Radostina Georgieva

Highly efficient magnetic stem cell labeling with citrate-coated superparamagnetic iron oxide nanoparticles for MRI tracking

Tracking of transplanted stem cells is essential to monitor safety and efficiency of cell-based therapies. Magnetic resonance imaging (MRI) offers a very sensitive, repetitive and non-invasive in vivo detection of magnetically labeled cells but labeling with commercial superparamagnetic iron oxide nanoparticles (SPIONs) is still problematic because of low labeling efficiencies and the need of potentially toxic transfection agents. In this study, new experimental citrate-coated SPIONs and commercial Endorem and Resovist SPIONs were investigated comparatively in terms of in vitro labeling efficiency, effects on stem cell functionality and in vivo MRI visualization. Efficient labeling of human mesenchymal stem cells (MSCs) without transfection agents was only achieved with Citrate SPIONs. Magnetic labeling of human MSCs did not affect cell proliferation, presentation of typical cell surface marker antigens and differentiation into the adipogenic and osteogenic lineages. However, chondrogenic differentiation and chemotaxis were significantly impaired with increasing SPION incorporation. Transplanted SPION-labeled MSCs were visualized in vivo after intramuscular injection in rats by 7T-MRI and were retrieved ex vivo by Prussian Blue and immunohistochemical stainings. Though a careful titration of SPION incorporation, cellular function and MRI visualization is essential, Citrate SPIONs are very efficient intracellular magnetic labels for in vivo stem cell tracking by MRI.

Surface-modified loaded human red blood cells for targeting and delivery of drugs

Red blood cells (RBCs) are natural carriers which can be used for targeted drug delivery. Conditions during loading and surface modification are essential for carrier-RBC preparation for specifically targeted drug delivery. Therefore, human RBCs were loaded with albumin and magnetic nanoparticles (NPs) by different hypotonic haemolysis procedures and compared based on loading efficiency and membrane damage. Samples were analysed by flow cytometry and confocal microscopy. The optimized loading procedure resulted in 90% albumin-loaded carrier-RBCs with <4% Annexin V binding and 263u2009pg iron per RBC after loading with iron oxide NPs. Albumin-loaded RBCs were subsequently surface conjugated with insulin and IgG via biotin–streptavidin. Insulin-conjugated carrier-RBCs were observed to attach and to be internalized by cultured endothelial cells. Uptake was not observed for carrier-RBCs non-specifically modified with IgG. Attachment of other peptides with high specificity will open novel opportunities for targeting various cells, tissues and for crossing biological barriers.

New 4-Maleamic Acid and 4-Maleamide Peptidyl Chalcones as Potential Multitarget Drugs for Human Prostate Cancer

ABSTRACTPurposeThe objective of this study was to investigate the effect of new 4-maleamic acid and 4-maleamide peptidyl chalcone derivatives against human prostate cancer in vitro and in vivo.MethodsFrom a series of 21 chalcones, the effects of the three best inhibitors of PC-3 and LNCaP cell viability on growth, including cell cycle changes, adhesion, migration, and cell invasion, as well as their ability to inhibit angiogenesis, clonogenic activity, and matrix metalloproteinases MMP-2 and MMP-9, were tested. The effects in vivo were studied in PC-3 and LNCaP xenografts.ResultsThree of the examined chalcones reduced cell viability in both cell lines in a strong dose- and time-dependent manner. An inhibition of the cell cycle progress was observed. These changes were accompanied with the inhibition of cell adhesion, migration, and invasion as well as with reduced neovascularization in chick embryos, tumor colony formation, and MMP-9 activity. The in vivo results demonstrated the strong activity of these structures as inhibitors of tumor development in nude mice compared to non-treated animals.ConclusionThe results suggest the multitarget efficacy of 4-maleamic acid and 4-maleamide peptidyl chalcones against human prostate cancer cells and emphasize the potential therapeutic relevance of these compounds.

