Polymer-induced adhesion of endothelial cells.

The effects of neutral dextran concentration and molecular mass on the adhesion of endothelial cells (EC) to siliclad-covered glass surfaces were studied using interference reflection microscopy (IRM). Results indicate that close contact of the EC to the glass slides is markedly enhanced in the presence of 500 kDa dextran, with this increase reflected by both the speed of forming close contact as well as the size of the contact area. This increased adhesion is attributed to the reduction in surface concentrations of large polymers and, therefore, to the attractive forces caused by depletion interaction. Our findings suggest that depletion could play an important role in cell-cell or cell-surface interactions via accelerating and enhancing close contacts. This interaction should thus be considered in vivo and in vitro for specific potential applications, such as cell culture and cell adhesion to biomimetic surfaces. It should therefore be of particular interest in a wide range of biomedical applications.

Detection of acquired radioresistance in breast cancer cell lines using Raman spectroscopy and machine learning.

Radioresistance-a living cell's response to, and development of resistance to ionising radiation-can lead to radiotherapy failure and/or tumour recurrence. We used Raman spectroscopy and machine learning to characterise biochemical changes that occur in acquired radioresistance for breast cancer cells. We were able to distinguish between wild-type and acquired radioresistant cells by changes in chemical composition using Raman spectroscopy and machine learning with 100% accuracy. In studying both hormone receptor positive and negative cells, we found similar changes in chemical composition that occur with the development of acquired radioresistance; these radioresistant cells contained less lipids and proteins compared to their parental counterparts. As well as characterising acquired radioresistance in vitro, this approach has the potential to be translated into a clinical setting, to look for Raman signals of radioresistance in tumours or biopsies; that would lead to tailored clinical treatments.

Depletion interaction forces contribute to erythrocyte-endothelial adhesion in diabetes.

Abnormal adhesion of red blood cells (RBC) to the endothelium has been linked to the pathophysiology of several diseases associated with vascular disorders. Various biochemical changes on the outer membrane of RBC, as well as plasma protein levels, have been identified as possibly playing key roles, but the detailed interplay between plasma factors and cellular factors often remains unclear. In this work, we identified an alternative pathway by demonstrating that non-adsorbing macromolecules can also have a marked impact on the adhesion efficiency of RBC from patients with type 2 Diabetes (T2DM) to endothelial cells (EC). RBC isolated from blood samples of T2DM patients were suspended in isotonic solutions of dextran in order to mimic the impact of non-adsorbing macromolecules. Static and continuous flow adhesion assays were used to determine the adhesion behavior of T2DM RBC with EC and the results compared with those of normal controls. We found that the presence of non-adsorbing molecules promotes an increase in T2DM RBC - EC adhesion and that these RBC exhibit much greater adhesion than normal red cells. Our results thus suggest that the depletion mechanism might be an alternative phenomenon through which plasma proteins could cause enhanced RBC-EC adhesion in diabetes mellitus. These findings contribute towards the comprehensive understanding of pathophysiological mechanisms of vascular complications in diabetes and other diseases with similar vascular sequelae.

Phenazine methosulphate-treated red blood cells activate NF-κB and upregulate endothelial ICAM-1 expression.

Although enhanced Red Blood Cell (RBC) - Endothelial Cell (EC) interaction, as well as RBC induced EC activation, have been extensively studied in several RBC-linked pathologies, the specific individual effects of oxidatively modified RBC on EC activation has not yet been documented. However, increasing evidence in both experimental and clinical studies suggests that oxidatively modified RBC could be considered potential pathogenic determinants in several acute and chronic diseases displaying systemic oxidative stress. Therefore, the present study aimed to explore the specific effects of oxidized RBC interaction with endothelial cells on intracellular signaling pathways that promote EC activation. RBC were exposed to oxidative stress induced by phenazine methosulphate (PMS). It is shown that the interaction of oxidatively modified RBC with cultured human umbilical vein endothelial cells (HUVEC) results in: a) EC activation as indicated by the increased surface expression of intercellular adhesion molecule -1 (ICAM-1); b) the activation of transcription factor NF-κB, an indicator of cellular oxidant stress. These results emphasize the specific contribution of oxidatively modified RBC interaction to EC activation and their possible pathological role in vascular diseases and oxidative stress.

