Molecules Of Different Molecular Weights Research Articles

Neutral polymers partial exclusion in ionic solutions near charged membranes.

Neutral polymers have become a useful tool in biophysics. They are currently used in a variety of single-channel conductance measurements, to change solution conductivity and viscosity, to induce osmotic stress in polymer-excluded regions, or simply as crowding agents. Many experiments involve the proximity of neutral polymers to charged membranes or their confinement in aqueous pores formed by protein channels with charged residues. A well-known phenomenon relevant to this problem is the polarization of a neutral particle with dielectric properties different from the surrounding medium under the effect of an electric field. This polarization causes a dielectrophoretic force when inhomogeneous electric fields act on the particle and it has been extensively used in many biomedical applications involving particle trapping and cell separation, among others. In addition, whenever a neutral particle perturbs the equilibrium electric double layer near a charged surface, the osmotic pressure gradient acts as a net repulsive force that pushes the particle away from that surface. Both the dielectrophoretic force and the hydrostatic force contribute to exclude a neutral particle from a charged surface, provided the polarizability is lower in the particle than in the solution. Here we report analytical predictions and simulations of the exclusion of polyethylene glycol (PEG) molecules of different molecular weight from charged surfaces. Molecular dynamics of PEG near charged DPPS membranes and neutral DPPC membranes show a region of PEG exclusion near the membrane-solution interface for charged membranes. The simulations also point to a preferential alignment of PEG molecules near the charged membrane. The simulations are confirmed by preliminary neutron reflectometry data, which are consistent with an exclusion region of concentrated PEG solutions near the highly charged silicon dioxide/water interface.

The Impact of PEGylation on Cellular Uptake and In Vivo Biodistribution of Gold Nanoparticle MRI Contrast Agents.

Gold nanoparticles (GNPs) have immense potential in biomedicine, but understanding their interactions with serum proteins is crucial as it could change their biological profile due to the formation of a protein corona, which could then affect their ultimate biodistribution in the body. Grafting GNPs with polyethylene glycol (PEG) is a widely used practice in research in order to decrease opsonization of the particles by serum proteins and to decrease particle uptake by the mononuclear phagocyte system. We investigated the impact of PEGylation on the formation of protein coronae and the subsequent uptake by macrophages and MDA-MB-231 cancer cells. Furthermore, we investigated the in vivo biodistribution in xenograft tumor-bearing mice using a library of 4 and 10 nm GNPs conjugated with a gadolinium chelate as MRI contrast agent, cancer-targeting aptamer AS1411 (or CRO control oligonucleotide), and with or without PEG molecules of different molecular weight (Mw: 1, 2, and 5 kDa). In vitro results showed that PEG failed to decrease the adsorption of proteins; moreover, the cellular uptake by macrophage cells was contingent on the different configurations of the aptamers and the length of the PEG chain. In vivo biodistribution studies showed that PEG increased the uptake by tumor cells for some GNPs, albeit it did not decrease the uptake of GNPs by macrophage-rich organs.

Synthesis and Polyelectrolyte Functionalization of Hollow Fiber Membranes Formed by Solvent Transfer Induced Phase Separation.

Ultrafiltration membranes are important porous materials to produce freshwater in an increasingly water-scarce world. A recent approach to generate porous membranes is solvent transfer induced phase separation (STrIPS). During STrIPS, the interplay of liquid–liquid phase separation and nanoparticle self-assembly results in hollow fibers with small surface pores, ideal structures for applications as filtration membranes. However, the underlying mechanisms of the membrane formation are still poorly understood, limiting the control over structure and properties. To address this knowledge gap, we study the nonequilibrium dynamics of hollow fiber structure evolution. Confocal microscopy reveals the distribution of nanoparticles and monomers during STrIPS. Diffusion simulations are combined with measurements of the interfacial elasticity to investigate the effect of the solvent concentration on nanoparticle stabilization. Furthermore, we demonstrate the separation performance of the membrane during ultrafiltration. To this end, polyelectrolyte multilayers are deposited on the membrane, leading to tunable pores that enable the removal of dextran molecules of different molecular weights (>360 kDa, >60 kDa, >18 kDa) from a feed water stream. The resulting understanding of STrIPS and the simplicity of the synthesis process open avenues to design novel membranes for advanced separation applications.

