ß-Lactamase-Responsive Hydrogel Drug Delivery Platform for Bacteria-Triggered Cargo Release.

Antibiotic resistance is a growing public health threat that complicates the treatment of infections. ß-Lactamase enzymes, which hydrolyze the ß-lactam ring present in many common antibiotics, are a major cause of this resistance and are produced by a broad range of bacterial pathogens. Here, we developed hydrogels that degrade specifically in the presence of ß-lactamases and ß-lactamase-producing bacteria as a platform for bacteria-triggered drug delivery. A maleimide-functionalized ß-lactamase-cleavable cephalosporin was used as a crosslinker in the fabrication of hydrogels through end-crosslinked polymerization with multiarm thiol-terminated poly(ethylene glycol) macromers via Michael-type addition. We demonstrated that only hydrogels containing the responsive crosslinker were degraded by ß-lactamases and ß-lactamase-producing bacteria in vitro and in an ex vivo porcine skin infection model. Fluorescent polystyrene nanoparticles, encapsulated in the hydrogels as model cargo, were released at rates that closely tracked hydrogel wet mass loss, confirming ß-lactamase-triggered controlled cargo release. Nonresponsive hydrogels, lacking the ß-lactam crosslinker, remained stable in the presence of ß-lactamases and ß-lactamase-producing bacteria and exhibited no change in mass or nanoparticle release. Furthermore, the responsive hydrogels remained stable in non-ß-lactamase enzymes, including collagenases and lipases. These hydrogels have the potential to be used as a bacteria-triggered drug delivery system to control unnecessary exposure to encapsulated antimicrobials, which can provide effective infection treatment without exacerbating resistance.

ß-Lactamase triggered visual detection of bacteria using cephalosporin functionalized biomaterials.

We report the conjugation of a chromogenic cephalosporin ß-lactamase (ßL) substrate to polymers and integration into biomaterials for facile, visual ßL detection. Identification of these bacterial enzymes, which are a leading cause of antibiotic resistance, is critical in the treatment of infectious diseases. The ßL substrate polymer conjugate undergoes a clear to deep yellow color change upon incubation with common pathogenic Gram-positive and Gram-negative bacteria species. We have demonstrated the feasibility of formulating hydrogels with the ßL substrate covalently tethered to a poly(ethylene glycol) (PEG) polymer matrix, exhibiting a visible color change in the presence of ßLs. This approach has the potential to be used in diagnostic biomaterials for point-of-care detection of ßL-producing bacteria, helping combat the spread of drug resistant microbes.

Layer-by-Layer Biomaterials for Drug Delivery.

Controlled drug delivery formulations have revolutionized treatments for a range of health conditions. Over decades of innovation, layer-by-layer (LbL) self-assembly has emerged as one of the most versatile fabrication methods used to develop multifunctional controlled drug release coatings. The numerous advantages of LbL include its ability to incorporate and preserve biological activity of therapeutic agents; coat multiple substrates of all scales (e.g., nanoparticles to implants); and exhibit tuned, targeted, and/or responsive drug release behavior. The functional behavior of LbL films can be related to their physicochemical properties. In this review, we highlight recent advances in the development of LbL-engineered biomaterials for drug delivery, demonstrating their potential in the fields of cancer therapy, microbial infection prevention and treatment, and directing cellular responses. We discuss the various advantages of LbL biomaterial design for a given application as demonstrated through in vitro and in vivo studies.

Controlled delivery of a protein tyrosine phosphatase inhibitor, SHP099, using cyclodextrin-mediated host-guest interactions in polyelectrolyte multilayer films for cancer therapy.

The Src homology 2 domain containing protein tyrosine phosphatase-2 (SHP2) is a key enzyme in pathways regulating tumor growth signaling, and recently gained interest as a promising anticancer drug target. Many SHP2 inhibitors are currently under development, including SHP099, which has shown potent anticancer activity at low concentrations in vivo. In this work, we developed multilayer coatings for localized delivery of SHP099 to improve upon current cancer therapies. Layer-by-layer self-assembly was used to develop films composed of chitosan and poly-carboxymethyl-ß-cyclodextrin (PßCD) for the delivery of SHP099. SHP099 was successfully loaded into multilayer films via host-guest interactions with PßCD. Nuclear magnetic resonance spectroscopy confirmed the occurrence of this supramolecular assembly by identifying the interaction of specific terminal SHP099 protons with the protons of the CD. SHP099 release from assembled films was detected over 96 hours, and was found to inhibit colony formation of human breast adenocarcinoma cells in vitro. These multilayer films have the potential to be used in a range of anticancer applications and overcome common complications of systemic chemotherapeutic administration, while maximizing SHP099 efficacy.

Influence of poly-l-lysine molecular weight on antibacterial efficacy in polymer multilayer films.

Antibacterial coatings can prevent and treat medical device-associated infections. We examined the antibacterial properties of coatings assembled from poly-l-lysine (PLL) and hyaluronic acid (HA). PLL/HA films were fabricated using layer-by-layer assembly with three different PLL MWs, differentiated by number of repeat units, that is, 33, 91, and 407 (denoted by PLL30 , PLL90 , and PLL400 ). Films assembled with all three PLL MWs completely inhibited the growth of planktonic, gram-positive Staphylococcus aureus and methicillin-resistant S. aureus and gram-negative Pseudomonas aeruginosa and Escherichia coli over a 24-h exposure. All three film architectures also inhibited S. aureus attachment by ~60-70% compared to non-film-coated surfaces, likely attributed to significant film hydration and electrostatic repulsion due to HA. The true differences in antibacterial efficacy between different PLL MWs were observed upon repeated exposure of PLL/HA to S. aureus every 24 h. We found that PLL400 films lost the ability to inhibit planktonic S. aureus growth after one use while PLL30 and PLL90 films were effective over 4-5 and 9-13 repeated exposures, respectively. Our experiments indicated that differences in efficacy were related to low in-film mobility of PLL400 and also agreed with dissolution timescales for PLL30 and PLL90 films. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1324-1339, 2019.

Autocrine signaling is a key regulatory element during osteoclastogenesis.

Osteoclasts are responsible for bone destruction in degenerative, inflammatory and metastatic bone disorders. Although osteoclastogenesis has been well-characterized in mouse models, many questions remain regarding the regulation of osteoclast formation in human diseases. We examined the regulation of human precursors induced to differentiate and fuse into multinucleated osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL). High-content single cell microscopy enabled the time-resolved quantification of both the population of monocytic precursors and the emerging osteoclasts. We observed that prior to induction of osteoclast fusion, RANKL stimulated precursor proliferation, acting in part through an autocrine mediator. Cytokines secreted during osteoclastogenesis were resolved using multiplexed quantification combined with a Partial Least Squares Regression model to identify the relative importance of specific cytokines for the osteoclastogenesis outcome. Interleukin 8 (IL-8) was identified as one of RANKL-induced cytokines and validated for its role in osteoclast formation using inhibitors of the IL-8 cognate receptors CXCR1 and CXCR2 or an IL-8 blocking antibody. These insights demonstrate that autocrine signaling induced by RANKL represents a key regulatory component of human osteoclastogenesis.