Radical-Mediated Degradation of Thiol-Maleimide Hydrogels.

Michael addition between thiol- and maleimide-functionalized molecules is a long-standing approach used for bioconjugation, hydrogel crosslinking, and the functionalization of other advanced materials. While the simplicity of this chemistry enables facile synthesis of hydrogels, network degradation is also desirable in many instances. Here, the susceptibility of thiol-maleimide bonds to radical-mediated degradation is reported. Irreversible degradation in crosslinked materials is demonstrated using photoinitiated and chemically initiated radicals in hydrogels and linear polymers. The extent of degradation is shown to be dependent on initiator concentration. Using a model linear polymer system, the radical-mediated mechanism of degradation is elucidated, in which the thiosuccinimide crosslink is converted to a succinimide and a new thioether formed with an initiator fragment. Using laser stereolithography, high-fidelity spatiotemporal control over degradation in crosslinked gels is demonstrated. Ultimately, this work establishes a platform for controllable, radical-mediated degradation in thiol-maleimide hydrogels, further expanding their versatility as functional materials.

Advancing Bone-Targeted Drug Delivery: Leveraging Biological Factors and Nanoparticle Designs to Improve Therapeutic Efficacy.

Designing targeted drug delivery systems to effectively treat bone diseases ranging from osteoporosis to nonunion bone defects remains a significant challenge. Previously, nanoparticles (NPs) self-assembled from diblock copolymers of poly(styrene-alt-maleic anhydride)-b-poly(styrene) (PSMA-b-PS) delivering a Wnt agonist were shown to effectively target bone and improve healing via the introduction of a peptide with high affinity to tartrate-resistant acid phosphatase (TRAP), an enzyme deposited by the osteoclasts during bone remodeling. Despite these promising results, the underlying biological factors governing targeting and subsequent drug delivery system (DDS) design parameters have not been examined to enable the rational design to improve bone selectivity. Therefore, this work investigated the effect of target ligand density, the treatment window after injury, specificity of TRAP binding peptide (TBP), the extent of TRAP deposition, and underlying genetic factors (e.g., mouse strain differences) on TBP-NP targeting. Data based on in vitro binding studies and in vivo biodistribution analyses using a murine femoral fracture model suggest that TBP-NP-TRAP interactions and TBP-NP bone accumulation were ligand-density-dependent; in vitro, TRAP affinity was correlated with ligand density up to the maximum of 200,000 TBP ligands/NP, while NPs with 80,000 TBP ligands showed 2-fold increase in fracture accumulation at day 21 post injury compared with that of untargeted or scrambled controls. While fracture accumulation exhibited similar trends when injected at day 3 compared to that at day 21 postfracture, there were no significant differences observed between TBP-functionalized and control NPs, possibly due to saturation of TRAP by NPs at day 3. Leveraging a calcium-depletion diet, TRAP deposition and TBP-NP bone accumulation were positively correlated, confirming that TRAP-TBP binding leads to TBP-NP bone accumulation in vivo. Furthermore, TBP-NP exhibited similar bone accumulation in both C57BL/6 and BALB/c mouse strains versus control NPs, suggesting the broad applicability of TBP-NP regardless of the underlying genetic differences. These studies provide insight into TBP-NP design, mechanism, and therapeutic windows, which inform NP design and treatment strategies for fractures and other bone-associated diseases that leverage TRAP, such as marrow-related hematologic diseases.

Engineering models of head and neck and oral cancers on-a-chip.

Head and neck cancers (HNCs) rank as the sixth most common cancer globally and result in over 450 000 deaths annually. Despite considerable advancements in diagnostics and treatment, the 5-year survival rate for most types of HNCs remains below 50%. Poor prognoses are often attributed to tumor heterogeneity, drug resistance, and immunosuppression. These characteristics are difficult to replicate using in vitro or in vivo models, culminating in few effective approaches for early detection and therapeutic drug development. Organs-on-a-chip offer a promising avenue for studying HNCs, serving as microphysiological models that closely recapitulate the complexities of biological tissues within highly controllable microfluidic platforms. Such systems have gained interest as advanced experimental tools to investigate human pathophysiology and assess therapeutic efficacy, providing a deeper understanding of cancer pathophysiology. This review outlines current challenges and opportunities in replicating HNCs within microphysiological systems, focusing on mimicking the soft, glandular, and hard tissues of the head and neck. We further delve into the major applications of organ-on-a-chip models for HNCs, including fundamental research, drug discovery, translational approaches, and personalized medicine. This review emphasizes the integration of organs-on-a-chip into the repertoire of biological model systems available to researchers. This integration enables the exploration of unique aspects of HNCs, thereby accelerating discoveries with the potential to improve outcomes for HNC patients.

