scholarly journals Regulation Of Endocytosis By Ultrasound And Microbubble Treatment: Potential For Controlled Cellular Delivery Of Drugs To Cancer Cells

2021 ◽  
Author(s):  
Farnaz Fekri
Keyword(s):  
Targeted Delivery ◽  
Intracellular Delivery ◽  
Drug Uptake ◽  
Limiting Factor ◽  
Dependent Manner ◽  
Cellular Delivery ◽  
Plasma Membrane Permeability ◽  
Bulk Fluid ◽  
Biological Barriers ◽  
Endocytic Pathways

The delivery of therapeutics across biological barriers is a limiting factor in achieving ideal pharmacologic responses in patients. Modulating endocytic mechanisms with targeted, clinically-relevant interventions can increase intracellular delivery across biological barriers, and improve the efficacy of drugs. Ultrasound-microbubble (USMB) is a novel targeted delivery strategy that has shown promising potential in both diagnostic and therapeutic applications. The collective behaviour of microbubbles in the acoustic field can increase the plasma membrane permeability of surrounding cells, and enhance the delivery of therapeutics across biological barriers. USMB achieves the intracellular delivery of drugs through sonoporation and modulation of endocytic pathways, but the type of endocytic pathways and the mechanisms of activation were not known. I identified that, under distinct regulations, USMB enhances the rate of both clathrin-mediated endocytosis, as well as a non-receptor-mediated pathway responsible for internalizing bulk fluid into cells. I discovered that lysosome exocytosis and acidsphingomyelinase are required for the regulation of the clathrin-mediated pathway but not fluidphase endocytosis following USMB treatment. Given the potential of the clathrin-independent pathway to form high capacity carries for the uptake of fluids and therapeutics into cells, I aimed to identify the molecular identity of the proteins that drive the formation of non-clathrin coated vesicles following USMB treatment. I established that flotillins contribute to the USMB-induced vesicular uptake of fluid into cells, a phenomenon that depends on palmitoyltransferase DHHC5 and the Src-family kinase Fyn. Furthermore, I confirmed that USMB treatment can enhance the intracellular delivery of chemotherapeutic drugs such as cisplatin, and improve its therapeutic efficacy in a flotillin-dependent manner. This project established that both clathrin-mediated endocytosis and flotillin-dependent endocytosis can be modulated by clinically-relevant USMB treatments to enhance drug uptake and efficacy, revealing an important new strategy for targeted drug delivery in cancer treatment.

scholarly journals Regulation Of Endocytosis By Ultrasound And Microbubble Treatment: Potential For Controlled Cellular Delivery Of Drugs To Cancer Cells

2021 ◽  
Author(s):  
Farnaz Fekri
Keyword(s):  
Targeted Delivery ◽  
Intracellular Delivery ◽  
Drug Uptake ◽  
Limiting Factor ◽  
Dependent Manner ◽  
Cellular Delivery ◽  
Plasma Membrane Permeability ◽  
Bulk Fluid ◽  
Biological Barriers ◽  
Endocytic Pathways