Non-adsorbing macromolecules promote endothelial adhesion of erythrocytes with reduced sialic acids.

BACKGROUND: Abnormal adhesion of red blood cells (RBCs) to vascular endothelium is often associated with reduced levels of sialic acids on RBC membranes and with elevated levels of pro-adhesive plasma proteins. However, the synergistic effects of these two factors on the adhesion are not clear. In this work, we tested the hypothesis that macromolecular depletion interaction originating from non-adsorbing macromolecules can promote the adhesion of RBCs with reduced sialic acid content to the endothelium. METHODS: RBCs are treated with neuraminidase to specifically remove sialic acids from their surface followed by the evaluation of their deformability, zeta potential and membrane proteins. The adhesion of these enzyme-treated RBCs to cultured human umbilical vein endothelial cells (ECs) is studied in the presence of 70 or 500kDa dextran with a flow chamber assay. RESULTS: Our results demonstrate that removal of sialic acids from RBC surface can induce erythrocyte adhesion to endothelial cells and that such adhesion is significantly enhanced in the presence of high-molecular weight dextran. The adhesion-promoting effect of dextran exhibits a strong dependence on dextran concentration and molecular mass, and it is concluded to originate from macromolecular depletion interaction. CONCLUSION: These results suggest that elevated levels of non-adsorbing macromolecules in plasma might play a significant role in promoting endothelial adhesion of erythrocytes with reduced sialic acids. GENERAL SIGNIFICANCE: Our findings should therefore be of great value in understanding abnormal RBC-EC interactions in pathophysiological conditions (e.g., sickle cell disease and diabetes) and after blood transfusions.

Layer-by-layer nanoparticles as an efficient siRNA delivery vehicle for SPARC silencing.

Efficient and safe delivery systems for siRNA therapeutics remain a challenge. Elevated secreted protein, acidic, and rich in cysteine (SPARC) protein expression is associated with tissue scarring and fibrosis. Here we investigate the feasibility of encapsulating SPARC-siRNA in the bilayers of layer-by-layer (LbL) nanoparticles (NPs) with poly(L-arginine) (ARG) and dextran (DXS) as polyelectrolytes. Cellular binding and uptake of LbL NPs as well as siRNA delivery were studied in FibroGRO cells. siGLO-siRNA and SPARC-siRNA were efficiently coated onto hydroxyapatite nanoparticles. The multilayered NPs were characterized with regard to particle size, zeta potential and surface morphology using dynamic light scattering and transmission electron microscopy. The SPARC-gene silencing and mRNA levels were analyzed using ChemiDOC western blot technique and RT-PCR. The multilayer SPARC-siRNA incorporated nanoparticles are about 200 nm in diameter and are efficiently internalized into FibroGRO cells. Their intracellular fate was also followed by tagging with suitable reporter siRNA as well as with lysotracker dye; confocal microscopy clearly indicates endosomal escape of the particles. Significant (60%) SPARC-gene knock down was achieved by using 0.4 pmole siRNA/µg of LbL NPs in FibroGRO cells and the relative expression of SPARC mRNA reduced significantly (60%) against untreated cells. The cytotoxicity as evaluated by xCelligence real-time cell proliferation and MTT cell assay, indicated that the SPARC-siRNA-loaded LbL NPs are non-toxic. In conclusion, the LbL NP system described provides a promising, safe and efficient delivery platform as a non-viral vector for siRNA delivery that uses biopolymers to enhance the gene knock down efficiency for the development of siRNA therapeutics.