Photosensitizer-loaded electrospun chitosan-based scaffolds for photodynamic therapy and tissue engineering.

Novel chitosan-based nanofibrous composite materials containing different amounts of the photosensitizer Photosens were obtained by electrospinning and were characterized by scanning electron microscopy and by confocal laser scanning microscopy. The release of Photosens from the materials was investigated in water and in phosphate-buffered saline. A noncancerous (MC3T3-E1 murine osteoblasts) and a cancerous [T-47D (mammary gland)] cell line were cultivated on Photosens-containing scaffolds, and cell growth and metabolic activity were examined by confocal laser scanning microscopy and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide assay, respectively. The viability of both cell lines on Photosens-containing fibers decreased in a spatial manner upon laser irradiation of an appropriate wavelength and power density. Interestingly, the noncancerous MC3T3-E1 cells grown on Photosens -containing scaffolds were less affected by the irradiation. We conclude that the Photosens-containing electrospun chitosan nanofibers described here are of potential interest for biomedical applications, particularly topical photodynamic therapy and tissue engineering.

Novel Hemoglobin Particles—Promising New-Generation Hemoglobin-Based Oxygen Carriers

During the last 30 years, artificial oxygen carriers have been investigated intensively with the aim to develop universal blood substitutes. Favorably, hemoglobin-based oxygen carriers (HBOCs) are expected to meet the sophisticated requirements. However, the HBOCs tested until now show serious side effects, which resulted in failure of clinical trials and Food and Drug Administration disapproval. The main problem consists in vasoconstriction triggered by nitric oxide (NO) scavenging or/and oxygen oversupply in the pre-capillary arterioles. HBOCs with a size between 100u2009nm and 1u2009µm and high oxygen affinity are needed. Here we present a highly effective and simple fabrication procedure, which can provide hemoglobin particles (HbPs) with a narrow size distribution of around 700u2009nm, nearly uniform morphology, high oxygen affinity, and low immunogenicity. Isolated mouse glomeruli are successfully perfused with concentrated HbP suspensions without any observable vasoconstriction of the afferent arterioles. The results suggest no oxygen oversupply and limited NO scavenging by these particles, featuring them as a highly promising blood substitute.

Light-induced antibacterial activity of electrospun chitosan-based material containing photosensitizer.

Increasing antimicrobial resistance requires the development of novel materials and approaches for treatment of various infections. Utilization of photodynamic therapy represents an advanced alternative to antibiotics and metal-based agents. Here, we report the fabrication of electrospun material that possesses benefits of both topical antimicrobial and photodynamic therapies. This material combines chitosan, as a biocompatible polymer, and a second generation photosensitizer. The incorporation of photosensitizer doesnt affect the material morphology and its nearly uniform distribution in fibers structure was observed by confocal Raman microscopy. Owing to photosensitizer the prepared material exhibits the light-induced and spatially limited antimicrobial activity that was demonstrated against Staphylococcus aureus, an important etiological infectious agent. Such material can be potentially used in antibacterial therapy of chronic wounds, infections of diabetic ulcers, and burns, as well as rapidly spreading and intractable soft-tissue infections caused by resistant bacteria.

Physical attachment of fluorescent protein particles to atomic force microscopy probes in aqueous media: Implications for surface pH, fluorescence, and mechanical properties studies

Transfer of a fluorescently labeled protein particle from a surface to a microsized scanning probe has been induced by repetitive scanning in aqueous medium. The so‐attached particle can in turn act as a probing tool to study particle–substrate and particle–particle interactions. Attachment of the fluorescent particle occurs at the apical region of an atomic force microscope (AFM) cantilever tip and it endures repetitive loading–unloading cycles against the sample surface. Fluorescence microscopy has been used to address the exact location of the attached particle in the cantilever and to identify the moment when the particle contacts the sample. Moreover, we have observed that fluorescence intensity at the contact point is lower when the probing particle contacts another fluorescent particle than when it contacts the nonfluorescent substrate. The change in fluorescence is attributed to local changes of pH and interparticle‐quenching of fluorophores in the contact region. These findings are promising since they constitute a chemical‐free way to attach bioparticles to AFM probes under fisiological conditions. The atomic force microscopy combined with fluorescence microscopy provides a straight forward method to study particle/particle and particle/substrate interactions, as well as to investigate mechanical properties of biocolloids. Microsc. Res. Tech. 73:746–751, 2010.

