One-Pot Microfluidics to Engineer Chitosan Nanoparticles Conjugated with Antimicrobial Peptides Using "Photoclick" Chemistry: Validation Using the Gastric Bacterium Helicobacter pylori.

Surface bioconjugation of antimicrobial peptides (AMP) onto nanoparticles (AMP-NP) is a complex, multistep, and time-consuming task. Herein, a microfluidic system for the one-pot production of AMP-NP was developed. Norbornene-modified chitosan was used for NP production (NorChit-NP), and thiolated-AMP was grafted on their surface via thiol-norbornene "photoclick" chemistry over exposure of two parallel UV LEDs. The MSI-78A was the AMP selected due to its high activity against a high priority (level 2) antibiotic-resistant gastric pathogen: Helicobacter pylori (H. pylori). AMP-NP (113 ± 43 nm; zeta potential 14.3 ± 7 mV) were stable in gastric settings without a cross-linker (up to 5 days in pH 1.2) and bactericidal against two highly pathogenic H. pylori strains (1011 NP/mL with 96 µg/mL MSI-78A). Eradication was faster for H. pylori 26695 (30 min) than for H. pylori J99 (24 h), which was explained by the lower minimum bactericidal concentration of soluble MSI-78A for H. pylori 26695 (32 µg/mL) than for H. pylori J99 (128 µg/mL). AMP-NP was bactericidal by inducing H. pylori cell membrane alterations, intracellular reorganization, generation of extracellular vesicles, and leakage of cytoplasmic contents (transmission electron microscopy). Moreover, NP were not cytotoxic against two gastric cell lines (AGS and MKN74, ATCC) at bactericidal concentrations. Overall, the designed microfluidic setup is a greener, simpler, and faster approach than the conventional methods to obtain AMP-NP. This technology can be further explored for the bioconjugation of other thiolated-compounds.

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.

Extracellular matrix electrospun membranes for mimicking natural renal filtration barriers.

Kidney diseases are recognized as a major health problem, which affect 10% of the population. Because currently available therapies have many limitations, some tissue engineering strategies have been emerging as promising approaches in this field. In this work, porcine kidneys were decellularized to obtain decellularized kidney extracellular matrix (dKECM).1 Our results demonstrate a successful protocol of decellularization characterized by the removal of nucleic acid material and preservation of collagen and glycosaminoglycans. Blends of polycaprolactone (PCL)2 and dKECM were prepared by electrospinning and characterized. The biological performance of the membranes was tested with a human kidney cell line (HK-2) for 7 days. It was observed that cellular metabolic activity, proliferation and protein content increased with an increase in dKECM concentrations (30, 50 and 70%). Additionally, the expression of zona occludens-1 was revealed on dKECM-containing membranes but not on pure PCL membranes. To the best of our knowledge this is the first time that natural extracellular matrix is used to mimic the kidney basement membrane as an in vitro model. This could be a valuable tool for regenerative nephrology and may have an impact on the development of kidney advanced therapies in the future.

Development of non-orthogonal 3D-printed scaffolds to enhance their osteogenic performance.

Three-dimensional (3D)-printed polycaprolactone (PCL)-based scaffolds have been extensively proposed for Tissue Engineering (TE) applications. Currently, the majority of the scaffolds produced are not representative of the complex arrangement of natural structures, since the internal morphologies follow an orthogonal and regular pattern. In order to produce scaffolds that more closely replicate the structure of the extracellular matrix (ECM) of tissues, herein both circular and sinusoidal scaffolds were fabricated and compared to their conventional orthogonal counterparts. This is an innovative, versatile and efficient strategy to 3D print PCL scaffolds with unique curved geometries. The morphology and the mechanical behavior of the scaffolds were assessed. The biological response was analyzed by evaluating the cell seeding efficiency, cell adhesion, proliferation, and osteogenic activity of an osteoblastic-like cell line seeded in these scaffolds. The scaffolds were designed and produced to have a similar porosity of about 56%. The non-orthogonal structures demonstrated lead to higher values of Young's modulus, both under dry conditions and when immersed in PBS. Moreover, the biological data corroborate that non-orthogonal scaffolds influence the cellular responses in a positive manner, namely in the osteogenic activity when compared with the orthogonal controls. These results suggest that introducing less orthogonal elements, which better mimic the tissue ECM and architecture, may have a positive influence on the cellular behavior, being a potential strategy to address bone tissue engineering applications.