Deformable Nanovesicles Synthesized through an Adaptable Microfluidic Platform for Enhanced Localized Transdermal Drug Delivery.

Phospholipid-based deformable nanovesicles (DNVs) that have flexibility in shape offer an adaptable and facile method to encapsulate diverse classes of therapeutics and facilitate localized transdermal delivery while minimizing systemic exposure. Here we report the use of a microfluidic reactor for the synthesis of DNVs and show that alteration of input parameters such as flow speeds as well as molar and flow rate ratios increases entrapment efficiency of drugs and allows fine-tuning of DNV size, elasticity, and surface charge. To determine the ability of DNV-encapsulated drug to be delivered transdermally to a local site, we synthesized, characterized, and tested DNVs carrying the fluorescently labeled hydrophilic bisphosphonate drug AF-647 zoledronate (AF647-Zol). AF647-Zol DNVs were lyophilized, resuspended, and applied topically as a paste to the calvarial skin of mice. High-resolution fluorescent imaging and confocal microscopy revealed significant increase of encapsulated payload delivery to the target tissue-cranial bone-by DNVs as compared to nondeformable nanovesicles (NVs) or aqueous drug solutions. Interestingly, NV delivery was not superior to aqueous drug solution. Our studies show that microfluidic reactor-synthesized DNVs can be produced in good yield, with high encapsulation efficiency, reproducibility, and stability after storage, and represent a useful vehicle for localized transdermal drug delivery

Augmentation of IFN-γ by bone marrow derived immune cells in the presence of severe suppression of IFN-γ in gingivae induced by zoledronic acid and denosumab in Hu-BLT mice model of ONJ

IntroductionThe potential mechanisms governing drug induced osteonecrosis of the jaw (ONJ) is not well understood, and is one of the objectives of this study. Thus, we tested the release of IFN-γ within different immune compartments including bone marrow and gingivae upon treatment with zoledronic acid (ZOL) and denosumab which are known to induce ONJ in susceptible individuals.MethodsWe used humanized-BLT mouse model for the in-vivo studies reported in this paper. To determine the effects of zoledronic acid and denosumab on IFN-γ secretion and NK cell-mediated cytotoxicity; peripheral blood, bone marrow, spleen and gingiva were obtained after the injection of ZOL and denosumab in mice.ResultsPercentages of B cells are much higher in wild-type mice whereas the proportions of immune subsets in humans and reconstituted hu-BLT peripheral-blood are similar. Therefore, hu-BLT mice are preferable model to study human disease, in particular, immune-pathologies induced by ZOL and denosumab. Both agents resulted in a severe suppression of IFN-γ in the gingiva, whereas they heightened the release of IFN-γ and NK cell-mediated cytotoxicity by the BM-derived immune cells. ZOL increased the IFN-γ secretion by the spleen and peripheral blood immune cells, whereas denosumab decreased the release IFN-γ by these cells significantly.DiscussionZOL and denosumab may likely suppress IFN-γ secretion in gingiva through different mechanisms. In addition, to the suppression of IFN-γ secretion, denosumab mediated effect could in part be due to the decrease in the bone resorptive function of osteoclasts due to the induction of antibody dependent cellular cytotoxicity and lysis of osteoclasts, whereas ZOL is able to mediate cell death of osteoclasts directly. Suppression of IFN-gamma in gingiva is largely responsible for the inhibition of immune cell function, leading to dysregulated osteoblastic and osteoclastic activities. Restoration of IFN-gamma in the local microenvironment may result in establishment of homeostatic balance in the gingiva and prevention of osteonecrosis of jaw

Deformable Nanovesicles Synthesized through an Adaptable Microfluidic Platform for Enhanced Localized Transdermal Drug Delivery

Phospholipid-based deformable nanovesicles (DNVs) that have flexibility in shape offer an adaptable and facile method to encapsulate diverse classes of therapeutics and facilitate localized transdermal delivery while minimizing systemic exposure. Here we report the use of a microfluidic reactor for the synthesis of DNVs and show that alteration of input parameters such as flow speeds as well as molar and flow rate ratios increases entrapment efficiency of drugs and allows fine-tuning of DNV size, elasticity, and surface charge. To determine the ability of DNV-encapsulated drug to be delivered transdermally to a local site, we synthesized, characterized, and tested DNVs carrying the fluorescently labeled hydrophilic bisphosphonate drug AF-647 zoledronate (AF647-Zol). AF647-Zol DNVs were lyophilized, resuspended, and applied topically as a paste to the calvarial skin of mice. High-resolution fluorescent imaging and confocal microscopy revealed significant increase of encapsulated payload delivery to the target tissue—cranial bone—by DNVs as compared to nondeformable nanovesicles (NVs) or aqueous drug solutions. Interestingly, NV delivery was not superior to aqueous drug solution. Our studies show that microfluidic reactor-synthesized DNVs can be produced in good yield, with high encapsulation efficiency, reproducibility, and stability after storage, and represent a useful vehicle for localized transdermal drug delivery

Augmentation of IFN-γ by bone marrow derived immune cells in the presence of severe suppression of IFN-γ in gingivae induced by zoledronic acid and denosumab in Hu-BLT mice model of ONJ

