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Biofilm-Infected Human Clusteroid Three-Dimensional Coculture Platform to Replace Animal Models in Testing Antimicrobial Nanotechnologies

Wang, Anheng; Weldrick, Paul; Madden, Leigh; Paunov, Vesselin N.


Anheng Wang

Paul Weldrick

Vesselin N. Paunov


Microbial biofilms are a major concern in wound care, implant devices, and organ infections. Biofilms allow higher tolerance to antibacterial drugs, can impair wound healing, and potentially lead to sepsis. There has been a recent focus on developing novel nanocarrier-based delivery vehicles to enhance the biofilm penetration of traditional antibacterial drugs. However, a feasible in vitro human skin model to mimic the biofilm formation and its treatment for clearance have not yet been reported. The study describes the benefits of using an innovative bacterial biofilm-infected keratinocyte clusteroid model for the first time. It paves a new way for testing innovative nanomedicine delivery systems in a rapid and reproducible way on a realistic human cell-based platform, free of any animal testing. Herein, we have developed a novel composite 3D biofilm/human keratinocyte clusteroid coculture platform, which was used to measure biofilm clearance efficiency of nanoparticle (NP)-based therapeutics. We tested this model by treating the biofilm-infected 3D coculture layers by a ciprofloxacin-loaded Carbopol nanogel, surface-functionalized by the cationic protease Alcalase. We measured the antibacterial efficiency of the NP treatment on clearing Staphylococcus aureus and Pseudomonas aeruginosa biofilms on the 3D keratinocyte clusteroid/biofilm coculture model. Our experiments showed that these bacteria can successfully infect the 3D layer of keratinocyte clusteroids and produce a stable biofilm. The biofilms were efficiently cleared by treatment with a formulation of 0.0032wt% ciprofloxacin-loadedin 0.2 wt % Carbopol NPs surface-functionalized with 0.2 wt % Alcalase. Taken together, these promising results demonstrate that our coculture model can be exploited as a novel platform for testing the biofilm-eliminating efficiency of various NP formulations emulating skin and wound infections and could have wider applicability to replace animal models in similar experiments. This 3D cell culture-based platform could help in developing more effective antibacterial agents for clinical applications of antiplaque dental treatments, implants, infection control, and wound dressings.


Wang, A., Weldrick, P., Madden, L., & Paunov, V. N. (in press). Biofilm-Infected Human Clusteroid Three-Dimensional Coculture Platform to Replace Animal Models in Testing Antimicrobial Nanotechnologies. ACS Applied Materials & Interfaces,

Journal Article Type Article
Acceptance Date Mar 25, 2021
Online Publication Date May 6, 2021
Deposit Date May 7, 2021
Publicly Available Date May 7, 2022
Journal ACS Applied Materials & Interfaces
Print ISSN 1944-8244
Electronic ISSN 1944-8252
Publisher American Chemical Society
Peer Reviewed Peer Reviewed
Keywords Biofilms; Antimicrobial agents; Nanogels; Bacteria; Layers
Public URL


Article (<nobr>13.6 Mb</nobr>)

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©2021 The authors. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder

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