Optimized Enzyme-Linked Immunosorbent Assay for Anti-PEG Antibody Detection in Healthy Donors and Patients Treated with PEGylated Liposomal Doxorubicin.

There is considerable interest in quantifying anti-PEG antibodies, given their potential involvement in accelerated clearance, complement activation, neutralization, and acute reactions associated with drug delivery systems. Published and commercially available anti-PEG enzyme-linked immunosorbent assays (ELISAs) differ significantly in terms of reagents and conditions, which could be confusing to users who want to perform in-house measurements. Here, we optimize the ELISA protocol for specific detection of anti-PEG IgG and IgM in sera from healthy donors and in plasma from cancer patients administered with PEGylated liposomal doxorubicin. The criterion of specificity is the ability of free PEG or PEGylated liposomes to inhibit the ELISA signals. We found that coating high-binding plates with monoamine methoxy-PEG5000, as opposed to bovine serum albumin-PEG20000, and blocking with 1% milk, as opposed to albumin or lysozyme, significantly improve the specificity, with over 95% of the signal being blocked by competition. Despite inherent between-assay variability, setting the cutoff value of the optical density at the 80th percentile consistently identified the same subjects. Using the optimized assay, we longitudinally measured levels of anti-PEG IgG/IgM in cancer patients before and after the PEGylated liposomal doxorubicin chemotherapy cycle (1 month apart, three cycles total). Antibody titers did not show any increase but rather a decrease between treatment cycles, and up to 90% of antibodies was bound to the infused drug. This report is a step toward harmonizing anti-PEG assays in human subjects, emphasizing the cost-effectiveness and optimized specificity.

Highly aminated iron oxide nanoworms for simultaneous manufacturing and labeling of chimeric antigen receptor T cells.

Cell based therapies including chimeric antigen receptor (CAR) T cells are promising for treating leukemias and solid cancers. At the same time, there is interest in enhancing the functionality of these cells via surface decoration with nanoparticles (backpacking). Magnetic nanoparticle cell labeling is of particular interest due to opportunities for magnetic separation, in vivo manipulation, drug delivery and magnetic resonance imaging (MRI). While modification of T cells with magnetic nanoparticles (MNPs) was explored before, we questioned whether MNPs are compatible with CAR-T cells when introduced during the manufacturing process. We chose highly aminated 120 nm crosslinked iron oxide nanoworms (CLIO NWs, ~36,000 amines per NW) that could efficiently label different adherent cell lines and we used CD123 CAR-T cells as the labeling model. The CD123 CAR-T cells were produced in the presence of CLIO NWs, CLIO NWs plus protamine sulfate (PS), or PS only. The transduction efficiency of lentiviral CD123 CAR with only NWs was ~23% lower than NW+PS and PS groups (~33% and 35%, respectively). The cell viability from these three transduction conditions was not reduced within CAR-T cell groups, though lower compared to non-transduced T cells (mock T). Use of CLIO NWs instead of, or together with cationic protamine sulfate for enhancement of lentiviral transduction resulted in comparable levels of CAR expression and viability but decreased the proportion of CD8+ cells and increased the proportion of CD4+ cells. CD123 CAR-T transduced in the presence of CLIO NWs, CLIO NWs plus PS, or PS only, showed similar level of cytotoxicity against leukemic cell lines. Furthermore, fluorescence microscopy imaging demonstrated that CD123 CAR-T cells labeled with CLIO NW formed rosettes with CD123+ leukemic cells as the non-labeled CAR-T cells, indicating that the CAR-T targeting to tumor cells has maintained after CLIO NW labeling. The in vivo trafficking of the NW labeled CAR-T cells showed the accumulation of CAR-T labeled with NWs primarily in the bone marrow and spleen. CAR-T cells can be magnetically labeled during their production while maintaining functionality using the positively charged iron oxide NWs, which enable the in vivo biodistribution and tracking of CAR-T cells.

Complement Inhibitors Block Complement C3 Opsonization and Improve Targeting Selectivity of Nanoparticles in Blood.

