scholarly journals Survival and Proliferation under Severely Hypoxic Microenvironments Using Cell-Laden Oxygenating Hydrogels

2021 ◽  
Vol 12 (2) ◽  
pp. 30
Author(s):  
Shabir Hassan ◽  
Berivan Cecen ◽  
Ramon Peña-Garcia ◽  
Fernanda Roberta Marciano ◽  
Amir K. Miri ◽  
...  
Keyword(s):  
Prolonged Release ◽  
Therapeutic Approaches ◽  
Encapsulated Cells ◽  
Hypoxic Conditions ◽  
Skeletal Myoblasts ◽  
Artificial Tissue ◽  
Delivery Platform ◽  
Living Tissues

Different strategies have been employed to provide adequate nutrients for engineered living tissues. These have mainly revolved around providing oxygen to alleviate the effects of chronic hypoxia or anoxia that result in necrosis or weak neovascularization, leading to failure of artificial tissue implants and hence poor clinical outcome. While different biomaterials have been used as oxygen generators for in vitro as well as in vivo applications, certain problems have hampered their wide application. Among these are the generation and the rate at which oxygen is produced together with the production of the reaction intermediates in the form of reactive oxygen species (ROS). Both these factors can be detrimental for cell survival and can severely affect the outcome of such studies. Here we present calcium peroxide (CPO) encapsulated in polycaprolactone as oxygen releasing microparticles (OMPs). While CPO releases oxygen upon hydrolysis, PCL encapsulation ensures that hydrolysis takes place slowly, thereby sustaining prolonged release of oxygen without the stress the bulk release can endow on the encapsulated cells. We used gelatin methacryloyl (GelMA) hydrogels containing these OMPs to stimulate survival and proliferation of encapsulated skeletal myoblasts and optimized the OMP concentration for sustained oxygen delivery over more than a week. The oxygen releasing and delivery platform described in this study opens up opportunities for cell-based therapeutic approaches to treat diseases resulting from ischemic conditions and enhance survival of implants under severe hypoxic conditions for successful clinical translation.

scholarly journals Biochemical, Ameliorative and Cytotoxic Effects of Newly Synthesized Curcumin Microemulsions: Evidence from In Vitro and In Vivo Studies

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 817
Author(s):  
Abbas Rahdar ◽  
Mohammad Reza Hajinezhad ◽  
Saman Sargazi ◽  
Maryam Zaboli ◽  
Mahmood Barani ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Density Functional ◽  
Liver Enzymes ◽  
Elevated Serum ◽  
Ethyl Butyrate ◽  
Polymeric Chain ◽  
Prolonged Release ◽  
Advanced Therapies

Curcumin is known to exhibit antioxidant and tissue-healing properties and has recently attracted the attention of the biomedical community for potential use in advanced therapies. This work reports the formulation and characterization of oil-in-water F127 microemulsions to enhance the bioavailability of curcumin Microemulsions showed a high encapsulation efficiency and prolonged release. To investigate the interactions of curcumin with one unit of the polymeric chain of surfactant F127, ethyl butyrate, and sodium octanoate, as well as the interaction between ethyl butyrate and one unit of the F127 polymer chain, the Density Functional Theory (DFT) calculations at the M06-2X level of theory, were performed in water solution. The MTT assay was used to assess the cytotoxicity of free and encapsulated curcumin on non-malignant and malignant cell lines. Combination effects were calculated according to Chou-Talalay’s principles. Results of in vitro studies indicated that MCF7 and HepG2 cells were more sensitive to curcumin microemulsions. Moreover, a synergistic relationship was observed between curcumin microemulsions and cisplatin in all affected fractions of MCF7 and HepG2 cells (CI < 0.9). For in vivo investigation, thioacetamide-intoxicated rats received thioacetamide (100 mg/kg Sc) followed by curcumin microemulsions (30 mg/kg Ip). Thioacetamide-intoxicated rats showed elevated serum liver enzymes, blood urea nitrogen (BUN), and creatinine levels, and a significant reduction in liver superoxide dismutase (SOD) and catalase (CAT) activities (p < 0.05). Curcumin microemulsions reduced liver enzymes and serum creatinine and increased the activity of antioxidant enzymes in thioacetamide-treated rats in comparison to the untreated thioacetamide-intoxicated group. Histopathological investigations confirmed the biochemical findings. Overall, the current results showed the desirable hepatoprotective, nephroprotective, and anti-cancer effects of curcumin microemulsions.

