University of Groningen Adaptive antimicrobial nanocarriers for the control of infectious biofilms Liu, Yong

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(1)University of Groningen. Adaptive antimicrobial nanocarriers for the control of infectious biofilms Liu, Yong. IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.. Document Version Publisher's PDF, also known as Version of record. Publication date: 2019 Link to publication in University of Groningen/UMCG research database. Citation for published version (APA): Liu, Y. (2019). Adaptive antimicrobial nanocarriers for the control of infectious biofilms. University of Groningen.. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.. Download date: 27-06-2021.

(2) Adaptive Antimicrobial Nanocarriers for the Control of Infectious Biofilms Yong Liu.

(3) Adaptive Antimicrobial Nanocarriers for the Control of Infectious Biofilms By Yong Liu University Medical Center Groningen, University of Groningen Groningen, The Netherlands. Copyright © 2019 by Yong Liu Cover designed by Yong Liu Printed by IPSKAMP printing ISBN (printed version) 978-94-034-1607-6 ISBN (electronic version) 978-94-034-1606-9.

(4) Adaptive Antimicrobial Nanocarriers for the Control of Infectious Biofilms PhD thesis. to obtain the degree of PhD at the University of Groningen on the authority of the Rector Magnificus Prof. E. Sterken and in accordance with the decision by the College of Deans. This thesis will be defended in public on Monday 27 May 2019, at 12.45 hours. by. Yong Liu born on 17 June 1987 in Sichuan, China.

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(6) Supervisors Prof. H.J. Busscher Prof. H.C. van der Mei Prof. Y. Ren Prof. Linqi Shi. Assessment Committee Prof. W.J. Quax Prof. P.H. Elsinga Prof. H. Chen.

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(8) Paranimfen:. Damla Keskin Lu Yuan.

(9) To my dearest family!.

(10) Contents Introduction Nanotechnology‐Based Antimicrobials and Delivery Systems for Biofilm‐ Infection Control (Chem. Soc. Rev. 2019, 48, 428–446. IF: 40.182). 1. General Aim of This Thesis. 23. Chapter 2. Surface-Adaptive, Antimicrobially Loaded, Micellar Nanocarriers with Enhanced Penetration and Killing Efficiency in Staphylococcal Biofilms (ACS Nano 2016, 10, 4779−4789. IF: 13.709). 27. Chapter 3. Eradication of Multidrug-Resistant Staphylococcal Infections by LightActivatable Micellar Nanocarriers in a Murine Model (Adv. Funct. Mater. 2017, 27, 1701974. IF: 13.325). 47. Chapter 4. Nanocarriers with Conjugated Antimicrobials to Eradicate Pathogenic Biofilms Evaluated in Murine In Vivo and Human Ex Vivo Infection Models (Acta Biomater. 2018, 79, 331–343. IF: 6.383). 71. Chapter 5. Dual-antimicrobial Conjugates in Leukocyte-like Nanocarriers to Eradicate Intra-macrophageal Staphylococci (to be submitted). 103. Chapter 6. General discussion: Perspectives on and Need to Develop New Infection Control Strategies (submitted for publication in: Advances in antimicrobial and osteoinductive studies, Bingyun Li, Thomas Fintan Moriarty, Thomas Webster, Malcolm Xing (eds.), Springer International Publishing AG). 131. Summary. 143. Samenvatting. 147. Acknowledgements. 153. Chapter 1.

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(12) Chapter 1. 1. CHAPTER 1 General Introduction and Aim of This Thesis. Liu, Y.; Shi, L.; Su, L.; van der Mei, H. C.; Jutte, P. C.; Ren, Y.; Busscher, H. J. Nanotechnology-Based Antimicrobials and Delivery Systems for Biofilm-Infection Control. Chem. Soc. Rev. 2019. 48, 428–446. Reproduced with permission from Royal Society of Chemistry. 1.

(13) General Introduction and Aim of This Thesis. 1. 2.

(14) Chapter 1. 1. Nanotechnology-based antimicrobials and delivery systems for biofilminfection control Since the existence of human life, bacterial infections have posed a challenge to life. Over the past centuries, new ways to control human infections have been welcomed with great enthusiasm and subsequently been put on hold for various reasons. In old Chinese, Roman and Egyptian medicine, infections were treated with herbs, copper and mercury salts. Such treatments were little efficacious and not without side effects. In the late eighteenth century, hope was focused upon treating infections with probiotics, “health-promoting” bacteria, and in 1908 the Nobel prize for medicine was awarded to Elie Metchnikoff for his groundbreaking work on the application of lactic acid bacteria to enhance health and longevity in Bulgarian peasant populations. The development of probiotics was arrested however, because probiotics could by far not match the efficacy of penicillin, incidentally discovered by Alexander Flemming in 1923, another Nobel laureate in the field of infection control. Many new antibiotics have since been developed and for several decades antibiotics have been considered the final solution in the fight against biofilm-associated bacterial infections occurring across the human body (Figure 1).1 However overuse, not only with respect to the prevention and treatment of human infection but also in veterinary medicine and farming, have led to acquisition of mutations causing development of bacterial strains and species that are intrinsically resistant to all known antibiotics. New antibiotics are only sparsely developed because of the high costs involved and regulatory scrutiny, while at the same time it can be questioned how long it will take before bacteria adapt themselves to new antibiotics and become resistant.2 The problem of “intrinsic antimicrobial-resistance” (Figure 2A) is not the only problem however, that hampers infection control. Already in 1684, Van Leeuwenhoek used his own invented microscopes to describe that the vinegar with which he rinsed his teeth, killed only “those animals that are on the outside of the scurf”.3 This, over three hundred years old expression, relates to the second, still unsolved problem in the control of human bacterial infection: the poor penetration of an antimicrobial into what we now call “an infectious biofilm” (see Figure 2B). By far, most infections are caused by bacteria in their adhering, biofilm-mode of growth.2 Adhesion of bacteria, be it to the surface of other bacteria, bone or teeth or the surface of biomaterials implants and devices, stimulates bacteria to develop “emergent” properties, other than the properties they possess when “planktonically” suspended in a fluid phase. Bacteria respond to their adhering state by switching on a high number of genes that provide them with new, emergent properties, as summarized in Figure 3 and actually creating an organism fully incomparable with its planktonic counterpart. Whereas on the one hand, this may seem to invalidate all planktonic research, the ease at which planktonic evaluations can be done as compared to biofilm research, makes planktonic evaluations of new antimicrobials a useful screening tool because absence of planktonic efficacy, generally implies absence of antimicrobial efficacy against tougher to kill bacteria in a biofilm-mode of growth. The trigger for developing emergent properties in adhering bacteria is thought to come from nanoscopic cell wall deformation under the influence of the adhesion forces exrted upon an adhering bacterium by the surface to which it adheres to form an infectious biofilm on.4 The most notable emergent property is the ability of adhering bacteria to embed themselves in a selfproduced matrix of extracellular polymeric substances (EPS), composed of proteins, polysaccharides, humic acids and eDNA. EPS acts as a glue holding biofilm-bacteria together and protecting them against the host immune system and environmental challenges.1,2 Although the EPS-matrix contains water-filled channels required for the exchange of nutrients and metabolic waste-products with their environment, most antimicrobials have difficulty penetrating EPS, either through size limitations or adsorption to the EPS-matrix or to bacterial cell surfaces that can be highly negatively charged at physiological pH, but becoming more positively charged with decreasing pH inside a biofilm (Figure 2B).5 Whereas we now know this is the reason. 3. 1.

