Clearance of pathological antibodies using biomimetic nanoparticles

Significance

The selective depletion of disease-causing antibodies using nanoparticles offers a new model in the management of type II immune hypersensitivity reactions. The demonstration of pathophysiologically inspired nanoengineering serves as a valuable prototype for additional therapeutic improvements with the goal of minimizing therapy-related adverse effects. Through the use of cell membrane-cloaked nanoparticles, nanoscale decoys with strong affinity to pathological antibodies can be administered to disrupt disease processes in a minimally toxic manner. These biomimetic nanoparticles enable indiscriminate absorption of pathological antibodies regardless of their epitope specificities. This particular approach offers much promise in treating antibody-mediated autoimmune diseases, in which target antigens on susceptible cells can vary from patient to patient.

Abstract

Pathological antibodies have been demonstrated to play a key role in type II immune hypersensitivity reactions, resulting in the destruction of healthy tissues and leading to considerable morbidity for the patient. Unfortunately, current treatments present significant iatrogenic risk while still falling short for many patients in achieving clinical remission. In the present work, we explored the capability of target cell membrane-coated nanoparticles to abrogate the effect of pathological antibodies in an effort to minimize disease burden, without the need for drug-based immune suppression. Inspired by antibody-driven pathology, we used intact RBC membranes stabilized by biodegradable polymeric nanoparticle cores to serve as an alternative target for pathological antibodies in an antibody-induced anemia disease model. Through both in vitro and in vivo studies, we demonstrated efficacy of RBC membrane-cloaked nanoparticles to bind and neutralize anti-RBC polyclonal IgG effectively, and thus preserve circulating RBCs.

Type II immune hypersensitivities are driven by pathological antibodies targeting self-antigens, either naturally occurring or due to exposure to an exogenous substance present on the cellular exterior or ECM. This disease type makes up many of the most prevalent autoimmune diseases, including pernicious anemia, Grave disease, and myasthenia gravis, as well as autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (1–4). In addition, these diseases may occur after the administration of a new drug or following certain infections. Currently, therapies for these immune-mediated diseases remain relatively nonspecific via broad immune suppression (5). For instance, comprehensive immune suppression through systemic glucocorticoids (i.e., prednisone, methylprednisolone), cytotoxic drugs (i.e., cyclophosphamide, methotrexate, azathioprine), and monoclonal antibodies (i.e., rituximab, belimumab, infliximab) dominate treatment regimens to prevent further tissue destruction (6–8). Although this approach to therapy is effective for some patients in achieving remission, its efficacy remains variable and there is a well-established risk of adverse side effects, highlighting the need for better tailored therapies (9, 10).

The development of nanoparticle therapeutics has sparked new hope for the treatment of various important human diseases. Herein, we demonstrate the application of a biomimetic nanoparticle for the clearance of pathological antibodies using an established murine model of antibody-induced anemia (11). This disease may be idiopathic, as in AIHA, or drug induced, as in drug-induced anemia (DIA). In both cases, however, autoantibodies attack surface antigens present on RBCs. Therapy for AIHA is relatively standardized, with patients starting on systemic steroids and escalating to cytotoxic drugs and B cell-depleting monoclonal antibodies, and then possibly splenectomy based on patient response to therapy (12, 13). The shortcoming of suppressing the immune system with drug-based therapies is the considerable iatrogenic risk associated with nonspecific therapy and heightened susceptibility to severe infections following spleen removal (9, 10, 14). DIA, which can be the result of drug-hapten antibodies or drug-independent autoantibodies, is treated much the same way, with discontinuance of the offending drug and, much more often than in AIHA, performance of blood transfusions (15, 16). A subsequent limitation of repeated transfusions of packed RBCs is that although they revive tissue perfusion, they carry the risks of hemolytic transfusion reactions, the formation of alloantibodies, and iron toxicity (17–19).

It has previously been shown that mammalian cellular membrane, from both nucleated and nonnucleated cells, can be fused onto polymeric nanoparticle substrates to form stable core/shell structures (20, 21). These particles have been shown to retain and present natural cell membrane and surface antigens (22), which bare the target epitopes involved in antibody-mediated cellular clearance found in AIHA and DIA. To demonstrate the interception of pathological antibodies, we used RBC membrane-cloaked nanoparticles, herein denoted RBC antibody nanosponge(s) (ANS), to serve as alternative targets for anti-RBC antibodies and preserve circulating RBCs (Fig. 1). Unlike conventional immune therapy, these biomimetic nanoparticles have no drug payload to suppress normal lymphocytes or immune effector cells. Additionally, unlike blood transfusions, which serve as a replacement therapy, the RBC-ANS serves to deplete circulating antibody levels, without contributing further toxic metabolites due to the hemolysis of transfused cells. Moreover, it has been demonstrated in animal models of autoimmune diseases that the primary target antigens can vary and shift over the course of the diseases (23). Exploiting target cell membranes in their entirety overcomes the varying antigen specificities and presents a previously unidentified approach in intercepting the autoreactive antibody mechanism of type II immune hypersensitivity reactions.

