Intracellular calprotectin (S100A8/A9) facilitates DNA damage responses and promotes apoptosis in head and neck squamous cell carcinoma.

OBJECTIVES: In head and neck squamous cell carcinoma (HNSCC), poor prognosis and low survival rates are associated with downregulated calprotectin. Calprotectin (S100A8/A9) inhibits cancer cell migration and invasion and facilitates G2/M cell cycle arrest. We investigated whether S100A8/A9 regulates DNA damage responses (DDR) and apoptosis in HNSCC after chemoradiation. MATERIALS AND METHODS: Human HNSCC cases in TCGA were analyzed for relationships between S100A8/A9 and expression of apoptosis-related genes. Next, S100A8/A9-expressing and non-expressing carcinoma lines (two different lineages) were exposed to genotoxic agents and assessed for 53BP1 and γH2AX expression and percent of viable/dead cells. Finally, S100A8/A9-wild-type and S100A8/A9null C57BL/6j mice were treated with 4-NQO to induce oral dysplastic and carcinomatous lesions, which were compared for levels of 53BP1. RESULTS: In S100A8/A9-high HNSCC tumors, apoptosis-related caspase family member genes were upregulated, whereas genes limiting apoptosis were significantly downregulated based on TCGA analyses. After X-irradiation or camptothecin treatment, S100A8/A9-expressing carcinoma cells (i.e., TR146 and KB-S100A8/A9) showed significantly higher 53BP1 and γH2AX expression, DNA fragmentation, proportions of dead cells, and greater sensitivity to cisplatin than wild-type KB or TR146-S100A8/A9-KD cells. Interestingly, KB-S100A8/A9Δ113-114 cells showed similar 53BP1 and γH2AX levels to S100A8/A9-negative KB and KB-EGFP cells. After 4-NQO treatment, 53BP1 expression in oral lesions was significantly greater in calprotectin+/+ than S100A8/A9null mice. CONCLUSIONS: In HNSCC cells, intracellular calprotectin is strongly suggested to potentiate DDR and promote apoptosis in response to genotoxic agents. Hence, patients with S100A8/A9-high HNSCC may encounter more favorable outcomes because more tumor cells enter apoptosis with increased sensitivity to chemoradiation therapy.

In vitro physicochemical characterization of five root canal sealers and their influence on an ex vivo oral multi-species biofilm community.

AIM: To evaluate the physicochemical properties of five root canal sealers and assess their effect on an ex vivo dental plaque-derived polymicrobial community. METHODOLOGY: Dental plaque-derived microbial communities were exposed to the sealers (AH Plus [AHP], GuttaFlow Bioseal [GFB], Endoseal MTA [ESM], Bio-C sealer [BCS] and BioRoot RCS [BRR]) for 3, 6 and 18 h. The sealers' effect on the biofilm biomass and metabolic activity was quantified using crystal violet (CV) staining and MTT assay, respectively. Biofilm community composition and morphology were assessed by denaturing gradient gel electrophoresis (DGGE), 16S rRNA sequencing and scanning electron microscopy. The ISO6876:2012 specifications were followed to determine the setting time, radiopacity, flowability and solubility. Obturated acrylic teeth were used to assess the sealers' effect on pH. Surface chemical characterization was performed using SEM with coupled energy-dispersive spectroscopy. Data normality was assessed using the Shapiro-Wilk test. One-way anova and Tukey's tests were used to analyze data from setting time, radiopacity, flowability and solubility. Two-way anova and Dunnett's tests were used for the data analysis from CV, MTT and pH. 16S rRNA sequencing data were analyzed for alpha (Shannon index and Chao analysis) and beta diversity (Bray-Curtis dissimilarities). Differences in community composition were evaluated by analysis of similarity (p < .05). RESULTS: The sealers significantly influenced microbial community composition and morphology. All sealers complied with ISO6876:2012 requirements for setting time, radiopacity and flowability. Although only AHP effectively reduced the biofilm biomass, all sealers, except BRR, reduced biofilm metabolic activity. CONCLUSION: Despite adequate physical properties, none of the sealers tested prevented biofilm growth. Significant changes in community composition were observed. If observed in vivo, these changes could affect intracanal microbial survival, pathogenicity and treatment outcomes.

Calprotectin (S100A8/A9) Is an Innate Immune Effector in Experimental Periodontitis.