PEG Equilibrium Partitioning in the α-Hemolysin Channel: Neutral Polymer Interaction with Channel Charges.

We study the interaction of neutral polyethylene glycol (PEG) molecules of different molecular weights (MWs) with the charged residues of the α-hemolysin channel secreted by Staphylococcus aureus. Previously reported experiments of PEG equilibrium partitioning into this nanopore show that the charge state of the channel changes the ability of PEG entry in an MW-dependent manner. We explain such an effect by parameter-free calculations of the PEG self-energy from the channel 3D atomic structure that include repulsive dielectrophoretic and hydrostatic forces on the polymer. We found that the pH-induced shift in the measured free energy of partitioning ΔΔGexp from single-channel conductance measurements agrees with calculated energy changes ΔΔEcalc. Our results show that the PEG-sizing technique may need corrections in the case of charged biological pores.

Evaluation of green synthetic depressants for sulfide flotation: Synthesis, characterization and floatation performance to pyrite and chalcopyrite

Native starch has a complex molecular structure that limits its ability as a selective depressant in sulfide flotation separations. This research study was conducted primarily to develop a series of starch-based molecules of different molecular weights with the aim of obtaining the most selective depressant of pyrite in chalcopyrite flotation. Five low molecular weight molecules were produced by the oxidative treatment of starch using H2O2 as the oxidant and FeSO4·7H2O as the catalyst. Oxidation treatment resulted in molecular modification of starch, producing low molecular weight molecules of grafted polar groups with increased reaction time. The molecules developed were investigated by microflotation to obtain the most selective and effective depressant for pyrite than chalcopyrite. The optimal and most selective depressant was named tricarboxystarch (sodium salt), also known as TCSS, based on the results of characterization measurements. The inhibitory effect of tricarboxystarch on pyrite was far more selective and effective than native starch. Good selective separation between pyrite and chalcopyrite was achieved by adding tricarboxystarch to a low pH solution. Tricarboxystarch contained three –COOH and several –OH groups in the skeleton, providing excellent depression of pyrite. This study is a basic research that sheds new light on the development and optimization of organic polymers for flotation technology.

A transistor-based label-free immunosensor for rapid detection of tau protein

Tau protein in cerebrospinal fluid (CSF) is a central and relevant biomarker of Alzheimer’s disease (AD) that correlates with the severity of dementia. Unfortunately, so far, direct label-free detection of tau remains a challenge. Here, we present a transistor-based biosensor that detects the net charge of tau protein directly under physiological conditions. To achieve this, readily available whole anti-tau IgG antibodies are co-immobilized on the sensor surface with polyethylene glycol (PEG) molecules of different molecular weight. We show that by increasing the PEG size from 10 kDa to 20 kDa, the electrical response upon binding of tau improves significantly. These results support recent theoretical work that predicted larger PEGs to form a thicker surface layer with a higher detectable analyte charge. With 20 kDa PEG, we demonstrate label-free tau detection in a wide concentration range with detection limits <1 pM in 150 mM buffer and cell culture media, as well as < 10 pM in artificial CSF. This purely electrical method allows fast and simple tau detection within 30 min without sample processing, washing steps, or labeled detection antibodies. By exchanging the capture antibody, the platform is also amenable to different biomarkers and may enable future diagnostic tools for AD and other diseases.

Distribution and Diffusion of Macromolecule Delivery to the Brain via Focused Ultrasound using Magnetic Resonance and Multispectral Fluorescence Imaging

