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Cancer stem cells (CSCs) have been much proposed as potential tumor eradication targets since they possess highly tumorigenic qualities. However, efficient and fast enrichment of CSCs for cancer biology study and drug screening has been challenging. CD44 is a cell surface receptor for hyaluronic acid (HA) and has been reported as an important CSC marker. Here, we show a simple and label-free method for the enrichment of CSCs highly expressing CD44 using enzymatically crosslinked HA hydrogels. HA hydrogels were formed with different crosslink densities to modulate the interaction between the CD44 and HA chains. We show that HA hydrogels with defined crosslink densities isolated cancer cells expressing high CD44 from breast cancer cell lines in a facile, efficient manner. The enriched cells exhibited CSC-like characteristics such as high expression of CSC markers (octamer-binding transcription factor 4 (OCT4) and aldehyde dehydrogenase 1 (ALDH1)), enhanced tumorsphere formation and chemoresistance. The enriched cells also displayed strong tumorigenicity, metastatic potential and poor survival in vivo. The HA hydrogel provides a simple, fast and efficient platform for CSC enrichment and promotes new anticancer strategies that target breast CSCs. STATEMENT OF SIGNIFICANCE: There is strong interest in developing isolation methods for cancer stem cells (CSCs), due in growing desire for CSC eradication for promising cancer therapy. Tumor sphere formation and fluorescence-activated cell sorting have been widely used for CSC isolation, while these methods require cultivation for several days and labelling of cell surface proteins, respectively. A simple and label-free method for breast CSC isolation is developed using HA-based hydrogels with tunable crosslink density. The efficient enrichment of breast CSCs is achieved by HA-CD44 specific interaction, which is controlled by hydrogel crosslink density. We believe that the simple approach that isolates cells with CSC-like characteristics would facilitate the anticancer drug development and cancer research.

Multidrug resistant (MDR) Klebsiella pneumoniae is a major cause of healthcare-associated infections around the world, with attendant high rates of morbidity and mortality. Progressive reduction in potency of antibiotics capable of treating MDR K. pneumoniae infections - including lung infection - as a consequence of escalating drug resistance provides the motivation to develop drug candidates targeting MDR K. pneumoniae. We recently reported degradable broad-spectrum antimicrobial guanidinium-functionalized polycarbonates with unique antimicrobial mechanism - membrane translocation followed by precipitation of cytosolic materials. These polymers exhibited high potency against bacteria with negligible toxicity. The polymer with ethyl spacer between the quanidinium group and the polymer backbone (pEt_20) showed excellent in vivo efficacy for treating MDR K. pneumoniae-caused peritonitis in mice. In this study, the structures of the polymers were optimized for the treatment of MDR Klebsiella pneumoniae lung infection. Specifically, in vitro antimicrobial activity and selectivity of guanidinium-functionalized polycarbonates containing the same number of guanidinium groups but of a shorter chain length and a structural analogue containing a thiouronium moiety as the pendent cationic group were evaluated. The polymers with optimal compositions and varying hydrophobicity were assessed against 25 clinically isolated K. pneumonia strains for antimicrobial activity and killing kinetics. The results showed that the polymers killed the bacteria more efficiently than clinically used antibiotics, and repeated use of the polymers did not cause drug resistance in K. pneumonia. Particularly, the polymer with butyl spacer (pBut_20) self-assembled into micelles at high concentrations, where the hydrophobic component was shielded in the micellar core, preventing interacting with mammalian cells. A subtle change in the hydrophobicity increased the antimicrobial activity while reducing in vivo toxicity. The in vivo efficacy studies showed that pBut_20 alleviated K. pneumonia lung infection without inducing damage to major organs. Taken together, pBut_20 is promising for treating MDR Klebsiella pneumoniae lung infection in vivo. STATEMENT OF SIGNIFICANCE: Multidrug resistant (MDR) Klebsiella pneumoniae is a major cause of healthcare-associated infections, with attendant high rates of morbidity and mortality. The progressive reduction in antibiotics capable of treating MDR K. pneumoniae infections - including lung infection - as a consequence of escalating drug resistance rates provides the motivation to develop drug candidates. In this study, we report a degradable guanidinium-functionalized polycarbonate with unexpected antimicrobial activity and selectivity towards MDR Klebsiella pneumoniae. A subtle change in polymer hydrophobicity increases antimicrobial activity while reducing in vivo toxicity due to self-assembly at high concentrations. The polymer with optimal composition alleviates Klebsiella pneumonia lung infection without inducing damage to major organs. The polymer is promising for treating MDR Klebsiella pneumoniae lung infection in vivo.

