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Aim: The purpose of this study was to evaluate the antifungal activity of midazolam, alone and in association with azoles, against isolates of clinical Candida spp. in planktonic and biofilm form. Materials & methods: The antifungal activity was observed using the broth microdilution technique. Flow cytometry tests were performed to investigate the probable mechanism of action and the comet test and cytotoxicity test were applied to evaluate DNA damage. Results: Midazolam (MIDAZ) showed antifungal activity against planktonic cells (125-250 μg/ml) and reduced the viability of Candida spp. biofilms (125 a 2500 μg/ml). The interaction of MIDAZ against Candida spp. biofilms was observed through scanning electron microscopy, causing alteration of their appearance. Therefore, MIDAZ has antifungal potential against Candida spp.

Patients with diabetes mellitus (DM) have defective healing of bone fractures. It was previously shown that nonviral gene delivery of plasmid DNA (pDNA) that independently encodes bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2), acts synergistically to promote bone regeneration in a DM animal model. Additionally, both insulin (INS) and the hormonally active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1α,25(OH) 2 D 3 ) (VD3) have independently been shown to play key roles in regulating bone fracture healing in DM patients. However, these individual therapies fail to adequately stimulate bone regeneration, illustrating a need for novel treatment of bone fractures in diabetic patients. Here, the ability of local delivery of INS and VD3 along with BMP-2 and FGF-2 genes is investigated to promote bone formation ectopically in Type-2 diabetic rats. A composite consisting of VD3 and INS is developed that contains poly(lactic-co-glycolic acid) microparticles (MPs) embedded in a fibrin gel surrounded by a collagen matrix that is permeated with polyethylenimine (PEI)-(pBMP-2+pFGF-2) nanoplexes. Using a submuscular osteoinduction model, it is demonstrated that local delivery of INS, VD3, and PEI-(pBMP-2+pFGF-2) significantly improves bone generation compared to other treatments, thusimplicating this approach as a method to promote bone regeneration in DM patients with bone fractures., (© 2020 Wiley-VCH GmbH.)

Hydrogen sulfide (H 2 S) has emerged as a gaseous mediator capable of exhibiting many beneficial properties including cytoprotection, anti-inflammation, and vasodilation. The study presented here provides characterization of a poly(lactic acid) polymer with a functionalized 4-hydroxythiobenzamide (PLA-4HTB) capable of extended H 2 S release. The polymer was used to fabricate microparticles that can be potentially loaded with a drug allowing for co-release of the drug and H 2 S. Microparticles with the average diameter of 500 ± 207 nm were fabricated and shown to release 77.0 ± 1.76 µM of H 2 S over 4 weeks (release of H 2 S from 1 mg of particles). To test for the antioxidant properties of the PLA-4HTB microparticles, human embryonic kidney 293 cells were first incubated with PLA-4HTB microparticles and then oxidative stress was induced using CoCl 2 . Particle suspensions of 1 mg/mL were shown to protect cells resulting in reactive oxygen species (ROS) levels of superoxide that were similar to that of the control group. The microparticles fabricated from the PLA-4HTB released H 2 S over a sustained period of weeks to months, while providing protection from ROS. The microparticles described in this article represent a new platform technology that could be used to prevent and treat diseases caused by oxidative damage.