Focused ultrasound (FUS), in combination with microbubble contrast agents, can be used to transiently open the blood–brain barrier (BBB) to allow intravascular agents to cross into the brain. Often, FUS is carried out in conjunction with magnetic resonance imaging (MRI) to evaluate BBB opening to gadolinium-based MRI contrast agents. Although MRI allows direct visualization of the distribution of gadolinium-based contrast agents in the brain parenchyma, it does not allow measurements of the distribution of other molecules crossing the BBB. Therapeutic molecules (e.g., monoclonal antibodies) are much different in size than MRI contrast agents and have been found to have different distributions in the brain after FUS-mediated BBB opening. In the work described here, we combined in vivo MRI and ex vivo multispectral fluorescence imaging to compare the distributions of MRI contrast and dextran molecules of different molecular weights (3, 70 and 500 kDa) after FUS-mediated BBB opening through a range of ultrasound pressures (0.18–0.46 MPa) in laboratory mice. The volume of brain exposed was calculated from the MRI and fluorescence images and was significantly dependent on both molecular weight and ultrasound pressure. Diffusion coefficients of the different-molecular-weight dextran molecules in the brain parenchyma were also calculated from the fluorescence images and were negatively correlated with the molecular weight of the dextran molecules. The results of this work build on a body of knowledge that is critically important for the FUS technique to be used in clinical delivery of therapeutics to the brain.

Polycaprolactone porous template facilitates modulated release of molecules from alginate hydrogels

The development and study of advanced methods to encapsulate and provide a controlled release of drugs and bioactive agents provide the opportunity to deliver active factors in situ. This acute delivery requires systems to be able to precisely control the release rate, however this has been challenging to achieve using a single component system. We developed novel Polycaprolactone (PCL) porous templates to control the release of encapsulated alginate hydrogel containing water-soluble molecules of different molecular weights. The presence of molecule-loaded alginate within the PCL porous templates was confirmed by infrared spectroscopy, fluorescence imaging and scanning electron microscopy. The influence of the molecular weight of the encapsulated molecule was investigated. The release profile of PCL-alginate displayed significant improvement on slowing the release of molecules of different molecular weight when compared with alginate alone. Based on the results, we can suggest that PCL porous templates can be a promising candidate to form composite delivery systems carrying high molecular weight hydrophilic agents for tissue engineering and drug delivery applications.

Optimisation of HPMC ophthalmic inserts with sustained release properties as a carrier for thermolabile therapeutics

A methodology was developed and optimised for the preparation of a new drug delivery system (DDS) with sustained release properties to allow ocular protein delivery and to limit destructive production steps during manufacturing. Elevated temperatures, shear forces and an oxidative environment should be avoided in order to prevent denaturation or oxidation of proteins. An aqueous HPMC solution was prepared using heat and casted into small semi-rod-shaped PVC blisters. The polymer solution was allowed to cool down and was partially dehydrated at room temperature. A drug solution containing glycerol, drug and water was subsequently added to rehydrate the partially dehydrated polymer matrix at a temperature of 2°C. Several parameters of the production process were varied to determine their influence on the release kinetics from HPMC inserts from three different molecules of different molecular weight. This production method was further optimised in order to shorten the rehydration time from weeks to days, while eliminating heat and shear forces on the selected drug molecules sodium fluorescein, lysozyme and albumin. Slow release kinetics were achieved for sodium fluorescein and lysozyme as model drug molecules. The higher molecular weight of albumin prevented a good penetration into the insert during the rehydration process resulting in predominantly burst release. The biocompatibility of a viscous HPMC solution was evaluated on SV40-human corneal epithelial cells with PrestoBlue® and no cytotoxic effects were observed.

Massive Parallel Analysis of Single Cells in an Integrated Microfluidic Platform.

New tools that facilitate the study of cell-to-cell variability could help uncover novel cellular regulation mechanisms. We present an integrated microfluidic platform to analyze a large number of single cells in parallel. To isolate and analyze thousands of individual cells in multiplexed conditions, our platform incorporates arrays of microwells (7 pL each) in a multilayered microfluidic device. The device allows the simultaneous loading of cells into 16 separate chambers, each containing 4640 microwells, for a total of 74 240 wells per device. We characterized different parameters important for the operation of the microfluidic device including flow rate, solution exchange rate in a microchamber, shear stress, and time to fill up a single microwell with molecules of different molecular weight. In general, after ∼7.5 min of cell loading our device has an 80% microwell occupancy with 1-4 cells, of which 36% of wells contained a single cell. To test the functionality of our device, we carried out a cell viability assay with adherent and nonadherent cells. We also studied the production of neutrophil extracellular traps (NETs) from single neutrophils isolated from peripheral blood, observing the existence of temporal heterogeneity in NETs production, perhaps having implications in the type of the neutrophil response to an infection or inflammation. We foresee our platform will have a variety of applications in drug discovery and cellular biology by facilitating the characterization of phenotypic differences in a monoclonal cell population.