scholarly journals A therapeutic vascular conduit to support in vivo cell-secreted therapy

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Edward X. Han ◽  
Hong Qian ◽  
Bo Jiang ◽  
Maria Figetakis ◽  
Natalia Kosyakova ◽  
...  
Keyword(s):  
Vascular Graft ◽  
Large Scale ◽  
Biological Effects ◽  
Therapeutic Protein ◽  
Close Proximity ◽  
Delivery Platform ◽  
Stage Renal Disease ◽  
End Stage

AbstractA significant barrier to implementation of cell-based therapies is providing adequate vascularization to provide oxygen and nutrients. Here we describe an approach for cell transplantation termed the Therapeutic Vascular Conduit (TVC), which uses an acellular vessel as a scaffold for a hydrogel sheath containing cells designed to secrete a therapeutic protein. The TVC can be directly anastomosed as a vascular graft. Modeling supports the concept that the TVC allows oxygenated blood to flow in close proximity to the transplanted cells to prevent hypoxia. As a proof-of-principle study, we used erythropoietin (EPO) as a model therapeutic protein. If implanted as an arteriovenous vascular graft, such a construct could serve a dual role as an EPO delivery platform and hemodialysis access for patients with end-stage renal disease. When implanted into nude rats, TVCs containing EPO-secreting fibroblasts were able to increase serum EPO and hemoglobin levels for up to 4 weeks. However, constitutive EPO expression resulted in macrophage infiltration and luminal obstruction of the TVC, thus limiting longer-term efficacy. Follow-up in vitro studies support the hypothesis that EPO also functions to recruit macrophages. The TVC is a promising approach to cell-based therapeutic delivery that has the potential to overcome the oxygenation barrier to large-scale cellular implantation and could thus be used for a myriad of clinical disorders. However, a complete understanding of the biological effects of the selected therapeutic is absolutely essential.

scholarly journals MBRS-48. IDENTIFICATION OF NOVEL THERAPEUTIC APPROACHES FOR MYC-DRIVEN MEDULLOBLASTOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii406-iii406
Author(s):  
Kübra Taban ◽  
David Pauck ◽  
Mara Maue ◽  
Viktoria Marquardt ◽  
Hua Yu ◽  
...  
Keyword(s):  
New Drugs ◽  
Current Therapy ◽  
Cancer Drug ◽  
Mrna Levels ◽  
Cell Models ◽  
Therapeutic Approaches ◽  
Group 3 ◽  
Novel Therapeutic

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children and is frequently metastatic at diagnosis. Treatment with surgery, radiation and multi-agent chemotherapy may leave survivors of these brain tumors with long-term deficits as a consequence. One of the four consensus molecular subgroups of MB is the MYC-driven group 3 MB, which is the most malignant type and has a poor prognosis under current therapy. Thus, it is important to discover more effective targeted therapeutic approaches. We conducted a high-throughput drug screening to identify novel compounds showing efficiency in group 3 MB using both clinically established inhibitors (n=196) and clinically-applicable compounds (n=464). More than 20 compounds demonstrated a significantly higher anti-tumoral effect in MYChigh (n=7) compared to MYClow (n=4) MB cell models. Among these compounds, Navitoclax and Clofarabine showed the strongest effect in inducing cell cycle arrest and apoptosis in MYChigh MB models. Furthermore, we show that Navitoclax, an orally bioavailable and blood-brain barrier passing anti-cancer drug, inhibits specifically Bcl-xL proteins. In line, we found a significant correlation between BCL-xL and MYC mRNA levels in 763 primary MB patient samples (Data source: “R2 https://hgserver1.amc.nl”). In addition, Navitoclax and Clofarabine have been tested in cells obtained from MB patient-derived-xenografts, which confirmed their specific efficacy in MYChigh versus MYClow MB. In summary, our approach has identified promising new drugs that significantly reduce cell viability in MYChigh compared to MYClow MB cell models. Our findings point to novel therapeutic vulnerabilities for MB that need to be further validated in vitro and in vivo.

scholarly journals 708 Application of a novel mSENS drug delivery technology for mRNA therapeutics

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A750-A750
Author(s):  
Sojin Lee ◽  
Joon Young Park ◽  
Goo-Young Kim ◽  
Sang Woo Jo ◽  
Minhyuk Yun ◽  
...  
Keyword(s):  
Particle Size ◽  
Protein Expression ◽  
Cancer Vaccine ◽  
Encapsulation Efficiency ◽  
Tumor Model ◽  
Cell Responses ◽  
Delivery Platform ◽  
Mrna Therapeutics