(15) General Introduction and Aim of This Thesis. A Caries Gr+: Streptococcus mutans, Streptococcus sobrinus Periodontitis-Gingivitis Gr–: Porphyromonas gingivalis, Prevotella intermedia. 1. Otitis media Gr–: Haemophilus influenza, Moraxella catarrhalis Gr+: Streptococcus pneumoniae Cystic fibrosis pneumonia Gr–: Pseudomonas aeruginosa, Burkholderia cepacia. Tonsillitis Gr+: Corynebacterium diphtheriae Gr–: Neisseria gonorrhoeae Biliary tract infections Gr–: Escherichia coli, Enterobacter spp., Klebsiella spp., Pseudomonas spp.. Endocarditis Gr+: Streptococci, Staphylococcus aureus. Musculoskeletal infections Gr+: Staphylococcus aureus. Urinary tract infections Gr–: Escherichia coli Gr+: Staphylococcus saprophyticus. Burn wound infections Gr–: Pseudomonas aeruginosa Gr+: Staphylococcus aureus. Prostatitis Gr–: Escherichia coli Gr+: Staphylococcus aureus. Pyelonephritis Gr–: Escherichia coli Biomaterial-associated infection Gr+: Staphylococcus aureus, Staphylococcus epidermidis Gr–: Pseudomonas aeruginosa, Escherichia coli. Diabetic foot ulcers Gr+: Staphylococcus aureus Gr–: Pseudomonas aeruginosa Fasciitis Gr+: Staphylococcus aureus. B Gram-positive bacteria. Lipoteichoic acid. Peptidoglycan. Gram-negative bacteria. Teichoic acid. Lipopolysaccharide Outer membrane Peptidoglycan. Cell membrane. Cell membrane. Figure 1. (A) Examples of human bacterial infections occurring across the human body, illustrating the variety of different strains and species involved. (B) Gram-positive (Gr+) bacteria possess a single, inner lipid-membrane and a relatively thick, rigid peptidoglycan layer, while Gram-negative (Gr-) bacteria have a much thinner peptidoglycan layer in between an inner and outer lipidmembrane. This difference can affect the interaction of antimicrobials with the cell wall. why Van Leeuwenhoek complained about the lack of penetration of vinegar into the biofilm on his teeth, frustratingly the poor penetration of antimicrobials into infectious biofilms still constitutes a major problem to 4.

(16) Chapter 1. A. Antimicrobial-susceptible bacteria. Antimicrobial-resistant bacteria. Planktonic bacteria. 1. Planktonic bacteria Bacteria in a biofilm mode of growth. EPS. Substratum Poor antimicrobial penetration. B EPS. Physiological pH 7.4. Diffusion. Live bacteria. Water channel. Dead bacteria. -. Antimicrobials. pH < 6 Substratum. Figure 2. Intrinsic antimicrobial-resistance and poor penetration of antimicrobials into biofilms form the two main reasons for the recalcitrance of infectious biofilms to antimicrobial treatment. (A) Antimicrobial-susceptible bacteria are killed planktonically, while only biofilm bacteria at the outside of the biofilm can be reached and killed. Antimicrobial-resistant bacteria are neither killed in suspension (“planktonic-mode”) nor at the outside of a biofilm (compare left and right panels). (B) Regardless of intrinsic antimicrobial resistance, a second, general reason of recalcitrance is the poor penetration of antimicrobials through a biofilm to its depth due to reduced diffusion of antimicrobials and adsorption to the bacterially-produced, protective matrix of extracellular polymeric substances (EPS). The EPS-matrix allows transport of nutrients and metabolic wasteproducts through water channels, but severely hampers antimicrobial transport and only bacteria at the outside of a biofilm are killed by environmental antimicrobials, provided antimicrobialsusceptibility of the bacterial inhabitants of the biofilm. Note that pH in biofilms is lower than the physiological one outside an infectious biofilm. Details not drawn to scale. membrane. This difference can affect the interaction of antimicrobials with the cell wall. be solved in the control of human bacterial infections, next to intrinsic antimicrobial-resistance. The recalcitrance of infectious-biofilms to antimicrobials can be partly overcome by the use of local antimicrobial delivery systems, such as antibiotic-releasing bone cements or hydrogels to create high local antimicrobial concentrations that enhance penetration and killing. As a severe drawback, such delivery systems often show an initially high burst-release, followed by a low tail-release requiring many years for complete delivery of their entire antimicrobial cargo, which yields the danger of promoting the development of antimicrobial-resistance. With the steadily increasing number of multi-drug resistant bacterial strains, new. 5.

(17) General Introduction and Aim of This Thesis. 1. Planktonic bacterium. NEW EMERGENT PROPERTIES of ADHERING BACTERIA: - EPS production - social cooperation, communication and gene exchange - spatial organization of mixed and mono-species biofilms - resource capture and enzyme retention - desiccation tolerance - slow growing states (“dormancy”) and cells tolerant to antimicrobials (“persisters”) - enhanced survival of antimicrobial exposure. Altered gene expression in adhering bacteria. Adhesion force, causing cell wall deformation. 5- 500 nm. Figure 3. Ilustration of adhesion-force induced gene expression in adhering bacteria relative to planktonic ones4 due to nanoscopic cell wall deformation and summary of resulting emergent properties.2. NANOPARTICLES. SURFACE. INTERIOR. SIZE. SHAPE. Rods. Figure 4. Size and shape, surface and interior properties of NPs important for their use in biofilminfection control. infection-control measures are direly needed. Hopes are focused nowadays for instance, on antimicrobial peptides, either synthetic or naturally derived.6 Antimicrobial peptides can be potent and have been present. 6.

(18) Chapter 1. A. Stealth properties. 1. Uncharged Poly(ethylene glycol) (PEG). Dextran. Zwitterionic. Poly(2-methacryloyloxyethyl phosphorylcholine). B. Poly(sulfobetaine). Poly(carboxybetaine). pH responsiveness Imidazole Amine Polyhistidine pKa ~ 6.5. Poly(β-amino ester) pKa ~ 7.0. Amide. Alkoxyphenyl acylsulfonamide pKa ~ 6.5. Acylsulfonamide pKa ~ 4.5. Amino-acid. Amino acid. Amino+acid. Amine+acid. Figure 5. (A) Polymers used to equip NP surfaces with stealth properties. (B) Building blocks used to provide NPs with pH-responsive and charge-reversible properties, together with their pKa values. Red and blue colored moieties possess negatively- or positively-charged groups, respectively. Amino- or amide-groups will become protonated and adapt a positive-charge when the surrounding pH drops below their pKa. as part of the human natural defense system since the first existence of life, without stimulating any bacterial resistance. Yet, it is sometimes doubted whether this will remain to be the case, once clinical treatment of infection becomes large-scale based on the use of antimicrobial peptides. Phage therapy is also regarded as a possibility for treating recalcitrant infections, but detailed understanding of the interactions between phages, infecting bacteria and the human host is still lacking, impeding large-scale clinical use. With respect to biomaterial-associated infection of implants such as artificial hips and knees, non-adhesive, drug-releasing. 7.

