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Wound infection treatment with antimicrobial peptides (AMPs) is still not a reality, due to the loss of activity in vivo. Unlike the conventional strategy of encapsulating AMPs on nanoparticles (NPs) leaving activity dependent on the release profile, this work explores AMP grafting to poly(d,l-lactide-co-glycolide)-polyethylene glycol NPs (PLGA-PEG NPs), whereby AMP exposition, infection targeting and immediate action are promoted. NPs are functionalized with MSI-78(4-20), an equipotent and more selective derivative of MSI-78, grafted through a thiol-maleimide (Mal) Michael addition. NPs with different ratios of PLGA-PEG/PLGA-PEG-Mal are produced and characterized, with 40%PLGA-PEG-Mal presenting the best colloidal properties and higher amounts of AMP grafted as shown by surface charge (+8.6 ± 1.8 mV) and AMP quantification (326 μg mL−1, corresponding to 16.3 μg of AMP per mg of polymer). NPs maintain the activity of the free AMP with a minimal inhibitory concentration (MIC) of 8-16 μg mL−1 against Pseudomonas aeruginosa, and 16-32 μg mL−1 against Staphylococcus aureus. Moreover, AMP grafting accelerates killing kinetics, from 1-2 h to 15 min for P. aeruginosa and from 6-8 h to 0.5-1 h for S. aureus. NP activity in a simulated wound fluid is maintained for S. aureus and decreases slightly for P. aeruginosa. Furthermore, NPs do not demonstrate signs of cytotoxicity at MIC concentrations. Overall, this promising formulation helps unleash the full potential of AMPs for the management of wound infections.

Bacterial biofilms account for 80% of all chronic infections, with cells being up to 1000 times more resistant to antibiotics than their planktonic counterparts. The recently discovered ability of Helicobacter pylori to form biofilms once again highlights why this bacterium is one of the most successful human pathogens. The current treatments failure rate reaches 40% of cases, emphasizing that new therapeutic options are a pressing need. Nanostructured lipid carriers (NLC), with and without docosahexaenoic acid (DHA), were very effective against H. pylori planktonic cells but their effect on H. pylori biofilms was unknown. Here, DHA-loaded NLC (DHA-NLC) and NLC without any drug (blank NLC) were tested on an optimized H. pylori in vitro floating mature biofilm model. DHA-NLC and blank NLC reduced the total biofilm biomass and had a bactericidal effect against both biofilm and planktonic bacteria in all the concentrations tested (0.125-2 mg/mL). DHA-NLC achieved biofilm biomass reduction in a concentration ~ 8 times lower than blank NLC (0.125 vs 1 mg/mL, respectively). Both NLC were bactericidal at the lowest concentration tested (0.125 mg/mL) although with different efficiency, i.e. a decrease of ∼6 log

MSI-78A (Pexiganan A) is one of the few antimicrobial peptides (AMPs) able to kill Helicobacter pylori, a pathogenic bacterium that colonizes the gastric mucosa of half of the world's population. Antibiotics fail in 20–40% of H. pylori-infected patients, reinforcing the need for alternative treatments. Herein, a bioengineered approach was developed. MSI-78A with a C-terminal cysteine was grafted onto chitosan microspheres (AMP-ChMic) by thiol-maleimide (Michael-addition) chemistry using a long heterobifunctional spacer (NHS-PEG113-MAL). Microspheres with ∼4 µm diameter (near H. pylori length) and stable at low pH were produced by spray drying using a chitosan solution with an incomplete genipin crosslinking. A 3 × 10−5 µg AMP/microsphere grafting was estimated/confirmed by UV/Vis and FTIR spectroscopies. AMP-ChMic were bactericidal against H. pylori J99 (highly pathogenic human strain) at lower concentrations than the free peptide (∼277 µg grafted MSI-78A-SH/mL vs 512 µg free MSI-78A-SH/mL), even after pre-incubation in simulated gastric conditions with pepsin. AMP-ChMic killed H. pylori by membrane destabilization and cytoplasm release in a ratio of ∼10 bacteria/microsphere. This can be attributed to H. pylori attraction to chitosan, facilitating the interaction of grafted AMP with bacterium membrane. Overall, it was demonstrated that the peptide-microsphere conjugation chemistry did not compromise the MSI-78A antimicrobial activity, instead it boosted its bactericidal performance against H. pylori. Statement of significance Half of the world's population is infected with Helicobacter pylori, a gastric bacterium that is responsible for 90% of non-cardia gastric cancers. Therefore, H. pylori eradication is now advocated in all infected individuals. However, available antibiotic therapies fail in up to 40% patients. Antimicrobial peptides (AMPs) are appealing alternatives to antibiotics, but their high susceptibility in vivo limits their clinical translation. AMP immobilization onto biomaterials surface will overcome this problem. Herein, we demonstrate that immobilization of MSI-78A (one of the few AMPs with activity against H. pylori) onto chitosan microspheres (AMP-ChMic) enhances its anti-H. pylori activity even at acidic pH (gastric settings). These results highlight the strong potential of AMP-ChMic as an antibiotic alternative for H. pylori eradication.

A PrS/pDNA/RAPA nanocomposite coating of a drug-coated balloon inhibits neointimal hyperplasia and facilitates endothelial regeneration after balloon-induced vascular injuries.

