Sodium alginate/polycaprolactone co-axial wet-spun microfibers modified with N-carboxymethyl chitosan and the peptide AAPV for Staphylococcus aureus and human neutrophil elastase inhibition in potential chronic wound scenarios.

In chronic wound (CW) scenarios, Staphylococcus aureus-induced infections are very prevalent. This leads to abnormal inflammatory processes, in which proteolytic enzymes, such as human neutrophil elastase (HNE), become highly expressed. Alanine-Alanine-Proline-Valine (AAPV) is an antimicrobial tetrapeptide capable of suppressing the HNE activity, restoring its expression to standard rates. Here, we proposed the incorporation of the peptide AAPV within an innovative co-axial drug delivery system, in which the peptide liberation was controlled by N-carboxymethyl chitosan (NCMC) solubilization, a pH-sensitive antimicrobial polymer effective against Staphylococcus aureus. The microfibers' core was composed of polycaprolactone (PCL), a mechanically resilient polymer, and AAPV, while the shell was made of the highly hydrated and absorbent sodium alginate (SA) and NCMC, responsive to neutral-basic pH (characteristic of CW). NCMC was loaded at twice its minimum bactericidal concentration (6.144 mg/mL) against S. aureus, while AAPV was loaded at its maximum inhibitory concentration against HNE (50 µg/mL), and the production of fibers with a core-shell structure, in which all components could be detected (directly or indirectly), was confirmed. Core-shell fibers were characterized as flexible and mechanically resilient, and structurally stable after 28-days of immersion in physiological-like environments. Time-kill kinetics evaluations revealed the effective action of NCMC against S. aureus, while elastase inhibitory activity examinations proved the ability of AAPV to reduce HNE levels. Cell biology testing confirmed the safety of the engineered fiber system for human tissue contact, with fibroblast-like cells and human keratinocytes maintaining their morphology while in contact with the produced fibers. Data confirmed the engineered drug delivery platform as potentially effective for applications in CW care.

Antibacterial and hemostatic capacities of cellulose nanocrystalline-reinforced poly(vinyl alcohol) electrospun mats doped with Tiger 17 and pexiganan peptides for prospective wound healing applications.

Infection is a major issue in chronic wound care. Different dressings have been developed to prevent microbial propagation, but an effective, all-in-one (cytocompatible, antimicrobial and promoter of healing) solution is still to be uncovered. In this research, polyvinyl alcohol (PVA) nanofibrous mats reinforced with cellulose nanocrystal (CNC), at 10 and 20% v/v ratios, were produced by electrospinning, crosslinked with glutaraldehyde vapor and doped with specialized peptides. Crosslinking increased the mats' fiber diameters but maintained their bead-free morphology. Miscibility between polymers was confirmed by Fourier-transform infrared spectroscopy and thermal evaluations. Despite the incorporation of CNC having reduced the mats' mechanical performance, it improved the mats' surface energy and its structural stability over time. Pexiganan with an extra cysteine group was functionalized onto the mats via hydroxyl- polyethylene glycol 2-maleimide, while Tiger 17 was physisorbed to preserve its cyclic conformation. Antimicrobial assessments demonstrated the peptide-doped mat's effectiveness against Staphylococcus aureus and Pseudomonas aeruginosa; pexiganan contributed mostly for such outcome. Tiger 17 showed excellent capacity in accelerating clotting. Cytocompatibility evaluations attested to these mats' safety. C90/10 PVA/CNC mats were deemed the most effective from the tested group and, thus, a potentially effective option for chronic wound treatments.

Helicobacter pylori biofilms are disrupted by nanostructured lipid carriers: A path to eradication?