Integrating osteoimmunology and nanoparticle-based drug delivery systems for enhanced fracture healing.

Fracture healing is a complex interplay of molecular and cellular mechanisms lasting from days to weeks. The inflammatory phase is the first stage of fracture healing and is critical in setting the stage for successful healing. There has been growing interest in exploring the role of the immune system and novel therapeutic strategies, such as nanoparticle drug delivery systems in enhancing fracture healing. Advancements in nanotechnology have revolutionized drug delivery systems to the extent that they can modulate immune response during fracture healing by leveraging unique physiochemical properties. Therefore, understanding the intricate interactions between nanoparticle-based drug delivery systems and the immune response, specifically macrophages, is essential for therapeutic efficacy. This review provides a comprehensive overview of the relationship between the immune system and nanoparticles during fracture healing. Specifically, we highlight the influence of nanoparticle characteristics, such as size, surface properties, and composition, on macrophage activation, polarization, and subsequent immune responses. IMPACT STATEMENT: This review provides valuable insights into the interplay between fracture healing, the immune system, and nanoparticle-based drug delivery systems. Understanding nanoparticle-macrophage interactions can advance the development of innovative therapeutic approaches to enhance fracture healing, improve patient outcomes, and pave the way for advancements in regenerative medicine.

Bone-Targeted Nanoparticle Drug Delivery System-Mediated Macrophage Modulation for Enhanced Fracture Healing.

Despite decades of progress, developing minimally invasive bone-specific drug delivery systems (DDS) to improve fracture healing remains a significant clinical challenge. To address this critical therapeutic need, nanoparticle (NP) DDS comprised of poly(styrene-alt-maleic anhydride)-b-poly(styrene) (PSMA-b-PS) functionalized with a peptide that targets tartrate-resistant acid phosphatase (TRAP) and achieves preferential fracture accumulation has been developed. The delivery of AR28, a glycogen synthase kinase-3 beta (GSK3ß) inhibitor, via the TRAP binding peptide-NP (TBP-NP) expedites fracture healing. Interestingly, however, NPs are predominantly taken up by fracture-associated macrophages rather than cells typically associated with fracture healing. Therefore, the underlying mechanism of healing via TBP-NP is comprehensively investigated herein. TBP-NPAR28 promotes M2 macrophage polarization and enhances osteogenesis in preosteoblast-macrophage co-cultures in vitro. Longitudinal analysis of TBP-NPAR28 -mediated fracture healing reveals distinct spatial distributions of M2 macrophages, an increased M2/M1 ratio, and upregulation of anti-inflammatory and downregulated pro-inflammatory genes compared to controls. This work demonstrates the underlying therapeutic mechanism of bone-targeted NP DDS, which leverages macrophages as druggable targets and modulates M2 macrophage polarization to enhance fracture healing, highlighting the therapeutic benefit of this approach for fractures and bone-associated diseases.

Development of a Nanoparticle-Based Tendon-Targeting Drug Delivery System to Pharmacologically Modulate Tendon Healing.