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 investigate 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 anti-RBC binding capacity compared with when the incubation was performed in buffer alone (Fig. 2F). These results are indicative of relatively low nonspecific antibody–nanoparticle binding interactions and demonstrate the necessity for antigen–antibody concordance to achieve neutralization.

Fig. 2.

In vitro characterization of RBC-ANS. (A) TEM image demonstrates the core/shell structure of RBC-ANS. (Scale bar: 150 nm.) (B) Size and surface zeta-potential of pure PLGA cores, RBC-ANS, and RBC-ANS bound with anti-RBCs. (C) RBC-ANS (250 μg) incubated with five serial dilutions of fluorescent anti-RBCs demonstrated particle saturation at ∼27 μg of antibody, corresponding to a particle/antibody mass ratio of ∼9:1. (D) Equivalent amounts of RBC-ANS incubated with anti-RBCs or anti-Fc demonstrated significantly greater specific binding compared with nonspecific binding. The corresponding PEG-NP incubated with anti-RBCs served as a negative control. (E) Comparison of anti-RBC binding kinetics with a fixed amount of RBC-ANS or RBC ghosts. (Inset) Relative binding capacity of RBC-ANS vs. RBC ghosts at saturation. (F) Relative binding capacity of RBC-ANS in PBS vs. RBC-ANS in serum at saturation.

To characterize the binding stability and competitive binding capacity further, we varied the amounts of RBC-ANS mixed with a constant amount of fluorescent anti-RBCs in 5 vol% RBC solution. To assess in vitro binding stability, RBC-ANS was preincubated with anti-RBCs before mixing with 5 vol% RBC solution (Fig. 3 A and B), and to test competitive binding capacity, RBC-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 a reduction of RBC-bound antibody signal by ∼40% and ∼80% at equivalent RBC-ANS doses, respectively. To correlate dose dependence to relevant diagnostic parameters clinically, we completed an Ig agglutination test, which is equivalent to the qualitative direct antiglobulin test that is a gold standard laboratory diagnostic test often used in the diagnosis of AIHA (25, 26). By varying the dose of RBC-ANS from 0 to 250 μg, we demonstrated a dose-dependent neutralization of anti-RBCs (primary antibody) as evidenced by the progressive decrease of RBC agglutination upon addition of an agglutinating secondary antibody (Fig. 3 E–I).

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 μg of FITC–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: 0 μg, 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.)

After confirming in vitro that RBC-ANS could selectively bind anti-RBCs, we next assessed the ability of the particles to retain antibodies durably in vivo. A previously described anemia disease model, induced through i.p. injection of anti-RBCs, was used in the study (11). Five hundred micrograms of anti-RBCs, a sufficient amount to induce acute anemia, was injected i.p. into mice in the control group. Following the injection, the antibodies could diffuse across the peritoneal membrane, bind to circulating RBCs, and induce their clearance. Mice in the treatment group received the same dose of anti-RBCs incubated beforehand for 5 min at 37 °C with 5 mg of RBC-ANS. The relevant clinical parameters used for monitoring anemia responses, including RBC count, hemoglobin level, and hematocrit, of each group were then assessed daily for 4 d. Comparison of the hematological parameters between the control and treatment groups showed that anti-RBCs preincubated with RBC-ANS was less effective in inducing an anemic response (Fig. 4). Mice in the treatment group possessed a higher RBC count, hemoglobin content, and hematocrit throughout the duration of the study. All parameters were consistent with control mice that had not been challenged with anti-RBCs but had their blood drawn daily. This result suggests that the anti-RBCs were trapped by the RBC-ANS and were precluded from binding to circulating RBCs. The experiment demonstrates the feasibility of using target cell-mimicking nanoparticles to neutralize pathological antibodies.

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 per deciliter), and hematocrit (C, %) of the mice.