Upregulated in inflammation, calprotectin (complexed S100A8 and S100A9; S100A8/A9) functions as an innate immune effector molecule, promoting inflammation, and also as an antimicrobial protein. We hypothesized that antimicrobial S100A8/A9 would mitigate change to the local microbial community and promote resistance to experimental periodontitis in vivo. To test this hypothesis, S100A9-/- and wild-type (WT; S100A9+/+) C57BL/6 mice were compared using a model of ligature-induced periodontitis. On day 2, WT mice showed fewer infiltrating innate immune cells than S100A9-/- mice; by day 5, the immune cell numbers were similar. At 5 days post ligature placement, oral microbial communities sampled with swabs differed significantly in beta diversity between the mouse genotypes. Ligatures recovered from molar teeth of S100A9-/- and WT mice contained significantly dissimilar microbial genera from each other and the overall oral communities from swabs. Concomitantly, the S100A9-/- mice had significantly greater alveolar bone loss than WT mice around molar teeth in ligated sites. When the oral microflora was ablated by antibiotic pretreatment, differences disappeared between WT and S100A9-/- mice in their immune cell infiltrates and alveolar bone loss. Calprotectin, therefore, suppresses emergence of a dysbiotic, proinflammatory oral microbial community, which reduces innate immune effector activity, including early recruitment of innate immune cells, mitigating subsequent alveolar bone loss and protecting against experimental periodontitis.

Aggregated Solution Morphology of Poly(acrylic acid)-Poly(styrene) Block Copolymers Improves Drug Supersaturation Maintenance and Caco-2 Cell Membrane Permeation.

Amorphous solid dispersions of polymers and drugs have been shown to improve supersaturation maintenance of poorly water-soluble drugs. Herein, amorphous spray-dried dispersions (SDDs) of poly(acrylic acid)-polystyrene (PS-b-PAA) diblock copolymers with differing degrees of polymerization were prepared in aggregated and nonaggregated states with the Biopharmaceutical Classification System Class II drug, probucol (PBC). Specifically, PS90-b-PAA15, PS90-b-PAA80, PS38-b-PAA220, and PS38-b-PAA320 amphiphilic block polymers that covered a compositional range in the area of oral drug delivery were prepared to examine the role of molecular weight and controlled aggregation in promoting drug supersaturation and maintenance. In addition, hydrophilic homopolymers PAA20, PAA96, PAA226, and PAA392 were prepared as controls to evaluate the role of the block copolymer-based SDDs in PBC solubilization. Characterization such as powder X-ray diffraction, scanning electron microscopy, and dissolution tests under nonsink conditions were then performed to evaluate the SDDs. When comparing the block copolymer systems, polymers that were preaggregated into micellular structures prior to spray drying with the drug promoted higher drug solubility and maintenance than when the drug was formulated with molecularly dissolved PS-PAA block polymer. Interestingly, the aggregated PS90-b-PAA80 SDD with 25 wt % PBC achieved 100% burst release and maintained full supersaturation of PBC at pH 6.5 (physiological pH in the small intestine). Dissolution studies conducted at the pH of the stomach (pH = 1.2) show that a minimal amount of drug (∼10 µg/mL) was released, which could be used for protecting drugs from acidic environments (stomach) before reaching the small intestine. To evaluate drug bioavailability, in vitro Caco-2 cell assays were performed, which reveal that PAA-based excipients do not hinder drug permeation across the epithelial membrane and that PS90-b-PAA80 SDD with 25 wt % PBC achieved the highest membrane permeability coefficient. This work demonstrates that block copolymer-based SDDs capable of preaggregating into nanostructures may be a tunable drug-delivery platform that can improve solubility and supersaturation maintenance of Class II pharmaceutics while also not prohibiting bioavailability through model intestinal membranes. Indeed, this concept may be extended to accommodate a myriad of pharmaceutical and excipient structures.

Ternary Composite Nanofibers Containing Chondroitin Sulfate Scavenge Inflammatory Chemokines from Solution and Prohibit Squamous Cell Carcinoma Migration.

Recessive dystrophic epidermolysis bullosa (RDEB), an inherited disease featuring blistering wounds, causes constant inflammation that leads to the eventual development of an aggressive form of squamous cell carcinoma (RDEB SCC). The persistence of inflammatory chemokines such as MCP-1 and Il-8 in RDEB wounds may foster RDEB SCC carcinogenesis. We report the production of ternary composite nanofibers containing pullulan, chondroitin sulfate, and tannic acid as RDEB wound dressings. The swellable fibers are stable to hydration and absorb ∼500% their weight in water. The fibers remove ∼99% of MCP-1 from solution in <2 h. Scavenged media did not promote RDEB SCC migration.

Molecular Additives Significantly Enhance Glycopolymer-Mediated Transfection of Large Plasmids and Functional CRISPR-Cas9 Transcription Activation Ex Vivo in Primary Human Fibroblasts and Induced Pluripotent Stem Cells.