Tendon regeneration following acute injury is marred by a fibrotic healing response that prevents complete functional recovery. Despite the high frequency of tendon injuries and the poor outcomes, including functional deficits and elevated risk of re-injury, there are currently no pharmacological therapies in clinical use to enhance the healing process. Several promising pharmacotherapies have been identified; however, systemic treatments lack tendon specificity, resulting in poor tendon biodistribution and perhaps explaining the largely limited beneficial effects of these treatments on the tendon healing process. To address this major unmet need, we leveraged our existing spatial transcriptomics dataset of the tendon healing process to identify an area of the healing tendon that is enriched for expression of Acp5. Acp5 encodes tartrate-resistant acid phosphatase (TRAP), and we demonstrate robust TRAP activity in the healing tendon. This unexpected finding allowed us to refine and apply our existing TRAP binding peptide (TBP) functionalized nanoparticle (NP) drug delivery system (DDS) to facilitate improved delivery of systemic treatments to the healing tendon. To demonstrate the translational potential of this drug delivery system, we delivered the S100a4 inhibitor, Niclosamide to the healing tendon. We have previously shown that genetic knockdown of S100a4 enhances tendon healing. While systemic delivery of Niclosamide did not affect the healing process, relative to controls, TBP-NP delivery of Niclosamide enhanced both functional and mechanical outcome measures. Collectively, these data identify a novel tendon-targeting drug delivery system and demonstrate the translational potential of this approach to enhance the tendon healing process.

Slow hydrogel matrix degradation enhances salivary gland mimetic phenotype.

We recently developed a salivary gland tissue mimetic (SGm), comprised of salivary gland cells encapsulated in matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) hydrogels within arrays of ∼320 µm diameter spherical cavities molded in PDMS. The SGm provides a functional and physiologically relevant platform well-suited to high-throughput drug screening for radioprotective compounds. However, the utility of the SGm would benefit from improved retention of acinar cell phenotype and function. We hypothesized that tuning biochemical cues presented within the PEG hydrogel matrix would improve maintenance of acinar cell phenotype and function by mimicking the natural extracellular matrix microenvironment of the intact gland. Hydrogels formed using slower-degrading MMP-sensitive peptide crosslinkers showed >2-fold increase in sphere number formed at 48 h, increased expression of acinar cell markers, and more robust response to calcium stimulation by the secretory agonist, carbachol, with reduced SGm tissue cluster disruption and outgrowth during prolonged culture. The incorporation of adhesive peptides containing RGD or IKVAV improved calcium flux response to secretory agonists at 14 days of culture. Tuning the hydrogel matrix improved cell aggregation, and promoted acinar cell phenotype, and stability of the SGm over 14 days of culture. Furthermore, combining this matrix with optimized media conditions synergistically prolonged the retention of the acinar cell phenotype in SGm. STATEMENT OF SIGNIFICANCE: Salivary gland (SG) dysfunction occurs due to off-target radiation due to head and neck cancer treatments. Progress in understanding gland dysfunction and developing therapeutic strategies for the SG are hampered by the lack of in vitro models, as salivary gland cells rapidly lose critical secretory function within 24 hours in vitro. Herein, we identify properties of poly(ethylene glycol) hydrogel matrices that enhance the secretory phenotype of SG tissue mimetics within the previously-described SG-microbubble tissue chip environment. Combining slow-degrading hydrogels with media conditions optimized for secretory marker expression further enhanced functional secretory response and secretory marker expression.

Zwitterionic peptides: Tunable next-generation stealth nanoparticle modifications.

Adsorption of proteins to nanoparticles (NPs), a complex process that results in a protein corona, is controlled by NP surface properties that define NP interactions in vivo. Efforts to control adsorbed protein quantity through surface modification have led to improvements in circulation time or biodistribution. Still, current approaches have yet to be identified to control adsorbed protein identities within the corona. Here, we report the development and characterization of diverse zwitterionic peptides (ZIPs) for NP anti-fouling surface functionalization with specific and controllable affinity for protein adsorption profiles defined by ZIP sequence. Through serum exposure of ZIP-conjugated NPs and proteomics analysis of the resulting corona, we determined that protein adsorption profiles depend not on the exact composition of the ZIPs but on the sequence and order of charges along the sequence (charge motif). These findings pave the way for developing tunable ZIPs to orchestrate specific ZIP-NP protein adsorption profiles as a function of ZIP charge motif to better control cell and tissue specificity and pharmacokinetics and provide new tools for investigating relationships between protein corona and biological function. Furthermore, overall ZIP diversity enabled by the diversity of amino acids may ameliorate adaptive immune responses.

Impact of Nanoparticle Physicochemical Properties on Protein Corona and Macrophage Polarization.