To validate the clinical relevance of the RBC-ANS further, we administered daily injections of low-dose anti-RBCs i.p. to maintain a sustained level of the antibodies for anemia progression. RBC-ANS was injected i.v. with the aim of neutralizing the circulating antibodies and retarding anemia development. PEG-NP of analogous size was also administered as a control. Mice were divided into RBC-ANS plus anti-RBC, PEG-NP plus anti-RBC, and PBS plus anti-RBC groups. All mice received 200 μg of anti-RBCs daily through i.p. injection, followed by i.v. injection of 2 mg of either RBC-ANS, PEG-NP, or PBS daily for 4 d. Blood was obtained daily for the duration of the experiment to assess RBC count, hemoglobin level, and hematocrit. Starting from day 2, significant benefit in anemia-related parameters was observed in the RBC-ANS–treated group compared with PEG-NP control mice and vehicle-only mice (Fig. 5). The inability of PEG-NP to prevent anemia further supports the antigen-specific clearance of anti-RBCs mediated by RBC-ANS as opposed to the preservation of RBCs via saturation of the mononuclear phagocyte system (27, 28). To help assess the safety of the RBC-ANS approach, we also examined the autologous anti-RBC serum titers in mice 6 wk following RBC-ANS treatment. ELISA assessment of autoantibodies against mouse RBCs showed no observable elevation of autologous anti-RBC responses in mice receiving RBC-ANS treatment compared with the controls. The result confirms that the RBC-ANS/anti-RBC complex does not potentiate a humoral immune response against particle-associated membrane antigens (Fig. 6).

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.

Fig. 6.

RBC-ANS does not elicit autoimmune antibodies against RBCs. Six weeks following administration, ELISA analysis of serum from mice receiving RBC-ANS plus anti-RBCs, anti-RBCs alone, or PBS (Blank) showed no observable elevation of anti-RBC titer compared with the positive control.

Discussion

Autoimmune diseases, which include type II, type III, and type IV immune hypersensitivity reactions, are known to attack almost every body tissue, make up over 50 diseases, and contribute to over $65 billion in health care costs annually (29). AIHA was attributed to an autoantibody in 1904 by Donath and Landsteiner, and the mechanism of extravascular hemolysis was described by Metchinkoff in 1905, making it the first disease known to be caused by this mechanism (30). Although the etiology is often idiopathic, it can be induced by drugs (cephalosporins, chemotherapies, and quinines), as well as by malignancies and viral infections (25, 26, 30). Despite the differences in etiology, the final common disease pathway is the generation of antibodies against RBC membrane components, typically rhesus group and glycophorins, by a B-lymphocyte population that has lost self-tolerance to RBC surface antigen(s) (31). Most commonly, the pathological mechanism is IgG-mediated attack that leads to the opsonization of RBCs for extravascular destruction by phagocytes. Alternatively, AIHA can also be induced by IgM-mediated attack on RBCs, which causes RBC intravascular hemolysis via activation of the complement system (26, 30, 32, 33). Even though autoantibodies have long been recognized to play a significant role in the disease, to our knowledge, therapies specifically directed at these pathological antibodies were not previously explored. Existing AIHA therapy continues to target upstream disease mechanisms through reliance on broad immune suppression, blood transfusions, or splenectomy for refractory cases (12, 15). This treatment paradigm holds true for other type II immune hypersensitivities, which are also managed with broad immune suppression, such as using systemic glucocorticoids or cytotoxic drugs (3, 34).

Although efficacious for many patients, systemic steroids carry some of the highest risks of iatrogenic illness. Adverse effects of therapy include steroid myopathy, nosocomial infection, aseptic bone necrosis, accelerated osteoporosis, weight gain, metabolic derangements, and Cushingoid appearance (35, 36). In addition to these side effects, if steroid therapy fails, a patient may need to undergo surgery or systemic B-cell depletion with monoclonal antibodies or cytotoxic drugs, with side effects of severe infection, antibody transfusion reactions, and even the development of malignancies (10, 37). Given this landscape, it is meaningful to continue development of innovative therapeutic strategies to manage disease burden while minimizing iatrogenic risk. Nanoparticles have already shown promise in reducing the risk of systemic toxicity of chemotherapy while increasing efficacy both in emerging literature and clinically (38–40). We demonstrated that nanoparticles can be engineered to intercept binding between pathological antibodies and their target cells to have a favorable impact on disease status. This particular approach offers a unique therapeutic intervention for type II immune hypersensitivity reactions by targeting a final pathological mechanism and presents an attractive alternative to broad-spectrum immune suppression.