Covalent conjugation of antimicrobial peptides (AMP) is considered an effective approach to overcome their potential degradation in vivo. As the high cost of AMP-based therapies is one of the major hurdles towards their clinical application, it is urgently needed to explore high yield reactions. Herein, the highly efficient thiol-norbornene photoclick chemistry (TNPC) was explored for AMP grafting onto chitosan. Norbornenes were introduced onto chitosan (NorChit) in an aqueous/organic system, followed by UV-triggered conjugation of N- and C-terminus cysteine-modified Dhvar5 (NorChit-Dhvar5). Up to 0.38 norbornene groups per chitosan repeating unit and up to 43% conjugation yield in NorChit-Nt-Dhvar5 (80 μmol of Dhvar5/g) were achieved, while in NorChit-Ct-Dhvar5 conjugation yield was 30% (55 μmol of Dhvar5/g). Finally, NorChitDhvar5 ultrathin films showed up to a 35% reduction of total adhered Gram-positive Staphylococcus epidermidis and increased the adhesion and killing of Gram-negative Pseudomonas aeruginosa compared to unmodified chitosan. Moreover, NorChit-Dhvar5 was noncytotoxic to human neonatal dermal fibroblasts, according to ISO 10993-1. Overall, our findings indicate TNPC as a high yield strategy for AMP grafting onto norbornene-functionalized biopolymers towards the fabrication of antibacterial biomaterials. Work financially supported by Portuguese funds through FCT-MCTES as part of project “AntINFECT“ (POCI-01-0145-FEDER-031781). FCT-MCTES is further acknowledged for funding LAQV-REQUIMTE Research Unit (UIDB/50006/2020). P.A. thanks FCT for the doctoral grant SFRH/BD/145471/2019 financially supported by national (Norte 2020 Framework) and European Union funds (ESF – European Social Fund). F.C. acknowledges the Junior Researcher contract (CEECIND/01921/2017) funded by national funds through FCT, I. P. V.L. acknowledges her contract in the framework of the project PTDC/NAN-MAT/30898/2017 (FCT - Portuguese funds).

Hospital-acquired infections are still a major concern worldwide, being frequently related to bacterial biofilm formation on medical devices, and thus difficult to eradicate with conventional antimicrobial treatments. Therefore, infection-preventive solutions based on natural polymers are being investigated. Recently, a marine cyanobacterium-derived polymeric coating (CyanoCoating) has demonstrated great anti-adhesive potential when immobilized onto gold model substrates. In this work, we took this technology a step closer to an industrial application by covalently immobilizing CyanoCoating onto medical grade polyurethane (PU). This immobilization was developed through the introduction of linkable moieties onto PU inert surface using different pre-treatments. Besides the application of polydopamine (pDA) linker layer, other processes frequently found in industrial settings, as atmospheric plasma (using O2 or N2 as reactive gases) and ozone surface activations, were evaluated. From all the pre-treatments tested, the ozone activation was the most promising since the obtained coating not only revealed a homogeneous distribution, but also significantly reduced the adhesion of two relevant etiological bacteria in static conditions (the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli). Moreover, it also impaired E. coli biofilm formation under simulated urinary tract dynamic conditions, reinforcing the potential of CyanoCoating as an antibiotic-free alternative to mitigate medical device-associated infections, particularly in the urinnary tract. This work was financially supported by the Project AntiBioCoat - Anti-adhesive Biopolymer Coating (LCF/TR/CI18/50030020), from CaixaImpulse 2018, la Caixa Bank Foundation/EIT-Health and by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-028779 (PTDC/BIA MIC/28779/2017) and of the project “Institute for Research and Innovation in Health Sciences” (UID/BIM/04293/2019). This research was also funded by Base Funding -UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE - funded by national funds through the FCT/MCTES (PIDDAC) and project CVMAR+i—Industrial Innovation and Marine Biotechnology Valorization”, funded by INTERREG V Espanha Portugal (POCTEP) (0302_CVMAR_I_1_P). The authors acknowledge the support of the i3S Scientific Platforms Biointerfaces and Nanotechnology, and Biosciences Screening, member of the PPBI (PPBI-POCI-01-0145-FEDER-022122), CeNTI - Centre of Nanotechnology and Smart Materials, Famalicão, Portugal, for technical assistance with atmospheric plasma system and ozone generator, and to Centro de Materiais da Universidade do Porto (CEMUP), Porto, Portugal, for technical assistance with XPS, and Bruna Costa for all the support. We would also like to acknowledge the support from the ENIUS COST Action (CA16217). L.C.G. and F.C. acknowledge the Junior Researcher contracts (CEECIND/01700/2017, CEECIND/01921/2017) funded by national funds through FCT, I. P.

G. Julius Vancso, Jian Ji, Kazuhiko Ishihara, M. Cristina L. Martins and Shaoyi Jiang introduce the Journal of Materials Chemistry B themed collection on bioinspired surfaces engineering for biomaterials.