Macrophages, the major component of the mononuclear phagocyte system, uptake and clear systemically administered nanoparticles (NPs). Therefore, leveraging macrophages as a druggable target may be advantageous to enhance NP-mediated drug delivery. Despite many studies focused on NP-cell interactions, NP-mediated macrophage polarization mechanisms are still poorly understood. This work aimed to explore the effect of NP physicochemical parameters (size and charge) on macrophage polarization. Upon exposure to biological fluids, proteins rapidly adsorb to NPs and form protein coronas. To this end, we hypothesized that NP protein coronas govern NP-macrophage interactions, uptake, and subsequent macrophage polarization. To test this hypothesis, model polystyrene NPs with various charges and sizes, as well as NPs relevant to drug delivery, were utilized. Data suggest that cationic NPs potentiate both M1 and M2 macrophage markers, while anionic NPs promote M1-to-M2 polarization. Additionally, anionic polystyrene nanoparticles (APNs) of 50 nm exhibit the greatest influence on M2 polarization. Proteomics was pursued to further understand the effect of NPs physicochemical parameters on protein corona, which revealed unique protein patterns based on NP charge and size. Several proteins impacting M1 and M2 macrophage polarization were identified within cationic polystyrene nanoparticles (CPNs) corona, while APNs corona included fewer M1 but more M2-promoting proteins. Nevertheless, size impacts protein corona abundance but not identities. Altogether, protein corona identities varied based on NP surface charge and correlated to dramatic differences in macrophage polarization. In contrast, NP size differentially impacts macrophage polarization, which is dominated by NP uptake level rather than protein corona. In this work, specific corona proteins were identified as a function of NP physicochemical properties. These proteins are correlated to specific macrophage polarization programs and may provide design principles for developing macrophage-mediated NP drug delivery systems.

Drug Delivery Approaches to Improve Tendon Healing.

Tendon injuries disrupt the transmission of forces from muscle to bone, leading to chronic pain, disability, and a large socioeconomic burden. Tendon injuries are prevalent; there are over 300,000 tendon repair procedures a year in the United States to address acute trauma or chronic tendinopathy. Successful restoration of function after tendon injury remains challenging clinically. Despite improvements in surgical and physical therapy techniques, the high complication rate of tendon repair procedures motivates the use of therapeutic interventions to augment healing. While many biological and tissue engineering approaches have attempted to promote scarless tendon healing, there is currently no standard clinical treatment to improve tendon healing. Moreover, the limited efficacy of systemic delivery of several promising therapeutic candidates highlights the need for tendon-specific drug delivery approaches to facilitate translation. This review article will synthesize the current state-of-the-art methods that have been used for tendon-targeted delivery through both systemic and local treatments, highlight emerging technologies used for tissue-specific drug delivery in other tissue systems, and outline future challenges and opportunities to enhance tendon healing through targeted drug delivery.

Hydrogel Swelling-Mediated Strain Induces Cell Alignment at Dentin Interfaces.

Cell and tissue alignment is a defining feature of periodontal tissues. Therefore, the development of scaffolds that can guide alignment of periodontal ligament cells (PDLCs) relative to tooth root (dentin) surfaces is highly relevant for periodontal tissue engineering. To control PDLC alignment adjacent to the dentin surface, poly(ethylene glycol) (PEG)-based hydrogels were explored as a highly tunable matrix for encapsulating cells and directing their activity. Specifically, a composite system consisting of dentin blocks, PEG hydrogels, and PDLCs was created to control PDLC alignment through hydrogel swelling. PDLCs in composites with minimal hydrogel swelling showed random alignment adjacent to dentin blocks. In direct contrast, the presence of hydrogel swelling resulted in PDLC alignment perpendicular to the dentin surface, with the degree and extension of alignment increasing as a function of swelling. Replicating this phenomenon with different molds, block materials, and cells, together with predictive modeling, indicated that PDLC alignment was primarily a biomechanical response to swelling-mediated strain. Altogether, this study describes a novel method for inducing cell alignment adjacent to stiff surfaces through applied strain and provides a model for the study and engineering of periodontal and other aligned tissues.