The delivery of therapeutics across biological barriers is a limiting factor in achieving ideal pharmacologic responses in patients. Modulating endocytic mechanisms with targeted, clinically-relevant interventions can increase intracellular delivery across biological barriers, and improve the efficacy of drugs. Ultrasound-microbubble (USMB) is a novel targeted delivery strategy that has shown promising potential in both diagnostic and therapeutic applications. The collective behaviour of microbubbles in the acoustic field can increase the plasma membrane permeability of surrounding cells, and enhance the delivery of therapeutics across biological barriers. USMB achieves the intracellular delivery of drugs through sonoporation and modulation of endocytic pathways, but the type of endocytic pathways and the mechanisms of activation were not known. I identified that, under distinct regulations, USMB enhances the rate of both clathrin-mediated endocytosis, as well as a non-receptor-mediated pathway responsible for internalizing bulk fluid into cells. I discovered that lysosome exocytosis and acidsphingomyelinase are required for the regulation of the clathrin-mediated pathway but not fluidphase endocytosis following USMB treatment. Given the potential of the clathrin-independent pathway to form high capacity carries for the uptake of fluids and therapeutics into cells, I aimed to identify the molecular identity of the proteins that drive the formation of non-clathrin coated vesicles following USMB treatment. I established that flotillins contribute to the USMB-induced vesicular uptake of fluid into cells, a phenomenon that depends on palmitoyltransferase DHHC5 and the Src-family kinase Fyn. Furthermore, I confirmed that USMB treatment can enhance the intracellular delivery of chemotherapeutic drugs such as cisplatin, and improve its therapeutic efficacy in a flotillin-dependent manner. This project established that both clathrin-mediated endocytosis and flotillin-dependent endocytosis can be modulated by clinically-relevant USMB treatments to enhance drug uptake and efficacy, revealing an important new strategy for targeted drug delivery in cancer treatment.

scholarly journals Self-assembling ferritin-dendrimer nanoparticles for targeted delivery of nucleic acids to myeloid leukemia cells

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Federica Palombarini ◽  
Silvia Masciarelli ◽  
Alessio Incocciati ◽  
Francesca Liccardo ◽  
Elisa Di Fabio ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Targeted Delivery ◽  
Myeloid Leukemia ◽  
Electrostatic Interactions ◽  
Archaeoglobus Fulgidus ◽  
Leukemia Cells ◽  
Cellular Delivery ◽  
Self Assembling ◽  
Wide Range ◽  
Hybrid Nanoparticle

Abstract Background In recent years, the use of ferritins as nano-vehicles for drug delivery is taking center stage. Compared to other similar nanocarriers, Archaeoglobus fulgidus ferritin is particularly interesting due to its unique ability to assemble-disassemble under very mild conditions. Recently this ferritin was engineered to get a chimeric protein targeted to human CD71 receptor, typically overexpressed in cancer cells. Results Archaeoglobus fulgidus chimeric ferritin was used to generate a self-assembling hybrid nanoparticle hosting an aminic dendrimer together with a small nucleic acid. The positively charged dendrimer can indeed establish electrostatic interactions with the chimeric ferritin internal surface, allowing the formation of a protein-dendrimer binary system. The 4 large triangular openings on the ferritin shell represent a gate for negatively charged small RNAs, which access the internal cavity attracted by the dense positive charge of the dendrimer. This ternary protein-dendrimer-RNA system is efficiently uptaken by acute myeloid leukemia cells, typically difficult to transfect. As a proof of concept, we used a microRNA whose cellular delivery and induced phenotypic effects can be easily detected. In this article we have demonstrated that this hybrid nanoparticle successfully delivers a pre-miRNA to leukemia cells. Once delivered, the nucleic acid is released into the cytosol and processed to mature miRNA, thus eliciting phenotypic effects and morphological changes similar to the initial stages of granulocyte differentiation. Conclusion The results here presented pave the way for the design of a new family of protein-based transfecting agents that can specifically target a wide range of diseased cells. Graphic abstract

Targeted delivery of a SNARE protease to sensory neurons using a single chain antibody (scFv) against the extracellular domain of P2X3 inhibits the release of a pain mediator

2014 ◽  
Vol 462 (2) ◽  
pp. 247-256 ◽  
Author(s):  
Hui Ma ◽  
Jianghui Meng ◽  
Jiafu Wang ◽  
Stephen Hearty ◽  
J. Oliver Dolly ◽  
...  
Keyword(s):  
Sensory Neurons ◽  
Targeted Delivery ◽  
Intracellular Delivery ◽  
Extracellular Domain ◽  
Single Chain ◽  
Single Chain Antibody

A single-chain antibody was generated against an extracellular domain of human P2X3 as a targeting moiety. It was conjugated with a pain therapeutic SNARE protease derived from BoNT/A to demonstrate its intracellular delivery into pain-sensing neurons.