The development of antibiotics resistance has made multidrug-resistant (MDR) bacterial infection one of the most serious global health issues. Photothermal therapy (PTT) is an emerging therapeutic mode which can be applied to bacterial infection without inducing resistance. Moreover, enhanced therapeutic efficacy and less tissue damage can be realized with NIR-II fluorescence imaging (FLI) guided PTT. Herein, a polymeric luminogen with aggregation-induced emission (AIEgens) characteristics, poly(dithieno[3,2-b:2',3'-d]pyrrole-benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole)) (PDTPTBT), was synthesized and used as a photothermal agent for PTT of bacterial infections. PDTPTBT was encapsulated into liposomes (L-PDTPTBT) for improved water dispersibility. Upon 808 nm NIR irradiation, L-PDTPTBT can eliminate multiple bacteria including the Gram-positive methicillin-resistant Staphylococcus aureus and Enterococcus faecalis, the Gram-negative Escherichia coli and Pseudomonas aeruginosa. Serious damage of bacterial membrane and leakage of cytoplasm is observed after photothermal treatment using L-PDTPTBT. The potential of the formulation has been demonstrated in two infected animal models: (i) a subcutaneous abscess model and (ii) a diabetic skin infection model. In the diabetic skin infection model, the death of mice is largely suppressed and the wounds can heal more quickly with treatment of L-PDTPTBT under NIR irradiation. The excellent photothermal bactericidal ability and low cytotoxicity make L-PDTPTBT potential candidate for treating MDR bacterial infections in the future.

The multifunctional properties of antimicrobial peptides (AMPs) make them attractive candidates for the treatment of various diseases. AMPs are considered as alternatives to antibiotics due to the increasing number of multidrug-resistant (MDR) bacteria. However, bare AMPs have limited therapeutic potentials due to a low residence time in the blood circulatory system and susceptibility to proteases and an alkaline wound environment. These limitations are the major hurdles for AMPs to succeed as commercial drugs. In contrast, AMP-based materials, for instance, NPs, hydrogels, electrospun fibres, dressings and implants, could overcome these challenges and provide therapeutic efficacies to the conjugated AMPs superior to those of bare AMPs in different disease models. In this review, we discuss the preparation of different compositions of AMP-based materials and their therapeutic potential for the treatment of microbial infections in the brain, eyes, mouth, skin, lungs, and gastrointestinal and urinary tracts. Apart from antimicrobial potential, the applications of AMP-based materials in the regeneration of skin/bone, prevention of implant-associated infections, detection/imaging of bacteria, cancer therapy and gene delivery are discussed in this review. Lastly, we discuss different challenges that hinder the commercialization of AMP-based materials. Overall, this review provides a comprehensive account of the current progress and prospects of AMP-based materials for clinical applications.

TreatingHelicobacter pylori(H. pylori) infections has been a never-ending challenge, which has contributed to the high incidence of gastric cancer. The antibiotics commonly used are not reaching the infection site in its active state and in a concentration high enough to effectively kill the bacteria. In this context, amoxicillin-loaded lipid nanoparticles with carefully chosen materials were developed, namely dioleoylphosphatidylethanolamine (DOPE) as a targeting agent and Tween®80 and linolenic acid as antimicrobial agents. This work shows the ability of these nanoparticles in (i) targeting the bacteria (imaging flow cytometry) and inhibiting their adhesion to MKN-74 cells (bacteria-gastric cells adhesion model); (ii) killing the bacteria even as an antibiotic-free strategy (time-kill kineticstudies, scanning electron microscopy, and bacterial membrane permeability studies); (iii)overcoming gastrointestinal features using a newly developedin vitroinfection model that includes both physical (epithelial cells and mucus) and the chemical (acid medium) barriers; and in (iv) being incorporated in a floating system that can increase the retention time at the stomach. Overall, this work presents an effective nanosystem to deal with the ulcer-bug. Besides, it also provides two innovative tools transferable to other fields-anin vitroinfection model and a floating system to incorporate nanoparticles.

As we approach the end of the antibiotic era, newer therapeutic options, such as antimicrobial peptides (AMPs), are in urgent demand. AMP surface grafting onto biomaterials has been described as a good strategy to overcome problems associated with their in vivo stability. Helicobacter pylori is among the bacteria that pose greatest threat to human health, being MSI-78A one of the few bactericidal AMPs against this bacterium. Here, we report that MSI-78A grafted onto model surfaces (Self-Assembled Monolayers –SAMs), in a concentration of 30.3 ± 1.2 ng/cm2 determined by quartz crystal microbalance with dissipation (QCM-D), was able to kill, by contact, 98% of planktonic H. pylori in only 2 h. This fact was not verified against the control bacteria (Staphylococcus epidermidis), although the minimal inhibitory concentration (MIC) of MSI-78A in solution is much lower for S. epidermidis (2 µg/mL) than for H. pylori (64 µg/mL). Our results also demonstrated that, in opposite to other bacteria, H. pylori cells were attracted to ethylene glycol terminated (antiadhesive) surfaces, which can explain the high bactericidal potential of grafted MSI-78A. This proof of concept study establishes the foundations for development of MSI-78A grafted nanoparticles for gastric infection management within a targeted nanomedicine concept. This article is a result of the project NORTE-01-0145-FEDER-00012, (NORTE 2020) and FCT/MCTES-through the UID/BIM/04293/2019, PTDC/CTM-BIO/4043/2014, UID/QUI/50006/2013 (LAQV-REQUIMTE) projects. Claudia Monteiro acknowledges FCT for the SFRH/BPD/79439/2011 grant.