Geometric topographies are known to influence cellular differentiation toward specific phenotypes, but to date the range of features and type of substrates that can be easily fabricated to study these interactions is somewhat limited. In this study, an emerging technology, two-photon polymerization, is used to print topological patterns with varying feature-size and thereby study their effect on cellular differentiation. This technique offers rapid manufacturing of topographical surfaces with good feature resolution for shapes smaller than 3 µm. Human-induced pluripotent stem cells, when attached to these substrates or a non-patterned control for 1 week, express an array of genetic markers that suggest their differentiation toward a heterogeneous population of multipotent progenitors from all three germ layers. Compared to the topographically smooth control, small features (1.6 µm) encourage differentiation toward ectoderm while large features (8 µm) inhibit self-renewal. This study demonstrates the potential of using two-photon polymerization to study and control stem cell fate as a function of substrate interactions. The ability to tailor and strategically design biomaterials in this way can enable more precise and efficient generation or maintenance of desired phenotypes in vitro and in vivo., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Controlled drug delivery systems have been utilized to enhance the therapeutic effects of many drugs by delivering drugs in a time-dependent and sustained manner. Here, with the aid of 3D printing technology, drug delivery devices were fabricated and tested using a model drug (fluorophore: rhodamine B). Poly(ethylene glycol) dimethacrylate (PEGDMA) devices were fabricated using a two-photon polymerization (TPP) system and rhodamine B was homogenously entrapped inside the polymer matrix during photopolymerization. These devices were printed with varying porosity and morphology using varying printing parameters such as slicing and hatching distance. The effects of these variables on drug release kinetics were determined by evaluating device fluorescence over the course of one week. These PEGDMA-based structures were then investigated for toxicity and biocompatibility in vitro, where MTS assays were performed using a range of cell types including induced pluripotent stem cells (iPSCs). Overall, tuning the hatching distance, slicing distance, and pore size of the fabricated devices modulated the rhodamine B release profile, in each case presumably due to resulting changes in the motility of the small molecule and its access to structure edges. In general, increased spacing provided higher drug release while smaller spacing resulted in some occlusion, preventing media infiltration and thus resulting in reduced fluorophore release. The devices had no cytotoxic effects on human embryonic kidney cells (HEK293), bone marrow stromal stem cells (BMSCs) or iPSCs. Thus, we have demonstrated the utility of two-photon polymerization to create biocompatible, complex miniature devices with fine details and tunable release of a model drug., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Melanoma is an incurable disease for which alternative treatments to chemotherapy alone are sought. Here, using a melanoma model, we investigated the antitumor potential of combining ultrasound (US) with poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with doxorubicin (DOX). The aim was to achieve synergistic tumoricidal activity through direct and indirect US-mediated damage of tumor cells combined with sustained and potentially controllable release (when combined with US) of DOX from microspheres. An in vitro release assay demonstrated an ability of US to affect the release kinetics of DOX from DOX-loaded PLGA microspheres by inducing a 12% increase in the rate of release. In vitro viability assays demonstrated that combining US with DOX-loaded PLGA microspheres resulted in synergistic tumor cell (B16-F10 melanoma cells) killing. Melanoma-bearing mice were treated intratumorally with DOX (8 µg)-loaded microspheres and subjected to US treatment at the tumor site. This treatment could significantly extend survival (mean survival (MS) = 22.1 days) compared to untreated mice (MS = 10.4 days) and most other treatments, such as blank microspheres plus US (MS = 11.5 days) and DOX (8 µg)-loaded microspheres alone (MS = 13 days). The findings that immune checkpoint blockade did not significantly extend survival of mice treated with DOX (8 µg)-loaded microspheres plus US, and that tumor-free ("cured") mice were not protected from subsequent tumor rechallenge suggests minimal involvement of the adaptive immune response in the observed antitumor activity. Nevertheless, the synergistic increase in survival of melanoma-challenged mice treated with the combination of US and DOX-loaded microspheres implicates such a treatment methodology as a promising additional tool for combatting otherwise currently incurable cancers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Uterine serous carcinoma, one of the most aggressive types of endometrial cancer, is characterized by poor outcomes and mutations in the tumour suppressor p53. Our objective was to engender synthetic lethality to paclitaxel (PTX), the frontline treatment for endometrial cancer, in tumours with mutant p53 and enhance the therapeutic efficacy using polymeric nanoparticles (NPs). First, we identified the optimal NP formulation through comprehensive analyses of release profiles and cellular-uptake and cell viability studies. Not only were PTX-loaded NPs superior to PTX in solution, but the combination of PTX-loaded NPs with the antiangiogenic molecular inhibitor BIBF 1120 (BIBF) promoted synthetic lethality specifically in cells with the loss-of-function (LOF) p53 mutation. In a xenograft model of endometrial cancer, this combinatorial therapy resulted in a marked inhibition of tumour progression and extended survival. Together, our data provide compelling evidence for future studies of BIBF- and PTX-loaded NPs as a therapeutic opportunity for LOF p53 cancers.