Farnesol delivery via polymeric nanoparticle carriers inhibits cariogenic cross-kingdom biofilms and prevents enamel demineralization.

Streptococcus mutans and Candida albicans are frequently detected together in the plaque from patients with early childhood caries (ECC) and synergistically interact to form a cariogenic cross-kingdom biofilm. However, this biofilm is difficult to control. Thus, to achieve maximal efficacy within the complex biofilm microenvironment, nanoparticle carriers have shown increased interest in treating oral biofilms in recent years. Here, we assessed the anti-biofilm efficacy of farnesol (Far), a hydrophobic antibacterial drug and repressor of Candida filamentous forms, against cross-kingdom biofilms employing drug delivery via polymeric nanoparticle carriers (NPCs). We also evaluated the effect of the strategy on teeth enamel demineralization. The farnesol-loaded NPCs (NPC+Far) resulted in a 2-log CFU/mL reduction of S. mutans and C. albicans (hydroxyapatite disc biofilm model). High-resolution confocal images further confirmed a significant reduction in exopolysaccharides, smaller microcolonies of S. mutans, and no hyphal form of C. albicans after treatment with NPC+Far on human tooth enamel (HT) slabs, altering the biofilm 3D structure. Furthermore, NPC+Far treatment was highly effective in preventing enamel demineralization on HT, reducing lesion depth (79% reduction) and mineral loss (85% reduction) versus vehicle PBS-treated HT, while NPC or Far alone had no differences with the PBS. The drug delivery via polymeric NPCs has the potential for targeting bacterial-fungal biofilms associated with a prevalent and costly pediatric oral disease, such as ECC.

Optimizing Soluble Cues for Salivary Gland Tissue Mimetics Using a Design of Experiments (DoE) Approach.

The development of therapies to prevent or treat salivary gland dysfunction has been limited by a lack of functional in vitro models. Specifically, critical markers of salivary gland secretory phenotype downregulate rapidly ex vivo. Here, we utilize a salivary gland tissue chip model to conduct a design of experiments (DoE) approach to test combinations of seven soluble cues that were previously shown to maintain or improve salivary gland cell function. This approach uses statistical techniques to improve efficiency and accuracy of combinations of factors. The DoE-designed culture conditions improve markers of salivary gland function. Data show that the EGFR inhibitor, EKI-785, maintains relative mRNA expression of Mist1, a key acinar cell transcription factor, while FGF10 and neurturin promote mRNA expression of Aqp5 and Tmem16a, channel proteins involved in secretion. Mist1 mRNA expression correlates with increased secretory function, including calcium signaling and mucin (PAS-AB) staining. Overall, this study demonstrates that media conditions can be efficiently optimized to support secretory function in vitro using a DoE approach.

Tissue Engineered Neurovascularization Strategies for Craniofacial Tissue Regeneration.

Craniofacial tissue injuries, diseases, and defects, including those within bone, dental, and periodontal tissues and salivary glands, impact an estimated 1 billion patients globally. Craniofacial tissue dysfunction significantly reduces quality of life, and successful repair of damaged tissues remains a significant challenge. Blood vessels and nerves are colocalized within craniofacial tissues and act synergistically during tissue regeneration. Therefore, the success of craniofacial regenerative approaches is predicated on successful recruitment, regeneration, or integration of both vascularization and innervation. Tissue engineering strategies have been widely used to encourage vascularization and, more recently, to improve innervation through host tissue recruitment or prevascularization/innervation of engineered tissues. However, current scaffold designs and cell or growth factor delivery approaches often fail to synergistically coordinate both vascularization and innervation to orchestrate successful tissue regeneration. Additionally, tissue engineering approaches are typically investigated separately for vascularization and innervation. Since both tissues act in concert to improve craniofacial tissue regeneration outcomes, a revised approach for development of engineered materials is required. This review aims to provide an overview of neurovascularization in craniofacial tissues and strategies to target either process thus far. Finally, key design principles are described for engineering approaches that will support both vascularization and innervation for successful craniofacial tissue regeneration.