(19) General Introduction and Aim of This Thesis. 1. and contact-killing coatings are under design.1 Last but not least, nanotechnology is looked at as an extremely promising way to create new antimicrobials and delivery systems, able to penetrate biofilms and kill multi-drug resistant strains. 1.1. Nanoparticle properties for biofilm-infection control Table 1. Nanotechnology-based antimicrobials in a biofilm. Nanoparticles and biofilm inhabitants not drawn to scale Nanoparticle Proposed Mechanism of Advantages Disadvantages (NP) Antimicrobial Action Metal-based nanocomposites Ag and other heavy metal NPs. Au NPs. • Killing major Gr+ and Gr– planktonic pathogens • Easy to synthesize through Ag NP physical, chemical or ROS biological methods • Diverse formulations possible (e.g. coating or Transmembrane emulsion) Mitochondrial Membrane electron transport • Synergistic effects with damage interruption damage antibiotics • Easy to synthesize through Au NP NIR light physical, chemical or biological methods Local heat • Easy-tunable surface properties and shape (e.g. spheres, tubes, or flowers) • Photothermal properties NIR Membrane and EPS damage Protein damage DNA damage. Ag+ ion. • Mechanism of biofilm penetration and killing not fully understood • Potentially cytotoxic. • Killing only demonstrated for a limited number of strains • Potentially cytotoxic • Poor storage stability • Costly. Carbon-based nanomaterials Graphene-based NPs. Protein damage DNA damage. Phospholipid extraction ROS Membrane puncturing. • Bacterial killing efficacy • Diverse formulations possible (e.g. coating or emulsion) • Electronic, thermal and mechanical properties. • Poor storage stability • Potentially cytotoxic. • Tunable surface properties • Easy to synthesize and modify •Can be made biocompatible and biodegradable. • Poor storage stability due to positively-charged surfaces • Limited number of polymers available • Potential toxic solvent residuals. Mitochondrial Membrane damage damage. Polymer-based nanoparticles Natural and synthetic polymeric NPs. Polymeric NP EPS damage . . . Membrane damage. 8. .

(20) Chapter 1. Nanoparticles (NPs) can be synthesized by a wide variety of methods, including mechanical stretching, soft lithography, microfluidics or self-assembly using different materials like inorganic, small molecules, macromolecules and polymers. Size and shape, surface and interior properties of resulting NPs are important to consider with respect to the control of biofilm-infection (Figure 4). 1.1.1. Size. NP size is crucial for their penetration into biofilms and should not exceed the dimensions of water-filled channels in biofilms (Figure 2B). Estimates of the dimensions of water-filled channels in biofilms are hard to give, because the definition of a water-filled “channel” and its distinction from water-filled pores and voids is not clear. However, dimensions of channels and pores generally range from 10 nm up to a few μm’s, with the nanometer-size estimate referring more accurately to the water-filled connections in bacterial aggregates in a biofilm and the micrometer-sized one referring to pores and voids. Also, the structure and resulting bacterial density in a biofilm depend on nutrient availability and absence or presence of fluid shear during growth. In line, fluorescent NPs with stealth properties and diameters up to 130 nm have been shown to penetrate bacterial biofilms without adsorbing to the EPS-matrix or bacterial cell surfaces, but with strongly reduced diffusion coefficients due to water structuring in narrow channels.7,8 Also, such NPs after intravascular administration can circulate longtime in the blood before excretion through the urinary pathway. NPs with diameters less than 5 nm are more readily cleared from the body, but can have enhanced antimicrobial efficacy, sometimes at the expense of their compatibility with tissue cells, since they can internalize into mammalian cells and produce reactive oxygen species ,causing cell death.9 Apart from impeding biofilm penetration, NPs with diameters above 500 nm may activate the complement system, resulting in rapid recognition by the immune system and clearance from the blood.9 In summary, it may be concluded that the ideal diameter for NPs in biofilm-infection control ranges between 5 and 100-200 nm, but not exceeding 500 nm (Figure 4). 1.1.2. Surface. Whereas several human infection sites, like burn-wounds, diabetic foot ulcers or biofilms in the oral cavity can be reached without systemic administration of antimicrobials, internal infections like endocarditis or musculoskeletal infections can only be reached through the bloodstream. This requires NPs loaded with antimicrobials to be blood compatible, without adsorbing abundant blood proteins that alter their surface properties or activating the blood clotting cascade. Stealth transport through blood can be achieved by decorating NPs with different types of hydrophilic polymers (Figure 5A). Stealth properties of NP drugcarriers at the same time make them biocompatible with host tissue cells and allow NPs loaded with antimicrobials to penetrate into infectious biofilms. Effective NP penetration in infectious biofilms however, also requires their substantive presence without wash-out. Several surface chemistries are available to target NPs towards a biofilm and stimulate their accumulation inside the biofilm that make use of the acidic pH inside biofilms with respect to the physiological pH of 7.4 outside the biofilm (see Figure 2B), by provoking a surface charge- reversal of the NPs from negative to positive charge (Figure 5B) to create electrostatic doublelayer attraction with negatively-charged bacterial cell surfaces, according to the DLVO-theory of colloidal stability, named after Derjaguin-Landau-Verwey and Overbeek.4 NPs functionalized with positively-charged molecules, such as quaternary ammonium compounds, cannot be administered through the blood circulation to reach an infectious biofilm, because of their fast opsonization and recognition by host immune cells9 and their applicability is limited to infectious biofilms that can be externally reached. Strong, localized electrostatic double-layer attraction upon contact of such NP surfaces with bacteria can yield severe membrane damage, and subsequent cell death (“contact-killing”). Contact-killing of infectious bacteria can only be done within a small window of positive charge densities. Too low positive charge densities do not yield contact-killing, while too high charge densities harm mammalian cell membranes as well.. 9. 1.

(21) General Introduction and Aim of This Thesis. A. Coupling of stabilizing agents. 1. OH O. HO. OH. OH. O HO. OH. HO. Ag. OH. HO. OH O HO. OH O OH OH O OH. HO. HO. OH O HO. O HO OH. O. HO. O. O OH HO. OH. OH O. OH O O. OH OH OH. HO. O. OH OH. OH. O OH. -cyclodextrin. B. Polyphenol. Coupling of antibiotics O OH. NH2 HO. O. N. Ag O. S. H3N. NH2. O N. OH H2N. CH3. O N S O O. HN O. O. H2N. O. O. Aztreonam. NH3 H. H H H N S. O. N O O. O. Ampicillin. HO O. Figure 6. Functionalized molecular moieties used for Ag NPs coating. (A) Chemical structures of β-cyclodextrin and polyphenols used to stabilize colloidal Ag NPs by coupling through hydrogen-bonding. (B) Chemical structures of antimicrobials can be used synergistically with Ag NPs to enhance bacterial killing by chelating antimicrobial carboxyl/amino groups on the Ag NPs. 1.1.3. Shape. Shape, especially of functionalized NPs, can further enhance their contact-killing ability since shape can increase local adhesion forces that may yield changes in membrane surface tension and subsequent membrane damage. Nano-knives or nano-blades, owing to graphene oxide sharp edges, can puncture bacterial cell membranes causing leakage of intracellular constituents and cell death.10,11 NPs also possess the unique quality to generate high amounts of heat upon near-infrared (NIR) irradiation and obtain temperatures that can range up to 70-80 °C owing to plasmon resonance. When generated localized inside a biofilm, such temperature increases, though involving small amounts of heat, are sufficient to kill bacteria inside an infectious biofilm.12 NPs are also known for their extensive ion-release that can yield bacterial cell death with an impact of shape. An increased number of active facets, like in truncated triangular silver nanoplates was more effective in reducing the viability of planktonic Escherichia coli than silver nanorods or silver ions. The antibacterial efficacy of zinc oxides nanoflowers decreased from petal flowers, to fusiform flowers and rod flowers due to differences in surface area and porosity (Table 1). NP shape furthermore determines blood-circulation lifetimes, protein adsorption, tissue cell damage and uptake by immune cells.9 Spherical NPs have highest cellular uptake with little membrane damage, while rod- or otherwise shaped NPs have a larger contact area with cell membrane receptors, thus creating more membrane damage and possibly chronic inflammation.13. 10.