Redox-Sensitive Ultrashort Peptide Hydrogel with Tunable Mechanical Properties for Anti-Tumor Drug Delivery

2020 ◽  
Vol 16 (11) ◽  
pp. 1588-1599
Author(s):  
Yiping Li ◽  
Ying Zhu ◽  
Shiyao Luo ◽  
Yue He ◽  
Zhewei Huang ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Targeted Delivery ◽  
Subcutaneous Tissue ◽  
Drug Loading ◽  
Gelation Time ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
The Cross ◽  
Hydrogel System

In this study, we report a new ultrashort peptide (LOC), which forms a redox-sensitive hydrogel after cross-linking with the mild oxidant H2 O2 and used it for tumor-targeted delivery of doxorubicin hydrochloride (DOX). LOC gelled within a few minutes in low-concentration H2 O2 solution. The concentration of H2 O2 significantly altered the gelation time and mechanical properties of the hydrogel. The in vitro micromorphology, secondary structure and rheology characterization of cross-linked hydrogels confirmed the sensitivity and injectability to reducing agent. The cross-linked hydrogel had a strong drug loading capacity, and the drug was released in a GSH concentration-dependent manner, following the Fick diffusion model. In addition, the cross-linked hydrogel showed no cytotoxicity to normal fibroblasts, and no damage to the subcutaneous tissue of mice was observed. In vitro cytotoxicity experiments showed that the DOX-hydrogel system exhibited good anti-cancer efficacy. In vivo studies using 4T1 tumor-bearing mice showed that the DOX-hydrogel system had a significant inhibitory effect on tumors. Therefore, the newly designed redox-sensitive hydrogel can effectively enhance the therapeutic efficacy of DOX and reduce toxicity, making it an attractive biological material.

scholarly journals Global Expression Profiling and Physiological Characterization of Corynebacterium glutamicum Grown in the Presence of l-Valine

2003 ◽  
Vol 69 (5) ◽  
pp. 2521-2532 ◽  
Author(s):  
C. Lange ◽  
D. Rittmann ◽  
V. F. Wendisch ◽  
M. Bott ◽  
H. Sahm
Keyword(s):  
Corynebacterium Glutamicum ◽  
Expression Profiling ◽  
Large Subunit ◽  
Mrna Level ◽  
Acetohydroxyacid Synthase ◽  
Limiting Factor ◽  
Unexpected Result ◽  
Dependent Manner ◽  
Wild Type ◽  
Concentration Dependent Manner

ABSTRACT Addition of l-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 ΔilvA ΔpanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of l-isoleucine could relieve the valine effect on VAL1 whereas l-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.

scholarly journals PLA-PEG Nanoparticles Improve the Anti-Inflammatory Effect of Rosiglitazone on Macrophages by Enhancing Drug Uptake Compared to Free Rosiglitazone

Materials ◽  
2018 ◽  
Vol 11 (10) ◽  
pp. 1845 ◽  
Author(s):  
Giovanna Giacalone ◽  
Nicolas Tsapis ◽  
Ludivine Mousnier ◽  
Hélène Chacun ◽  
Elias Fattal
Keyword(s):  
Polymeric Nanoparticles ◽  
Heart Diseases ◽  
The United States ◽  
Drug Uptake ◽  
Dependent Manner ◽  
Raw264.7 Macrophages ◽  
Efficient Delivery ◽  
Anti Inflammatory ◽  
Atherosclerosis Treatment ◽  
Inflammatory Effect