BackgroundSuccessful clinical translation of mRNA therapeutics requires an appropriate delivery strategy to overcome instability of mRNA and facilitate cellular uptake into the cells.1 Several lipid based nanoparticle approaches that encapsulate mRNA, notably lipid nanoparticle (LNP), have been developed, but their efficiency for delivery to certain target tissues and toxicity profiles still have room for improvement. The application of a novel polymer based nanoparticle technology platform, so called Stability Enhanced Nano Shells (SENS) for mRNA (mSENS) as a mRNA delivery platform for a cancer vaccine was demonstrated.MethodsThe physicochemical properties of mSENS formulation, particle size and encapsulation efficiency, were characterized using dynamic light scattering (DLS) and gel retardation assay. Using luciferase-encoding mRNA, the protein expression levels in vitro and in vivo were evaluated by luciferase assay or bioluminescence imaging (BLI), respectively. For cancer vaccine studies, antigen (tyrosinase-related protein 2 (Trp-2))-specific T cell responses were assessed by immunophenotyping mouse splenocytes using flow cytometry and by the enzyme-linked immunosorbent spot (ELISPOT) assay. The anti-tumor efficacy was studied in B16F10 lung tumor model in C57BL/6 mice. Liver and systemic toxicity of mSENS treated mice was evaluated through blood chemistry and complete blood count (CBC) tests.ResultsA library of mSENS formulations complexed with luciferase-encoding mRNA, were characterized for their particle size, surface charge, encapsulation efficiency, colloidal stability, and in vitro and in vivo luciferase protein expression level. Upon systemic administration in mice, varying biodistribution profiles were observed, implicating the potential for tailored delivery to target tissues. Particularly, cancer vaccine application was further developed leveraging the formulation with preferential spleen delivery. Following vaccination with Trp-2 mRNA encapsulated with mSENS (Trp-2 mRNA-mSENS) in B16F10 tumor bearing mice, strong Trp-2 antigen-specific IFN-γ T-cell responses were observed. Generated anti-tumor immunity also marked suppression of B16F10 lung tumors were observed in Trp-2-mSENS immunized mice compared to non-immunized controls, demonstrating the potential of mSENS as a mRNA delivery platform for the application for vaccine.ConclusionsProprietary biodegradable polymer based-mSENS platform offers an attractive delivery strategy for mRNA by tailoring to specific therapeutic applications. Depending on the application, whether it’s a vaccine or protein replacement, a rationally designed mSENS formulation can efficiently distribute mRNA to specific tissues. In particular, application of a splenic mSENS formulation for a cancer vaccine has been demonstrated in murine tumor model. In summary, mRNA delivery through mSENS platform is expected to provide significant opportunities in clinical development for mRNA therapeutics.Ethics ApprovalThe study was approved by Samyang Biopharmaceuticals’ IACUC (Institutional Animal Care and Use Committee), approval number SYAU-2027.ReferencePiotr S. Kowalski, Arnab Rudra, Lei Miao, and Daniel G. Anderson, delivering the messenger: advances in technologies for therapeutic mRNA delivery. Molecular Therapy Vol. 27 No 4 April 2019.

scholarly journals miR-206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro

2019 ◽  
Author(s):  
Roza K. Przanowska ◽  
Ewelina Sobierajska ◽  
Zhangli Su ◽  
Kate Jensen ◽  
Piotr Przanowski ◽  
...  
Keyword(s):  
Muscle Function ◽  
Skeletal Muscle Regeneration ◽  
C2c12 Myoblasts ◽  
Skeletal Myoblasts ◽  
Microrna Targets ◽  
Microrna Family ◽  
Differentiation Pathways

AbstractmiR-206, miR-1a-1 and miR-1a-2 are induced during differentiation of skeletal myoblasts and promote myogenesis in vitro. miR-206 is required for skeletal muscle regeneration in vivo. Although this microRNA family is hypothesized to play an essential role in differentiation, a triple knockout of the three genes has not been done to test this hypothesis. We report that triple KO C2C12 myoblasts generated using CRISPR/Cas9 method differentiate despite the expected de-repression of the microRNA targets. Surprisingly, their mitochondrial function is diminished. Triple KO mice demonstrate partial embryonic lethality, most likely due to the role of miR-1a in cardiac muscle differentiation. Two triple KO mice survive and grow normally to adulthood with smaller myofiber diameter and diminished physical performance. Thus, unlike other microRNAs important in other differentiation pathways, the miR-206 family is not absolutely essential for myogenesis and is instead a modulator of optimal differentiation of skeletal myoblasts.