(22) Chapter 1. B MIC (nM). A. 6000. NP4. 1. 4000. NP1 2000. NP5. NP2. NP3. 0 2.0. 3.0. 4.0. 5.0. Hydrophobicity (log P). C. D 2 nm Au-NS. 6 nm Au-SN. 2 nm Au-SN. 6 nm Au-NS. Au-NS. Au-SN. Figure 7. (A) Molecular structures of different cationic- and hydrophobic-ligands on Au nanoparticles (Au NPs). (B) Minimal inhibitory concentrations (MIC) of Au NPs bearing different cationic- and hydrophobic-ligands against an E. coli strain as a function of the hydrophobic value (log P) of the end groups. Reprinted with permission from ref. 21. Copyright 2014, American Chemical Society. (C, D) Chemical structure of zwitterionic ligands used for Au NP modification and morphological damage to P. aeruginosa membrane structures after exposure to 2 and 6 nm Au−SN (panel C) and Au−NS (panel D) NPs. Red arrows indicate membrane damage. Scale bars equal 200 nm. Reprinted with permission from ref. 22. Copyright 2016, American Chemical Society. 1.1.4. Interior. Mesoporous silica NPs, dendrimers, as well as the hydrophobic or hydrophilic core-structures in polymeric micelles or liposomes respectively, can be loaded with antimicrobials, photosensitizers or enzymes (Table 2) to either allow them to freely circulate in the blood without causing damage or better penetrate a biofilm. Therewith poorly water-soluble drugs can be transported to infection sites that are otherwise beyond reach.14 Hydrophobic antimicrobials like Triclosan, protoporphyrin IX, farnesol and essential oils can be transported as cargo in the hydrophobic core of micelles, while the hydrophilic core of many liposomes is suitable for loading with hydrophilic drugs. Multi-charged drugs can be encapsulated into oppositely-charged carriers through electrostatic double-layer interactions, although these can become unstable in high ionic-strength conditions, as in blood, saliva or urine. For gaseous nitric oxide, carriers with secondary amino groups are necessary to react with nitric oxide under high pressure (normally 5 atmosphere) to form amino diazeniumdiolates, overcoming the short lifetime of nitric oxide. Loading of NPs with antimicrobials has as a disadvantage that they may inadvertently lose their cargo underway to an infection site, which can be prevented by conjugating the antimicrobial to nanocarriers through biodegradable, chemical bonds. Since many bacterial strains and species produce lipases, amongst which esterase, esterase linkages are ideally suitable for conjugating antimicrobials to nanocarriers, as the bond is only degraded once the nanocarrier is inside a biofilm. 1.2. Nanotechnology-based new antimicrobials 1.2.1. Metal-based nanocomposites. 11.

(23) General Introduction and Aim of This Thesis. Natural polymeric NPs. A. 1. Unstructured NPs Hydrophilic domain Hydrophobic domain. Chitosan. ε-Polylysine. Synthetic polymeric NPs. B. Polymeric micelles Hydrophilic shell Hydrophobic core. Random copolymers. Polycarbonate. Polymersomes Hydrophilic core Hydrophilic periphery Hydrophobic shell Random copolymers. Dendrimers Hydrophilic shell. Antimicrobial peptides. Glycopeptides. Figure 8. Schematic presentations of polymer-based nanoparticles used as new antimicrobials in infection control. (A) Unstructured NPs made of natural polymers. (B) Synthetic polymeric NPs, including polymeric micelles, polymersomes and dendrimers. Note the hydrophilic/ hydrophobic structuring of the polymeric nanoparticles. Metal oxides nanoparticles. Metal oxides (ZnO, TiO2, SiO2, MgO, CaO, CuO, Fe3O4) NPs utilize antimicrobial mechanisms involving (1) toxic heavy metal ions release, (2) bacterial cell membrane dissociation and subsequent leakage of intracellular content, (3) reactive oxygen species production (Table 1).15 Metal oxide NPs show good antimicrobial effects on drug-resistant strains as well as prevention of biofilm formation and eradication. Notably, of all the above mentioned metal oxides NPs, superparamagnetic iron oxide NPs (SPIONs), have been recognized as a promising tool for site-specific treatment of antibioticresistant Staphylococcus aureus biofilms. Together with metals including iron, zinc, and silver, SPIONs can be targeted to an infection site in a magnetic field, demonstrating superior efficacy to antibiotics or metal salts. 12.

(24) Chapter 1. against bacteria in a biofilm-mode of growth.16 Also metal ion-conjugated SPIONs showed good penetration and eradication of both Gram-positive methicillin-resistant S. aureus (MRSA) and Gram-negative (i.e. E. coli and Pseudomonas aeruginosa) biofilms.17 However, especially at higher concentrations, metal oxide NPs can be cytotoxic, limiting their applicability, that might be overcome by ion-doping or polymer-conjugation. Artificial enzymes mimics such as intrinsic peroxidase-like, catalytic iron oxide (CAT-NPs) can penetrate S. mutans biofilms to cause bacterial death within acidic niches of caries-causing biofilms.14 Ag nanoparticles. Ag NPs antibacterial effects are mostly due to their dissolution and release of Ag+ ions, which can enter a target bacterium and subsequently induce generation of intracellular reactive oxygen species and bacterial lysis (Table 1). However, underway in an infectious biofilm Ag+ ions can interact with components of the EPS-matrix (e.g. amine, thiols or carboxylates), preventing their penetration. Nevertheless, Ag NPs with diameters less than 20 nm effectively penetrated E. coli and Pseudomonas fluorescens biofilms.18 Ag NPs are prone to aggregation, reducing their antimicrobial efficacy and therefore surface functionalization before application is important to (1) prevent aggregation and enhance their killing efficacy in biofilms, and (2) reduce their cellular uptake and cytotoxicity. Natural and environmentally-benign compounds like β-cyclodextrin and polyphenols (Figure 6A) constitute effective stabilizing agents enhancing bacterial killing in biofilms and reducing cytotoxicity of Ag NPs.19 Antimicrobials or antibiotics can also be chelated to Ag NPs (Figure 6B) through coordinate bonds between carboxyl/amino groups of an antibiotic. Chelated antimicrobials are easily released due to their weak binding and high ion release from Ag NPs with their high volume to area ratio in combination with chelated and subsequently released aztreonam or ampicillin killed P. aeruginosa biofilms or planktonic S. aureus, that could not be killed by the antibiotic alone.20 Au nanoparticles. Au NPs possess similar antimicrobial mechanisms as many other metal-based NPs, but in addition can inhibit intra-cellular ATP synthesis and tRNA binding,15 while intra-cellular uptake can be adjusted by changing their surface charge or size. Cationically- and hydrophobically-modified Au NPs (Figure 7A) effectively killed different strains of clinical, multidrug resistant pathogens in their planktonicmode of growth.21 The antimicrobial efficacy of cationically- and hydrophobically-modified Au NPs increased with the hydrophobic value of the ligand end-group (Figure 7B). Surprisingly, 6 nm Au NPs functionalized with zwitterionic surfaces more efficiently disrupted bacterial membranes of planktonic Gram-positive and Gram-negative bacteria than 2 nm Au NPs (see Figures 7C and 7D for an example), regardless of the type of zwitterionic ligands used.22 Beside their intrinsic antimicrobial properties, Au NPs are also photothermal, generating high, localized amounts of heat upon NIR irradiation. NIR can activate Au NPs to release vibrational energy (“heat”) to the environment yielding high local temperatures up to 70-80 °C that can cause bacterial killing (Table 1). The highly-localized temperature increase by activated, surface-adaptive and charge-responsive photothermal Au NPs killed staphylococci in their biofilm-mode of growth, while not affecting healthy tissues that required much more heat production due to their larger size than bacteria.12 1.2.2. Carbon-based nanomaterials. Graphene materials. Graphene-based NPs have antimicrobial properties that depends mostly on their size and shape.10 Graphene-based NPs are hydrophobic with limited water-solubility and stability and are consequently mostly applied as contact-killing coatings on surfaces. However, 1D-carbon nanotubes and 2D-graphene nanosheets fixed on substratum surfaces only demonstrated limited contact-killing and minor prevention of biofilm formation (Table 1). 0D-graphene oxide quantum dots (GQDs) possess higher peroxidase-like activity than 2D-graphene due to their high electron transport properties. In presence of a low level of H2O2, peroxidase-like GQDs produced hydroxyl radicals, inducing lysis of Gram-negative E. coli and Gram-positive S. aureus, preventing the formation of biofilms. Graphene materials have been suggested. 13. 1.