Among cardiovascular diseases, atherosclerosis remains the first cause of death in the United States of America and Europe, as it leads to myocardial infarction or stroke. The high prevalence of heart diseases is due to the difficulty in diagnosing atherosclerosis, since it can develop for decades before symptoms occur, and to the complexity of the treatment since targets are also important components of the host defenses. The antidiabetics thiazolidinediones, among which is rosiglitazone (RSG), have demonstrated anti-atherosclerotic effect in animal models, and are therefore promising candidates for the improvement of atherosclerosis management. Nevertheless, their administration is hindered by the insurgence of severe side effects. To overcome this limitation, rosiglitazone has been encapsulated into polymeric nanoparticles, which permit efficient delivery to its nuclear target, and selective delivery to the site of action, allowing the reduction of unwanted effects. In the present work, we describe nanoparticle formulation using polylactic acid (PLA) coupled to polyethylene glycol (PEG), their characterization, and their behavior on RAW264.7 macrophages, an important target in atherosclerosis treatment. RSG nanocarriers showed no toxicity on cells at all concentrations tested, an anti-inflammatory effect in a dose-dependent manner, up to 5 times more efficient than the free molecule, and an increased RSG uptake which is consistent with the effect shown. These biodegradable nanoparticles represent a valid tool to be further investigated for the treatment of atherosclerosis.

Biological barriers to cellular delivery of lipid-based DNA carriers

1999 ◽  
Vol 38 (3) ◽  
pp. 291-315 ◽  
Author(s):  
Marcel B Bally ◽  
Pierrot Harvie ◽  
Frances M.P Wong ◽  
Spencer Kong ◽  
Ellen K Wasan ◽  
...  
Keyword(s):  
Cellular Delivery ◽  
Biological Barriers ◽  
Dna Carriers

Nitrogen supply controls vegetative growth, biomass and nitrogen allocation for grapevine (cv. Shiraz) grown in pots

2015 ◽  
Vol 42 (1) ◽  
pp. 105 ◽  
Author(s):  
Aurélie Metay ◽  
Jessica Magnier ◽  
Nicolas Guilpart ◽  
Angélique Christophe
Keyword(s):  
Vegetative Growth ◽  
Limiting Factor ◽  
Dependent Manner ◽  
Leaf Emergence ◽  
N Supply ◽  
Secondary Axis ◽  
Axis Elongation ◽  
Primary Axis ◽  
N Deficiency ◽  
Area Expansion

Maintaining grapevine productivity with limited inputs is crucial in Mediterranean areas. Apart from water, nitrogen (N) is also an important limiting factor in grape growing. The effects of N deficiency on grapevine growth were investigated in this study. Two-year-old Vitis vinifera L.cv. Shiraz plants grafted on 110 R were grown in pots placed outside and exposed to various N supplies (0, 0.6, 1.2, 2.4 and 12 g plant–1) under well-watered conditions. At veraison, plants were harvested and organs separately dried, weighed and analysed for N. During plant growth, the length of the primary and secondary axes and the number of leaves on them were recorded. The N content of leaves was also analysed at three phenological stages (flowering, bunch closure and veraison). All growth processes were inhibited by N deficiency in an intensity-dependent manner. Quantitative relationships with N supply were established. Vegetative growth responded negatively to N stress when comparing control N supply with no N supply: primary axis elongation (–61%), leaf emergence on the primary axis (–47%), leaf emergence on the secondary axis (–94%) and lamina area expansion (–45%). Significant differences on the plant N status were observed from flowering onwards which might be useful for managing fertilisation.

scholarly journals Histones Induce the Release of Extracellular Hemoglobin and Red Blood Cell-Derived Microvesicles with Procoagulant Activity

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2514-2514
Author(s):  
Denis Noubouossie ◽  
Juraj Sokol ◽  
Mark Gerard Piegore ◽  
Anton Ilich ◽  
Michael Wayne Henderson ◽  
...  
Keyword(s):  
Cell Membranes ◽  
Cell Activation ◽  
Calcium Ionophore ◽  
Lipid Vesicles ◽  
Morbidity And Mortality ◽  
Limiting Factor ◽  
Dependent Manner ◽  
Histone Binding ◽  
Extracellular Histones ◽  
Free Plasma