Complement is one of the critical branches of innate immunity that determines the recognition of engineered nanoparticles by immune cells. Antibody-targeted iron oxide nanoparticles are a popular platform for magnetic separations, in vitro diagnostics, and molecular imaging. We used 60 nm cross-linked iron oxide nanoworms (CLIO NWs) modified with antibodies against Her2/neu and EpCAM, which are common markers of blood-borne cancer cells, to understand the role of complement in the selectivity of targeting of tumor cells in whole blood. CLIO NWs showed highly efficient targeting and magnetic isolation of tumor cells spiked in lepirudin-anticoagulated blood, but specificity was low due to high uptake by neutrophils, monocytes, and lymphocytes. Complement C3 opsonization in plasma was predominantly via the alternative pathway regardless of the presence of antibody, PEG, or fluorescent tag, but was higher for antibody-conjugated CLIO NWs. Addition of various soluble inhibitors of complement convertase (compstatin, soluble CD35, and soluble CD55) to whole human blood blocked up to 99% of the uptake of targeted CLIO NWs by leukocytes, which resulted in a more selective magnetic isolation of tumor cells. Using well-characterized nanomaterials, we demonstrate here that complement therapeutics can be used to improve targeting selectivity.

The Interplay Between Blood Proteins, Complement, and Macrophages on Nanomedicine Performance and Responses.

In the blood, depending on their physicochemical characteristics, nanoparticles attract a wide range of plasma biomolecules. The majority of blood biomolecules bind nonspecifically to nanoparticles. On the other hand, biomolecules such as pattern-recognition complement-sensing proteins may recognize some structural determinants of the pristine surface, causing complement activation. Adsorption of nonspecific blood proteins could also recruit natural antibodies and initiate complement activation, and this seems to be a global process with many preclinical and clinical nanomedicines. We discuss these issues, since complement activation has ramifications in nanomedicine stability and pharmacokinetics, as well as in inflammation and disease progression. Some studies have also predicted a role for complement systems in infusion-related reactions, whereas others show a direct role for macrophages and other immune cells independent of complement activation. We comment on these discrepancies and suggest directions for exploring the underlying mechanisms.

Clickable Methyltetrazine-Indocarbocyanine Lipids: A Multicolor Tool Kit for Efficient Modifications of Cell Membranes.

Cell-based therapeutics are one of the most promising and exciting breakthroughs in modern medicine. Modification of the cell surface with ligands, biologics, drugs, and nanoparticles can further enhance the functionality. Previously, we described the synthesis of a dioctadecyl indocarbocyanine Cy3 analog (aminomethyl-DiI) for efficient and stable modification (painting) of mouse erythrocytes with small molecules, enzymes, and biologics. Here, we synthesized a near-infrared aminomethyl dioctadecyl derivative of Cy7 (aminomethyl-DOCy7) and systematically compared it to aminomethyl-DiI as an anchor for the modification of human erythrocytes, Jurkat cells, and primary T cells with immunoglobulin G. To enable copper-free click chemistry modification of cell membranes, we conjugated a methyltetrazine (MTz) group to the amino-indocyanine lipids via a polyethylene glycol (PEG) linker. DOCy7-PEG3400-MTz showed over 99% modification efficiency of human red blood cells (RBCs) at 25 µM. Reaction of trans-cyclooctene (TCO) modified immunoglobulin G (IgG) with DOCy7-PEG4-MTz-modified RBCs (2-step method) resulted in ∼80,000 IgG molecules per erythrocyte, whereas modification with a preconjugated DOCy7-PEG3400-IgG construct (1-step method) resulted in ∼20,000 IgG molecules per erythrocyte as detected by immuno dot-blot. The number of IgG/RBC was controlled by the concentration of IgG. The incubation of RBCs with DiI-PEG3400-MTz resulted in a similar number of IgG/RBC. Modification of the T-lymphocyte cell line Jurkat with IgG resulted in ∼1 × 106 IgG/cell with the 1-step and 2-step methods, and the efficiency was similar for DOCy7 and DiI constructs. Finally, we used DOCy7 and DiI constructs to demonstrate efficient modification of primary CD3+T cells from healthy donors. In conclusion, click indocarbocyanine conjugates represent a novel multicolor chemical biology tool kit for efficient surface modification of different cells types and can be used for potential imaging and drug delivery applications involving engineered cells.