(25) General Introduction and Aim of This Thesis. A. Quantity Adsorbed (cm3/g STP). 1. B –OH. –NH2. –P(CH3)O3H. Relative pressure (P/P0). C. E. F. G. NO Release (ppm mg–1 s–1). Peptide release (%). D. Si-OH Adsorption Si-OH Desorption Si-P(CH3)O3H Adsorption Si-P(CH3)O3H Desorption. Time (h). Time (h). Figure 9. Mesoporous silica NPs used in tandem delivery of antimicrobials. (A) Chemical structures of tetraethylorthosilicate N-methylaminopropyltrimethoxysilane and 3-(trimethoxysilyl)propylmethyl phosphonate monomers to create neutral, positively-charged and negatively-charged NP surfaces respectively, suitable for antimicrobial carriage. (B) Liquid nitrogen adsorption–desorption isotherms for two differently modified mesoporous silica NPs. Reprinted with permission from ref. 36. Copyright 2017, John Wiley and Sons. (C) Brunauer–Emmett–Teller (BET) surface area, pore volume and antimicrobial peptide loading efficiencies (w/w%) of differently modified silica NPs (see also panel A). BET surface areas and pore volumes were derived from liquid N2 adsorption branch according to BET (see also panel B). (D) Electrostatic double-layer interaction of negatively-charged phosphonated. 14.

(26) Chapter 1. for application in infected wound healing and oral biofilm control. In a murine model, GQD-impregnated wound-dressings showed enhanced antimicrobial activity in combination with low concentration of H2O2 when applied to infected wounds.23 Carbon quantum dots. Carbon quantum dots (CQDs, also called C-dots), have extremely small size, usually less than 10 nm and are commonly synthesized via hydrothermal methods and can be modified with diverse functional groups.24 Naturally occurring polyamines25 and solid ammonium citrate have been used to create CQDs and when modified with spermidine, carry a high positive charge with antimicrobial effects against planktonic Gram-positive (S. aureus, Bacillus subtilis) and Gram-negative (E. coli, P. aeruginosa) Table 2 Nanotechnology-based antimicrobials delivery systems in biofilms. Nanoparticles and biofilm inhabitants not drawn to scale Nanoparticle Proposed Mechanisms of Advantages Disadvantages (NP) Drug Release Mesoporous Silica NPs. Biofilm Conjugated drug. 1. Free drug. Pore-opening. • Tunable particle size • Non-specific target • Stable and rigid framework • Poor storage stability • High surface area and large pore volume • Unique porous structure. 2. Gate-keeper. Bacterium Triggered release. Liposomes Bacterium Hydrophobic drug. 1. • Fusion with bacterial membrane • Reduce side effects. • Mechanism of biofilm penetration and killing not fully known • Poor storage stability • Non-specific target. • Tunable core properties • Easy to synthesize and modify • Can be made biocompatible and biodegradable. • Poor storage stability • Non-specific target • Potential toxic solvent residuals. Fusion. 2 Hydrophilic drug. Triggered release. Polymer-based NPs 1 Hydrophobic drug. Diffusion Core erosion. Hydrophilic drug. 2. Triggered release. silica NPs with cationic antimicrobial lactoferrin‐dKK peptides. (E) Release of antimicrobial peptides from phosphonated mesoporous silica NPs (see also panel D) into phosphate buffered saline. Reprinted with permission from ref. 36. Copyright 2017, John Wiley and Sons. (F) N-diazeniumdiolate structures on amine-functionalized silica NPs and subsequent NO release. (G) Proton-triggered NO release as a function of time for N-diazeniumdiolate silica NPs with different size. Reprinted with permission from ref. 37. Copyright 2011, American Chemical Society.. 15. 1.

(27) General Introduction and Aim of This Thesis. A. C. Neutral lipids. 1. N. H3N. N. Head. O P O O O. O O C R1 O O C R2. Phosphatidyl choline. O P O O. O O C R1 O O C R2. Phosphatidyle thanolamine. O. O. Cationic lipids. Tail. Head. O P O O. O H N C R1 HO. O. Tail. N. HO. R1. HO R1. H3N OH. Sphingomyelin. H N. H N. R2. O O. Cholesterol. O. B. Anionic lipids Head H3N COO. HO HO HO. OH. HO OH. O P O O O. R1. H N. Tail O O C R1 O O C R2. O P O O O OH. O O C R1 O O C R2. O P O O O. O O C R1 O O C R2. R2 O. Phosphatidyl serine. O O C R1 O O C R2. N. Phosphatidyl glucose N. Phosphatidyl glycerol. R1 R2. Figure 10. Lipids used for liposome preparation. (A, B) Chemical structures of the neutral (A) and anionic (B) phospholipids used for fusing liposome preparation. Phospholipids consist of a hydrophilic head group, determining the charge properties of the liposomes and a hydrophobic tail composed of uncharged fatty acids (R1 and R2). (C) Chemical structures of cationic, nonphospolipids, used in liposome preparation. pathogens. Spermidine-modified CQDs cause damage to the bacterial membrane (see also Table 1) with low cytotoxicity and hemolysis rate. Also, spermidine-modified CQDs induced opening of corneal epithelial tight-junctions to increase the efficacy of topical treatment of bacterial keratitis in a rabbit model.24 CQDs obtained by burning Lactobacillus plantarum to ashes, prevented E. coli biofilm formation,26 while also showing low cytotoxicity. Amine-functionalized QCDs made fluorescent with lauryl betaine have shown bacterial imaging capability, that can potentially be used to diagnose infection and localize infection sites.27 1.2.3. Polymer-based nanoparticles. Natural polymeric nanoparticles. Natural polymers, such as chitosan or ε-polylysine can be used to prepare unstructured positively-charged and antimicrobial nanoparticles (Figure 8A). Among them, chitosan is most commonly used. Chitosan is a natural polymer, consisting N-acetyl-glucosamine residues and glucosamine residues arranged in random order (Figure 8A). Most amino groups on free glucosamine will become protonated and hence positively charged at pH < 6.5, as in infectious biofilms.28 Positively-charged chitosan chains can interact with negatively-charged bacterial cell surfaces, causing membrane damage, leakage of intracellular components, inhibition of protein synthesis and mRNA transcription by binding to DNA.. 16.