Controlling the Integration of Polyvinylpyrrolidone onto Substrate by Quartz Crystal Microbalance with Dissipation To Achieve Excellent Protein Resistance and Detoxification.

Blood purification systems, in which the adsorbent removes exogenous and endogenous toxins from the blood, are widely used in clinical practice. To improve the protein resistance of and detoxification by the adsorbent, researchers can modify the adsorbent with functional molecules, such as polyvinylpyrrolidone (PVP). However, achieving precise control of the functional molecular density, which is crucial to the activity of the adsorbent, remains a significant challenge. In the present study, we prepared a model system for blood purification adsorbents in which we controlled the integration density of PVP molecules of different molecular weights on an Au substrate by quartz crystal microbalance with dissipation (QCM-D). We characterized the samples with atomic force microscopy, X-ray photoelectron spectroscopy, and QCM-D and found that the molecular density and the chain length of the PVP molecules played important roles in determining the properties of the sample. At the optimal condition, the modified sample demonstrated strong resistance to plasma proteins, decreasing the adsorption of human serum albumin (HSA) and fibrinogen (Fg) by 92.5% and 79.2%, respectively. In addition, the modified sample exhibited excellent detoxification, and the adsorption of bilirubin increased 2.6-fold. Interestingly, subsequent atomistic molecular dynamics simulations indicated that the favorable interactions between PVP and bilirubin were dominated by hydrophobic interactions. An in vitro platelet adhesion assay showed that the adhesion of platelets on the sample decreased and that the platelets were maintained in an inactivated state. The CCK-8 assay indicated that the modified sample exhibited negligible cytotoxicity to L929 cells. These results demonstrated that our method holds great potential for the modification of adsorbents in blood purification systems.

Erythrocyte lysis and Xenopus laevis oocyte rupture by recombinant Plasmodium falciparum hemolysin III.

Malaria kills more than 1 million people per year worldwide, with severe malaria anemia accounting for the majority of the deaths. Malaria anemia is multifactorial in etiology, including infected erythrocyte destruction and decrease in erythrocyte production, as well as destruction or clearance of noninfected erythrocytes. We identified a panspecies Plasmodium hemolysin type III related to bacterial hemolysins. The identification of a hemolysin III homologue in Plasmodium suggests a potential role in host erythrocyte lysis. Here, we report the first characterization of Plasmodium falciparum hemolysin III, showing that the soluble recombinant P. falciparum hemolysin III is a pore-forming protein capable of lysing human erythrocytes in a dose-, time-, and temperature-dependent fashion. The recombinant P. falciparum hemolysin III-induced hemolysis was partially inhibited by glibenclamide, a known channel antagonist. Studies with polyethylene glycol molecules of different molecular weights indicated a pore size of approximately 3.2 nm. Heterologous expression of recombinant P. falciparum hemolysin III in Xenopus oocytes demonstrated early hypotonic lysis similar to that of the pore-forming aquaporin control. Live fluorescence microscopy localized transfected recombinant green fluorescent protein (GFP)-tagged P. falciparum hemolysin III to the essential digestive vacuole of the P. falciparum parasite. These transfected trophozoites also possessed a swollen digestive vacuole phenotype. Native Plasmodium hemolysin III in the digestive vacuole may contribute to lysis of the parasitophorous vacuole membrane derived from the host erythrocyte. After merozoite egress from infected erythrocytes, remnant P. falciparum hemolysin III released from digestive vacuoles could potentially contribute to lysis of uninfected erythrocytes to contribute to severe life-threatening anemia.

