University of Groningen
Magnetic Nanoparticles for the Control of Infectious Biofilms Quan, Kecheng
DOI:
10.33612/diss.170829667
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Publication date: 2021
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Quan, K. (2021). Magnetic Nanoparticles for the Control of Infectious Biofilms. University of Groningen. https://doi.org/10.33612/diss.170829667
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Summary
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Magnetic-targeting of antimicrobial nanoparticles is one of the strategies considered for treating biofilm-related infections. Magnetic-targeting is promising but also holds limitations that need to be solved or circumvented before magnetic-targeting can be used in clinical infection-control.
In Chapter 1.1, we reviewed possibilities and impossibilities of the application of magnetic-targeting for the control of infectious biofilms. Targeting of chemotherapeutics towards a tumor site by magnetic nanocarriers had already been considered promising in tumor-control. Since, magnetic nanoparticles are also considered for use in infection-control as a new means to prevent antimicrobial resistance from becoming the number one cause of death by the year 2050. To this end, magnetic nanoparticles can either be loaded with an antimicrobial for use as a delivery vehicle or modified to acquire intrinsic antimicrobial properties. Magnetic nanoparticles can also be used for the local generation of heat to kill infectious microorganisms. Although appealing for tumor- and infection-control, injection in the blood circulation may yield reticuloendothelial uptake and physical obstruction in organs that yield reduced targeting efficiency. This can be prevented with suitable surface modification. However, precise techniques to direct magnetic nanoparticles towards a target site are lacking. The problem of precise targeting is aggravated in infection-control due to the micrometer-size of infectious biofilms, as opposed to targeting of nanoparticles towards centimeter-sized tumors. This review aims to identify possibilities and impossibilities of magnetic targeting of nanoparticles for infection-control. We first review targeting techniques and the spatial resolution they can achieve as well as surface-chemical modifications of magnetic nanoparticles to enhance their targeting efficiency and antimicrobial efficacy. It is concluded that targeting problems encountered in tumor control using magnetic nanoparticles, are neglected in most studies on their potential application in infection-control. Currently biofilm targeting by smart, self-adaptive and pH-responsive, antimicrobial nanocarriers for instance, seems easier to achieve than magnetic targeting. This leads to the conclusion, that magnetic targeting of nanoparticles for the control of micrometer-sized infectious biofilms may be less promising than initially expected. However, using propulsion
Summary
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rather than precise targeting of magnetic nanoparticles in a magnetic field to traverse through infectious-biofilms can create artificial channels for enhanced antibiotic transport. This is identified as a more feasible, innovative application of magnetic nanoparticles in infection-control than precise targeting and distribution of magnetic nanoparticles over the depth of a biofilm.
Considering the possibilities of creating artificial channels in infectious biofilms for enhanced antibiotic transport, we performed a second review on the role of water-filled channels in biofilms (Chapter 1.2). InfraRed- (IR) and Nuclear Magnetic Resonance (NMR)-based techniques provide direct evidence of water presence in biofilms. Comparison of the weight of hydrated and dried biofilms have demonstrated that biofilms can comprise over 70 wt% water. Bacteria in biofilms are glued together in a matrix of self-produced, extracellular polymeric substances (EPS). Water in the biofilm matrix, i.e. outside bacterial cells, occurs as bound and free water. Bound water can be found adsorbed to bacteria and matrix components and possesses different IR and NMR signatures than free water. Microscopically observable, open structures in biofilms are assumed to be filled with free water. Bacteria are actively involved in the formation of water-filled structures, that can be transient due to blocking by bacterial growth, visco-elastic collapse or additional formation of EPS. Literature refers to water-filled structures in biofilms as “channels” and “pores”, without making a clear distinction between the two. Based on this review, we propose to distinguish channels and pores based on function and dimensions. Channels allow convective-diffusional transport of nutrients, waste-products and other molecules through a biofilm. In order to meet the requirements set to a channel as a means of transport, the cargo should not adsorb to the channel walls and the channel should have a large length/width ratio. Pores mainly serve to store e.g. nutrients and dilute waste-products or antimicrobials. As compared with channels, pores should thus have a length/width ratio of around unity. The understanding provided here on the occurrence, physico-chemical properties and role of water in biofilms, can be employed in industrial and environmental applications to optimize bioreactor yields as well as to enhance penetration of antimicrobials for eradication of biofilms, including pathogenic, infectious biofilms in the human body.
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From the first literature review (Chapter 1.1), it can be concluded that the difficulties involved in precise magnetic targeting of nanoparticles to a micrometer-sized infection-site are grossly underestimated. Therefore, the aim of this thesis was to investigate whether and how magnetic nanoparticles can be used to facilitate antimicrobial penetration using magnetic targeting for enhanced eradication of infectious biofilms, but without the need of precise targeting at the micrometer level.