Layer-by-layer polyelectrolyte-polyester hybrid microcapsules for encapsulation and delivery of hydrophobic drugs.

A two-step process is developed to form layer-by-layer (LbL) polyelectrolyte microcapsules, which are able to encapsulate and deliver hydrophobic drugs. Spherical porous calcium carbonate (CaCO3) microparticles were used as templates and coated with a poly(lactic acid-co-glycolic acid) (PLGA) layer containing hydrophobic compounds via an in situ precipitation gelling process. PLGA layers that precipitated from N-methyl-2-pyrrolidone (NMP) had a lower loading and smoother surface than those precipitated from acetone. The difference may be due to different viscosities and solvent exchange dynamics. In the second step, the successful coating of multilayer polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) onto the PLGA coated CaCO3 microparticles was confirmed with AFM and ζ-potential studies. The release of a model hydrophobic drug, ibuprofen, from these hybrid microcapsules with different numbers of PAH/PSS layers was investigated. It was found that the release of ibuprofen decreases with increasing layer numbers demonstrating the possibility to control the release of ibuprofen with these novel hybrid microcapsules. Besides loading of hydrophobic drugs, the interior of these microcapsules can also be loaded with hydrophilic compounds and functional nanoparticles as demonstrated by loading with Fe3O4 nanoparticles, forming magnetically responsive dual drug releasing carriers.

Surface functionalization of nanoparticles to control cell interactions and drug release.

Nanoparticles made from poly(dl-lactide-co-glycolide) (PLGA) are used to deliver a wide range of bioactive molecules, due to their biocompatibility and biodegradability. This study investigates the surface modification of PLGA nanoparticles via the layer-by-layer (LbL) deposition of polyelectrolytes, and the effects of these coatings on the release behavior, cytotoxicity, hemolytic activity, and cellular uptake efficiency. PLGA nanoparticles are modified via LbL adsorption of two polyelectrolyte pairs: 1) poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) and 2) poly(L-lysine hydrobromide) (PLL) and dextran sulfate (DES). It is demonstrated that both PAH/PSS and PLL/DES coatings suppress the burst release usually observed for unmodified PLGA nanoparticles and that the release behavior can be adjusted by changing the layer numbers, layer materials, or by crosslinking the layer constituents. Neither bare nor polyelectrolyte-modified PLGA nanoparticles show any signs of cytotoxicity. However, nanoparticles with a positively charged polyelectrolyte as the outermost layer induce hemolysis, whereas uncoated particles or particles with a negatively charged polyelectrolyte as the outermost layer show no hemolytic activity. Furthermore, particles with either PAH or PLL as the outermost layer also demonstrate a higher uptake efficiency by L929 fibroblast cells, due to a higher cell-particle affinity. This study suggests that LbL coating of PLGA nanoparticles can control the release behavior of bioactive molecules as well as the surface activity, therefore providing a promising strategy to enhance the efficiency of nanoparticulate drug-delivery systems.

Engineering of erythrocyte-based drug carriers: control of protein release and bioactivity.

This work reports the fabrication of layer-by-layer (LbL) polyelectrolyte coated erythrocyte carriers that provide a simple means for controlling the burst and subsequent release of lysozyme. Erythrocytes were loaded with RITC-lysozyme as model compound via the hypotonic dialysis method. An encapsulation efficiency of 41.6% and a loading amount of 12.7 pg/cell was achieved. It is demonstrated that these carriers maintain their shape and integrity similar to natural erythrocytes after the encapsulation procedures, and achieve a uniform distribution of the encapsulated lysozyme. The erythrocyte carriers were fixed with glutaraldehyde and then successfully coated with biocompatible polyelectrolytes, poly-L: -lysine hydrobromide and dextran sulfate, using the LbL method. It is demonstrated that the release profile of the encapsulated macromolecule can be regulated by adjusting the number of polyelectrolyte layers. Furthermore by adjusting the concentrations of the cross linking agent the activity of the encapsulated lysozyme can be well preserved. These core-shell microcapsules, consisting of erythrocytes loaded with bioactive substances and coated with a polyelectrolyte multilayer shell, hold promise for a new type of biocompatible and biodegradable drug delivery system.