Staphylococcus aureus (S. aureus) is considered by the World Health Organization as a high priority pathogen for which new therapies are needed. This is particularly important for biofilm implant-associated infections once the only available treatment option implies a surgical procedure combined with antibiotic therapy. Consequently, these infections represent an economic burden for Healthcare Systems. A new strategy has emerged to tackle this problem: for small bugs, small particles. Here, we describe how nanotechnology-based systems have been studied to treat S. aureus biofilms. Their features, drawbacks and potentialities to impact the treatment of these infections are highlighted. Furthermore, we also outline biofilm models and assays required for preclinical validation of those nanosystems to smooth the process of clinical translation., Staphylococcus aureus biofilms are a major cause for implant-associated infections. The lack of an effective treatment and the escalating numbers of antibiotic-resistance events are prompting the research toward alternative nano-based strategies.

Bacterial biofilms are a major health concern, mainly due to their contribution to increased bacterial resistance to well-known antibiotics. The conventional treatment of biofilms represents a challenge, and frequently, eradication is not achieved with long-lasting administration of antibiotics. In this context, the present work proposes an innovative therapeutic approach that is focused on the encapsulation of N-acetyl-l-cysteine (NAC) into lipid nanoparticles (LNPs) functionalized with d-amino acids to target and disrupt bacterial biofilms. The optimized formulations presented a mean hydrodynamic diameter around 200 nm, a low polydispersity index, and a high loading capacity. These formulations were stable under storage conditions up to 6 months. In vitro biocompatibility studies showed a low cytotoxicity effect in fibroblasts and a low hemolytic activity in human red blood cells. Nevertheless, unloaded LNPs showed a higher hemolytic potential than NAC-loaded LNPs, which suggests a safer profile of the latter. The in vitro antibiofilm efficacy of the developed formulations was tested against Staphylococcus epidermidis (Gram-positive) and Pseudomonas aeruginosa (Gram-negative) mature biofilms. The results showed that the NAC-loaded LNPs were ineffective against S. epidermidis biofilms, while a significant reduction of biofilm biomass and bacterial viability in P. aeruginosa biofilms were observed. In a more complex therapeutic approach, the LNPs were further combined with moxifloxacin, revealing a beneficial effect between the LNPs and the antibiotic against P. aeruginosa biofilms. Both alone and in combination with moxifloxacin, unloaded and NAC-loaded LNPs functionalized with d-amino acids showed a great potential to reduce bacterial viability, with no significant differences in the presence or absence of NAC. However, the presence of NAC in NAC-loaded functionalized LNPs shows a safer profile than the unloaded LNPs, which is beneficial for an in vivo application. Overall, the developed formulations present a potential therapeutic approach against P. aeruginosa biofilms, alone or in combination with antibiotics. ispartof: ACS APPLIED MATERIALS & INTERFACES vol:13 issue:36 pages:42329-42343 ispartof: location:United States status: published

The lack of small-diameter vascular grafts (inner diameter

Antibiotic resistance is increasing and new strategies are needed to fight infection. Advanced materials are promising tools that can be combined with innovative alternatives to conventional antibiotics to allow more targeted and efficient treatment. In this work, we explored the activity against Staphylococcus epidermidis (S. epidermidis) of the &alpha, helical antimicrobial peptide (AMP) MSI-78(4-20) (KFLKKAKKFGKAFVKIL) when covalently bound to a chitosan coating. The AMP MSI-78(4-20) (17 mer) is an improved version of its parent MSI-78 (22 mer, commercially known as Pexiganan), a cost-effective short AMP, which was demonstrated to be as effective as MSI-78 and less toxic to eukaryotic cells. An MSI-78(4-20)&ndash, chitosan coating could be applied in several infection scenarios, ranging from bone implants to wound dressings, as chitosan possesses osteoconductive and hemostatic properties. Cysteine-modified MSI-78(4-20) was covalently immobilized onto the chitosan coating through a succinimidyl-[(N-maleimidopropionamido)-octaethyleneglycol] ester (SM(PEG)8), a heterobifuncional crosslinker, with N-hydroxysuccinimide (NHS) ester and maleimide groups, by its N- and C- termini. The MSI-78(4-20)&ndash, chitosan coating demonstrated bactericidal properties independently of the tethering site and an improved performance in the presence of plasma proteins, which mimics conditions that will be encountered in vivo. This AMP&ndash, chitosan coating has therefore great potential for applications in medical devices such as implants or even wound dressings.

Catheter-associated urinary tract infections (CAUTIs) represent about 40% of all healthcare-associated infections. Herein, the authors report the further development of an infection preventive anti-adhesive coating (CyanoCoating) meant for urinary catheters, and based on a natural polymer released by a marine cyanobacterium. CyanoCoating performance was assessed against relevant CAUTI etiological agents, namely Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus (MRSA), and Candida albicans in the presence of culture medium or artificial urine, and under biofilm promoting settings. CyanoCoating displayed a broad anti-adhesive efficiency against all the uropathogens tested (68&ndash, 95%), even in the presence of artificial urine (58&ndash, 100%) with exception of P. mirabilis in the latter condition. Under biofilm-promoting settings, CyanoCoating reduced biofilm formation by E. coli, P. mirabilis, and C. albicans (30&ndash, 60%). In addition, CyanoCoating prevented large crystals encrustation, and its sterilization with ethylene oxide did not impact the coating stability. Therefore, CyanoCoating constitutes a step forward for the implementation of antibiotic-free alternative strategies to fight CAUTIs.