Encapsulation of Primary Salivary Gland Acinar Cell Clusters and Intercalated Ducts (AIDUCs) within Matrix Metalloproteinase (MMP)-Degradable Hydrogels to Maintain Tissue Structure and Function.

Progress in the development of salivary gland regenerative strategies is limited by poor maintenance of the secretory function of salivary gland cells (SGCs) in vitro. To reduce the precipitous loss of secretory function, a modified approach to isolate intact acinar cell clusters and intercalated ducts (AIDUCs), rather than commonly used single cell suspension, is investigated. This isolation approach yields AIDUCs that maintain many of the cell-cell and cell-matrix interactions of intact glands. Encapsulation of AIDUCs in matrix metalloproteinase (MMP)-degradable PEG hydrogels promotes self-assembly into salivary gland mimetics (SGm) with acinar-like structure. Expression of Mist1, a transcription factor associated with secretory function, is detectable throughout the in vitro culture period up to 14 days. Immunohistochemistry also confirms expression of acinar cell markers (NKCC1, PIP and AQP5), duct cell markers (K7 and K5), and myoepithelial cell markers (SMA). Robust carbachol and ATP-stimulated calcium flux is observed within the SGm for up to 14 days after encapsulation, indicating that secretory function is maintained. Though some acinar-to-ductal metaplasia is observed within SGm, it is reduced compared to previous reports. In conclusion, cell-cell interactions maintained within AIDUCs together with the hydrogel microenvironment may be a promising platform for salivary gland regenerative strategies.

Nanoparticle-Mediated Delivery of Micheliolide Analogs to Eliminate Leukemic Stem Cells in the Bone Marrow.

Micheliolide (MCL) is a naturally occurring sesquiterpene lactone that selectively targets leukemic stem cells (LSCs), which persist after conventional chemotherapy for myeloid leukemias, leading to disease relapse. To overcome modest MCL cytotoxicity, analogs with ≈two-threefold greater cytotoxicity against LSCs are synthesized via late-stage chemoenzymatic C-H functionalization. To enhance bone marrow delivery, MCL analogs are entrapped within bone-targeted polymeric nanoparticles (NPs). Robust drug loading capacities of up to 20% (mg drug mg-1 NP) are obtained, with release dominated by analog hydrophobicity. NPs loaded with a hydrolytically stable analog are tested in a leukemic mouse model. Median survival improved by 13% and bone marrow LSCs are decreased 34-fold following NPMCL treatments versus controls. Additionally, selective leukemic cell and LSC cytotoxicity of the treatment versus normal hematopoietic cells is observed. Overall, these studies demonstrate that MCL-based antileukemic agents combined with bone-targeted NPs offer a promising strategy for eradicating LSCs.

Macrophage depletion increases target specificity of bone-targeted nanoparticles.

Despite efforts to achieve tissue selectivity, the majority of systemically administered drug delivery systems (DDSs) are cleared by the mononuclear phagocyte system (MPS) before reaching target tissues regardless of disease or injury pathology. Previously, we showed that while tartrate-resistant acid phosphatase (TRAP) binding peptide (TBP)-targeted polymeric nanoparticles (TBP-NP) delivering a bone regenerative Wnt agonist improved NP fracture accumulation and expedited healing compared with controls, there was also significant MPS accumulation. Here we show that TBP-NPs are taken up by liver, spleen, lung, and bone marrow macrophages (Mϕ), with 76 ± 4%, 49 ± 11%, 27 ± 9%, and 92 ± 5% of tissue-specific Mϕ positive for NP, respectively. Clodronate liposomes (CLO) significantly depleted liver and spleen Mϕ, resulting in 1.8-fold and 3-fold lower liver and spleen and 1.3-fold and 1.6-fold greater fracture and naïve femur accumulation of TBP-NP. Interestingly, depletion and saturation of MPS using 10-fold greater TBP-NP doses also resulted in significantly higher TBP-NP accumulation at lungs and kidneys, potentially through compensatory clearance mechanisms. The higher NP dose resulted in greater TBP-NP accumulation at naïve bone tissue; however, other MPS tissues (i.e., heart and lungs) exhibited greater TBP-NP accumulation, suggesting uptake by other cell types. Most importantly, neither Mϕ depletion nor saturation strategies improved fracture site selectivity of TBP-NPs, possibly due to a reduction of Mϕ-derived osteoclasts, which deposit the TRAP epitope. Altogether, these data support that MPS-mediated clearance is a key obstacle in robust and selective fracture accumulation for systemically administered bone-targeted DDS and motivates the development of more sophisticated approaches to further improve fracture selectivity of DDS.