Feraheme (Ferumoxytol) Is Recognized by Proinflammatory and Anti-inflammatory Macrophages via Scavenger Receptor Type AI/II.

Feraheme (ferumoxytol), a negatively charged, carboxymethyl dextran-coated ultrasmall superparamagnetic iron oxide nanoparticle (USPIO, 30 nm, -16 mV), is clinically approved as an iron supplement and is used off-label for magnetic resonance imaging (MRI) of macrophage-rich lesions, but the mechanism of recognition is not known. We investigated mechanisms of uptake of Feraheme by various types of macrophages in vitro and in vivo. The uptake by mouse peritoneal macrophages was not inhibited in complement-deficient serum. In contrast, the uptake of larger and less charged SPIO nanoworms (60 nm, -5 mV; 120 nm, -5 mV, respectively) was completely inhibited in complement deficient serum, which could be attributed to more C3 molecules bound per nanoparticle than Feraheme. The uptake of Feraheme in vitro was blocked by scavenger receptor (SR) inhibitor polyinosinic acid (PIA) and by antibody against scavenger receptor type A I/II (SR-AI/II). Antibodies against other SRs including MARCO, CD14, SR-BI, and CD11b had no effect on Feraheme uptake. Intraperitoneally administered PIA inhibited the peritoneal macrophage uptake of Feraheme in vivo. Nonmacrophage cells transfected with SR-AI plasmid efficiently internalized Feraheme but not noncharged ultrasmall SPIO of the same size (26 nm, -6 mV), suggesting that the anionic carboxymethyl groups of Feraheme are responsible for the SR-AI recognition. The uptake by nondifferentiated bone marrow derived macrophages (BMDM) and by BMDM differentiated into M1 (proinflammatory) and M2 (anti-inflammatory) types was efficiently inhibited by PIA and anti-SR-AI/II antibody. Interestingly, all BMDM types expressed similar levels of SR-AI/II. In conclusion, Feraheme is efficiently recognized via SR-AI/II but not via complement by different macrophage types. The recognition by the common phagocytic receptor has implications for specificity of imaging of macrophage subtypes.

Evaluation of Targeting Efficiency of Joints with Anticollagen II Antibodies.

Diseases of the joints affect over 10% of the world's population, resulting in significant morbidity. There is an unmet need in strategies for specific delivery of therapeutics to the joints. Collagen type II is synthesized by chondrocytes and is mainly restricted to the cartilage and tendons. Arthrogen-CIA is a commercially available anticollagen II antibody cocktail that reacts with 5 different epitopes on human, bovine, and mouse collagen II. Arthrogen has been used for induction of experimental rheumatoid arthritis (RA) in mice because of high complement activation on the cartilage surface. Native collagen II might serve as a useful target for potential delivery of therapeutics to the joint. To evaluate the efficiency and specificity of targeting collagen II, Arthrogen was labeled with near-infrared (NIR) dye IRDye 800 or IRDye 680. Using ex vivo NIR imaging, we demonstrate that Arthrogen efficiently and specifically accumulated in the limb joints regardless of the label dye or injection route (intravenous and subcutaneous). After subcutaneous injection, the mean fluorescence of the hind limb joints was 19 times higher than that of the heart, 8.7 times higher than that of the liver, and 3.7 times higher than that of the kidney. Control mouse IgG did not show appreciable accumulation. Microscopically, the antibody accumulated on the cartilage surface of joints and on endosteal surfaces. A monoclonal antibody against a single epitope of collagen II showed similar binding affinity and elimination half-life, but about three times lower targeting efficiency than Arthrogen in vitro and ex vivo, and about two times lower targeting efficiency in vivo. We suggest that an antibody against multiple epitopes of collagen II could be developed into a highly effective and specific targeting strategy for diseases of the joints or spine.