The propagation of human embryonic stem cells (hESCs) in three-dimensional (3D) scaffolds facilitates the cell expansion process and supplies pluripotent cells of high quality for broad-spectrum applications in regenerative medicine. Herein, we report an enzyme-mediated hyaluronic acid–tyramine (HA–Tyr) hydrogel that encapsulated and propagated hESCs in 3D. HA–Tyr hydrogels were formed by crosslinking the tyramine moieties with horseradish peroxidase (HRP) and hydrogen peroxide (H 2 O 2 ). By changing the HRP and H 2 O 2 concentration, we prepared HA–Tyr hydrogels of different mechanical strength and studied the self-renewal properties of hESCs in these scaffolds. We observed that both the chemical composition and mechanical strength of substrates were important factors affecting cell proliferation and pluripotency. The HA–Tyr hydrogel with a compressive modulus of ∼350 Pa supported the proliferation of hESCs at the pluripotent state in both mTeSR1 medium and mouse embryonic fibroblast (MEF)-conditioned medium. Immunohistochemical analyses revealed that hESCs proliferated well and formed spheroid structures in 3D, without undergoing apoptosis. The hESCs cultured in HA–Tyr hydrogels showed high expression of CD44 and pluripotency markers. These cells exhibited the capability to form cell derivatives of all three embryonic germ layers in vitro and in vivo . In addition, the genetic integrity of the hESCs was unaffected in the 3D cultivation system. Statement of Significance The scope of this study is to provide a stable 3D cultivation system for the expansion of human embryonic stem cells (hESCs) towards clinical applications. We report an enzyme mediated hyaluronic acid–tyramine (HA–Tyr) hydrogel that encapsulated and propagated hESCs in 3D. Unlike other HA-based photo-crosslinked hydrogel systems reported, we investigated the effects of mechanical strength of hydrogels on the self-renewal properties of hESCs in 3D. Then, we characterized hESCs cultured in hydrogels with lower mechanical strength that best supported the self-renewal of hESCs. Hence, we demonstrated a reliable approach for the controlled propagation of hESCs in 3D. We believe that such an approach would facilitate the development of stem cell-based therapy towards clinical applications.

Amphotericin B (AmB), a poorly soluble and toxic antifungal drug, was encapsulated into polymeric micelles self-assembled from phenylboronic acid-functionalized polycarbonate/PEG (PEG-PBC) and urea-functionalized polycarbonate/PEG (PEG-PUC) diblock copolymers via hydrogen-bonding, boronate ester bond, and/or ionic interactions between the boronic acid group in the micellar core and amine group in AmB. Three micellar formulations were prepared: AmB/B micelles using PEG-PBC, AmB/U micelles using PEG-PUC and AmB/B+U mixed micelles using 1:1 molar ratio of PEG-PBC and PEG-PUC. The average particle sizes of the micelles were in the range of 54.4–84.8 nm with narrow size distribution and zeta potentials close to neutral. UV–Vis absorption analysis indicated that AmB/B micelles significantly reduced AmB aggregation status due to the interactions between AmB and the micellar core, while Fungizone® and AmB/U micelles had no effect. AmB/B+U mixed micelles exerted an intermediate effect. Both AmB/B micelles and AmB/B+U mixed micelles showed sustained drug release, with 48.6 ± 2.1% and 59.2 ± 1.8% AmB released respectively after 24 h under sink conditions, while AmB/U micelles displayed a burst release profile. All AmB-loaded micelles showed comparable antifungal activity to free AmB or Fungizone®, while AmB/B micelles and AmB/B+U mixed micelles were much less hemolytic than other formulations. Histological examination showed that AmB/B and AmB/B+U micelles led to a significantly lower number of apoptotic cells in the kidneys compared to Fungizone®, suggesting reduced nephrotoxicity of the micellar formulations in vivo. These phenylboronic acid-functionalized polymeric micelle systems are promising drug carriers for AmB to reduce non-specific toxicities without compromise in antifungal activity. Statement of Significance There is a pressing need for a novel and cost-effective delivery system to reduce the toxicity induced by the antifungal agent, amphotericin B (AmB). In this study, phenylboronic acid-functionalized polycarbonate/PEG diblock copolymers were used to fabricate micelles for improved AmB-micelle interaction via the manipulation of hydrogen-bonding, boronate ester bond, ionic and hydrophobic interactions. Compared to free AmB and Fungizone®, the resultant micellar systems displayed improved stability while reducing non-specific toxicities without a compromise in antifungal activity. These findings demonstrate the potential of biodegradable functional polycarbonate micellar systems as promising carriers of AmB for the treatment of systemic fungal infections.