We report a simple technique for the high throughput generation of tissue spheroids based on the encapsulation of dispersed adherent cells in particle-stabilized water-in-water emulsions.

Most current methods for the preparation of tissue spheroids require complex materials, involve tedious physical steps and are generally not scalable. We report a novel alternative, which is both inexpensive and up-scalable, to produce large quantities of viable human keratinocyte cell clusters (clusteroids). The method is based on a two-phase aqueous system of incompatible polymers forming a stable water-in-water (w/w) emulsion, which enabled us to rapidly fabricate cell clusteroids from HaCaT cells. We used w/w Pickering emulsion from aqueous solutions of the polymers dextran (DEX) and polyethylene oxide (PEO) and a particle stabilizer based on whey protein (WP). The HaCaT cells clearly preferred to distribute into the DEX-rich phase and this property was utilized to encapsulate them in the water-in-water (DEX-in-PEO) emulsion drops then osmotically shrank to compress them into clusters. Prepared formulations of HaCaT keratinocyte clusteroids in alginate hydrogel were grown where the cells percolated to mimic 3D tissue. The HaCaT cell clusteroids grew faster in the alginate film compared to the individual cells formulated in the same matrix. This methodology could potentially be utilised in biomedical applications.

Antimicrobial resistance (AMR) is one of the major threats to modern healthcare. Many types of bacteria have developed resistance to multiple antibiotic treatments, while additional antibiotics have not been recently brought to market. One approach to counter AMR based on the beta-lactamase enzyme has been to use cotreatments of an antibiotic and an inhibitor, to enhance the antibiotic action. Here, we aimed to enhance this technique by developing nanocarriers of two cationic beta-lactam class antibiotics, amoxicillin, and ticarcillin, combined with a beta-lactamase inhibitor, clavulanic acid, which can potentially overcome this type of AMR. We demonstrate for the first time that beta-lactamase inhibitor-loaded nanocarriers in cotreatments with either free or nanocarrier-loaded beta-lactam antibiotics can enhance their effectiveness further than when used alone. We use surfacefunctionalized shellac-/Poloxamer 407-stabilized antibiotic nanocarriers on Pseudomonas aeruginosa, which is susceptible to ticarcillin but is resistant to amoxicillin. We show an amplification of the antibiotic effect of amoxicillin and ticarcillin loaded in shellac nanoparticles, both alone and as a cotreatment with free or nanocarrier-loaded clavulanic acid. We also report a significant increase in the antimicrobial effects of clavulanic acid loaded in such nanocarriers as a cotreatment. We explain the increased antimicrobial activity of the cationically functionalized antibiotic-loaded nanoparticles with electrostatic attraction to the bacterial cell wall, which delivers higher local antibiotic and inhibitor concentrations. The effect is due to the accumulation of the clavulanic acid-loaded nanocarriers on the bacterial cell walls that allows a higher proportion of the inhibitor to engage with the produced intracellular betalactamases. These nanocarriers were also found to have a very low cytotoxic effect against human keratinocytes, which shows great potential for overcoming enzyme-based AMR.

We have developed and tested a novel ELISA-like approach for bacterial detection based upon selective adhesion of targeted bacteria to microwells with prefabricated bacterial bioimprints. Bioimprints were produced from three bacterial species; Escherichia coli (Gram-negative), Rhodococcus rhodochrous (Gram-positive) and Sarcina aurantiaca (Gram-negative), by using molding with curable silicone from dense layers of bacterial cells deposited on a glass substrate. We demonstrated that the surface functionalized whole cell bioimprints were able to selectively recognize and bind their own bacterial cell type. In order to detect target bacteria that are bound to the matching bioimprint, we also developed silica nanoparticles dual-functionalized with (3-glycidyloxypropyl)trimethoxysilane (GLYMO) coupled with 4-hydroxyphenylboronic acid (4-HPBA), SiO 2 NPs/GLYMO/4-HPBA, which were further conjugated with horseradish peroxidase (HRP). Bacterial detection was demonstrated to work in the established ELISA-like protocol using the colorimetric reaction of the conjugated HRP with 3,3′,5,5′tetramethylbenzidine (TMB). The bioimprints were used instead of capture antibodies and HRP-coated dual functionalized silica nanoparticles instead of a secondary antibody with TMB as the enzyme-converted reagent, producing a colored byproduct. This bacterial bioimprint-based detection method does not rely on any antibodies, uses stable and inexpensive reagents, and could potentially find application for rapid diagnostics of bacterial pathogens in clinical samples at the point of care. K E Y W O R D S bacteria, bioimprints, ELISA, functionalized silica nanoparticles, horse radish peroxidase 1 INTRODUCTION Bacterial infections remain as a serious issue in modern health care. [1-3] In recent years, this need for the rapid diagnosis of bacterial infections has resulted in the This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.