Specific binding of red blood cells to endothelial cells is regulated by nonadsorbing macromolecules.

Abnormal adhesion of red blood cells to the endothelium has been linked to the pathophysiology of several diseases associated with vascular disorders. Various biochemical changes, including phosphatidylserine exposure on the outer membrane of red blood cells as well as plasma protein levels, have been identified as being likely to play a key role, but the detailed interplay between plasma factors and cellular factors remains unknown. It has been proposed that the adhesion-promoting effect of plasma proteins originates from ligand interaction, but evidence substantiating this assumption is often missing. In this work, we identified an alternative pathway by demonstrating that nonadsorbing macromolecules can also have a marked impact on the adhesion efficiency of red blood cells with enhanced phosphatidylserine exposure to endothelial cells. It is concluded that this adhesion-promoting effect originates from macromolecular depletion interaction and thereby presents an alternative mechanism by which plasma proteins could regulate cell-cell interactions. These findings should thus be of potential value for a detailed understanding of the pathophysiology of diseases associated with vascular complications and might be applicable to a wide range of cell-cell interactions in plasma or plasma-like media.

Non-adsorbing macromolecules in plasma induce erythrocyte adhesion to the endothelium.

Red blood cell (RBC) adhesion to the endothelium is usually insignificant. However, an enhanced adhesion can be observed in various pathological conditions such as diabetes mellitus or sickle cell disease, which is often accompanied by elevated levels of pro-adhesive plasma proteins such as fibrinogen. In the past, these proteins have only been considered to act as ligands, cross-linking the corresponding receptors on adjacent cells, but the detailed underlying mechanism often remained obscure. This work demonstrates that the presence of non-adsorbing polymers in plasma can also enhance the adhesion efficiency of RBCs to endothelial cells (ECs) through depletion interaction. Furthermore, adhesion of RBCs to ECs may be likewise promoted by the protein fibrinogen through depletion interaction. We propose an alternative mechanism for the pro-adhesive effects of plasma proteins and indicate that depletion interaction might play a significant role for the stabilization and destabilization of blood flow in health and disease.

Influence of layer-by-layer (LbL) assembled CaCO(3)-carriers on macrophage signaling cascades.

Numerous drawbacks in the current medical treatment of chronic inflammations still require the design of sensitive and gentle methods without side effects. Layer-by-layer (LbL) coated microcarriers loaded with a cocktail of anti-inflammatory substances are supposed to be a new challenge for the medical treatment of immunoreactive cells such as macrophages and polymorphonuclear leukocytes (PMN). Nevertheless, microcarrier application requires biocompatibility of the system itself. Therefore, the aim of this study was to investigate microcarrier CaCO(3) systems LbL coated with biopolymers and a lipid bilayer, respectively, regarding the maintenance of the release of pro-inflammatory cytokines as TNFα and IL1ß at normal levels. Only marginal increases after microcarrier interaction were allowed. The required microcarrier optimization results in the maximum use of a carrier/cell ratio of 1:1 for biopolymer-coated carriers and a carrier/cell ratio up to 5:1 for lipid-bilayer-coated carriers during the coincubation with macrophage-like cells. Low formation of reactive oxygen species (ROS) could not be maintained by either reduced carrier/cell ratios or by a surface lipid bilayer.

Erythrocyte-endothelium adhesion can be induced by dextran.

