466 research outputs found
Target-sensitive immunoerythrocytes: interaction of biotinylated red blood cells with immobilized avidin induces their lysis by complement
AbstractRed blood cells (RBC) coated with antibody (immunoerythrocytes) may be useful for drug targeting. Previously we have developed a methodology for avidin (streptavidin)-mediated attachment of biotinylated antibodies (b-Ab) to biotinylated RBC (B-RBC). We have observed that binding of avidin to B-RBC in suspension leads to their complement-mediated lysis by autologous serum. In the present work we have studied the interaction of B-RBC, which are not complement susceptible, with immobilized avidin and their consequent susceptibility to lysis by complement. B-RBC adhered tightly to avidin-coated surfaces and were rendered susceptible to lysis by autologous serum. A long biotin ester provided more effective binding of the B-RBC to immobilized avidin and greater lysis by complement, than a short biotin ester. Based on these results, we have hypothesized that targeting of serum-stable drug-loaded B-RBC attained by step-wise administration of b-Ab and streptavidin may provide target-sensitive lysis of B-RBC. To confirm this hypothesis, we have studied b-Ab and streptavidin mediated targeting of B-RBC to immobilized antigen. Step-wise addition of biotinylated antibody, avidin or streptavidin and b-RBC caused specific binding of B-RBC to immobilized antigen and their subsequent lysis by autologous serum. Therefore, our results obtained in an in vitro model demonstrate that B-RBC might be used for targeting and local release of drug
Red blood cells: Supercarriers for drugs, biologicals, and nanoparticles and inspiration for advanced delivery systems
Red blood cells (RBCs) constitute a unique drug delivery system as a biologic or hybrid carrier capable of greatly enhancing pharmacokinetics, altering pharmacodynamics (for example, by changing margination within the intravascular space), and modulating immune responses to appended cargoes. Strategies for RBC drug delivery systems include internal and surface loading, and the latter can be performed both ex vivo and in vivo. A relatively new avenue for RBC drug delivery is their application as a carrier for nanoparticles. Efforts are also being made to incorporate features of RBCs in nanocarriers to mimic their most useful aspects, such as long circulation and stealth features. RBCs have also recently been explored as carriers for the delivery of antigens for modulation of immune response. Therefore, RBC-based drug delivery systems represent supercarriers for a diverse array of biomedical interventions, and this is reflected by several industrial and academic efforts that are poised to enter the clinical realm
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Anti-Inflammatory Effect of Targeted Delivery of SOD to Endothelium: Mechanism, Synergism with NO Donors and Protective Effects In Vitro and In Vivo
Pro-inflammatory activation of vascular endothelium is implicated in pathogenesis of severe conditions including stroke, infarction and sepsis. We have recently reported that superoxide dismutase (SOD) conjugated with antibodies (Ab/SOD) that provide targeted delivery into endothelial endosomes mitigates inflammatory endothelial activation by cytokines and agonists of Toll-like receptors (TLR). The goal of this study was to appraise potential utility and define the mechanism of this effect. Ab/SOD, but not non-targeted SOD injected in mice alleviated endotoxin-induced leukocyte adhesion in the cerebral vasculature and protected brain from ischemia-reperfusion injury. Transfection of endothelial cells with SOD, but not catalase inhibited NFκB signaling and expression of Vascular Cell Adhesion Molecule-1 induced by both cytokines and TLR agonists. These results affirmed that Ab/SOD-quenched superoxide anion produced by endothelial cells in response to proinflammatory agents mediates NFκB activation. Furthermore, Ab/SOD potentiates anti-inflammatory effect of NO donors in endothelial cells in vitro, as well as in the endotoxin-challenged mice. These results demonstrate the central role of intracellular superoxide as a mediator of pro-inflammatory activation of endothelium and support the notion of utility of targeted interception of this signaling pathway for management of acute vascular inflammation
Non-affinity factors modulating vascular targeting of nano- and microcarriers
Particles capable of homing and adhering to specific vascular biomarkers have potential as fundamental tools in drug delivery for mediation of a wide variety of pathologies, including inflammation, thrombosis, and pulmonary disorders. The presentation of affinity ligands on the surface of a particle provides a means of targeting the particle to sites of therapeutic interest, but a host of other factors come into play in determining the targeting capacity of the particle. This review presents a summary of several key considerations in nano- and microparticle design that modulate targeted delivery without directly altering epitope-specific affinity. Namely, we describe the effect of factors in definition of the base carrier (including shape, size, and flexibility) on the capacity of carriers to access, adhere to, and integrate in target biological milieus. Furthermore, we present a summary of fundamental dynamics of carrier behavior in circulation, taking into account interactions with cells in circulation and the role of hemodynamics in mediating the direction of carriers to target sites. Finally, we note non-affinity aspects to uptake and intracellular trafficking of carriers in target cells. In total, recent findings presented here may offer an opportunity to capitalize on mitigating factors in the behavior of ligand-targeted carriers in order to optimize targeting
Bivalent engagement of endothelial surface antigens is critical to prolonged surface targeting and protein delivery in vivo