(28) Chapter 1. The antimicrobial efficacy of chitosan is generally stronger against Gram-positive bacteria than against Gramnegative ones.29 Chitosan NPs can be made by shifting the hydrophobic/hydrophilic domain ratio in chitosan and have better water solubility, surface area/volume ratio, surface charge density than molecular chitosan, and accordingly better antimicrobial efficacy. Chitosan NPs act against bacteria, fungi, and viruses.28 NPs prepared from low molecular weight chitosans have better antimicrobial activity (> 95% killing) than high molecular weight chitosans (20 to 25% killing) in S. mutans biofilms.30 Synthetic polymeric nanoparticles. Diverse amphiphilic block polymers have been synthesized with cationic and hydrophobic moieties that mimic antimicrobial peptides6 and can be easily assembled into nanostructured NPs, like polymeric micelles or vehicles. Polymeric micelles. Polymeric micelles are core-shell structures (Figure 8B) composed of amphiphilic block single-chain polymers that can contain a higher local concentration of cationic or hydrophobic moieties than can be comprised in single-chained polymers. Biodegradable polymeric micelles composed of cationic, amphiphilic polycarbonates inhibited the planktonic growth of methicillin-resistant S. aureus (MRSA), other Gram-positive bacteria and fungi without significant haemolytic activity or acute toxicity in a murine model.31 Polymeric micelles composed of linear-random copolymers possessing oligoethylene glycol (hydrophilic), amine (positive domain), and hydrophobic groups, killed Gram-negative P. aeruginosa and E. coli in their planktonic and biofilm-mode of growth.32 Polymersomes. Polymersomes or polymeric vehicles, distinguish themselves from micelles by the possession of a hydrophilic core comprised by a hydrophobic polymer shell and a hydrophilic periphery (Figure 8B). Polymersomes can be functionalized with cationic charge to render them antimicrobial, similarly as cationically modified polymeric micelles.33 Polymersomes are more promising for drug delivery than micelles as they can be loaded with both hydrophobic or hydrophilic drugs. Dendrimers. Dendrimers are star-shaped and highly branched synthetic molecules (Figure 8B), with an exact molecular weight, and a well-controllable and narrow size-distribution. Dendrimers can be composed of synthetic antimicrobial peptides with demonstrated efficacy against a wide variety of planktonic bacterial strains and species,34 or synthetic glycopeptides that can block specific lectins on P. aeruginosa to prevent its biofilm formation.35 1.4. Nanotechnology-based antimicrobials delivery systems NPs not only possess antimicrobial properties of their own, but can also be applied as antimicrobial delivery systems, particularly when core-structured. Below, we evaluate advantages and disadvantages of different types of antimicrobial nanocarriers with respect to infection control (see also Table 2). 1.4.1. Mesoporous silica nanocarriers. Mesoporous silica NPs are extremely suitable for carrying antimicrobials and other drugs. Loading efficiencies of antimicrobials carried in tandem with silica NPs are mostly affected by the electrostatic double-layer interactions between the antimicrobials and the silica surface. In order to achieve high drug loading efficacy, differently charged monomers can be applied to enhance the suitability of silica NPs to carry differently charged antimicrobials. (Figure 9A). By virtue of their high BET (Brunauer–Emmett–Teller) surface area and porosity (Figure 9B),36 mesoporous silica NPs can carry up to 10-fold more load than non-porous silica NPs, depending on their surface modification (Figure 9C). Release of antimicrobials carried in tandem with silica NPs carried through electrostatic double-layer (Figure 9D) is not very well controlled and can easily occur inadvertendly underway in the bloodstream to an infection site in the body (Figure 9E).36 Chemically conjugated antimicrobials are less prone to inadvertend release. Silica NPs consisting of N-diazeniumdiolates (Figure 9F) can release nitric oxide through proton-triggered release (Figure 9G).37 Nitric oxide-releasing silica NPs showed more than 99% killing of biofilm-grown P. aeruginosa, E. coli, S. aureus, Staphylococcus 17. 1.

(29) General Introduction and Aim of This Thesis. A Fibroblast viability (%). 20 99.5% Bacteria loss. 10. Biofilm Log10 CFU. 1. B. 0 -10 -20 PM pIX MS M+P P S M prior to plasma exposure after plasma exposure. Emulsion concentration (v/v %). D Zeta potential (mV). C. Figure 11. Micellar nanocarriers used in antimicrobial delivery. (A) Viability of 3T3 fibroblasts (green line) and colony forming units of biofilm P. aeruginosa after exposure to carvacrol-loaded polymeric nanocarriers at different emulsion concentrations. Reprinted with permission from ref. 44. Copyright 2015, American Chemical Society. (B) Diameters and zeta potentials of mixed shell polymeric micelles (MSPMs) with or without PpIX loading at pH 7.4 prior to and after 24 h exposure to 10% murine plasma. Reprinted with permission from ref. 7. Copyright 2017, John Wiley and Sons. (C) Zeta potentials of single (SSPMs) and MSPM as a function of pH, showing charge reversal of MSPMs upon decreasing pH. Reprinted with permission from ref. 45. Copyright 2016, American Chemical Society. (D) Cumulative release of Triclosan (T) from SSPMs and MSPMs at pH 7.4 in absence or presence of lipase. Reprinted with permission from ref. 45. Copyright 2016, American Chemical Society. epidermidis, and Candida albicans with only limited influence on fibroblast proliferation. Nitric oxide does not only kill but also disperses biofilm inhabitants, through free radical mechanisms similar, as antimicrobial mechanisms of inflammatory cells.37 Hydrophobic antimicrobials as cinnamaldehyde or Triclosan, need to be covalently conjugated into mesoporous silica NPs in order to achieve acceptable loading efficiencies. Cinnamaldehyde conjugated to silica NPs released under the acidic condition, eradicated pathogenic biofilms of E. coli, P. aeruginosa, methicillinresistant S. aureus and Enterobacter cloacae, while selectively promoting fibroblast proliferation.38 Triclosanbound to silica NPs through triclosan-(3-(triethoxysilyl)propyl) carbamate, possessed better antimicrobial efficacy against E. coli and S. aureus in their planktonic mode of growth than free Triclosan. Importantly, also last-resort hydrophilic antibiotics like vancomycin can be conjugated to amine-functionalized silica NP surfaces for in tandem carriage and bacterial killing. 1.4.2. Liposomes. Liposomes are the natural analogues of polymersomes, and accordingly have a similar structure (compare 18.

(30) Chapter 1. Safety parameters. Output parameters Biofilm thickness. Biofilm metabolic activity. In vitro experiments. Biofilm bioluminescence. Colony forming units in dispersed biofilms. Damage to cellular layers Hemolysis. Live-dead staining of intact biofilms and fluorescent imaging . Animal in vivo studies. Organ analysis. Histological analysis. Blood parameters. Weight loss, Temperature. Human clinical trials Duration of treatment. 1. Penetration and retention in biofilm. Inflammation Tissue damage Biodistribution Organ accumulation in non-infected control group. Eradication of infection by multidrug resistant strains Unexpected side effects Premature death Organ failure/damage Development of secondary infections Vomiting Rash Itching. Cost effectiveness. Figure 12. Output and safety assessment parameters in in vitro experiments, animal in vivo studies and human clinical trials, including output parameters exclusive to the three different levels of downward clinical translation and their overlap with other levels (double-lined, differently colored boxes). Figures 10 and 8B), with their hydrophilic core making them ideal for carriage of hydrophilic antimicrobials that can not be carried by micelles. Liposomes are exclusively made of phospholipids (Figure 10) and have the unique property of fusing with bacterial phospholipid bilayers to release their antimicrobial cargo directly into a bacterium (Table 2). Due to their inner hydrophilic core, most conventional antibiotics can be loaded into liposomes, while fusing of neutral and anionic liposomes (Figures 10A and 10B) with the phospholipid membrane has yielded superior killing of many Gram-positive and Gram-negative bacterial strains in their biofilm mode of growth. Owing to their chemical similarity with bacterial membranes, liposome fusion does not yield membrane disruption, but only creates transport channels.39 Penetration through the peptidoglycan layer is assumed to occur through pores. Cationically modified (Figure 10C), antibiotic loaded phosphatidylcholine liposomes are usually more strongly attracted to negatively charged bacterial cell surfaces with better penetration into biofilms and bacterial killing than their uncharged liposomal counterparts or simply dissolved antimicrobials.39 In order to achieve the triggered fusion with bacterial membranes and associated antimicrobial release, toxin or photothermal moieties have been incorporated in liposomes. Pore-forming toxins secreted by bacteria can damage the liposome membranes and trigger the release of the encapsulated antimicrobials, such as α-toxin secreted by methicillin-resistant S. aureus triggerd release of vancomycin from gold nanoparticlestabilized liposomes to inhibit growth of planktonic S. aureus.40 Similarly, damage done upon NIR irradiation to. 19.