Nanoparticle carrier co-delivery of complementary antibiofilm drugs abrogates dual species cariogenic biofilm formation in vitro.

BACKGROUND: Dental caries is a multifactorial disease caused by pathogenic biofilm. In particular, Streptococcus mutans synthesizes biofilm exopolysaccharides, while Candida albicans is associated with the development of severe carious lesions. AIM: This study aimed to prevent the formation of S. mutans and C. albicans biofilms by exploiting pH-sensitive nanoparticle carriers (NPCs) with high affinity to exopolysaccharides to increase the substantivity of multi-targeted antibiofilm drugs introduced topically in vitro. METHODS: Dual-species biofilms were grown on saliva-coated hydroxyapatite discs with sucrose. Twice-daily, 1.5 min topical treatment regimens of unloaded and drug-loaded NPC were used. Drugs included combinations of two or three compounds with distinct, complementary antibiofilm targets: tt-farnesol (terpenoid; bacterial acid tolerance, fungal quorum sensing), myricetin (flavonoid; exopolysaccharides inhibitor), and 1771 (lipoteichoic acid inhibitor; bacterial adhesion and co-aggregation). Biofilms were evaluated for biomass, microbial population, and architecture. RESULTS: NPC delivering tt-farnesol and 1771 with or without myricetin completely prevented biofilm formation by impeding biomass accumulation, bacterial and fungal population growth, and exopolysaccharide matrix deposition (vs. control unloaded NPC). Both formulations hindered acid production, maintaining the pH of spent media above the threshold for enamel demineralization. However, treatments had no effect on pre-established dual-species biofilms. CONCLUSION: Complementary antibiofilm drug-NPC treatments prevented biofilm formation by targeting critical virulence factors of acidogenicity and exopolysaccharides synthesis.

A novel peptide inhibitor of Dll4-Notch1 signalling and its pro-angiogenic functions.

BACKGROUND AND PURPOSE: The Dll4-Notch1 signalling pathway plays an important role in sprouting angiogenesis, vascular remodelling and arterial or venous specificity. Genetic or pharmacological inhibition of Dll4-Notch1 signalling leads to excessive sprouting angiogenesis. However, transcriptional inhibitors of Dll4-Notch1 signalling have not been described. EXPERIMENTAL APPROACH: We designed a new peptide targeting Notch signalling, referred to as TAT-ANK, and assessed its effects on angiogenesis. In vitro, tube formation and fibrin gel bead assay were carried out, using human umbilical vein endothelial cells (HUVECs). In vivo, Matrigel plug angiogenesis assay, a developmental retinal model and tumour models in mice were used. The mechanisms underlying TAT-ANK activity were investigated by immunochemistry, western blotting, immunoprecipitation, RT-qPCR and luciferase reporter assays. KEY RESULTS: The amino acid residues 179-191 in the G-protein-coupled receptor-kinase-interacting protein-1 (GIT1-ankyrin domain) are crucial for GIT1 binding to the Notch transcription repressor, RBP-J. We designed the peptide TAT-ANK, based on residues 179-191 in GIT1. TAT-ANK significantly inhibited Dll4 expression and Notch 1 activation in HUVECs by competing with activated Notch1 to bind to RBP-J. The analyses of biological functions showed that TAT-ANK promoted angiogenesis in vitro and in vivo by inhibiting Dll4-Notch1 signalling. CONCLUSIONS AND IMPLICATIONS: We synthesized and investigated the biological actions of TAT-ANK peptide, a new inhibitor of Notch signalling. This peptide will be of significant interest to research on Dll4-Notch1 signalling and to clinicians carrying out clinical trials using Notch signalling inhibitors. Furthermore, our findings will have important conceptual and therapeutic implications for angiogenesis-related diseases.