Plasma proteins have been identified to play a key role in the increased adhesiveness of red blood cells (RBC) to endothelial cells (EC) in various diseases associated with vascular complications. However, the underlying mechanisms on how plasma proteins facilitate adhesion remain unclear. In this study, we investigated if macromolecular depletion is able to induce adhesion of RBC to EC. RBC were suspended in solutions containing the neutral polyglucose dextran to mimic the effects of nonadsorbing macromolecules and the dynamics of RBC-EC adhesion were recorded using a parallel plate flow chamber system. Cell adhesion was markedly increased in the presence of dextran with a molecular mass larger than 70 kDa, with this increase reflected by both the number of cells adhering and the strength of the adhesion. This increased adhesiveness is attributed to reduced surface concentrations of the large polymers and hence attractive forces due to depletion interaction. Our results thus provide a rational explanation on how nonadsorbing plasma proteins could play a significant role in abnormal RBC-EC adhesion in vivo.

Colloidal DNA carriers for direct localization in cell compartments by pH sensoring.

Multifunctional colloidal microparticles allow the integration of various active agents as well as reporter molecules into one system without interfering combining delivery and sensing functions. In this study, calcium carbonate particles were functionalized with fluorescein isothiocyanate-labeled poly(allylamine hydrochloride) (FITC-PAH) allowing particle localization in cell compartments of different pH. Plasmid DNA (pEGFP-C1 and pDsRed1-N1) as a reporter agent for drug release in the cytoplasm and rhodamine-B-isothiocyanate-labeled protamine (RITC-PRM) were integrated into biocompatible and biodegradable PRM/DXS multilayers. The uptake and processing of the particles by HEK293T/17 cells were investigated via flow cytometry and confocal laser scanning microscopy. The presented data show a clear correlation between the fluorescence intensity of the FITC-labeled core, that is, the particle localization after cellular uptake, and the expression of fluorescent proteins by the cells without further cell staining. In conclusion, this particle design allows the simultaneous study of particle location and processing to monitor the transport and release of active agents and should thus be an invaluable tool for the study and design of nano- and microcarrier systems.

Role of macromolecular depletion in red blood cell adhesion.

The adhesion of red blood cells (RBCs) to cells or surfaces is of current basic science and clinical interest yet the details governing this process are still being explored. In this study, the effects of nonadsorbing polymers on the adhesion of RBC to albumin-coated glass have been investigated employing interference reflection microscopy. Our experimental results indicate that adhesion can be induced in the presence of dextran with a molecular mass >or=70 kDa and that the induced forces are strong enough to significantly suppress membrane undulations. The overall dependence of the adhesion energies on the polymer concentration is consistent with the assumption that macromolecular depletion induces this attractive interaction. In conclusion, our results indicate that depletion interaction might play a significant role in RBC adhesion via initiating close contacts, and thus suggest the importance of depletion forces for RBC interactions and its relevance to a wide variety of in vitro and in vivo cell-cell and cell-surface interactions.

Depletion of high molecular weight dextran from the red cell surface measured by particle electrophoresis.

The reversible aggregation of human red blood cells (RBC) by proteins or polymers continues to be of biological and biophysical interest, yet the mechanistic details governing the process are still being explored. A depletion model has been proposed for aggregation by the neutral polyglucose dextran and its applicability at high molecular weights has been recently documented. In the present study the depletion of high molecular weight dextrans on the red cell surface was measured as a function of polymer molecular mass (40 kDa-28 MDa), ionic strength (5 and 15 mM NaCl) and polymer concentration (< or =0.9 g/dL). The experimental data clearly indicate an increasing depletion effect with increasing molecular weight: the effects of medium viscosity on RBC mobility were markedly overestimated by the Helmholtz-Smoluchowski relation, with the difference increasing with dextran molecular mass. These results agree with the concept of polymer depletion near the RBC surface and lend strong support to a "depletion model" mechanism for dextran-mediated RBC aggregation. Our findings provide important new insight into polymer-RBC interactions and suggest the usefulness of this model for fundamental studies of cell-cell affinity and for the development of new agents to stabilize or destabilize specific bio-fluids.

Red blood cell adhesion can be reduced by non-reactive macromolecules.

