Edited by: Diana Boraschi, National Research Council, Italy
Reviewed by: Janos Szebeni, University of Miskolc, Hungary; Yang Li, University of Colorado Denver, USA
†Present address: Swetha Inturi, Noven Pharmaceuticals, Miami, FL, USA
Specialty section: This article was submitted to Inflammation, a section of the journal Frontiers in Immunology
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Balancing surface functionalization and low immune recognition of nanomedicines is a major challenge. Opsonization with the third component of the complement protein (C3) plays a major role in immune cell recognition of nanomedicines. We used dextran-coated superparamagnetic iron oxide nanoworms (SPIO NWs) to study the effect of surface functionalization on C3 opsonization in mouse serum and subsequent macrophage/leukocyte recognition
The success of systemically and locally administered nanomaterials largely depends on the ability of nanosized carriers to efficiently evade the immune system (
Superparamagnetic iron oxide (SPIO) nanoparticles have been used as magnetic resonance imaging (MRI) contrast agents and also as carriers for drug delivery (
Interestingly, modifying the surface dextran coating with epichlorohydrin (ECH) [resulting in poly-(2-hydroxypropyl ether) hydrogel] blocked mouse complement C3 opsonization and leukocyte uptake (
Dextran-coated SPIO NWs were prepared from 20 kDa dextran, FeCl2, and FeCl3 by a modified Molday and MacKenzie precipitation protocol (
In order to confirm the role of the LP as the inciting pathway of complement activation in mouse serum, SPIO NWs were incubated in validated sera obtained from mice deficient for various complement pathways (Figure
Next, we compared the efficiency of C3 opsonization of CL-NWs, CL-NWs-PEG, and CL-NWs-PEG-Ab with SPIO NWs. Particles were incubated in normal mouse serum or in sera deficient for the LP factors MBL-A/C and MASP-2, washed, loaded in the same amount on a gel, and analyzed by Western blotting. All CL-NWs formulations showed 80–85% decrease in C3 opsonization in normal serum compared with SPIO NWs (Figures
C3 is the critical opsonin mediating the uptake of foreign pathogens by macrophages and leukocytes (
Previously, we demonstrated that complement C3 mediates the uptake of SPIO NWs by blood leukocytes in mice (
Surface immobilized Abs and immune complexes, however, are known to trigger complement
Next, we sought an explanation to the observed lack of complement activation by CL-NWs-PEG-Ab (160 IgG/particle) in serum (Figure
Our studies demonstrate that surface modifications of nanoparticles have profound effect on complement C3 opsonization and the resultant immune uptake. Previous studies using PEGylated liposomes and nanoparticles showed loss of stealth properties after tethering of Abs and ligands (
Importantly, our study suggests that complement activation
All reagents used for NW synthesis including Fe salts and 20 kDa (range 15–25 kDa) dextran were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture media were purchased from Corning Life Sciences (Corning, NY, USA). Copper grids (300 mesh) were purchased from Electron Microscopy Sciences (Hatfield, PA, USA). Anti-HER2 Ab Herceptin® was generously donated by the Pharmacy of the University of Colorado Cancer Center, Anschutz Medical Campus. Anti-mouse anti-C3 Ab was purchased from MP Biomedicals (Solon, OH, USA). IRDye 800CW-labeled secondary Abs were purchased from Li-COR Biosciences (Lincoln, NE, USA). Purified anti-mouse/human CD11b Ab was purchased from BioLegend (San Diego, CA, USA). Anti-mouse F4/80 Ab was purchased from Caltag Medsystems Ltd. (Buckingham, UK). Hoechst 33342 for nuclei staining was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Abs for each experiment were diluted according to the recommendations from manufacturers. Mouse sera deficient for C3, MBL-A/C, MBL-A/C/factor D, and factor B were collected from mice that were bred in an animal vivarium at the University of Colorado Anschutz Medical Campus according to the Institutional Animal Care and Use Committee (IACUC) approved breeding protocol. Recombinant human hirudin (lepirudin, catalog No. ACM154) was obtained from Aniara Diagnostica, LLC (West Chester, OH, USA), reconstituted in water to 1 μg/μL (160 antithrombin units/μL), and stored aliquoted at −80°C. Lepirudin anticoagulated plasma (hereafter plasma) was obtained by collecting blood through the cardiac puncture (final lepirudin concentration 3–4 μg/mL) and centrifuging the tube at 2,000
Nanoworms were synthesized by a modified one-pot Molday and MacKenzie (
CL-NWs were aminated by adding different concentrations of ammonium hydroxide at 4°C overnight to form CL-NWs-NH2 and dialyzed for 24 h to remove free NH4OH. For the Ab conjugation, MAL-PEG-SVA (MAL-PEG-SVA, Laysan Bio) was reacted at excess with CL-NWs-NH2 at RT for 30 min to form CL-NWs-PEG-MAL or CL-NWs-PEG. Anti-HER2 Ab Herceptin® was reacted in the last step with MAL-PEG to form CL-NWs-PEG-Ab. NWs were purified by ultracentrifugation at 55,000 rpm, filtered through a 0.45 μm pore filter, and finally, stored in Dulbecco’s phosphate-buffered saline (DPBS) at pH 7.4 before use.