Through the stabilization of biological membrane on a polymeric nanoparticle substrate, we unveiled the ability of cell membrane-coated nanoparticles to serve as an antibody decoy to improve disease parameters. Our results indicate the ability of RBC-ANS to bind to anti-RBCs effectively and preclude its interaction with RBCs. The therapeutic potential of the proposed approach was validated in vivo with separate administration of anti-RBCs and RBC-ANS via i.p. and i.v. routes, respectively. Although the RBC-ANS reduced the antibody-mediated anemic response, equivalent doses of PEG-NP of analogous physicochemical properties failed to moderate the effect of the anti-RBCs. The outcome of the in vivo study further indicates that the improved hematological status upon RBC-ANS treatment was mediated by specific antibody–antigen interaction, rather than particle-mediated saturation of phagocytic cells (27, 28). We also established a lack of humoral response against the RBC membrane antigens following administration of RBC-ANS and anti-RBCs, which validates the safety of the approach, because the RBC-ANS, in the presence of anti-RBCs, did not potentiate an RBC autoantibody immune response. It has been previously reported that RBC membrane-coated nanoparticles are primarily metabolized in the liver (21, 41), where particulate metabolism generally promotes a tolerogenic immune response (42, 43). In addition, several reports have shown that antigen-laden polymeric nanoparticles, in the absence of immune adjuvants, are immune-tolerizing (44–46). Although rigorous immunological studies in more faithful AIHA animal models are warranted, the present study exhibits the feasibility of applying cell membrane-coated nanoparticles for clearing pathological antibodies. Adding promise to the approach is the demonstration of both nucleated and nonnucleated mammalian cell membranes that have been successfully stabilized by nanoparticle cores (20, 21). This capacity to functionalize particles, with a variety of multiantigen membranes, offers a platform for the development of a robust line of therapies against additional type II immune hypersensitivities.

Currently, the paradigm in targeted nanomedicine revolves around high-throughput screening for ligand-receptor recognition and the subsequent nanoparticle functionalization with specific targeting molecules (47, 48). With regard to type II immune hypersensitivity reactions, such a functionalization process could prove limited owing to the varying antigen specificities among pathological antibodies from patient to patient, such as in the case of AIHA (11, 23, 45). Through the appropriate application of biological membranes, which possess the diversity of surface antigens susceptible to pathological antibodies, biomimetic nanoparticles can be prepared in a facile manner for selective immunomodulation. Furthermore, drug-loaded cores or those made from different materials, such as metallic or inorganic nanoparticles, can be used to create multifunctional formulations. We believe the demonstration of pathophysiologically inspired nanoengineering serves as a valuable prototype for additional therapeutic advances, offering the opportunity for selective disease intervention while minimizing iatrogenic risks associated with many traditional drug-based therapies.

Materials and Methods

Preparation of RBC-ANS.

RBC-ANS was prepared following previously described methods (21). Briefly, ∼100-nm PLGA polymeric cores were prepared using 0.67 dL/g of carboxy-terminated 50:50 poly(_DL-lactide-co-glycolide) (LACTEL Absorbable Polymers) in a nanoprecipitation process. The PLGA polymer was first dissolved in acetone at a concentration of 10 mg/mL. One milliliter of the solution was then added rapidly to 3 mL of water. For fluorescently labeled formulations, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD; excitation/emission = 644/665 nm; Life Technologies) was loaded into the polymeric cores at 0.1 wt%. The mixture was then stirred in open air for 1 h and placed in a vacuum for another 3 h. The resulting nanoparticle solution was filtered using Amicon Ultra-4 Centrifugal Filters with a molecular mass cutoff of 10 kDa (Millipore). RBC membrane coating was then completed by fusing RBC membrane vesicles with PLGA particles via sonication using an FS30D bath sonicator at a frequency of 42 kHz and a power of 100 W for 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 Zetasizer, which showed an average hydrodynamic diameter of ∼100 nm and ∼115 nm before and after the membrane coating process, respectively. The structure of RBC-ANS was examined with TEM. A drop of the RBC-ANS solution at 100 μg/mL was deposited onto a glow-discharged, carbon-coated grid for 10 s and then rinsed with 10 drops of distilled water. A drop of 1 wt% uranyl acetate stain was added to the grid. The sample was then imaged using an FEI 200 kV Sphera microscope. PEG-NP was prepared using poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG-PLGA; Sigma–Aldrich). The PEG-PLGA polymer was dissolved in acetone at 10 mg/mL, and 1 mL of solution was added to 3 mL of water. For fluorescently labeled formulations, DiD was loaded into the polymeric cores at 0.1 wt%. The mixture was then stirred in open air for 1 h and subsequently placed in a vacuum for another 3 h.