The stiffness of the substrates has been found to have a strong effect on cell behaviors, especially on cell adhesion, which is the first cellular event when cells contact materials. Much effort has been made to develop the materials with controlled stiffness for regulating cell adhesion. However, most available strategies for controlling the stiffness of material surfaces are generally limited to be static, which means that the stiffness is fixed during cell adhesion. Herein, we developed polyelectrolyte multilayer films (PEMs), and their stiffness can be dynamically modulated by mild stimuli. The PEMs were made by alternative deposition of poly-L-lysine (PLL) and thiol group modified hyaluronan (HA-SH) using the layer-by-layer assembly technique. The (PLL/HA-SH) multilayers can be cross-linked via oxidation of thiol groups. After crosslinking, the stiffness was increased and the adhesion of fibroblast cells was promoted. The stiffness of the multilayer films can be down-regulated dynamically by adding glutathione (GSH) in the medium, leading to in situ reduction of cell adhesion. Our study provides a promising strategy for the development of material surfaces with dynamically changeable stiffness, which is of great potential in the field of cell-based biomaterials.

Helicobacter pylori infection is one of the major risk factors for gastric cancer development. Available antibiotic-based treatments not only fail in around 20% of patients but also have a severe negative impact on the gut microbiota. Recently, we demonstrated that nanostructured lipid carriers (NLC), even without any drug loaded, are bactericidal against H. pylori at low concentrations. This work aims to clarify NLC mode of action and to evaluate if their bactericidal effect is specific to H. pylori without affecting bacteria from microbiota. NLC were produced by hot homogenization followed by ultrasonication method, using Precirol®ATO5 and Miglyol®812 as lipids and Tween®60 as a surfactant. NLC were able to eradicate H. pylori without affecting the other tested bacteria (Lactobacillus, E. coli, S. epidermidis and S. aureus). Bioimaging assays demonstrated that NLC rapidly bind to and cross the H. pylori bacterial membrane, destabilizing and disrupting it, which leads to leakage of the cytoplasmic contents and consequent bacterial death. In an era where efficient alternatives to antibiotics are urgent, NLC are an interesting route to be explored in the quest for new antibiotic-free therapies to fight H. pylori infection.

Ana Paula Pêgo, M. Cristina L. Martins and Pedro L. Granja introduce the themed issue on the 31st Annual Conference of the European Society for Biomaterials (ESB 2021).

Helicobacter pylori is the etiologic agent of several gastric disorders. The growing rates of unsuccessfulness of available antibiotics-based therapy led to the need for non-antibiotic compounds for H. pylori treatment. The antibacterial activity of Eucalyptus nitens and Eucalyptus globulus outer bark lipophilic extracts were evaluated against a panel of H. pylori strains, as well as the role of pure triterpenic acids and synthetic mixtures mimicking the natural extracts. Best results were obtained with E. nitens outer bark extract (MIC = 64 μg/mL) and it was demonstrated that after 6 h of exposure to the MIC, there was a significant decrease (>50%) in H. pylori growth. Moreover, it was determined that the antibacterial effect of Eucalyptus spp. outer bark extracts was due to the synergetic effect of four triterpenic acids, namely betulinic, betulonic, ursolic and oleanolic acids. Our findings open new perspectives for development of anti-H. pylori therapies based on the use of these plant extracts.

Antimicrobial peptides (AMP) are promising alternatives to classical antibiotics, due to their high specificity and potency at low concentrations, and low propensity to elicit pathogen resistance. Immobilization of AMP onto biomaterials is an emergent field of research, towards creation of novel antimicrobial materials able to avoid formation of biofilms on the surfaces of medical devices. Herein, we report the chemical route towards one such material, where chitosan was used as biocompatible carrier for the covalent grafting of Dhvar-5, a well-known potent AMP, via the chemoselective ("click") Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The material's structure, as well as peptide loading, were confirmed by Fourier-transformed infra-red (FT-IR) and X-ray photoelectron (XPS) spectroscopies, and by Amino Acid Analysis (AAA), respectively. Results herein reported demonstrate that, with proper optimization, the "click" CuAAC is an attractive approach for the tethering of AMP onto chitosan, in order to create novel antimicrobial materials potentially valuable for biomedical applications.

International audience; Persistent Helicobacter pylori (H. pylori) infection is related to 90% of gastric cancers. With bacterial resistance rising and treatment inefficiency affecting 15% of the patients, alternative treatments urge. Chitosan microspheres (ChMics) have been proposed as an H. pylori-binding system. This work evaluates ChMics biocompatibility, mucopenetration and capacity to treat H. pylori infection in mice after oral administration. ChMics of different size (XL, ∼120 µm and XS, ∼40 µm) and degree of acetylation (6% and 16%) were developed and revealed to be able to adhere both human and mouse-adapted H. pylori strains without cytotoxicity towards human gastric cells. Ex vivo studies showed that smaller (XS) microspheres penetrate further within the gastric foveolae, suggesting their ability to reach deeply adherent bacteria. In vivo assays showed 88% reduction of infection when H. pylori-infected mice (C57BL/6) were treated with more mucoadhesive XL6 and XS6 ChMics. Overall, ChMics clearly demonstrate ability to reduce H. pylori gastric infection in mice, with chitosan degree of acetylation being a dominant factor over microspheres' size on H. pylori removal efficiency. These results evidence the strong potential of this strategy as an antibiotic-free approach to fight H. pylori infection, where microspheres are orally administered, bind H. pylori in the stomach, and remove them through the gastrointestinal tract. STATEMENT OF SIGNIFICANCE: Approximately 90% of gastric cancers are caused by the carcinogenic agent Helicobacter pylori, which infects >50% of the world population. Bacterial resistance, reduced antibiotic bioavailability, and the intricate distribution of bacteria in mucus and within gastric foveolae hamper the success of most strategies to fight H. pylori. We demonstrate that an antibiotic-free therapy based on bare chitosan microspheres that bind and remove H. pylori from stomach can achieve 88% reduction of infection from H. pylori-infected mice. Changing size and mucoadhesive properties, microspheres can reach different areas of gastric mucosa: smaller and less mucoadhesive can penetrate deeper into the foveolae. This promising, simple and inexpensive strategy paves the way for a faster bench-to-bedside transition, therefore holding great potential for clinical application.