To date, the mechanisms behind red blood cell (RBC) adhesion remain unclear. However, polymer depletion at the red cell surface has been shown to play a significant role. Interestingly, most previous studies have focused on the adhesion-promoting effects of one type of large polymer or plasma protein. However, the situation in vivo is more complex in that one needs to consider a mixture of various bio-macromolecules. To explore this complexity, Interference Reflection Microscopy was used to investigate how mixtures of various polymers affect RBC adhesion. RBC adhesion to albumin-coated glass coverslips was studied in the presence of two pro-adhesion polymers [dextran70 kDa and 35 kDa poly(ethylene glycol) (PEG 35)] with and without three types of smaller polymers: dextran 10 kDa, PEG 10 kDa and Poloxamer 188. Our findings show that the presence of small polymers can inhibit the adhesion-promoting effects of dextran 70 and PEG 35, with a more pronounced reduction for heterogeneous mixtures. Interpretation of our results in terms of the depletion model appears appropriate, in that our findings are consistent with the assumption that this reduction occurs because of an increase of small molecules in the depletion region. This study thus suggests that depletion interaction can control cell-cell interactions in complex environments (e.g., in vivo), and indicates that considering the interplay of all plasma constituents is important in order to understand the pathophysiology of diseases associated with cell adhesion and vascular complications.

Effects of dextran molecular weight on red blood cell aggregation.

The reversible aggregation of human red blood cells (RBC) by proteins or polymers continues to be of biologic and biophysical interest, yet the mechanistic details governing the process are still being explored. Although a depletion model with osmotic attractive forces due to polymer depletion near the RBC surface has been proposed for aggregation by the neutral polyglucose dextran, its applicability at high molecular mass has not been established. In this study, RBC aggregation was measured over a wide range of dextran molecular mass (70 kDa to 28 MDa) at concentrations

Depletion interactions in polymer solutions promote red blood cell adhesion to albumin-coated surfaces.

The effects of concentration and molecular weight of neutral dextrans on the adhesion of human red blood cells (RBC) to albumin-coated glass have been investigated using a parallel-plate flow chamber. Results indicate that the adhesion is markedly increased in the presence of 70 kDa and 500 kDa dextran, with this increase reflected by both the number of cells adhering and the strength of the adhesion. This increased adhesiveness is attributed to reduced surface concentrations of the large polymers and hence attractive forces due to depletion interaction. Depletion interaction brings the adjacent surfaces closer, leading to an increased number of binding sites available to the cell and thus more efficient and stronger adhesion of single cells. Our results suggest that depletion might play a role in other specific cell-cell or cell-surface interactions via initiating close contacts to allow specific binding.

Effects of neutral polymers on the mechanics of red blood cell adhesion onto coated glass surfaces.

BACKGROUND: Cell-cell and cell-surface adhesion modulated by water-soluble polymers continues to be of current interest, especially since prior reports have indicated a role for depletion-mediated attractive forces. OBJECTIVE: To determine the effects of concentration and molecular mass of the neutral polymer dextran (40 kDa to 28 MDa) on the adhesion of human red blood cells (RBC) to coated glass coverslips. METHODS: Confocal-reflection interference contrast microscopy (C-IRM), in conjunction with phase contrast imaging, was utilized to measure the adhesion dynamics and contact mechanics of RBC during the initial stages of cell contact with several types of substrates. RESULTS: Adhesion is markedly increased in the presence of dextran with a molecular mass ⩾ 70 kDa. This increased adhesiveness is attributed to reduced surface concentration of the large polymers and hence increased attractive forces due to depletion interaction. The equilibrium deformation of adhering RBC was modeled as a truncated sphere and the calculated adhesion energies were in close agreement with theoretical results. CONCLUSIONS: These results clearly demonstrate that polymer depletion can promote RBC adhesion to artificial surfaces and suggest that this phenomenon may play a role in other specific and non-specific cell-cell interactions, such as rouleau formation and RBC-endothelial cell adhesion.