Monitoring nutrient transport in tissue-engineered grafts

Limited nutrient diffusion in three-dimensional (3D) constructs is a major concern in tissue engineering. Therefore, monitoring nutrient availability and diffusion within a scaffold is an important asset. Since nutrients come in various forms, we have investigated the diffusion of the oxygen, luciferin and dextran molecules within tissue-engineered constructs using optical imaging technologies. First, oxygen availability and diffusion were investigated, using transgenic cell lines in which a hypoxia-responsive element drives expression of the green fluorescent protein gene. Using confocal imaging, we observed oxygen limitation, starting at around 200 µm from the periphery in the context of agarose gel with 1 million CHO cells. Diffusion of luciferin was monitored real-time in agarose gels using a cell line in which the luciferase gene was driven by a constitutively active CMV promoter. Gel concentration affected the diffusion rate of luciferin. Furthermore, we assessed the diffusion rates of fluorescent dextran molecules of different molecular weights in biomaterials by fluorescence recovery after photobleaching (FRAP) and observed that diffusion depended on both molecular size and gel concentration. In conclusion, we have validated a set of efficient tools to investigate molecular diffusion of a range of molecules and to optimize biomaterials design in order to improve nutrient delivery.

Tuneable nanochannel formation for sample-in/answer-out devices

Control of the dielectric breakdown of PDMS was achieved by limiting the current during the breakdown process. This enabled tuning of the nanochannel pore size and hence their permeability for molecules of different molecular weights. This method enabled the analysis of the drug quinine from whole blood in 3 min using a simple, disposable microfluidic device.

One‐Step Synthesis of PEGylated Gold Nanoparticles with Tunable Surface Charge

The present work reports a rapid, simple and efficient one‐step synthesis and detailed characterisation of stable aqueous colloids of gold nanoparticles (AuNPs) coated with unmodified poly(ethylene)glycol (PEG) molecules of different molecular weights and surface charges. By mixing and heating aqueous solutions of PEG with variable molecular chain and gold(III) chloride hydrate (HAuCl4) in the presence of NaOH, we have successfully produced uniform colloidal 5 nm PEG coated AuNPs of spherical shape with tunable surface charge and an average diameter of 30 nm within a few minutes. It has been found out that PEGylated AuNPs provide optical enhancement of the characteristic vibrational bands of PEG molecules attached to the gold surface when they are excited with both visible (532 nm) and NIR (785 nm) laser lines. The surface enhanced Raman scattering (SERS) signal does not depend on the length of the PEG molecular chain enveloping the AuNPs, and the stability of the colloid is not affected by the addition of concentrated salt solution (0.1 M NaCl), thus suggesting their potential use for in vitro and in vivo applications. Moreover, by gradually changing the chain length of the biopolymer, we were able to control nanoparticles’ surface charge from −28 to −2 mV, without any modification of the Raman enhancement properties and of the colloidal stability.

Engineering fibrotic tissue in pancreatic cancer: A novel three-dimensional model to investigate nanoparticle delivery

Pancreatic cancer contains both fibrotic tissue and tumor cells with embedded vasculature. Therefore anti-cancer nanoparticles need to extravasate from tumor vasculature and permeate thick fibrotic tissue to target tumor cells. To date, permeation of drugs has been investigated in vitro using monolayer models. Since three-dimensional migration of nanoparticles cannot be analyzed in a monolayer model, we established a novel, three-dimensional, multilayered, in vitro model of tumor fibrotic tissue, using our hierarchical cell manipulation technique with K643f fibroblasts derived from a murine pancreatic tumor model. NIH3T3 normal fibroblasts were used in comparison. We analyzed the size-dependent effect of nanoparticles on permeation in this experimental model using fluorescent dextran molecules of different molecular weights. The system revealed permeation decreased as number of layers of cultured cells increased, or as molecule size increased. Furthermore, we showed changes in permeation depended on the source of the fibroblasts. Observations of this sort cannot be made in conventional monolayer culture systems. Thus our novel technique provides a promising in vitro means to investigate permeation of nanoparticles in fibrotic tissue, when both type and number of fibroblasts can be regulated.

Impedance analysis of adherent cells after in situ electroporation: Non-invasive monitoring during intracellular manipulations

In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250 μm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature. The gold-film electrodes were also used to deliver well-defined AC voltage pulses of several volts amplitude and several hundred milliseconds duration to the adherent cells in order to achieve reversible membrane electroporation ( in situ electroporation = ISE). Electroporation-assisted introduction of membrane impermeable molecules into the cytoplasm was studied by using FITC-labeled dextran molecules of different molecular weights. Probes as big as 2 MDa were successfully loaded into the cells residing on the electrode surface. Time-resolved impedance measurements before and immediately after the electroporation pulse revealed the kinetics of membrane resealing as well as subsequent changes in cell morphology. Cells recovered from the electroporation pulse within less than 90 min. When membrane-impermeable, bioactive compounds like N 3 − or bleomycin were introduced into the cells by in situ electroporation, concomitant ECIS readings sensitively reported on the associated response of the cells to these toxins as a function of time (ISE-ECIS).