scholarly journals ROS-triggered self-accelerating drug release nanosystem with charge conversion for enhanced cancer therapy

2020 ◽  
Author(s):  
Xinyi Zhang ◽  
Tiantian Zhu ◽  
Yaxin Miao ◽  
Lu Zhou ◽  
Weifang Zhang
Keyword(s):  
Drug Release ◽  
Tumor Tissue ◽  
Anticancer Therapy ◽  
Cell Uptake ◽  
Oxygen Species ◽  
Anticancer Drug Delivery ◽  
Tocopheryl Succinate ◽  
Delivery Platform

Abstract Background: The enhancement tumor retention and of cellular uptake of drugs are important factors in maximizing anticancer therapy and minimizing side effects of encapsulated drugs. Herein, a delivery nanoplatform with a pH-triggered charge-reversal capability and self-amplifiable reactive oxygen species (ROS) level inducing drug release pattern was constructed by encapsulating doxorubicin (DOX) in pH/ROS-responsive polymeric micelle.Results: The surface charge of this system can be converted from negative to positive for enhanced tumor cell uptake in response to the weakly acidic tumor tissue. In addition, methionine-based system was dissociated in a ROS-rich intracellular environment, resulting in a phase transition and the release of DOX. Then, the exposed α-tocopheryl succinate (α-TOS) segments can be capable of producing ROS, which further induced the self-amplifiable disassembly of the micelles and drug release. Conclusions: We confirmed efficient DOX delivery into cancer cells, upregulation of tumoral ROS level and induction of the apoptotic capability in vitro. The system exhibited outstanding tumor inhibition capability in vivo, indicating that dual stimuli nanosytem would be great potential as an anticancer drug delivery platform.

scholarly journals Fetal Hemoglobin Rescues Ineffective Erythropoiesis in Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-15
Author(s):  
Sara El Hoss ◽  
Sylvie Cochet ◽  
Auria Godard ◽  
Hongxia Yan ◽  
Michaël Dussiot ◽  
...  
Keyword(s):  
Cell Death ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Ineffective Erythropoiesis ◽  
Hypoxic Conditions ◽  
Cd34 Cells ◽  
F Cells

Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a point mutation in the β globin gene resulting in a Glu-to-Val substitution at the 6th position of the β globin protein. The resulting abnormal hemoglobin (HbS) polymerizes under hypoxic conditions driving red blood cell (RBC) sickling (Pauling et al., 1949). While pathobiology of circulating RBCs has been extensively analyzed in SCD, erythropoiesis is surprisingly poorly documented. In β-thalassemia, ineffective erythropoiesis is characterized by high levels of apoptotic erythroblasts during the late stages of terminal differentiation, due to an accumulation of free β-globin chains (Arlet et al., 2016). Ineffective erythropoiesis is the major cause of anemia in β-thalassemia patients. In contrast, a marked decrease in life span of circulating red cells, a feature of sickle red cells, is considered to be the major determinant of chronic anemia in SCD. It is generally surmised that ineffective erythropoiesis contributes little to anemia. The bone marrow environment has been well documented to be hypoxic (0.1 to 6% O2) (Mantel et al., 2015). As hypoxia induces HbS polymerization, we hypothesized that cell death may occur in vivo because of HbS polymer formation in the late stages of differentiation characterized by high intracellular hemoglobin concentration. In the present study, using both in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. We explored the mechanistic basis of the ineffective erythropoiesis in SCD using biochemical, cellular and imaging techniques. In vitro erythroid differentiation using CD34+ cells isolated from SCD patients and from healthy donors was performed. A 2-phase erythroid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e. normoxia and partial hypoxia (5% O2). We found that hypoxia induces cell death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). This inference was supported by flow cytometry data showing higher percentages of dead cells within the non-F-cell population as compared to the F-cell population for SCD cells. Moreover, SCD dead cells showed higher levels of chaperon protein HSP70 in the cytoplasm than live cells, while no difference was detected between both subpopulations for control cells, suggesting that cell death of SCD erythroblasts was probably due to HSP70 cytoplasmic sequestration. This was supported by western-blot experiments showing less HSP70 in the nucleus of SCD erythroblasts under hypoxia, associated with decreased levels of GATA-1. At the molecular level, HSP70 was co-immunoprecipitated with HbS under hypoxia indicating that both proteins were in the same complex and suggesting interaction between HSP70 and HbS polymers in the cyotplasm. Importantly, we confirm these results in vivo by showing that in bone marrow of SCD patients (n = 5) cell loss occurs during terminal erythroid differentiation, with a significant drop in the cell count between the polychromatic and the orthochromatic stages (Figure 1). In order to specifically address the role of HbF in cell survival, we used a CRISPR-Cas9 approach to mimic the effect of hereditary persistence of fetal hemoglobin (HPFH). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1) (Weber, Frati, et al. 2020). As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control. These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions, clearly demonstrating the positive and selective effect of HbF on SCD cell survival (Figure 2). In summary, our study shows that HbF has a dual beneficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing ineffective erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rotavirus Replication Factories Are Complex Ribonucleoprotein Condensates