Abstract Background Histones are nuclear proteins that can be released extracellularly following cell death or certain mechanisms of cell activation. Infusion of high doses of histones into mice causes lethality. Circulating levels of extracellular histones are elevated in several disease states associated with inflammation and thrombosis. The mechanisms by which extracellular histones induce morbidity and mortality are not fully understood. Histones may activate or be toxic to several cell types in vitro. Most of these activities have been shown to be dependent on histone signaling through toll-like receptors (TLRs). Red blood cells (RBC) do not express TLRs. However, previous studies have reported that histones promote RBC aggregation, increase shear-dependent RBC fragility, reduce deformability under flow and cause hemolytic anemia in mice. Histones also induce calcium influx and promote PS expression on RBC membranes thereby supporting the prothrombinase activity and shortening clotting times in plasma. Histones may insert into cell membranes (independently of TLRs), and form pore-like structures similar to ionophore. The role of this process in histone-induced RBC toxicity has not been fully investigated. Objective We assessed the ability of individual histones to promote the release of extracellular hemoglobin (Ex-Hb) and RBC-derived microvesicles (RBC-MVs), as well as RBC-MVs' ability to initiate or propagate thrombin generation (TG) in MV-free plasma. Methods RBCs were isolated from citrated whole blood from healthy volunteers, and were re-suspended in buffer with or without 10uM A23187 (calcium ionophore), recombinant histones H2A, H2B, H3 or H4 for 1 hour at 37°C. To assess histone binding, RBCs were washed and dual-stained with fluorophore-conjugated anti-glycophorin A (GPA) and anti-panhistone antibodies. Stained RBCs were analyzed using imaging flow cytometry, and were considered positive for histone binding if the fluorescence intensity was > 95th percentile of RBCs processed in the absence of histones. RBCs were removed by centrifugation at 2,500g for 15 minutes x2. RBC-MVs present in the supernatant were pelleted by centrifugation at 20,000g for 30 min. Isolated RBC-MVs were then re-suspended in buffer to assess PS exposure using the microparticle activity assay (Hyphen Biomed). RBC-MVs were also re-suspended in re-calcified MV-free plasma supplemented with 4uM 41%PC/44%PE/15%PS lipid vesicles to assess their ability to initiate TG by monitoring the activity of thrombin on a fluorogenic substrate over time. To assess their ability to propagate TG, coagulation was initiated by 1pM tissue factor in MV-free plasma containing histone-induced RBC-MVs without addition of synthetic lipid vesicles. In this condition, phospholipids present on the surface of histone-induced RBC-MVs are the limiting factor for TG. The supernatant collected after pelleting RBC-MVs was used to measure Ex-Hb by absorbance at 540 nm. Results The percentage of RBCs positive for histone binding was 21.6%, 2.3%, 76.9% and 98.1% for H2A, H2B, H3 and H4, respectively. Representative images of RBC staining for GPA and histone H4 is shown in Figure 1A. All histone types induced the release of Ex-Hb (Fig. 1B) and RBC-MVs that possess PS activity (Fig. 1C) in a dose-dependent manner, with the highest PS activity observed with H2A. RBC-MVs released by H2A or H2B clearly initiated TG in MV-free normal pool plasma (Fig.1D). They also significantly enhanced TF-initiated TG in the absence of additional lipid vesicles. The release of Ex-Hb and procoagulant RBC-MVs induced by histones were re-capitulated by ionophore to a greater degree, as expected (Fig.1B-E). Conclusions All 4 types of core histones bind to RBCs and induce the release of Ex-Hb. Our findings suggest that despite their lower binding to RBCs, histones H2A and H2B may have a greater ability to induce the release of procoagulant RBC-MVs that can initiate and propagate TG in plasma. The reason for the differential toxicity of histone types on RBCs and the discrepancy between the relative effect of histone-induced RBC-MVs on PS equivalents and TG are unknown. Histone-induced RBC toxicity appears to mimic calcium ionophore, supporting the idea that histones may insert and form functional pores in cell membranes. The contribution of Ex-Hb and procoagulant RBC-MVs to the morbidity and mortality of extracellular histones in vivo requires further studies. Disclosures Monroe: Novo Nordisk A/S: Honoraria, Research Funding.

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