Nuclear abnormalities (micronuclei and meta-nuclear changes) have been used as biomarkers to identify cell damages. As children are more vulnerable to the adverse effects of pollution when compared to adults, assessing genetic damage caused by environmental influences is of great interest. As such, the objective was to determine metanuclear (karyolysis, pycnosis, karyorrhexis, binucleated cells, chromosome bridges and micronuclei) in cells from the oral mucosa of children associated with the school environment, gender, exposure to cigarette smoke and vehicular traffic. Analyses of nuclear abnormalities were performed in exfoliated buccal cells of children from two public schools located in Dourados - MS. The data were analyzed through Kruskal-Wallis test considering a significance level of 5% (p < .05). The results showed that children exposed to cigarette smoke presented higher levels of nuclear abnormalities than children who were not usually exposed to this type of mutagenic and genotoxic agent, suggesting that such contaminants are related to clastogenic and aneugenic effects on DNA. Moreover, female children had higher amounts of nuclear abnormalities when compared to male children. With regards to the school environment, the study results indicated statistical differences in of term chromosomal abnormalities for schools A and B. Thus, it was possible to determine that children exposed to cigarette smoke are susceptible to further genetic damage than unexposed children, and female children may be more susceptible to genotoxic and mutagenic agents. This study contributes to the current knowledge on the mutagenic characteristics of human cells, supporting the adoption of preventive Public Health measures.

Bone fracture healing impairment related to systemic diseases such as diabetes can be addressed by growth factor augmentation. We previously reported that growth factors such as fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) work synergistically to encourage osteogenesis in vitro. In this report, we investigated if BMP-2 and FGF-2 together can synergistically promote bone repair in a leporine model of diabetes mellitus, a condition that is known to be detrimental to union. We utilized two kinds of plasmid DNA encoding either BMP-2 or FGF-2 formulated into polyethylenimine (PEI) complexes. The fabricated nanoplexes were assessed for their size, charge, in vitro cytotoxicity, and capacity to transfect human bone marrow stromal cells (BMSCs). Using diaphyseal long bone radial defects in a diabetic rabbit model it was demonstrated that co-delivery of PEI-(pBMP-2+pFGF-2) embedded in collagen scaffolds resulted in a significant improvement in bone regeneration compared to PEI-pBMP-2 embedded in collagen scaffolds alone. This study demonstrated that scaffolds loaded with PEI-(pBMP-2+pFGF-2) could be an effective way of promoting bone regeneration in patients with diabetes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Controlled drug delivery systems, that include sequential and/or sustained drug delivery, have been utilized to enhance the therapeutic effects of many current drugs by effectively delivering drugs in a time-dependent and repeatable manner. In this study, with the aid of 3D printing technology, a novel drug delivery device was fabricated and tested to evaluate sequential delivery functionality. With an alginate shell and a poly(lactic-co-glycolic acid) (PLGA) core, the fabricated tubes displayed sequential release of distinct fluorescent dyes and showed no cytotoxicity when incubated with the human embryonic kidney (HEK293) cell line or bone marrow stromal stem cells (BMSC). The controlled differential release of drugs or proteins through such a delivery system has the potential to be used in a wide variety of biomedical applications from treating cancer to regenerative medicine.