RBC-ANS Binding Capacity and Specificity Studies.

Antibodies were first labeled with FITC. Specifically, 100 μL of polyclonal rabbit anti-mouse RBC IgG (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 7.8 μg of FITC-conjugated anti-Fc (Rockland Antibodies and Assays) to determine the specificity of RBC-ANS against anti-RBCs compared with control samples. To compare binding kinetics, serially diluted concentrations of 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 binding observed at saturation. Binding capacity was expressed as a ratio of the fluorescent signals at saturation. To test binding capacity in serum, RBC-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 a ratio of the fluorescent signals.

Competitive Binding Studies.

RBC-ANS was prepared at 1 mg/mL in 1× Dulbecco’s PBS (Gibco) and serially diluted to make five solutions (1 mg/mL, 500 μg/mL, 250 μg/mL, 100 μg/mL, and 50 μg/mL) with 1× PBS as a control. For the preincubation study, these solutions were combined with 50 μg of anti-RBCs and incubated for 2 min at 37 °C before the addition of 1 mL of washed 5 vol% mouse RBC solution. For the competitive coincubation study, RBC-ANS and 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 10 min at 37 °C and then washed three times in 1× PBS to remove supernatant thoroughly and collect RBC pellets. Flow cytometry was used to measure the FITC signal of the collected RBC population using a Becton Dickinson FACSCanto II. Flow cytometry data were analyzed using FlowJo software from Treestar.

RBC Agglutination Titration.

The experiment was carried out per the manufacturer’s instructions (Rockport Antibodies and Assays). Briefly, 100 μL of anti-RBCs (primary antibody) at 156 μg/mL was added to 100 μL of 5 vol% washed RBCs in 1× PBS, along with 62.5 μL of RBC-ANS (250 μg, 100 μg, 50 μg, 25 μg, or 0 μg) and incubated for 45 min at 37 °C. The RBC solution was then washed three times by centrifuging the sample at 3,500 × g for 1 min and exchanging the supernatant with 1× PBS each time. One hundred microliters of anti-Fc (agglutinating secondary antibody) at 156 μg/mL was added to each sample, which was then placed in an analog vortex mixer (Fisher Scientific) at 625 rpm for 5 min and then spun down at 3,500 × g for 20 s. The sample was then resuspended using a pipette to disrupt the pellet. For the negative control, 100 μL of 6% (wt/vol) BSA was used in lieu of secondary antibody. All samples were then viewed via a light microscope at 10× magnification and imaged via mounted camera.

In Vivo Stability of RBC-ANS and Anti-RBC Binding.

Following induction of anemia via i.p. injection of anti-RBCs, we randomly assorted 12 CD-1 mice (Charles River Laboratories) into two groups of six. The treatment group received 500 μg of anti-RBCs incubated with 5 mg of RBC-ANS for 5 min at 37 °C before injection, and anti-RBC only mice received anti-RBCs incubated in 1× PBS for 5 min. A control group of mice received injections of PBS only. A few drops of blood were collected from each mouse before injections on day 0 to establish starting blood counts, and this procedure was repeated on each day of the experiment. Samples were stored in potassium-EDTA Microvette tubes (Sarstedt) and vigorously mixed to prevent clotting. Samples were then run on the same day using a Drew Scientific Hemavet 950 (Erba Diagnostics), and RBC count, hemoglobin level, and hematocrit were recorded daily.

In Vivo Neutralization of Circulating Anti-RBCs by RBC-ANS.

Using the established i.p. model for antibody delivery, 30 mice were randomized to three groups of 10 mice. Each group of mice received a 100-μL i.p. injection of 2 mg/mL anti-RBCs on days 0, 1, 2, and 3. The treatment group also received a tail vein i.v. injection of 200 μL of RBC-ANS (10 mg/mL) in 1× PBS within 30 min of i.p. antibody delivery. The PEG-NP group received an equivalent 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 were recorded on days 0, 1, 2, 3, and 4.

Anti-RBC Autoimmune Study.

Six weeks after the in vivo neutralization studies, serum was collected from 12 mice (six in each group) and a standard ELISA was performed. Washed CD-1 mouse RBCs were plated at 1 × 10⁶ RBCs per well onto a Costar 96 well plate. One hundred microliters of collected serum was added in a sequence of six 1:5 dilutions. HRP-conjugated goat anti-mouse antibody IgG (Biolegend) was used to probe for bound antibodies. The plate was developed using 3,3′,5,5′-tetramethylbenzidine substrate, and 1 M HCl was used to stop the reaction. Absorbance was measured at 450 nm.