Targeted drug delivery to the endothelium has the potential to generate localized therapeutic effects at the blood- tissue interface. For some therapeutic cargoes, it is essential to maintain contact with the bloodstream to exert protective effects. The pharmacokinetics (PK) of endothelial surface- targeted affinity ligands and biotherapeutic cargo remain a largely unexplored area, despite obvious translational implications for this strategy. To bridge this gap, we site- specifically radiolabeled mono- (scFv) and bivalent (mAb) affinity ligands specific for the endothelial cell adhesion molecules, PECAM- 1 (CD31) and ICAM- 1 (CD54). Radiotracing revealed similar lung biodistribution at 30 minutes post- injection (79.3% ± 4.2% vs 80.4% ± 10.6% ID/g for αICAM and 58.9% ± 3.6% ID/g vs. 47.7% ± 5.8% ID/g for αPECAM mAb vs. scFv), but marked differences in organ residence time, with antibodies demonstrating an order of magnitude greater area under the lung concentration vs. time curve (AUCinf 1698 ± 352 vs. 53.3 ± 7.9 ID/g*hrs for αICAM and 1023 ± 507 vs. 114 ± 37 ID/g*hrs for αPECAM mAb vs scFv). A physiologically based pharmacokinetic model, fit to and validated using these data, indicated contributions from both superior binding characteristics and prolonged circulation time supporting multiple binding- detachment cycles. We tested the ability of each affinity ligand to deliver a prototypical surface cargo, thrombomodulin (TM), using one- to- one protein conjugates. Bivalent mAb- TM was superior to monovalent scFv- TM in both pulmonary targeting and lung residence time (AUCinf 141 ± 3.2 vs 12.4 ± 4.2 ID/g*hrs for ICAM and 188 ± 90 vs 34.7 ± 19.9 ID/g*hrs for PECAM), despite having similar blood PK, indicating that binding strength is more important parameter than the kinetics of binding. To maximize bivalent target engagement, we synthesized an oriented, end- to- end anti- ICAM mAb- TM conjugate and found that this therapeutic had the best lung residence time (AUCinf 253 ± 18 ID/g*hrs) of all TM modalities. These observations have implications not only for the delivery of TM, but also potentially all therapeutics targeted to the endothelial surface.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156501/3/fsb220760_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156501/2/fsb220760-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156501/1/fsb220760.pd
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Immunotargeting of tumor vasculature: preclinical development of novel antibody-based imaging and therapy against TEM1/CD248
The success of antibody-based theranostics depends on the identification of tumor specific biomarkers and the development of corresponding antibodies with high-affinity and specificity. Tumor endothelial marker-1 (TEM1) is highly expressed in tumor vasculature of multiple cancers but not in normal organs. The expression of TEM1 was first evaluated and confirmed by immunohistochemistry from 53 cases of metastatic serous ovarian cancer at HUP. TEM1 positive tumor stroma was observed in >95% of the cases studied. Hence, developing sensitive and effective theranostic agents against TEM1 are of utmost significance in improving diagnosis and treatment of ovarian cancer. Our goals are: 1) engineer TEM1-specific antibodies; 2) evaluate these engineered antibodies in imaging and immunotherapies in preclinical models. To generate TEM1-targeting agents, we designed a panel of multivalent fusion proteins from scFv78, a previously isolated single chain variable fragment specifically recognizing the extracellular domain of TEM1. scFv78 was fused with different huIgG1 Fc region (CH2-, CH3-, or hinge). Proteins were expressed in 293F cells and purified by affinity chromatography. All scFv78 variants exhibited comparable thermo and serum stability in vitro. Among them, the scFv78-Fc fusion (78Fc) has the highest affinity to TEM1 (Kd = 0.15nM, 15X higher than scFv78). Pharmacokinetics (PK) and biodistribution of the protein panel were evaluated in naïve and TEM1+ tumor bearing animals. 78Fc has a t1/2 of 5.1hr, which is suitable for in vivo therapeutic and imaging applications. Therefore, 78Fc was further developed as imaging tool and antibody-drug conjugate (ADC) based on its favorable affinity, stability, half-life and PK profile. In pilot studies with preclinical animal models of tumor vasculature, fluorophore- and [124-I]-labeled 78Fc demonstrated specific enrichment in TEM1+ grafts, but not in control tumor or other organs, by both optical and immunoPET imaging. In addition, 78Fc-MMAE conjugate exerted specific killing of TEM1+ cells. In summary, we have developed a panel of innovative theranostics agents targeting TEM1 on the vasculature of ovarian cancer and several other solid tumors. Our long term goal is to translate such combined approach into the clinic: Using TEM1-antibody as imaging tools to select, and monitor patients for TEM1-antibody based targeted therapies
Adaptation of Mycoplasma gallisepticum to unfavorable growth conditions: Changes in morphological and physiological characteristics
Adaptation of Mycoplasma gallisepticum to unfavorable growth conditions results in altered morphological and physiological characteristics of the cells. M. gallisepticum populations in a complete nutrient medium contain pear-shaped vegetative cells ( d - 0.3 μm; l - 0.8 μm) with pronounced polar and cytoskeletonlike structures. Such mycoplasma cells are able to induce damage in a bacterial genome, causing an SOS response of the test strain (Escherichia coli PQ37). In a starvation medium, M. gallisepticum produces nanoforms, small coccoid cells (d - 0.15-0.2 μm) without either polar or cytoskeleton-like structures. Unlike vegetative cells, nanoforms do not induce genome damage. Alleviation of unfavorable growth conditions results in a reversion of nanoforms to vegetative cells. © 2008 MAIK Nauka
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