Transmission electron microscopy imaging was conducted to visualize the NWs using FEI Tecnai Spirit BioTwin electron microscope (Electron Microscopy Facility at the University of Colorado Boulder) at a 100 kV working voltage. Size measurements were done in DPBS and zeta potential measurements were done in 0.1× DPBS at RT using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). The intensity-weighted size distribution peak value was used to report hydrodynamic diameters of the NWs. To quantify the Ab on the NWs, known amount of CL-NWs-PEG-Ab (0.2 μg Fe) was applied in triplicates onto a 0.2 μm pore nitrocellulose membrane (Bio-Rad). Standard dilutions of free trastuzumab were also applied onto the membrane to generate a standard curve. The membrane was blocked with 5% (w/w) non-fat dry milk in PBS-T (DPBS with 0.1% Tween® 20) for 1 h at RT and probed with IRDye 800CW-labeled antihuman Ab. The membrane was scanned with Odyssey infrared imager (Li-COR Biosciences, Lincoln, NE, USA). The integrated dot intensity in the scanned images was determined from 16-bit grayscale images using ImageJ software and plotted using Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA) to determine the number of Ab molecules per spot using the calibration curve. Concentration of particles per milligram Fe was determined with NanoSight (Malvern Instruments) and Fe concentration was determined with ferrozine iron assay as described before (
Superparamagnetic iron oxide and CL-NWs (10 μL of 1 mg/mL) were incubated with 30 μL of mouse serum or lepirudin plasma for 15 min at RT. At the end of incubation, particles were washed three times with 1× PBS by centrifugation at 100,000
Human breast cancer cell line SKBR-3 cells were maintained in McCoy’s 5A medium (ATCC) supplemented with 10% fetal bovine serum. Mouse peritoneal macrophages were obtained by peritoneal lavage with 5 mL ice cold PBS, post-mortem. For experiment, cells were plated into 96-well plate. For uptake experiments, NWs were preincubated for 15 m with WT mouse sera or C3 KO sera and added at 0.1 mg/mL Fe concentration to cells for 6 h. After the incubation, cells were washed using DPBS for three times, fixed in 4% paraformaldehyde at 4°C overnight, and then stained using Prussian blue for 1 h. Prussian blue staining is the standard method for detecting iron in cells and tissues, and has been extensively used for detecting uptake of iron oxide nanoparticles by our group and others (
Wild type and
The statistical analysis was performed using Prism 6 software. The differences between means of experimental groups were analyzed using a two-tailed parametric
This study was carried out in accordance with the guidelines of the University of Colorado Office of Animal Care. The protocol was approved by the University of Colorado IACUC.
GW designed and performed experiments, and analyzed data. JG performed experiments. SI performed experiments. BB performed experiments. NB analyzed data and provided reagents. VH analyzed data and provided reagents. SM designed experiments, analyzed data, and edited the paper. DS designed experiments, analyzed data, and wrote the paper.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer YL declared a shared affiliation, though no other collaboration, with several of the authors to the handling Editor, who ensured that the process nevertheless met the standards of a fair and objective review.
The study was funded by the University of Colorado Denver startup and NIH grants EB022040 and CA194058-01A1 to DS, and the Danish Agency for Science, Technology and Innovation (Det Strategiske Forskningsråd), reference 09-065746 as well as RiboBio Co. Ltd. (Guangzhou, China) to SM.
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