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 log10 for DHA-NLC and ∼5 log10 for blank NLC. In addition, the equivalent amount of free DHA (3.1 µM) only reduced bacterial viability in ∼2 log10, demonstrating the synergistic effect of DHA and NLC in the treatment of H. pylori biofilms. Nevertheless, although viable bacteria were not detected by colony forming unit (CFU) counting after treatment with both NLC, confocal microscopy imaging highlighted that some H. pylori cells remained alive. In addition, scanning electron microscopy (SEM) analysis confirmed an increase in bacteria with a coccoid morphology after treatment, suggesting a transition to a viable but non-culturable (VBNC) state. Altogether, it is herein established that NLC, even without any drug, are promising for the management of H. pylori bacteria organized in biofilms, opening new perspectives for the eradication of this gastric pathogen.

Tiger 17 and pexiganan as antimicrobial and hemostatic boosters of cellulose acetate-containing poly(vinyl alcohol) electrospun mats for potential wound care purposes.

In this research, we propose to engineer a nanostructured mat that can simultaneously kill bacteria and promote an environment conducive to healing for prospective wound care. Polyvinyl alcohol (PVA) and cellulose acetate (CA) were combined at different polymer ratios (100/0, 90/10, 80/20% v/v), electrospun and crosslinked with glutaraldehyde vapor. Crosslinked fibers increased in diameter (from 194 to 278 nm), retaining their uniform structure. Fourier-transform infrared spectroscopy and thermal analyses proved the excellent miscibility between polymers. CA incorporation incremented the fibers swelling capacity and reduced the water vapor and air permeabilities of the mats, preventing the excessive drying of wounds. The antimicrobial peptide cys-pexiganan and the immunoregulatory peptide Tiger 17 were incorporated onto the mats via polyethylene glycol spacer (hydroxyl-PEG2-maleimide) and physisorbed, respectively. Time-kill kinetics evaluations revealed the mats effectiveness against Staphylococcus aureus and Pseudomonas aeruginosa. Tiger 17 played a major role in accelerating clotting of re-calcified plasma. Data reports for the first time the collaborative effect of pexiganan and Tiger 17 against bacterial infections and in boosting hemostasis. Cytocompatibility data verified the peptide-modified mats safety. Croslinked 90/10 PVA/CA mats were deemed the most promising combination due to their moderate hydrophilicity and permeabilities, swelling capacity, and high yields of peptide loading.

Metabolic profile of Candida albicans and Candida parapsilosis interactions within dual-species biofilms.

Within the oral cavity, the ability of Candida species to adhere and form biofilms is well-recognized, especially when Candida albicans is considered. Lately, a knowledge gap has been identified regarding dual-species communication of Candida isolates, as a way to increase virulence, with evidences being collected to support the existence of interactions between C. albicans and Candida parapsilosis. The present work evaluated the synergistic effect of the two Candida species, and explored chemical interactions between cells, evaluating secreted extracellular alcohols and their relation with yeasts' growth and matrix composition. A total of four clinical strains of C. albicans and C. parapsilosis species, isolated from single infections of different patients or from co-infections of a same patient, were tested. It was found that dual-species biofilms negatively impacted the growth of C. parapsilosis and their biofilm matrix, in comparison with mono-species biofilms, and had minor effects on the biofilm biomass. Alcohol secretion revealed to be species- and strain-dependent. However, some dual-species cultures produced much higher amounts of some alcohols (E-nerolidol and E, E-Farnesol) than the respective single cultures, which proves the existence of a synergy between species. These results show evidence that interactions between Candida species affect the biofilm matrix, which is a key element of oral biofilms.

Grafting MSI-78A onto chitosan microspheres enhances its antimicrobial activity.

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.

Orally administrated chitosan microspheres bind Helicobacter pylori and decrease gastric infection in mice.

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.

Uncovering Akkermansia muciniphila resilience or susceptibility to different temperatures, atmospheres and gastrointestinal conditions.

Data regarding Akkermansia muciniphila viability under stress remains scarce despite its beneficial potential. Therefore, the main goal was to assess A. muciniphila culturability when exposed to different temperatures, atmospheres and gastrointestinal simulated conditions. Cultivable cell numbers A. muciniphila remain high after refrigerated and room temperatures oxygen exposure, and gastrointestinal passage.