Extensional rheology of DNA suspensions in microfluidic devices

Newtonian liquids that contain even small amounts (∼ppm) of flexible polymers can exhibit viscoelastic behavior in extensional flows. Here, the effects of the presence of DNA molecules in viscous fluids on the dynamics of filament thinning and drop breakup are investigated experimentally in a cross-slot microchannel. Both bulk flow and single molecule experiments are presented. Suspensions of DNA molecules of different molecular weights (MW) are used, namely λ-DNA (MW = 3 × 107) and T4 DNA (MW = 1 × 108). Results of both dilute (c/c* = 0.5) and semi-dilute (c/c* = 1) suspensions are compared to those of a viscous, Newtonian liquid. Results show that the dynamics of the high MW, semi-dilute suspension of T4 DNA are similar to viscoelastic fluids such as slow, exponential decay of the fluid thread and beads-on-a-string morphology. The exponential decay rate of the filament thickness is used to measure the steady extensional viscosity of all fluids. We find that the semi-dilute T4 DNA suspension exhibits extensional strain rate thinning extensional viscosity, while for all other fluids the extensional viscosity is independent of strain rate. Direct visualization of fluorescently labeled λ-DNA molecules using high-speed imaging shows that the strong flow in the thinning fluid threads provide sufficient forces to stretch the majority of DNA molecules away from their equilibrium coiled state. The distribution of molecular stretch lengths, however, is very heterogeneous due to molecular individualism and initial conditions.

Chitosan and its derivatives as drug penetration enhancers

The review deals with the penetration enhancement properties of chitosan and its derivatives for oral, nasal, buccal and vaginal drug delivery applications. Penetration enhancement through the ocular epithelium and through the skin are beyond the scope of this paper. Extensive experimental evidence has been given of the absorption enhancement properties of chitosan with regard to hydrophilic molecules of different molecular weights. The mechanisms involved in the enhancement effect of chitosan are different depending on whether the epithelia are endowed with tight junctions (intestinal and nasal) or lack tight junctions (buccal and vaginal). Chitosan and its derivatives, when applied in solution to the mucosal site, are capable of facilitating the paracellular route by opening the tight junctions or by interacting with extra-cellular matrix components. The added value of chitosan nanocarriers in promoting drug absorption is also considered. Nanocarriers are capable of being internalized by cells in different areas, thus promoting drug transport. Moreover their mucoadhesive properties and their ability to protect macromolecular drugs from degradation indirectly contribute to enhancing absorption.

Solute Transport in Intervertebral Disc

Loss of nutrient supply to the human intervertebral disc (IVD) cells is thought to be a major cause of disc degeneration in humans. To address this issue, transport of molecules of different size have been analyzed by a combination of experimental and modeling studies. Solute transport has been compared for steady-state and transient diffusion of several different solutes with molecular masses in the range 3-70 kDa, injected into parts of the disc where degeneration is thought most likely to occur first and into the blood supply to the disc. Diffusion coefficients of fluorescently tagged dextran molecules of different molecular weights have been measured in vitro using the concentration gradient technique in thin specimens of disc outer annulus and nucleus pulposus. Diffusion coefficients were found to decrease with molecular weight following a nonlinear relationship. Diffusion coefficients changed more rapidly for solutes with molecular masses less than 10 kDa. Although unrealistic or painful, solutes injected directly into the disc achieve the largest disc coverage with concentrations that would be high enough to be of practical use. Although more practical, solutes injected into the blood supply do not penetrate to the central regions of the disc and their concentrations dissipate more rapidly. Injection into the disc would be the best method to get drugs or growth factors to regions of degeneration in IVDs quickly; else concentrations of solute must be kept at a high value for several hours in the blood supply to the discs.