title: Anti-PEG antibodies alter the mobility and biodistribution of densely PEGylated nanoparticles in mucus.
abstract: Antibodies that specifically bind polyethylene glycol (PEG) can lead to rapid elimination of PEGylated therapeutics from the systemic circulation. We have recently shown that virus-binding IgG can immobilize viruses in mucus via multiple low-affinity crosslinks between IgG and mucins. However, it remains unclear whether anti-PEG antibodies in mucus may also alter the penetration and consequently biodistribution of PEGylated nanoparticles delivered to mucosal surfaces. We found that both anti-PEG IgG and IgM can readily bind nanoparticles that were densely coated with PEG polymer to minimize adhesive interactions with mucus constituents. Addition of anti-PEG IgG and IgM into mouse cervicovaginal mucus resulted in extensive trapping of mucus-penetrating PEGylated nanoparticles, with the fraction of mobile particles reduced from over 95% to only 34% and 7% with anti-PEG IgG and IgM, respectively. Surprisingly, we did not observe significant agglutination induced by either antibody, suggesting that particle immobilization is caused by adhesive crosslinks between mucin fibers and IgG or IgM bound to individual nanoparticles. Importantly, addition of corresponding control antibodies did not slow the PEGylated nanoparticles, confirming anti-PEG antibodies specifically bound to and trapped the PEGylated nanoparticles. Finally, we showed that trapped PEGylated nanoparticles remained largely in the luminal mucus layer of the mouse vagina even when delivered in hypotonic formulations that caused untrapped particles to be drawn by the flow of water (advection) through mucus all the way to the epithelial surface. These results underscore the potential importance of elucidating mucosal anti-PEG immune responses for PEGylated therapeutics and biomaterials applied to mucosal surfaces.
  
  
  
  PEG, generally considered a 'stealth' polymer, is broadly used to improve the circulation times and therapeutic efficacy of nanomedicines. Nevertheless, there is increasing scientific evidence that demonstrates both animals and humans can generate PEG-specific antibodies. Here, we show that anti-PEG IgG and IgM can specifically immobilize otherwise freely diffusing PEG-coated nanoparticles in fresh vaginal mucus gel ex vivo by crosslinking nanoparticles to the mucin mesh, and consequently prevent PEG-coated nanoparticles from accessing the vaginal epithelium in vivo. Given the increasing use of PEG coatings to enhance nanoparticle penetration of mucosal barriers, our findings demonstrate that anti-PEG immunity may be a potential concern not only for systemic drug delivery but also for mucosal drug delivery.
authors.affiliation: UNC/NCSU Joint Department of Biomedical Engineering, University of North Carolina - Chapel Hill, 125 Mason Farm Road, Chapel Hill, NC 27599, USA.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Henry
authors.affiliation: Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: Ying-Ying
authors.initials: YY
authors.lastname: Wang
authors.affiliation: Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina - Chapel Hill, 125 Mason Farm Road, Chapel Hill, NC 27599, USA.
authors.forename: Qi
authors.initials: Q
authors.lastname: Yang
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287, USA.
authors.forename: Thuy
authors.initials: T
authors.lastname: Hoang
authors.affiliation: Center for Nanomedicine, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287, USA.
authors.forename: Sumon
authors.initials: S
authors.lastname: Chattopadhyay
authors.affiliation: Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: Timothy
authors.initials: T
authors.lastname: Hoen
authors.affiliation: Center for Nanomedicine, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287, USA; Department of Ophthalmology, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287, USA.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Ensign
authors.affiliation: UNC/NCSU Joint Department of Biomedical Engineering, University of North Carolina - Chapel Hill, 125 Mason Farm Road, Chapel Hill, NC 27599, USA.
authors.forename: Kenetta L
authors.initials: KL
authors.lastname: Nunn
authors.affiliation: Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina - Chapel Hill, 125 Mason Farm Road, Chapel Hill, NC 27599, USA.
authors.forename: Holly
authors.initials: H
authors.lastname: Schroeder
authors.affiliation: Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina - Chapel Hill, 125 Mason Farm Road, Chapel Hill, NC 27599, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: McCallen
authors.affiliation: ReProtect, Inc., 703 Stags Head Road, Baltimore, MD 21286, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Moench
authors.affiliation: Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Cone
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, No. 128, Section 2, Academia Road, Taipei 11529, Taiwan.
authors.forename: Steve R
authors.initials: SR
authors.lastname: Roffler
authors.affiliation: Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina - Chapel Hill, 125 Mason Farm Road, Chapel Hill, NC 27599, USA; UNC/NCSU Joint Department of Biomedical Engineering, University of North Carolina - Chapel Hill, 125 Mason Farm Road, Chapel Hill, NC 27599, USA; Department of Microbiology & Immunology, University of North Carolina - Chapel Hill, 125 Mason Farm Road, Chapel Hill, NC 27599, USA. Electronic address: lai@unc.edu.
authors.forename: Samuel K
authors.initials: SK
authors.lastname: Lai
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D000918:Antibody Specificity / Q000187:drug effects; D002584:Cervix Uteri / Q000378:metabolism; D004058:Diffusion; D005260:Female; D005629:Frozen Sections; D051379:Mice; D009093:Mucus / Q000378:metabolism*; D053758:Nanoparticles / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000378:metabolism*; D014018:Tissue Distribution / Q000187:drug effects; D014621:Vagina / Q000378:metabolism
chemical_list: D000906:Antibodies; D011092:Polyethylene Glycols
keywords: Diffusion; IgG; IgM; Mucosal immunity; Nanomedicine; PEGylation