Variability of Complement Response toward Preclinical and Clinical Nanocarriers in the General Population.

Opsonization (coating) of nanoparticles with complement C3 component is an important mechanism that triggers immune clearance and downstream anaphylactic and proinflammatory responses. The variability of complement C3 binding to nanoparticles in the general population has not been studied. We examined complement C3 binding to dextran superparamagnetic iron oxide nanoparticles (superparamagnetic iron oxide nanoworms, SPIO NWs, 58 and 110 nm) and clinically approved nanoparticles (carboxymethyl dextran iron oxide ferumoxytol (Feraheme, 28 nm), highly PEGylated liposomal doxorubicin (LipoDox, 88 nm), and minimally PEGylated liposomal irinotecan (Onivyde, 120 nm)) in sera from healthy human individuals. SPIO NWs had the highest variation in C3 binding (n = 47) between subjects, with a 15-30 fold range in levels of C3. LipoDox (n = 12) and Feraheme (n = 18) had the lowest levels of variation between subjects (an approximately 1.5-fold range), whereas Onivyde (n = 18) had intermediate between-subject variation (2-fold range). There was no statistical difference between males and females and no correlation with age. There was a significant correlation in complement response between small and large SPIO NWs, which are similar structurally and chemically, but the correlations between SPIO NWs and other types of nanoparticles, and between LipoDox and Onivyde, were not significant. The calculated average number of C3 molecules bound per nanoparticle correlated with the hydrodynamic diameter but was decreased in LipoDox, likely due to the PEG coating. The conclusions of this study are (1) all nanoparticles show variability of C3 opsonization in the general population; (2) an individual's response toward one nanoparticle cannot be reliably predicted based on another nanoparticle; and (3) the average number of C3 molecules per nanoparticle depends on size and surface coating. These results provide new strategies to improve nanomedicine safety.

Cell-penetrating peptide CGKRK mediates efficient and widespread targeting of bladder mucosa following focal injury.

The bladder presents an attractive target for topical drug delivery. The barrier function of the bladder mucosa (urothelium) presents a penetration challenge for small molecules and nanoparticles. We found that focal mechanical injury of the urothelium greatly enhances the binding and penetration of intravesically-administered cell-penetrating peptide CGKRK (Cys-Gly-Lys-Arg-Lys). Notably, the CGKRK bound to the entire urothelium, and the peptide was able to penetrate into the muscular layer. This phenomenon was not dependent on intravesical bleeding and was not caused by an inflammatory response. CGKRK also efficiently penetrated the urothelium after disruption of the mucosa with ethanol, suggesting that loss of barrier function is a prerequisite for widespread binding and penetration. We further demonstrate that the ability of CGKRK to efficiently bind and penetrate the urothelium can be applied toward mucosal targeting of CGKRK-conjugated nanogels to enable efficient and widespread delivery of a model payload (rhodamine) to the bladder mucosa.

Dual-porosity hollow nanoparticles for the immunoprotection and delivery of nonhuman enzymes.

Although enzymes of nonhuman origin have been studied for a variety of therapeutic and diagnostic applications, their use has been limited by the immune responses generated against them. The described dual-porosity hollow nanoparticle platform obviates immune attack on nonhuman enzymes paving the way to in vivo applications including enzyme-prodrug therapies and enzymatic depletion of tumor nutrients. This platform is manufactured with a versatile, scalable, and robust fabrication method. It efficiently encapsulates macromolecular cargos filled through mesopores into a hollow interior, shielding them from antibodies and proteases once the mesopores are sealed with nanoporous material. The nanoporous shell allows small molecule diffusion allowing interaction with the large macromolecular payload in the hollow center. The approach has been validated in vivo using l-asparaginase to achieve l-asparagine depletion in the presence of neutralizing antibodies.