(31) General Introduction and Aim of This Thesis. liposomes equipped with photothermal cypate can trigger fusion and release of encapsulated antibiotics.41. 1. 1.4.3. Polymeric nanocarriers. Polymeric nanoparticles provide many possibilities for modification in order to achieve better targeting towards biofilms and responsive drug release owing to the enormous diversity in available polymers (Table 2). Natural polymeric nanocarriers. Nanoparticles composed of chitosan or its quaternary ammonium derivatives possess a positively-charged surface (Figure 8A), enhancing their accumulation in biofilms, although deep penetration may be hampered by electrostatic double-layer attraction underway in a biofilm. Once inside a biofilm, chitosan nanocarriers can act synergistically with their antimicrobial cargoes. Cargo can also comprise photodynamic molecules such as methylene blue, which can produce toxic singlet oxygen under UV light irradiation, demonstrating synergy with chitosan nanoparticles against S. aureus and P. aeruginosa biofilms.39 Vancomycin carrying gelatine nanocarriers have recently been demonstrated to possess antimicrobial efficacy, as evaluated in zebrafish larvae.42 Micellar nanocarriers. Many of the challenges in antimicrobial delivery deep inside a biofilm can be met by suitably designed micelles, while also long-term circulation in blood can be established by modification or suitable selection of the hydrophilic tail group (see Figure 8B). The hydrophobic core of micelles allows loading of poorly water-soluble antimicrobials like ciprofloxacin, killing S. aureus and P. aeruginosa biofilms43 or carvacrol oil, acting against P. aeruginosa without affecting the viability of fibroblasts in co-culture with bacteria (Figure 11A).44 Surface-adaptive mixed-shell polymeric micelles (MSPMs) composed of the copolymer poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) and poly (ε-caprolactone)-block-poly(β-amino ester) (PCLb-PAE) do not show significant changes in diameter and zeta potentials upon exposure to blood plasma (Figure 11B), indicative of absence of blood protein adsorption owing to their stealth properties,45 while their surface charge becomes positive (Figure 11C) upon and during stealth penetration in more acidic biofilms to allow accumulation without wash-out. Farnesol-loaded micelles degraded to release farnesol in the acidic environment of S. mutans biofilms, demonstrating 4-fold higher killing than farnesol in solution.46 Not only pH, but also bacterial enzymes can be used to trigger the release of antimicrobials loaded in micellar nanocarriers. Surface-adaptive, antimicrobialTable 3 Hurdles to be overcome in the translation from bench-to-bedside In nanotechnology In in vitro experiments science • Mechanisms of bacterial killing by new nanotechnology-based antimicrobial is imperative • Bacterial killing should be carried out in biofilms • Better realization of microbiological and clinical significance • More focus on specific infections rather than broad and general. 20. • Selection of the most appropriate pathogen for the type of infection aimed for • Comparison with current treatment modalities should be included • Better models are required to mimic the multi-factorial in vivo situation in relation with the type of infection aimed for. In animal in vivo studies • Selection of the most suitable animal species • Selection of the most appropriate pathogen for the type of infection aimed for • Comparison with current treatment modalities should be included • Translation of output parameters to human clinical benefits • Translation of output parameters to human safety. In human clinical trials • Limited availability of patients with a multi-drug resistant infection versus a suitable control group.

(32) Chapter 1. loaded PEG-b-PCL and PCL-b-PAE MSPMs facilitated fast penetration and accumulation of the MSPMs into staphylococcal biofilms and electrostatic double-layer targeting to bacterial cell surfaces, where bacterial lipases degraded the poly (ε-caprolactone) cores to release the antimicrobial cargo (Figure 11D). Triclosan-loaded MSPMs possess better killing efficacies for staphylococci both in their planktonic- and biofilm-mode of growth compared to Triclosan in solution and Triclosan loaded in single shell polymeric micelles.45 However, the main drawback of loading antimicrobials into micellar cores is the inadvertend loss of antimicrobial cargo underway in the blood circulation to the biofilm target site. In order to prevent this, antimicrobials can be conjugated into the surfactants making the micelles through linkages that can be degraded by bacterial enzymes once inside a biofilm.47 1.4.4. Dendrimeric nanocarriers. Polar hydroxyl or amino groups of dendrimers provide good opportunities for conjugation of antibiotics through ester or amide bonds. Amino groups on PAMAM dendrimers can react with carboxyl groups containing antibiotics like vancomycin to form amide bonds. Vancomycin-tethered PAMAM dendrimers showed an enhanced binding ability towards vancomycin-resistant S. aureus compared to vancomycin in solution, owing to the high local vancomycin density on the dendrimer surface. Similarly, nitric oxide-releasing PAMAM dendrimers enhanced killing efficacy against biofilms of P. aeruginosa and S. aureus.48 1.5. Nanotechnology-based infection control: the road from bench to bedside In all likelihood, chemists have already created many more nanotechnology-based antimicrobials considered promising to face the threat of untreatable infectious biofilms than will ever be downward-clinically translated to the bedside and be used to the benefit of patients. This is partly due to the fact that many academic solutions are driven by the incentive for researchers to write high impact papers rather than solving a problem and as a consequence, solutions proposed are complicated and impossible to upscale in a cost-effective manner allowing successful commercialization and clinical use. Regulatory requirements form a second important hurdle on the road towards clinical application, and in the USA the Food and Drug Administration requires demonstration of both human benefit and assessment of risk, while the European Union’s focus is on safety. Demonstration of risk and safety are both extremely costly, with in vitro experiments and animal in vivo studies each possessing their own specific shortcomings that, taken together, make the step to human clinical trials a huge one. To our knowledge, only tobramycin encapsulated in 1,2-dipalmitoyl-sn-glycero-3phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol sodium salt liposomes, called Fluidosomes™ are in a Phase-II trial for treating cystic fibrosis-associated respiratory tract infections.39 1.5.1. In vitro experiments. In vitro experiments can either be geared towards inhibition of biofilm growth (clinically captured under the denominator “prophylaxis”) or eradication of existing infectious biofilms (“therapy”). Although such experiments yield useful information on antimicrobial efficacy and mechanisms of action, results bear little or no relevance to the in vivo situation.49 In vitro biofilms are grown under optimal growth conditions, either under static conditions in wells, or in flow displacement systems with selected bacterial strains and species. However, even different isolates of the same strain, need not necessarily react similarly to an antimicrobial and the real challenge is the defeat of multidrug-resistant strains. To prevent bias by inappropriate pathogen selection, the Infectious Diseases Society of America suggests the use of a set of antibiotic-resistant bacteria, acronymically dubbed ESKAPE pathogens (Enterococcus faecium , S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa and Enterobacter spp.) for their ability to escape from antibiotics.50 Evaluation of antimicrobial efficacy in vitro can be done on the basis of several parameters (Figure 12) that are subsequently subjected to statistical analysis. However, in clinical microbiology statistically significant differences of a factor of 2 to 10 or arguably even 100, are meaningless. Clinical significance commences at minimally 3 to 4 log-unit. 21. 1.