Fast, efficient, and inexpensive methods for delivering functional nucleic acids to primary human cell types are needed to advance regenerative medicine and cell therapies. Plasmid-based gene editing (such as with CRISPR-Cas9) can require the delivery of plasmids that are large (∼9.5-13 kbp) in comparison to common reporter plasmids (∼5-8 kbp). To develop more efficient plasmid delivery vehicles, we investigated the effect of plasmid size on the transfection of primary human dermal fibroblasts (HDFs) and induced pluripotent stem cells (iPSCs) using a heparin-treated trehalose-containing polycation (Tr4-heparin). Transfections with 4.7 kbp to 10 kbp plasmids exhibited high rates of polyplex internalization with both plasmid sizes. However, transfection with the large plasmid was nearly eliminated in HDFs and significantly reduced in iPSCs. Molecular additives were used to probe intracellular barriers to transfection. Chloroquine treatments were used to destabilize endosomes, and dexamethasone and thymidine were used to destabilize the nuclear envelope. Destabilizing the nuclear envelope resulted in significantly increased large-plasmid-transfection, indicating that nuclear localization may be more difficult for large plasmids. To demonstrate the potential clinical utility of this formulation, HDFs and iPSCs were treated with to dexamethasone-Tr4-heparin polyplexes encoding dCas9-VP64, synthetic transcription activator, targeted to collagen type VII. These transfections enhanced collagen expression in HDFs and iPSCs by 5- and 20-fold, respectively, compared to an untransfected control and were the more effective than the Lipofectamine 2000 control. Functional plasmid transfection efficiency can be significantly improved by nuclear destabilization, which could lead to improved development of nonviral vehicles for ex vivo CRISPR-Cas9 gene editing.

Heparin Enhances Transfection in Concert with a Trehalose-Based Polycation with Challenging Cell Types.

Improving the delivery of nucleic acids to diverse tissue types in culture is important for translating genome editing for regenerative cell therapies. Herein, we examine the effect of transfection media additives, such as the sulfated glycosaminoglycan heparin, in dramatically increasing pDNA delivery efficiency and transgene expression in a wide variety of cell types. Polyplexes formed by combining pDNA and Tr4, a cationic glycopolymer containing repeated trehalose and pentaethylenetetramine groups, were treated with low concentrations of heparin prior to in vitro transfection with plasmid DNA. Polyplex formulations were found to be stable and form ternary complexes upon heparin addition according to dynamic light scattering and ethidium bromide dye exclusion assays. Heparin-coated polyplexes offer significant increases (approximately 4-fold) in GFP expression compared to polyplexes prepared with Tr4 only in primary fibroblasts, U87, and HepG2 cells. Heparin was also shown to increase GFP expression in a linear, dose-dependent manner. The heparin-treated Tr4 polyplexes exhibited more than 50% higher cellular internalization with HepG2 cells while showing minimal increases with U87 and primary fibroblasts. Pharmacological inhibition was used to further understand the endocytic pathways taken during transfection in the presence and absence of heparin. It was found that heparin-treated polyplexes are endocytosed primarily through macropinocytosis and clathrin-mediated pathways, while Tr4 polyplexes without heparin appear to be internalized primarily via caveolae. Heparin appears to also modify the nuclear localization behavior of Tr4 polyplexes, which likely contributes to increased efficiency and transgene expression.

Fast, Efficient, and Gentle Transfection of Human Adherent Cells in Suspension.

We demonstrate a highly efficient method for gene delivery into clinically relevant human cell types, such as induced pluripotent stem cells (iPSCs) and fibroblasts, reducing the protocol time by one full day. To preserve cell physiology during gene transfer, we designed a microfluidic strategy, which facilitates significant gene delivery in a short transfection time (<1 min) for several human cell types. This fast, optimized and generally applicable cell transfection method can be used for rapid screening of different delivery systems and has significant potential for high-throughput cell therapy applications.

Zr- and Hf-based nanoscale metal-organic frameworks as contrast agents for computed tomography.

Nanoscale metal-organic frameworks (NMOFs) of the UiO-66 structure containing high Zr (37 wt%) and Hf (57 wt%) content were synthesized and characterized, and their potential as contrast agents for X-ray computed tomography (CT) imaging was evaluated. Hf-NMOFs of different sizes were coated with silica and poly(ethylene glycol) (PEG) to enhance biocompatibility, and were used for in vivo CT imaging of mice, showing increased attenuation in the liver and spleen.

Lipid-Coated Nanoscale Coordination Polymers for Targeted Delivery of Antifolates to Cancer Cells.

Nanoscale coordination polymers (NCPs) have been demonstrated as an interesting platform for drug delivery, as they possess many advantages over small-molecule chemotherapeutics, such as high payloads, lower systemic toxicity, tunability, and enhanced tumor uptake. Existing formulations for the delivery of methotrexate (MTX), an antifolate cancer drug, have very low drug loadings. Herein, we report the incorporation of MTX as a building block in an NCP formulation with exceptionally high drug loadings (up to 79.1 wt%) and the selective delivery of the NCP to cancer cells. Encapsulation of the NCP in a functionalized lipid bilayer allows for targeted delivery and controlled release to cancer cells. A phosphor can be doped into the NCPs for monitoring particle uptake by optical imaging. The lipid-coated and anisamide-targeted NCPs have superior in vitro efficacy against acute lymphoblastic leukemia cells when compared to free drug.