Binding and isolation of tumor cells in biological media with perfluorocarbon microbubbles.

With the emerging interest in personalized medicine, there is strong demand for new technologies for clinical sample interrogation. Exfoliated tumor cells in variety of pathological samples (e.g., blood, bone marrow, urine) could provide invaluable information for diagnosis and prognosis of cancers. Here we describe a detailed method for capture and isolation of tumor cells in medium, blood, or large issue buffy coat using EpCAM-targeted buoyant microbubbles (MBs). Perflorohexane gas lipid shell MBs were prepared with emulsification method and conjugated with antibody as described by us before [25]. The binding of EpCAM-targeted MBs to A549 (human lung carcinoma) and 4T1 (mouse breast carcinoma) cells spiked into BSA/PBS or blood was more than 90%, which was comparable with commercial anti-EpCAM immunomagnetic beads (DynaBeads). Anti-EpCAM MBs efficiently (75-82%) isolated BxPC3 pancreatic tumor cells spiked into medium, blood or a buffy coat, within 15-30 min of incubation. We discuss MB parameters and experimental conditions critical to achieve efficient cells binding and isolation. In conclusion, MB-assisted cell isolation is a promising method for rapid enrichment of cells and biomarkers from biological samples.

Mechanisms of complement activation by dextran-coated superparamagnetic iron oxide (SPIO) nanoworms in mouse versus human serum.

BACKGROUND: The complement system is a key component of innate immunity implicated in the neutralization and clearance of invading pathogens. Dextran coated superparamagnetic iron oxide (SPIO) nanoparticle is a promising magnetic resonance imaging (MRI) contrast agent. However, dextran SPIO has been associated with significant number of complement-related side effects in patients and some agents have been discontinued from clinical use (e.g., Feridex™). In order to improve the safety of these materials, the mechanisms of complement activation by dextran-coated SPIO and the differences between mice and humans need to be fully understood. METHODS: 20 kDa dextran coated SPIO nanoworms (SPIO NW) were synthesized using Molday precipitation procedure. In vitro measurements of C3 deposition on SPIO NW using sera genetically deficient for various components of the classical pathway (CP), lectin pathway (LP) or alternative pathway (AP) components were used to study mechanisms of mouse complement activation. In vitro measurements of fluid phase markers of complement activation C4d and Bb and the terminal pathway marker SC5b-C9 in normal and genetically deficient sera were used to study the mechanisms of human complement activation. Mouse data were analyzed by non-paired t-test, human data were analyzed by ANOVA followed by multiple comparisons with Student-Newman-Keuls test. RESULTS: In mouse sera, SPIO NW triggered the complement activation via the LP, whereas the AP contributes via the amplification loop. No involvement of the CP was observed. In human sera the LP together with the direct enhancement of the AP turnover was responsible for the complement activation. In two samples out of six healthy donors there was also a binding of anti-dextran antibodies and C1q, suggesting activation via the CP, but that did not affect the total level of C3 deposition on the particles. CONCLUSIONS: There were important differences and similarities in the complement activation by SPIO NW in mouse versus human sera. Understanding the mechanisms of immune recognition of nanoparticles in mouse and human systems has important preclinical and clinical implications and could help design more efficient and safe nano-formulations.

Complement-dependent uptake of nanoparticles by blood phagocytes: brief overview and perspective.

Immune recognition and uptake of nanoparticles remain the hot topic in nanomedicine research. Complement is the central player in the immune recognition of engineered nanoparticles. Here, we summarize the accumulated knowledge on the role of complement in the interactions of nanomaterials with blood phagocytes. We describe the interplay between surface properties, complement opsonization, and immune uptake, primarily of iron oxide nanoparticles. We discuss the rigor of the published research and further identify the following knowledge gaps: 1) the role of complement in the variability of uptake of nanomaterials in healthy and diseased subjects, and 2) modulation of complement interactions to improve the performance of nanomaterials. Addressing these gaps is critical to improving translational chances of nanomaterials for drug delivery and imaging applications.

