study. Six weeks after the in vivo neutralization studies, Hughes Medical Institute Medical Research Fellowship. R.H.F. is supported by serum was collected from 12 mice (six in each group) and a standard ELISA was the Department of Defense through the National Defense Science and performed. Washed CD-1 mouse RBCs were plated at 1 × 106 RBCs per well Engineering Graduate Fellowship Program. B.T.L. is supported by NIH Training onto a Costar 96 well plate. One hundred microliters of collected serum was Grant R25CA153915 from the National Cancer Institute.

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