IntroductionThe potential mechanisms governing drug induced osteonecrosis of the jaw (ONJ) is not well understood, and is one of the objectives of this study. Thus, we tested the release of IFN-γ within different immune compartments including bone marrow and gingivae upon treatment with zoledronic acid (ZOL) and denosumab which are known to induce ONJ in susceptible individuals.MethodsWe used humanized-BLT mouse model for the in-vivo studies reported in this paper. To determine the effects of zoledronic acid and denosumab on IFN-γ secretion and NK cell-mediated cytotoxicity; peripheral blood, bone marrow, spleen and gingiva were obtained after the injection of ZOL and denosumab in mice.ResultsPercentages of B cells are much higher in wild-type mice whereas the proportions of immune subsets in humans and reconstituted hu-BLT peripheral-blood are similar. Therefore, hu-BLT mice are preferable model to study human disease, in particular, immune-pathologies induced by ZOL and denosumab. Both agents resulted in a severe suppression of IFN-γ in the gingiva, whereas they heightened the release of IFN-γ and NK cell-mediated cytotoxicity by the BM-derived immune cells. ZOL increased the IFN-γ secretion by the spleen and peripheral blood immune cells, whereas denosumab decreased the release IFN-γ by these cells significantly.DiscussionZOL and denosumab may likely suppress IFN-γ secretion in gingiva through different mechanisms. In addition, to the suppression of IFN-γ secretion, denosumab mediated effect could in part be due to the decrease in the bone resorptive function of osteoclasts due to the induction of antibody dependent cellular cytotoxicity and lysis of osteoclasts, whereas ZOL is able to mediate cell death of osteoclasts directly. Suppression of IFN-gamma in gingiva is largely responsible for the inhibition of immune cell function, leading to dysregulated osteoblastic and osteoclastic activities. Restoration of IFN-gamma in the local microenvironment may result in establishment of homeostatic balance in the gingiva and prevention of osteonecrosis of jaw

Neuronal PAS Domain 2 (Npas2)‐Deficient Fibroblasts Accelerate Skin Wound Healing and Dermal Collagen Reconstruction

The circadian clock, which consists of endogenous self-sustained and cell-autonomous oscillations in mammalian cells, is known to regulate a wide range of peripheral tissues. The unique upregulation of a clock gene, neuronal PAS domain protein 2 (Npas2), observed along with fibroblast aging prompted us to investigate the role of Npas2 in the homeostasis of dermal structure using in vivo and in vitro wound healing models. Time-course healing of a full-thickness skin punched wound exhibited significantly faster wound closure in Npas2-/- mice than wild-type (WT) C57Bl/6J mice. Dorsal skin fibroblasts isolated from WT, Npas2+/-, and Npas2-/- mice exhibited consistent profiles of core clock gene expression except for Npas2 and Per2. In vitro behavioral characterizations of dermal fibroblasts revealed that Npas2-/- mutation was associated with increased proliferation, migration, and cell contraction measured by floating collagen gel contraction and single-cell force contraction assays. Npas2 knockout fibroblasts carrying sustained the high expression level of type XII and XIV FAICT collagens and synthesized dermis-like thick collagen fibers in vitro. Confocal laser scanning microscopy demonstrated the reconstruction of dermis-like collagen architecture in the wound healing area of Npas2-/- mice. This study indicates that the induced Npas2 expression in fibroblasts may interfere with skin homeostasis, wound healing, and dermal tissue reconstruction, providing a basis for novel therapeutic target and strategy. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc

Neuronal PAS domain 2 (Npas2) facilitated osseointegration of titanium implant with rough surface through a neuroskeletal mechanism.

Titanium (Ti) biomaterials have been applied to a wide range of implantable medical devices. When placed in bone marrow, Ti-biomaterials integrate to the surrounding bone tissue by mechanisms that are not fully understood. We have previously identified an unexpected upregulation of circadian clock molecule neuronal PAS domain 2 (Npas2) in successfully integrated implant with a rough surface. This study aimed to elucidate the molecular mechanism of osseointegration through determining the role of Npas2. Human bone marrow stromal cells (BMSC) that were cultured on a Ti disc with SLA surface exhibited increased NPAS2 expression compared to BMSC cultured on a machined surface. A mouse model was developed in which miniature Ti implants were surgically placed into femur bone marrow. The implant push-out test and bone-to-implant contact measurements demonstrated the establishment of osseointegration in 3 weeks. By contrast, in Npas2 functional knockout (KO) mice, the implant push-out value measured for SLA surface Ti implant was significantly decreased. Npas2 KO mice demonstrated normal femur bone structure surrounding the Ti implant; however, the recovered implants revealed abnormal remnant mineralized tissue, which lacked dense collagen architecture typically found on recovered implants from wild type mice. To explore the mechanisms leading to the induced Npas2 expression, an unbiased chemical genetics analysis was conducted using mouse BMSC carrying an Npas2-reporter gene for high throughput screening of Library of Pharmacologically Active Compounds. Npas2 modulating compounds were found clustered in regulatory networks of the α2-adrenergic receptor and its downstream cAMP/CREB signaling pathway. Mouse primary BMSC exposed to SLA Ti disc significantly increased the expression of α2-adrenergic receptors, but the expression of β2-adrenergic receptor was unaffected. Our data provides the first evidence that peripheral clock gene component Npas2 plays a role in facilitating the enhanced osseointegration through neuroskeletal regulatory pathways induced by BMSC in contact with rough surface Ti implant