Biomedical devices in the blood flow disturb the fine-tuned balance of pro- and anti-coagulant factors in blood and vessel wall. Numerous technologies have been suggested to reduce coagulant and inflammatory responses of the body towards the device material, ranging from camouflage effects to permanent activity and further to a responsive interaction with the host systems. However, not all types of modification are suitable for all types of medical products. This review has a focus on application-oriented considerations of hemocompatible surface fittings. Thus, passive versus bioactive modifications are discussed along with the control of protein adsorption, stability of the immobilization, and the type of bioactive substance, biological or synthetic. Further considerations are related to the target system, whether enzymes or cells should be addressed in arterial or venous system, or whether the blood vessel wall is addressed. Recent developments like feedback controlled or self-renewing systems for drug release or addressing cellular regulation pathways of blood platelets and endothelial cells are paradigms for a generation of blood contacting devices, which are hemocompatible by cooperation with the host system. STATEMENT OF SIGNIFICANCE: This paper is part 4 of a series of 4 reviews discussing the problem of biomaterial associated thrombogenicity. The objective was to highlight features of broad agreement and provide commentary on those aspects of the problem that were subject to dispute. We hope that future investigators will update these reviews as new scholarship resolves the uncertainties of today.

Antimicrobial peptides (AMPs) have been described as one of the most promising compounds able to address one of the main health threats of the twenty-first century that is the continuous rise of multidrug-resistant microorganisms. However, despite the clear advantages of AMPs as a new class of antimicrobials, such as broad spectrum of activity, high selectivity, low toxicity and low propensity to induce resistance, only a small fraction of AMPs reported thus far have been able to successfully complete all phases of clinical trials and become accessible to patients. This is mainly related to the low bioavailability and still somewhat expensive production of AMP along with regulatory obstacles. This chapter offers an overview of selected AMPs that are currently in the market or under clinical trials. Strategies for assisting AMP industrial translation and major regulatory difficulties associated with AMP approval for clinical evaluation will be also discussed.

Human mesenchymal stem/stromal cells (hMSC) are promising therapeutic agents for regenerative medicine. However, therapeutic doses necessary for clinical application require in vitro expansion, ideally under Xeno-Free (XF) conditions to avoid the use of foetal bovine serum (FBS). We previously reported that hMSCs could be expanded using a pharmaceutical-grade human plasma-derived supplement for cell culture (SCC, Plastem®) combined with bFGF and TGFβ1, on fibronectin (Fn)-coated surfaces. hMSCs expansion may also be affected by the chemistry of the culture surface, which modulates protein adsorption at the cell-material interface and, consequently, cell behavior. This work aimed to evaluate the effect of surface chemistry on hMSCs behavior in SCC-based XF media. For that, self-assembled monolayers (SAMs) with hydrophobic (-CH3) and hydrophilic (neutral -OH, positively charged -NH3+ and negatively charged -COO-) groups were used as model surfaces. Under XF conditions, Fn coating showed to be necessary to improve hMSC adhesion (4 h) onto all surfaces, except for OH-SAMs, probably due to a low protein adsorption capacity characteristic of this surface. In terms of cell metabolic activity (5 days) on Fn-coated surfaces, an increase over time under XF conditions was observed in all SAMs except in CH3-SAMs, which can be attributed to strong and irreversible protein adsorption characteristic of hydrophobic surfaces. This trend was also observed under FBS conditions. Nevertheless, none of the surfaces improved hMSC metabolic activity, as compared with tissue-cultured surfaces. Overall, this work describes the role of surface chemistry in XF hMSC expansion.

Biomedical devices in the blood flow disturb the fine-tuned balance of pro- and anti-coagulant factors in blood and vessel wall. Numerous technologies have been suggested to reduce coagulant and inflammatory responses of the body towards the device material, ranging from camouflage effects to permanent activity and further to a responsive interaction with the host systems. However, not all types of modification are suitable for all types of medical products.This review has a focus on application-oriented considerations of hemocompatible surface fittings. Thus, passive versus bioactive modifications are discussed along with the control of protein adsorption, stability of the immobilization, and the type of bioactive substance,biological or synthetic. Further considerations are related to the target system, whether enzymes or cells should be addressed in arterial or venous system, or whether the blood vessel wall is addressed. Recent developments like feedback controlled or self-renewing systems for drug release or addressing cellular regulation pathways of blood platelets and endothelial cells are paradigms for a generation of blood contacting devices, which are hemocompatible by cooperation with the host system.