A 17-mer Membrane-Active MSI-78 Derivative with Improved Selectivity toward Bacterial Cells.

Antimicrobial peptides are widely recognized as an excellent alternative to conventional antibiotics. MSI-78, a highly effective and broad spectrum AMP, is one of the most promising AMPs for clinical application. In this study, we have designed shorter derivatives of MSI-78 with the aim of improving selectivity while maintaining antimicrobial activity. Shorter 17-mer derivatives were created by truncating MSI-78 at the N- and/or C-termini, while spanning MSI-78 sequence. Despite the truncations made, we found a 17-mer peptide, MSI-78(4-20) (KFLKKAKKFGKAFVKIL), which was demonstrated to be as effective as MSI-78 against the Gram-positive Staphylococcus strains tested and the Gram-negative Pseudomonas aeruginosa. This shorter derivative is more selective toward bacterial cells as it was less toxic to erythrocytes than MSI-78, representing an improved version of the lead peptide. Biophysical studies support a mechanism of action for MSI-78(4-20) based on the disruption of the bacterial membrane permeability barrier, which in turn leads to loss of membrane integrity and ultimately to cell death. These features point to a mechanism of action similar to the one described for the lead peptide MSI-78.

Antimicrobial properties of membrane-active dodecapeptides derived from MSI-78.

Antimicrobial peptides (AMPs) are a class of broad-spectrum antibiotics known by their ability to disrupt bacterial membranes and their low tendency to induce bacterial resistance, arising as excellent candidates to fight bacterial infections. In this study we aimed at designing short 12-mer AMPs, derived from a highly effective and broad spectrum synthetic AMP, MSI-78 (22 residues), by truncating this peptide at the N- and/or C-termini while spanning its entire sequence with 1 amino acid (aa) shifts. These designed peptides were evaluated regarding antimicrobial activity against selected gram-positive Staphylococcus strains and the gram-negative Pseudomonas aeruginosa (P. aeruginosa). The short 12-mer peptide CEM1 (GIGKFLKKAKKF) was identified as an excellent candidate to fight P. aeruginosa infections as it displays antimicrobial activity against this strain and selectivity, with negligible toxicity to mammalian cells even at high concentrations. However, in general most of the short 12-mer peptides tested showed a reduction in antimicrobial activity, an effect that was more pronounced for gram-positive Staphylococcus strains. Interestingly, CEM1 and a highly similar peptide differing by only one aa-shift (CEM2: IGKFLKKAKKFG), showed a remarkably contrasting AMP activity. These two peptides were chosen for a more detailed study regarding their mechanism of action, using several biophysical assays and simple membrane models that mimic the mammalian and bacterial lipid composition. We confirmed the correlation between peptide helicity and antimicrobial activity and propose a mechanism of action based on the disruption of the bacterial membrane permeability barrier.

The potential utility of chitosan micro/nanoparticles in the treatment of gastric infection.

Gastric infections are mainly caused by Helicobacter pylori (H. pylori), a bacterium that colonizes the gastric mucosa of over 50% of the world's population. Chronic H. pylori infection has been associated with gastric diseases such as chronic gastritis, peptic ulcer and gastric adenocarcinoma. Current eradication treatment relies on antibiotic-based therapies that are unsuccessful in approximately 20% of the patients. Chitosan, a natural and cationic polysaccharide has been investigated in the treatment of H. pylori infection. Due to its mucoadhesive properties, it has been used in the form of micro/nanoparticles, polyelectrolyte complexes or coatings as antibiotic encapsulation systems for gastric delivery, but alternative molecules may also be incorporated. It has been recently proposed that chitosan can also be used for H. pylori binding and scavenging from the host stomach due to its antimicrobial/binding properties. In this manuscript, a brief description of the use of chitosan in H. pylori treatment is reviewed.