Validation of dot blot immunoassay for measurement of complement opsonization of nanoparticles.

Complement plays a critical role in the immune response toward nanomaterials. The complement attack on a foreign surface results in the deposition of C3, assembly of C3 convertases, the release of anaphylatoxins C3a and C5a, and finally, the formation of membrane attack complex C5b-9. Various technologies can measure complement activation markers in the fluid phase, but measurements of surface C3 deposition are less common. Previously, we developed an ultracentrifugation-based dot blot immunoassay (DBI) to measure the deposition of C3 and other protein corona components on nanoparticles. Here, we validate the repeatability of the DBI and its correlation with pathway-specific and common fluid phase markers. Moreover, we discuss the advantages of DBI, such as cost-effectiveness and versatility, while addressing potential limitations. This study provides insights into complement activation at the nanosurface level, offering a valuable tool for nanomedicine researchers in the field.

Inhibition of acute complement responses towards bolus-injected nanoparticles using targeted short-circulating regulatory proteins.

Effective inhibition of the complement system is needed to prevent the accelerated clearance of nanomaterials by complement cascade and inflammatory responses. Here we show that a fusion construct consisting of human complement receptor 2 (CR2) (which recognizes nanosurface-deposited complement 3 (C3)) and complement receptor 1 (CR1) (which blocks C3 convertases) inhibits complement activation with picomolar to low nanomolar efficacy on many types of nanomaterial. We demonstrate that only a small percentage of nanoparticles are randomly opsonized with C3 both in vitro and in vivo, and CR2-CR1 immediately homes in on this subpopulation. Despite rapid in vivo clearance, the co-injection of CR2-CR1 in rats, or its mouse orthologue CR2-Crry in mice, with superparamagnetic iron oxide nanoparticles nearly completely blocks complement opsonization and unwanted granulocyte/monocyte uptake. Furthermore, the inhibitor completely prevents lethargy caused by bolus-injected nanoparticles, without inducing long-lasting complement suppression. These findings suggest the potential of the targeted complement regulators for clinical evaluation.

Nanoparticle-Binding Immunoglobulins Predict Variable Complement Responses in Healthy and Diseased Cohorts.

Systemic administration of nanomedicines results in the activation of the complement cascade, promoting phagocytic uptake and triggering proinflammatory responses. Identifying the biomarkers that can predict the "risk" of abnormally high complement responders can improve the safety and efficacy of nanomedicines. Polyethylene glycol (PEG) and dextran are two types of clinically approved polymer coatings that trigger complement activation. We performed a multifaceted analysis of the factors affecting the complement activation by PEGylated liposomal doxorubicin (PLD) and dextran-coated superparamagnetic iron oxide nanoworms (SPIO NWs) in plasma from patients with different inflammatory disease conditions and healthy donors. The complement activation (measured as deposition of the complement protein C3) varied greatly, with 29-fold and 26-fold differences for PLD and SPIO NWs, respectively. Chronic inflammation, acute infection, use of steroids, and sex had minor effects on the variable complement activation, whereas age inversely correlated with the complement activation. C-reactive protein level was not predictive of high (top 20th percentile) complement responses. Plasma concentrations of the main complement factors, as well as total IgG and IgM, showed no correlation with the activation by either nanoparticle. On the other hand, plasma concentrations of anti-PEG IgG and IgM showed a strong positive correlation with the activation by PLD. Particularly, titers of anti-PEG IgM showed the best predictive value for the "risk" of high complement activation by PLD. Titers of antidextran IgG and IgM showed a lower correlation with the activation by SPIO NWs and poor predictive value of the top 20% complement responses. Nanoparticle-bound immunoglobulins showed the best correlation with complement activation and a strong predictive value, supporting the critical role of immunoglobulins in inciting complement. The opsonization of PLD with C3 in plasma with high anti-PEG antibodies was predominantly via the alternative pathway. Characterizing the nature of nanoparticle-binding antibodies has important implications in mitigating and stratifying nanomedicine safety.