Antimicrobial peptides (AMPs) have been described as one of the most promising compounds able to address one of the main health threats of the twenty-first century that is the continuous rise of multidrug-resistant microorganisms. However, despite the clear advantages of AMPs as a new class of antimicrobials, such as broad spectrum of activity, high selectivity, low toxicity and low propensity to induce resistance, only a small fraction of AMPs reported thus far have been able to successfully complete all phases of clinical trials and become accessible to patients. This is mainly related to the low bioavailability and still somewhat expensive production of AMP along with regulatory obstacles. This chapter offers an overview of selected AMPs that are currently in the market or under clinical trials. Strategies for assisting AMP industrial translation and major regulatory difficulties associated with AMP approval for clinical evaluation will be also discussed.

Urinary catheters are extensively used in hospitals, being responsible for about 75% of hospital-acquired infections. In this work, a de novo designed antimicrobial peptide (AMP) Chain201D was studied in the context of urinary catheter-associated infections. Chain201D showed excellent antimicrobial activity against relevant ATCC strains and clinical isolates of bacteria and yeast and demonstrated high stability in a wide range of temperatures, pH and salt concentrations. Moreover, the bactericidal activity of Chain201D immobilized on a model surface was studied against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), some of the most prevalent strains found in urinary catheter-associated infections. Chain201D was successfully tethered to ((1-mercapto-11-undecyl)-(tetra(ethylene glycol) (EG4)) terminated self-assembled monolayers (SAMs), (EG4-SAMs), activated by 1,1'-Carbonyldiimidazole (CDI) at different concentrations. Chain201D surfaces can bind and kill by contact a high percentage of adherent bacteria. These achievements are obtained without any peptide modification (for chemoselective conjugation) and without the use of a spacer. Moreover, increased amounts of immobilized AMP lead to higher numbers of adhered/dead bacteria, revealing a concentration-dependent behaviour and demonstrating that Chain201D has excellent potential for developing antimicrobial urinary catheters. AntINFECT: Bioengineered Advanced Therapies for Problematic Infected Wounds POCI-01-0145-FEDER-031781) and Institute for Research and Innovation in Health Sciences (UID/BIM/04293/2019). C. Monteiro would like to acknowledge national funding through FCT – Fundação para a Ciência e a Tecnologia, I.P., provided by the contract-program and according to numbers 4, 5 and 6 of art. 23 of Law no. 57/2016 of 29th August, as amended by Law no. 57/2017 of 19th July. Professor Paula Gomes from Faculdade de Ciências da Universidade do Porto (FC-UP), Porto Peptide Synthesis Facility (POP-UP) for CEM 7 peptide. The funding received from Tekes, Finland (3331/31/2014) is thankfully acknowledged.

Bacterial infections are the most eminent public health challenge of the 21st century. The primary step leading to infection is bacterial adhesion to the surface of host cells or medical devices, which is mediated by a multitude of molecular interactions. At the interface of life sciences and physics, last years advances in atomic force microscopy (AFM)-based force spectroscopy techniques have made possible to measure the forces driving bacteria-cell and bacteria-materials interactions on a single molecule/cell basis (single molecule/cell force spectroscopy). Among the bacteria-(bio)materials surface interactions, the life-threatening infections associated to medical devices involving Staphylococcus aureus and Escherichia coli are the most eminent. On the other hand, Pseudomonas aeruginosa binding to the pulmonary and urinary tract or the Helicobacter pylori binding to the gastric mucosa, are classical examples of bacteria-host cell interactions that end in serious infections. As we approach the end of the antibiotic era, acquisition of a deeper knowledge of the fundamental forces involved in bacteria - host cells/(bio)materials surface adhesion is crucial for the identification of new ligand-binding events and its assessment as novel targets for alternative anti-infective therapies. This article aims to highlight the potential of AFM-based force spectroscopy for new targeted therapies development against bacterial infections in which adhesion plays a pivotal role and does not aim to be an extensive overview on the AFM technical capabilities and theory of single molecule force spectroscopy. The authors would like to thank FEDER – Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operational Program for Competitiveness and Internationalization (POCI), Portugal 2020 and NORTE-01-0145-FEDER-000012. Authors acknowledge FCT – Fundaç˜ ao para a Ciˆencia e a Tecnologia/ Minist´erio da Ciˆencia, Tecnologia e Inovaçao ˜ for funding through projects POCI-01- 0145-FEDER-007274, PyloriBinders – Helicobacter pylori specific biomaterials for antibiotic-free treatment/diagnostic of gastric infection (PTDC/CTM-BIO/4043/2014) and AntINFECT – Bioengineered Advanced Therapies for Problematic Infected Wounds (POCI-01-0145-FEDER031781).