2020 ◽  
Author(s):  
Florian Geiger ◽  
Guido Papa ◽  
William E. Arter ◽  
Julia Acker ◽  
Kadi L. Saar ◽  
...  
Keyword(s):  
Phase Separation ◽  
Unique Feature ◽  
Rna Viruses ◽  
Therapeutic Approaches ◽  
Subcellular Organelles ◽  
Selective Enrichment ◽  
Novel Therapeutic ◽  
Liquid Liquid Phase Separation

AbstractRNA viruses induce formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation. We demonstrate that rotavirus proteins NSP5 and NSP2 undergo phase separation in vitro and form RNA-rich condensates in vivo that can be reversibly dissolved by aliphatic diols. During infection, these RNA-protein condensates became less dynamic and impervious to aliphatic diols, indicating a transition from a liquid to solid state. Some aspects of assembly of rotavirus replication factories mirror the formation of cytoplasmic ribonucleoprotein granules, while the selective enrichment of viral transcripts appears to be a unique feature of these condensates. Such complex RNA-protein condensates that underlie replication of RNA viruses represent an attractive target for developing novel therapeutic approaches.

Adrenomedullin: angiogenesis and gene therapy

2005 ◽  
Vol 288 (6) ◽  
pp. R1432-R1437 ◽  
Author(s):  
Noritoshi Nagaya ◽  
Hidezo Mori ◽  
Shinsuke Murakami ◽  
Kenji Kangawa ◽  
Soichiro Kitamura
Keyword(s):  
Gene Transfer ◽  
Cell Biology ◽  
Therapeutic Potential ◽  
Lattice Structure ◽  
Rabbit Model ◽  
Mitogen Activated Protein Kinase ◽  
Tissue Ischemia ◽  
Hypoxic Conditions

Adrenomedullin (AM) is a potent, long-lasting vasodilator peptide that was originally isolated from human pheochromocytoma. AM signaling is of particular significance in endothelial cell biology since the peptide protects cells from apoptosis, promotes angiogenesis, and affects vascular tone and permeability. The angiogenic effect of AM is mediated by activation of Akt, mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2, and focal adhesion kinase in endothelial cells. Both AM and its receptor, calcitonin receptor-like receptor, are upregulated through a hypoxia-inducible factor-1-dependent pathway under hypoxic conditions. Thus AM signaling plays an important role in the regulation of angiogenesis in hypoxic conditions. Recently, we have developed a nonviral vector, gelatin. Positively charged gelatin holds negatively charged plasmid DNA in its lattice structure. DNA-gelatin complexes can delay gene degradation, leading to efficient gene transfer. Administration of AM DNA-gelatin complexes induces potent angiogenic effects in a rabbit model of hindlimb ischemia. Thus gelatin-mediated AM gene transfer may be a new therapeutic strategy for the treatment of tissue ischemia. Endothelial progenitor cells (EPCs) play an important role in endothelial regeneration. Interestingly, EPCs phagocytose ionically linked DNA-gelatin complexes in coculture, which allows nonviral gene transfer into EPCs. AM gene transfer into EPCs inhibits cell apoptosis and induces proliferation and migration, suggesting that AM gene transfer strengthens the therapeutic potential of EPCs. Intravenous administration of AM gene-modified EPCs regenerate pulmonary endothelium, resulting in improvement of pulmonary hypertension. These results suggest that in vivo and in vitro transfer of AM gene using gelatin may be applicable for intractable cardiovascular disease.

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