On the molecular interaction between albumin and ibuprofen: An AFM and QCM-D study.

The adsorption of proteins on surfaces often results in a change of their structural behavior and consequently, a loss of bioactivity. One experimental method to study interactions on a molecular level is single molecular force spectroscopy that permits to measure forces down to the pico-newton range. In this work, the binding force between human serum albumin (HSA), covalently immobilized on glutaraldehyde modified gold substrates, and ibuprofen sodium salt was studied by means of single molecular force spectroscopy. First of all, a protocol was established to functionalize atomic force microscopy (AFM) tips with ibuprofen. The immobilization protocol was additionally tested by quartz crystal microbalance with dissipation (QCM-D) and contact angle measurements. AFM was used to characterize the adsorption of HSA on gold substrates, which lead to a packed monolayer of thickness slightly lower than the reported value in solution. Finally, single molecule spectroscopy results were used to characterize the binding force between albumin and ibuprofen and calculate the distance of the transition state (0.6 nm) and the dissociation rate constant (0.055 s(-1)). The results might indicate that part of the adsorbed protein still preserves its functionality upon adsorption.

Inflammatory activation of human serum albumin- or ovalbumin-modified chitosan particles to macrophages and their immune response in human whole blood

Nanomaterials have been extensively used in the biomedical field. These nanoscale objects may either promote or restrain immune responses depending on their surface characteristics and compositions. In this study, chitosan (CS) particles prepared using an emulsion-crosslinking method were modified with different amounts of human serum albumin (HSA) and ovalbumin (OVA), resulting in four types of modified CS particles, i.e. CS@HSA-10, CS@HSA-57, CS@OVA-13 and CS@OVA-65, respectively. They had a similar size of about 150 nm in a dry state, and were swollen 2-3 fold in PBS. No significant cytotoxicity was determined toward in vitro cultured RAW264.7 and THP-1 cells. However, all the modified CS particles, in particular the OVA-modified ones (CS@OVA-13 and CS@OVA-65), induced significantly higher secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) compared with the negative control. In human whole blood, CS@OVA-13 and CS@OVA-65 were phagocytosed with a significantly higher ratio by granulocytes and monocytes, leading to the higher secretion of TNF-α, IL-1β and IL-8, and a larger extent of platelet activation than CS@HSA-10 and CS@HSA-57, respectively.

Preclinical In Vitro Safety Investigations of Submicron Sized Hemoglobin Based Oxygen Carrier HbMP-700: IN VITRO SAFETY INVESTIGATION OF HbMP-700

Hemoglobin-based oxygen carriers (HBOCs) are being developed as oxygen and plasma volume-expanding therapeutics though their potential to promote oxidative tissue injury and nitric oxide (NO) scavenging combined with vasoconstriction has raised safety concerns. Therefore, we focused on these aspects during preclinical studies performed with the recently introduced hemoglobin microparticles (HbMP-700). Besides oxidative stress, we investigated possible vasoconstrictory influence of HBOCs as well as genetic toxicity. The novel developed HbMP-700 presented here provides a high oxygen affinity which prevents premature oxygen oversupply and avoids vasoconstriction of small blood vessels in vitro. The size of these particles is 700 nm (larger than 100 nm and smaller than 1000 nm) in order to prevent penetration through the blood vessels endothelial gaps, NO-scavenging, and to avoid phagocytosis of large particles. We expect that the HbMP-700 meets the sophisticated requirements as a universal blood substitute.