In Chapter 2 we applied magnetic, antimicrobial-carrying nanoparticles to combat infectious bacterial biofilms, while directly targeting the nanoparticles to and distributing them over an infectious biofilm to yield optimal penetration and accumulation. Penetration and accumulation of antimicrobials over the thickness of a biofilm is a conditio sine qua non for effective killing of biofilm inhabitants. Simplified schematics on magnetic-targeting, always picture homogeneous distribution of magnetic, antimicrobial-carrying nanoparticles over the thickness of biofilms, but this is not easy to achieve. Here, gentamicin-carrying magnetic nanoparticles (MNPs-G) were synthesized through gentamicin-conjugation with iron-oxide nanoparticles and used to demonstrate the importance of their homogeneous distribution over the thickness of a biofilm. Diameters of MNPs-G were around 60 nm, well below the limit for reticulo-endothelial rejection. MNPs-G killed most ESKAPE-panel pathogens, including Escherichia coli, equally as well as gentamicin in solution. MNPs-G distribution in a Staphylococcus aureus biofilm was dependent on magnetic-field exposure time and most homogeneous after 5 min magnetic-field exposure. Exposure of biofilms to MNPs-G with 5 min magnetic-field exposure, not only yielded homogeneous distribution of MNPs-G, but concurrently better staphylococcal killing at all depths than MNPs, gentamicin in solution, and MNPs-G after other magnet-field exposure times. In summary, homogeneous distribution of gentamicin-carrying magnetic nanoparticles over the thickness of a staphylococcal biofilm was essential for killing biofilm inhabitants and required optimizing of the magnetic-field exposure time. This conclusion is important for further successful development of magnetic, antimicrobial-carrying nanoparticles towards clinical application.
Summary
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Since precise antimicrobial targeting of infectious biofilms by an applied magnetic field is hard to achieve, alternative ways to apply magnetic-targeting were explored. The poor penetrability of many biofilms contributes to the recalcitrance of infectious biofilms to antimicrobial treatment. In Chapter 3, we propose a new application for the use of magnetic nanoparticles in nanomedicine to create artificial channels in infectious biofilms to enhance antimicrobial penetration and bacterial killing that was inspired by the second review to the introduction of this thesis on water-filled channels and their function in a biofilm (Chapter 1.2). S. aureus biofilms were exposed to magnetic-iron-oxide nanoparticles (MIONPs), while magnetically forcing MIONP movement through the biofilm. Confocal Laser Scanning Microscopy demonstrated artificial channel digging perpendicular to the substratum surface. Artificial channel digging significantly (4-6 fold) enhanced biofilm penetration and bacterial killing efficacy by gentamicin in two S. aureus strains with and without the ability to produce extracellular polymeric substances. Herewith, this work provides a simple, new and easy way to enhance the eradication of infectious biofilms using MIONPs combined with clinically applied antibiotic therapies.
The impact of surface modification on MNPs and in vivo application of channel digging by magnetic-targeting were not addressed in Chapter 3. In particular, Chapter 3 left the question unanswered, whether interaction of magnetic nanoparticles with biofilm components impacts the efficacy of antibiotics after artificial channel digging. In Chapter 4, we functionalized MIONPs with polydopamine (PDA) to modify their interaction with staphylococcal pathogens and EPS and relate the interaction with in
vitro biofilm eradication by gentamicin after magnetic channel digging. PDA-modified
MIONPs had less negative zeta potentials than unmodified MIONPs due to the presence of amino groups and accordingly more interaction with negatively charged staphylococcal cell surfaces than unmodified MIONPs. Neither unmodified nor PDA-modified MIONPs interacted with EPS. Concurrently, use of non-interacting unmodified MIONPs for artificial channel digging in in vitro grown staphylococcal biofilms enhanced the efficacy of gentamicin more than the use of interacting, PDA-modified MIONPs. In vivo experiments in mice using a sub-cutaneous infection model
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confirmed that non-interacting, unmodified MIONPs enhanced eradication by gentamicin of S. aureus Xen36 biofilms about 10-fold. Combined with the high biocompatibility of magnetic nanoparticles, these results form an important step in understanding the mechanism of artificial channel digging in infectious biofilms for enhancing antibiotic efficacy in hard-to-treat infectious biofilms in patients.
Finally, a second alternative way to apply MNPs towards infection-control not requiring precise targeting, was explored. Biomaterial-associated infections constitute a special class of infection that can occur any time after surgical implantation of biomaterial implants and limits their success rates. On-demand, antimicrobial release coatings have been designed, but in vivo application of a release trigger is troublesome while low, inadvertent leakage can exhaust a coating. In Chapter 5, we apply a MNP coating to a biomaterial surface, that can be pulled-off in a magnetic field when an infectious biofilm has formed. MNPs remained stably adsorbed to a surface upon exposure to PBS for at least 50 days, did not promote bacterial adhesion or negatively affect interaction with tissue cells. Nanoparticles could be magnetically pulled-off a surface through an adhering biofilm, creating artificial water channels in the biofilm. At a MNP coating concentration of 0.64 mg cm-2, these artificial channels increased the
penetrability of S. aureus and Pseudomonas aeruginosa biofilms towards different antibiotics, yielding 10-fold more antibiotic killing of biofilm inhabitants than in absence of artificial channel digging. This innovative use of MNPs for the eradication of biomaterial-associated infections requires no precise targeting of MNPs and allows more effective use of existing antibiotics by breaking the penetration barrier of infectious biofilm upon demand after surgical implantation of a biomaterials implant.
In the General Discussion of this thesis (Chapter 6), their significance of magnetic channel digging for biofilm control is further discussed and questions left unanswered in this thesis are identified along with suggestions for future research.
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