(33) General Introduction and Aim of This Thesis. 1. reductions in viable counts due to short bacterial doubling time of around 20-40 min on average or ability of bacteria to remain dormantly present unsusceptible to antimicrobials, sometimes even for years. Note that 3 to 4 log-unit reductions represent percentage reductions of 99.9 and 99.99% respectively, while many papers in the field of new (nano-technology based) antimicrobial development report statistically significant reductions of less than 90%, i.e. only 1 log-unit and thus, are microbiologically insignificant.51 Moreover, these reductions as output parameters do not represent clinical benefit, which is defined in terms of percentages of patients cured with a new drug versus existing therapies and cost comparison. Also, the in vivo situation is an interplay between both blood and host immune components, tissue cells, bacteria and antimicrobials administered, which is hard to mimic in vitro. In vitro assessment of safety is equally difficult (see also Figure 12) and is usually done by exposing cellular layers to an antimicrobial while monitoring cell morphology and metabolic activity. Co-cultures comprised in 3D tissue models, transwell systems or microfluidic flow devices (“organ-on-a-chip”)52 more comprehensively model the in vivo situation, but it remains to be seen whether such advanced in vitro models will be able to replace in vivo animal experiments. 1.5.2. Animal in vivo studies. Animal experiments experience strong societal opposition and require extensive approval procedures, mostly along with demonstration of favorable in vitro results. Choice of the proper bacterial strains and species is equally important as in in vitro studies, while also the infection site and administration method of the antimicrobial (sub-cutaneous or intra-venous injection, oral administration) are important to consider.1 However, the most complicating factor is the choice of the animal to be used. Rats are notorious for their ability to cure an infection on their own without assistance of an antimicrobial, while in rabbits dosing of the challenge number of bacterial pathogens introduced needs to be well balanced in order to avoid premature death of the animal.49 Most infection studies are done in young, healthy mice or dogs, while infection occurs frequently in sick, elderly or immune-deficient humans that on top of all that often use multiple medications.49 These considerations make it doubtful whether animal experiments on new nanotechnology-based antimicrobials are truly helpful in predicting the outcome of human clinical trials. Yet, they better mimic the clinical situation than in vitro experiments, albeit with similar outcome parameters (see also Figure 12), that unfortunately do not directly reflect clinical benefit. Animal experiments give a good impression of possible adverse effects and biodistribution of a new antimicrobial through the body and its clearance from the body, but without the guarantee of being extrapolatable to the human situation. Concluding, despite these shortcomings, animal experiments will remain to form an indispensable step towards human clinical application particularly because opposite to human clinical trials, they offer the choice of creating an infection by a desired, preferentially multidrug-resistant pathogen. 1.5.3. Animal in vivo studies. The ethics of human clinical trials has undergone major changes over the past decades and has become much more strict.53 By comparison, in 1957, a human clinical trial was conducted to determine the virulence of Staphylococcus pyogenes (nowadays called S. aureus) for man in a wound-infection model, based on the realization that it was a fallacy to determine bacterial virulence based on arbitrarily chosen animal models. Also, at the time, the faith in antibiotics was still infinitely strong and, antibiotic resistance not yet a wellknown phenomenon. Thus purposely infecting humans with pathogenic bacteria for research purposes was considered ethically justified. However, particularly volunteers infected with staphylococci in presence of a biomaterials suture, became severely ill, and could only be cured after long-term antibiotic treatment. The authors describe that as a result they had “great difficulties in finding further volunteers”.54 Nowadays, human clinical trials are more strictly regulated, with restrictions to evaluate new antimicrobials becoming less strict for severely ill patients, like patients with diabetic foot ulcers to whom amputation is the sole alternative. However, 22.

(34) Chapter 1. clinical trials in which human volunteers are purposely infected with a specific pathogen as done in 1957, are out of the question. Yet, the need for effective new antimicrobials is largest for specific, multidrug-resistant pathogens. Therefore, the numbers of patients to be enrolled in order to demonstrate clinical benefit of a new antimicrobial are large, exceeding reality and requiring multi-center trials, involving doctors with different medical skills and traditions, patients with different life-styles and possibly other diseases underlying the infection. These considerations make patient availability and costs important factors in conducting meaningful human clinical trials and a hurdle on the road from bench to bedside that has to be taken one way or another, if we want nanotechnology-based antimicrobials to represent more than interesting science.. 1.6. Conclusions and perspective Numerous highly innovative, nanotechnology-based antimicrobials and delivery systems for infection control have been summarized in this chapter. However, working mechanisms of action have not always sufficiently determined, comprising definition of the type of infection aimed for and use of the right target strains (see Table 3). Moreover, since most human bacterial infections involve bacteria in a biofilm-mode of growth and while killing of planktonic bacteria is essential to demonstrate, more emphasis should be placed on the general behaviour of nanotechnology-based antimicrobials and delivery systems in biofilms under relevant conditions (see also Table 3). Whereas many papers include animal in vivo studies, often the study is not set up to combat a specific type of infection with the right administration procedure, infection site and pathogen. Other studies ignore the need for animal experiments as a necessary step towards human clinical trials without justification. Whereas by-passing animal experiments may be acceptable if advanced in vitro experiments are carried out with the aim of revealing mechanisms of killing, accounting for the multifactorial nature and human response to infection and safety assessment, will probably always necessitate animal studies. The perspectives of nanotechnology-based antimicrobials and delivery systems in infection control is promising, but it is important that evaluation of the assets of the field be evaluated in a more multidisciplinary approach to reach the ultimate goal of treating multidrug-resistant biofilm-associated infections. Time will have to tell however, whether also nanotechnology-based new antimicrobials will undergo the same faith as antibiotics and bacteria will find a way to effectively evade them when large-scale used.. 2. Aim of this thesis Infection by antibiotic-resistant bacteria is predicted to become the number one cause of death by the year 2050. It is extremely difficult to effectively treat infectious biofilms, because bacteria in a biofilm-mode of growth have embedded themselves in a protective, self-produced matrix that impedes antimicrobial penetration, on top of the problem of intrinsic antibiotic-resistance amongst bacterial pathogens. Therefore, it is a big challenge in the coming decades to develop new antimicrobials to eradicate infectious biofilms composed of multi-drug resistant bacteria. The aim of this thesis was to develop self-adaptive, antimicrobial polymeric nanocarriers with enhanced ability to penetrate and eradicate infectious biofilms by multi-drug resistant bacteria.. REFERENCES (1) Busscher, H. J.; van der Mei, H. C.; Subbiahdoss, Surface. Sci. Transl. Med. 2012, 4 (153), 153rv110. G.; Jutte, P. C.; van den Dungen, J. J. A. M.; Zaat, S. A. J.; (2) Flemming, H.-C.; Wingender, J.; Szewzyk, U.; Schultz, M. J.; Grainger, D. W. Biomaterial-Associated Steinberg, P.; Rice, S. A.; Kjelleberg, S. Biofilms: An Infection: Locating the Finish Line in the Race for the Emergent Form of Bacterial Life. Nat. Rev. Microbiol.. 23. 1.

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