Laminin immobilization into diverse biological and synthetic matrices has been explored to replicate the microenvironment of stem cell niches and gain insight into the role of extracellular matrix (ECM) on stem cell behavior. However, the site-specific immobilization of this heterotrimeric glycoprotein and, consequently, control over its orientation and bioactivity has been a challenge that has limited many of the explored strategies to date. In this work, we established an affinity-based approach that takes advantage of the native high affinity interaction between laminin and the human N-terminal agrin (hNtA) domain. This interaction is expected to promote the site-selective immobilization of laminin to a specific substrate, while preserving the exposure of its key bioactive epitopes. Recombinant hNtA (rhNtA) domain was produced with high purity (>90%) and successfully conjugated at its N-terminal with a thiol-terminated poly(ethylene glycol) (PEG) without affecting its affinity to laminin. Self-assembled monolayers (SAMs) of mono-PEGylated rhNtA on gold (mPEG rhNtA-SAMs) were then prepared to evaluate the effectiveness of this strategy. The site-specific immobilization of laminin onto mPEG rhNtA-SAMs was shown to better preserve protein bioactivity in comparison to laminin immobilized on SAMs of thiol-PEG-succinimidyl glutaramide (HS-PEG-SGA), used for the non-selective covalent immobilization of laminin, as evidenced by its enhanced ability to efficiently self-polymerize and mediate cell adhesion and spreading of human neural stem cells. These results highlight the potential of this novel strategy to be used as an alternative to the conventional immobilization approaches in a wide range of applications, including engineered coatings for neuroelectrodes and cell culture, as well as biofunctionalization of 3D matrices. The mass spectrometry technique was performed at the Proteomics i3S Scientific Platform ( Roteiro/0028/2013 ; LISBOA-01-0145-FEDER-022125 ) with the assistance of Hugo Osório. IRRAS analysis was performed at the Biointerfaces and Nanotechnology Scientific Platform, with the assistance of Ricardo Vidal. In Cell Analyzer experiments were performed at the Biosciences Screening i3S Scientific Platform , member of the PPBI ( PPBI-POCI-01-0145-FEDER-022122 ) with the assistance of André Maia. Confocal microscopy was conducted at the Bioimaging i3S Scientific Platform , member of the PPBI ( PPBI-POCI-01-0145-FEDER-022122 ), with the assistance of Maria Lázaro. This work was supported by projects NORTE-01-0145-FEDER-000008 and NORTE-01-0145-FEDER-000012 , supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation ( POCI ), Portugal 2020; by Portuguese funds through FCT/MCTES in the framework of the project “Institute for Research and Innovation in Health Sciences” ( POCI-01-0145-FEDER-007274 ) and by project COMBINE (Santa Casa da Misericordia de Lisboa – Prémio Neurociências Mello e Castro). D.B. was supported by FCT PhD Programs ( PD/BD/105953/2014 ) and Programa Operacional Potencial Humano (POCH), in the scope of the BiotechHealth Program (Doctoral Program on Cellular and Molecular Biotechnology Applied to Health Sciences), Programa FLAD Helthcare 2020 and the project PARES (Prémio Albino Aroso). Eduardo Conde-Sousa was supported by a post-doctoral grant of the project PPBI-POCI-01-0145-FEDER-022122 , in the scope of Fundação para a Ciência e Tecnologia, Portugal (FCT) National Roadmap of Research Infrastructures. Appendix A

Antimicrobial peptides (AMP) are powerful components of the innate immune system, as they display wide activity spectrum and low tendency to induce pathogen resistance. Hence, the development of AMP-based coatings is a very promising strategy to prevent biomaterials-associated infections. This work aims to investigate if Dhvar-5-chitosan conjugates, previously synthesized by us via azide-alkyne "click" reaction, can be applied as antimicrobial coatings. Ultrathin coatings were prepared by spin coater after dissolving Dhvar-5-chitosan conjugate powder in aqueous acetic acid. Peptide orientation and exposure from the surface was confirmed by ellipsometry and contact angle measurements. Bactericidal activity was evaluated against Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, the most prevalent pathogens in implant-associated infections. Results showed that Dhvar-5-chitosan coatings displayed bactericidal effect. Moreover, since Dhvar-5 has head-to-tail amphipathicity, it was clear that the bactericidal potency was dependent on which domain of the peptide (cationic or hydrophobic) was exposed. In this context, Dhvar-5 immobilized through its C-terminus (exposing its hydrophobic end) presented higher antimicrobial activity against Gram-positive bacteria and reduced adhesion of Gram-negative bacteria. This orientation-dependent antimicrobial activity was further corroborated by the anti-biofilm assay, as covalent immobilization of Dhvar-5 through its C-terminus provided anti-biofilm properties to the chitosan thin film. Immobilization of Dhvar-5 showed no cytotoxic effect against HFF-1 cells, as both metabolic activity and cell morphology were similar to control. In conclusion, Dhvar-5-chitosan coatings are promising antimicrobial surfaces without cytotoxic effects against human cells. STATEMENT OF SIGNIFICANCE: AMP-tethering onto ground biomaterial is still a poorly explored strategy in research. In this work, AMP-tethered ground chitosan is used to produce highly antibacterial ultrathin films. Powdered AMP-tethered chitosan appears as an alternative solution for antimicrobial devices production, as it is suitable for large scale production, being easier to handle for fabrication of different coatings and materials with antimicrobial properties and without inducing toxicity.

Optimized ratio in the codelivery of therapeutics is of crucial importance to promote the synergism rather than the antagonistic effects. In this study, a self-healing spongy coating was described to facilitate the surface-mediated delivery of drug "cocktails" proportionally. The formation of spongy structures within the coating was achieved by acidic treatment and freeze-drying. Various drug combinations can be readily integrated through wicking method and subsequent micropore self-healing. The ratio of drug loading can be precisely regulated by the composition of loading solution and the embedded drugs were released in proportion according to the initial ratio of drug combination.