There exists a dire need for improved therapeutics to achieve predictable bone regeneration. Gene therapy using non-viral vectors that are safe and efficient at transfecting target cells is a promising approach to overcoming the drawbacks of protein delivery of growth factors. Here, we investigated the transfection efficiency, cytotoxicity, osteogenic potential and in vivo bone regenerative capacity of chemically modified ribonucleic acid (cmRNA) (encoding BMP-2) complexed with polyethylenimine (PEI) and made comparisons with PEI complexed with conventional plasmid DNA (encoding BMP-2). The polyplexes were fabricated at an amine (N) to phosphate (P) ratio of 10 and characterized for transfection efficiency using human bone marrow stromal cells (BMSCs). The osteogenic potential of BMSCs treated with these polyplexes was validated by determining the expression of bone-specific genes, osteocalcin and alkaline phosphatase as well as through the detection of bone matrix deposition. Using a calvarial bone defect model in rats, it was shown that PEI-cmRNA (encoding BMP-2)-activated matrices promoted significantly enhanced bone regeneration compared to PEI-plasmid DNA (BMP-2)-activated matrices. Our proof of concept study suggests that scaffolds loaded with non-viral vectors harboring cmRNA encoding osteogenic proteins may be a powerful tool for stimulating bone regeneration with significant potential for clinical translation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Hydrogen sulfide is emerging as a critically important molecule in medicine, yet there are few methods for the long-term delivery of molecules that degrade to release H 2 S. In this paper the first long-term release of a thiobenzamide that degrades to release H 2 S is described. A series of polymers were synthesized by the copolymerization of L-lactide and a lactide functionalized with 4-hydroxythiobenzamide. A new method to attach functional groups to a derivative of L-lactide is described based on the addition of a thiol to an α,β-unsaturated lactide using catalytic I 2 . This reaction proceeded under mild conditions and did not ring-open the lactone. The copolymers had molecular weights from 8 to 88 kg mol -1 with PDIs below 1.50. Two sets of microparticles were fabricated from a copolymer; the average diameters of the microparticles were 0.53 and 12 μm. The degradation of the smaller microparticles was investigated in buffered water to demonstrate the slow release of thiobenzamide over 4 weeks. Based on the ability to synthesize polymers with different loadings of thiobenzamide and that thiobenzamide is a known precursor to H 2 S, these particles provide a polymer-based method to deliver H 2 S over days to weeks.

The current need for organ and tissue replacement, repair, and regeneration for patients is continually growing such that supply is not meeting demand primarily due to a paucity of donors as well as biocompatibility issues leading to immune rejection of the transplant. In order to overcome these drawbacks, scientists have investigated the use of scaffolds as an alternative to transplantation. These scaffolds are designed to mimic the extracellular matrix (ECM) by providing structural support as well as promoting attachment, proliferation, and differentiation with the ultimate goal of yielding functional tissues or organs. Initial attempts at developing scaffolds were problematic and subsequently inspired an interest in 3D printing as a mode for generating scaffolds. Utilizing three-dimensional printing (3DP) technologies, ECM-like scaffolds can be produced with a high degree of complexity, where fine details can be included at a micrometer level. In this Review, the criteria for printing viable and functional scaffolds, scaffolding materials, and 3DP technologies used to print scaffolds for tissue engineering are discussed. Creating biofunctional scaffolds could potentially help to meet the demand by patients for tissues and organs without having to wait or rely on donors for transplantation., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: The aim of this study was to investigate whether the hind limbs or intestinal tract is the most important initiator of the inflammatory response secondary aortic clamping and hind limb ischemia/reperfusion injury., Methods: Blood samples of Wistar rats obtained from posterior cava vein, portal vein, and heart cavity during either laparotomy (control group, n=8) or laparotomy + 2 h of aortic clamping and bilateral hind limb ischemia (ischemia group, n=8), or 2 h after ischemia and 2 h of reperfusion (ischemia-reperfusion group, n=8) were assayed for interleukin 6 (IL-6) and C-reactive protein (CRP)., Results: Serum IL-6 at the heart (223.6+/-197.9 [10-832] pg/mL) was higher (p<0.001) than at both portal (133.08+/-108.52 [4-372] pg/mL) and posterior cava veins (127.58+/-109.15 [8-388] pg/mL). CRP was not significant different among groups., Conclusion: The splanchnic region is also a source of inflammatory response secondary to ischemia and reperfusion of the hind limbs.